CHAPTER
58  MEDICAL BACTERIOLOGY
Geraldine S. Hall, Gail L. Woods
SPECIMEN PROCESSING, 1114
Gram Stain, 1114
Culture Techniques, 1115
MEDICALLY IMPORTANT
BACTERIA, 1116
Gram-Positive Cocci, 1116
Gram-Positive Rods, 1123
KEY POINTS
• Bacteria can be categorized based on the Gram stain reaction
(gram-positive or gram-negative), shape (cocci, bacilli, coccobacilli,
spirochete), preferred atmosphere (aerobic, microaerophilic,
anaerobic), and presence or absence of spores; they can be identified
on the basis of key biochemical tests, antigenic components (e.g.,
cell wall antigens, toxins), and/or molecular features.
• Among the gram-positive cocci, the most important human
pathogens (and the infections they commonly cause) are
Staphylococcus aureus (skin and soft tissue infections, bacteremia,
toxic shock syndrome), Streptococcus pyogenes (pharyngitis and its
nonsuppurative complications, skin and soft tissue infections),
Streptococcus agalactiae (neonatal bacteremia and meningitis),
Streptococcus pneumoniae (community-acquired pneumonia,
meningitis), and Enterococcus faecalis and Enterococcus faecium
(nosocomial urinary tract infections and bacteremia).
• Among gram-positive bacilli, the most important human pathogens
(and the infections they commonly cause) are Listeria monocytogenes
(meningitis, bacteremia), Nocardia species (pneumonia, soft tissue
infections, brain abscess), Bacillus anthracis (skin and soft tissue
infections, pneumonia; a bioterrorism agent), and Corynebacterium
diphtheriae (diphtheria), which is rarely encountered in the clinical
laboratory in the United States.
• Among gram-negative cocci, the most important human pathogens
(and the infections they commonly cause) are Neisseria meningitidis
(meningitis), Neisseria gonorrhoeae (gonorrhea), and Moraxella
catarrhalis.
• Gram-negative bacilli include Enterobacteriaceae, many of which are
normal flora in the gastrointestinal tract; nonfermentative gram-
negative bacilli (e.g., Pseudomonas aeruginosa and Acinetobacter
baumanii), which are found in the environment and cause human
infection when host defenses are compromised; halophilic organisms
(Vibrio species); microaerophilic bacteria (Campylobacter, Helicobacter);
fastidious organisms (Legionella species, Bordetella species, Francisella
tularensis, Brucella species, Haemophilus species); and miscellaneous
infrequently encountered bacteria.
• Among the Enterobacteriaceae, the most important human
pathogens (and the infections they commonly cause) are Escherichia
coli (urinary tract infection, diarrhea, bacteremia), Klebsiella
pneumoniae and Klebsiella oxytoca (urinary tract infection, pneumonia,
bacteremia), Proteus species (urinary tract infection), Salmonella
species (diarrhea, typhoid fever), Shigella species (diarrhea),
Enterobacter species (nosocomial pneumonia, urinary tract infection,
bacteremia), and Serratia species (nosocomial pneumonia).
• Among the anaerobes, the most important human pathogens (and
the infections they commonly cause) are the Bacteroides fragilis group
(intraabdominal infections, abscesses); Clostridium species, especially
Clostridium perfringens (soft tissue infections, food poisoning),
Clostridium tetani (tetanus), and Clostridium difficile (antibiotic-
associated diarrhea); and non–spore-forming gram-positive anaerobes
such as Actinomyces israelii and Propionibacterium acnes.
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Gram-Negative Bacteria— Gram-Negative Bacteria—The
Cocci, 1129 HACEK Bacteria, 1141
Gram-Negative Bacteria— Miscellaneous Gram-Negative
Bacilli, 1131 Bacilli, 1142
Gram-Negative Bacteria— Anaerobic Bacteria, 1147
Nonfermentative
SELECTED REFERENCES, 1152
Bacilli, 1134
A wide variety of bacterial species may be recovered from clinical speci-
mens. To appropriately assess the clinical significance of these organisms,
an understanding of the normal bacterial flora present at different ana-
tomic locations is essential. In some cases, the number of organisms
present can be extremely high—for example, 106 organisms/cm2 of skin,
109 organisms/mL of oral secretions, and 1011 organisms/g of colon con-
tents. It is important to obtain samples with minimal contamination from
the normal flora (Miller et al, 2007; Baron & Thomson, 2011). This may
be difficult but can be optimally achieved if proper procedures are fol-
lowed. These procedures, along with processing techniques that serve to
enhance recovery of pathogenic microorganisms, are discussed in Chapter
64. This chapter begins with a short discussion of laboratory procedures
used to process a specimen for bacterial culture, which is followed by a
more in-depth discussion of the bacterial species commonly considered to
be human pathogens.
SPECIMEN PROCESSING
GRAM STAIN
Few would disagree that direct examination of a specimen with Gram stain
is one of the most valuable procedures performed by the microbiology
laboratory. The Gram stain result rapidly provides information that is used
by the clinician for selecting appropriate antimicrobial therapy; it also
helps the laboratory technologist assess the quality of the specimen and
the extent to which certain organisms recovered in culture will be worked
up. Organisms present in abundant quantity in specimens containing many
white blood cells are given more attention than those that are present in
smaller numbers in the absence of white blood cells. Multiple specimens
positive for similar organisms in smears and cultures contribute to increased
clinical significance of the results.
To prepare a smear for staining, an aliquot of the most purulent or
bloody portion of the specimen is placed on a clean microscopic slide in a
manner that provides both thick and thin areas. For sterile body fluids, a
cytocentrifuge may be used to concentrate the specimen by 10 to 100 times
(Baron & Thomson, 2011). The material on the slide is allowed to air-dry,
is fixed with methanol or gentle heat, and then is stained with Gram stain
reagents (crystal violet, Gram iodine, alcohol, and safranin). Organisms
that have a gram-positive cell wall will resist decolorization with methanol
and will retain the purple color of the crystal violet; organisms that have
a gram-negative cell wall will be decolorized and will stain red with safra-
nin counterstain.
Stained smears are initially examined using a low-power objective to
look for large structures, such as nematode larvae, Curschmann’s spirals,
large granules, grains, bacterial microcolonies, or fungal forms. An oil
immersion lens is then used to assess the type of bacteria present. Because
105 organisms/mL must be present to see one organism per oil immersion
field (1000×), smears must be examined carefully to detect small numbers
of organisms.
The organisms observed should be evaluated for size, shape, and
Gram reaction, which should be reported with as much description as
possible; reporting the presence of gram-positive cocci in pairs that
resemble S. pneumoniae (Fig. 58-1) is more helpful than simply reporting

Figure 58-1  Gram stain of a sputum smear shows neutrophils, debris,
and gram-positive diplococci, suggestive of pneumococcal infection (oil
immersion).
gram-positive cocci in chains. White or red blood cells should be quanti-
fied and reported, along with any intracellular bacteria observed. Correla-
tion of Gram stain observations with culture results is a good way to
check on the quality of the stains and culture. Demonstration of many
bacteria on Gram stain that do not grow out in culture may indicate
unusual organisms that require more specialized media or the inability of
laboratory personnel to recognize certain colonial types in culture, or it
could suggest a false-positive Gram stain result caused by contamination
of reagents or collection materials, such as swabs, or incorrect interpreta-
tion of Gram stain results. Gram stain results could indicate the need to
inoculate additional media for a specific specimen. For example, finding
many gram-negative coccobacilli in a respiratory specimen could indicate
the need for a chocolate plate to recover Haemophilus spp., which would
not be recovered on a blood agar plate. Other stains, such as the acridine
orange stain, can be utilized for staining blood culture bottles, cerebro-
spinal fluid (CSF), or buffy coat preparations. This fluorescent stain pro-
vides a rapid and, at times, more sensitive stain for bacteria and fungi
(Mirrett et  al, 1982; Adler et  al, 2003). Bacteria and fungi will produce
an orange fluorescence, and mammalian cells will stain green. Some expe-
rience is required for accurate interpretation of the acridine orange stain,
and correct preparation of the smears is necessary to avoid excessive cel-
lular material, which can result in too much cellular DNA that can mask
the presence of any bacterial DNA.
Many laboratories use probes for identification of specific bacteria or
fungi in blood culture bottles. These are commercially available using a
fluorescent in situ hybridization (FISH) format (Advandx, Woburn, Mass.).
For example, when gram-positive cocci are seen in clusters on Gram stain
of a blood culture bottle, the QuickFISH BC probe can be used to dif-
ferentiate S. aureus from coagulase-negative staphylococci, or nonstaphy-
lococcal organisms. A multicenter study combined this probe with the
company’s mecA XpressFISH® assay that detects presence of mecA in S.
aureus (SA) to identify 209/211 methicillin-resistant SA (MRSA) within 2
hours of finding gram-positive cocci in the blood culture bottles (Salimnia
et al, 2014). If gram-positive cocci are seen in pairs and chains, a probe is
available that can specifically identify E. faecalis, the Enterococcus Quick
FISH BC. Only 2 discordants were found when the probe was used to
differentiate E. faecalis versus non–E. faecalis enterococci in 173 positive
blood cultures in a multicenter study (Deck et al, 2014). The newest of
these bacterial probes can be used to identify specific gram-negative bacilli
when these are seen on Gram stain of the positive blood culture bottle.
Yeast probes—PNA FISH-Yeast Traffic Light (YTL)—using the same
format are available to quickly and rapidly differentiate among Candida
spp. (Gorton et al, 2014)
More recently, two other techniques for rapid identification of bacteria
(and fungi) in positive blood culture bottles have become available.
One uses Matrix-Assisted Laser Desorption Ionization-Time-of-Flight
(MALDI-TOF) (bioMerieux MS or Bruker), and the other uses a molecu-
lar technique, Verigene (Nanosphere, Inc, Northbrook, Ill.) and FilmArray
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(BioFire, Salt Lake City, Utah) to identify bacteria quickly from positive
blood culture bottles. In a study comparing PNA FISH, Verigene, and
MALDI-TOF for the identification of gram-positive cocci, PNA FISH
and Verigene were 98% concordant with routine methods as compared to
MALDI-TOF, which was less sensitive, with 80% concordance to species
level and 88% to genus level (Martinez et al, 2014).
CULTURE TECHNIQUES
Media for culture are selected to provide the optimal conditions for growth
of pathogens commonly encountered at a particular site or in a particular
type of specimen. Consideration is given to special growth requirements
of bacteria associated with a given type of infection or to the necessity of
selecting certain pathogenic bacteria from a mixed population of indige-
nous flora. Therefore, the media chosen may include selective and dif-
ferential media, in addition to standard enrichment agar.
Blood-supplemented agar is a good general growth medium and can
be used to demonstrate the hemolytic action of colonies on the red blood
cells. Antibiotics or chemicals can be added to create a selective medium
such as colistin–nalidixic acid (CNA) agar or phenylethyl alcohol agar, both
of which are used to inhibit the growth of gram-negative bacilli, while
permitting gram-positive bacteria to grow. Heating the blood to make
chocolate agar and adding vitamin supplements creates an enriched
medium with available hemin (X factor) and nicotinamide adenine dinu-
cleotide (V factor) for the isolation of Haemophilus spp. and other fastidious
bacteria. Gram-negative bacilli may be separated from gram-positive
bacilli by using bile salts and dye in a medium such as MacConkey’s agar,
which additionally divides the colonies into lactose-positive and lactose-
negative colonies, thus making it both selective and differential. Guidelines
for the selection of media to be used with different types of specimens are
provided in Table 58-1.
Bacterial cultures are generally incubated at 35° C and are examined
PART 7
initially after 18 to 24 hours of incubation. Addition of 5% to 10% carbon
dioxide (CO2) may be essential or stimulatory to the growth of N. gonor­
rhoeae, Haemophilus influenzae, and S. pneumoniae, and should be used
whenever feasible. Exceptions to this recommendation are those cultures
on differential and selective media in which the pH alteration (which can
be affected by added CO2) is used to differentiate colony types (e.g., xylose-
lysine-deoxycholate [XLD] agar, Hektoen enteric [HE] agar).
For recovery of anaerobes, inoculated media should be placed into an
anaerobic environment as quickly as possible. Several types of anaerobic
culture systems are available. One of these is the anaerobic jar, in which
water is added to a CO2 and hydrogen (H2) generator package, and oxygen
(O2) is catalytically converted to water with palladium-coated alumina
pellets contained in a lid chamber. A modification of this system is a trans-
parent plastic bag containing its own gas generator and palladium catalyst
and designed to hold an agar plate; these are often referred to as anaerobic
Bio-Bags.
Another approach to anaerobic culture is the anaerobic glove box or
chamber, which consists of a large, clear plastic, airtight chamber filled
with an oxygen-free gas mixture of nitrogen, hydrogen, and carbon
dioxide. Specimens, plates, and tubes are introduced into or removed
from the chamber through a gas interchange lock. Anaerobiosis is main-
tained by palladium catalysts and the hydrogen gas in the chamber. All
manipulations within the chamber are done with neoprene gloves sealed
to the chamber wall or, for “gloveless” systems, through a hole with
sleeves that seal tightly around the forearms. The chambers contain inter-
nal incubators that maintain the incubation temperature. Each of the
anaerobe systems has its advantages and disadvantages, but all are equally
effective in isolating clinically significant anaerobic bacteria from speci-
mens. A system for processing anaerobic specimens under a constant
anaerobic environnent to reduce the chance of excess exposure to oxygen
is available in the Anoxamat system (Summanen et  al, 1999; Shahin
et  al, 2003).
Bacterial cultures should be examined routinely after 18 to 24 hours of
incubation. The exception to this is the anaerobe culture, which is gener-
ally examined at 48 hours to allow these slower-growing bacteria to
produce visible colonies. In general, solid media are held for 48 hours, with
liquid media held for an additional 24 to 48 hours. If this is different for
specific organisms, it will be mentioned in the text.
A preliminary report is issued when the culture is first examined; this
report is updated as additional information becomes available. Certain
results (e.g., positive blood or CSF Gram stain, isolation of an organism
requiring infection control measures) are reported to the health care pro-
vider as soon as the information becomes available. Final reports are issued
when all work on a culture has been completed.
1115
 TABLE 58-1
58  Medical Bacteriology
Guidelines for Media Selection for Various Specimens*
MEDIA FOR RECOVERY OF
MEDIA FOR RECOVERY OF AEROBIC AND FACULTATIVELY ANAEROBIC BACTERIA ANAEROBIC BACTERIA

Specimen BAP MAC or EMB CBA Broth† Other BAP‡ BBE PEA

Body cavities Consider the use

of BCB for large
volumes of fluids
Fluids
Cerebrospinal X X X (for shunt
specimens)
Peritoneal X X X BCB X X X
Pleural; pericardial X X X BCB X
Synovial X X X BCB
Wounds
Aspirate X X X X X X
Swab§ X X
Tissue# X X X X X X X
Respiratory Tract
Sputum X X X
Throat X
Bronchoalveolar lavage X X X CYE¶
Brush; washings X X X X¶ X¶ X¶
Nasal X
Genitourinary
Vaginal/rectal for group B Lim broth Selective or
chromogenic
Streptococcus (GBS) GBS media
Other X X X GC media** X X X
Cervix GC media
Urethra/penis GC media
Urine
Midvoid X X Screen; chromagar
Suprapubic aspirate X X
Feces X EB HE or XLD; Campy
Eye X X X†† X††
Ear; internal aspirate X X X
Vascular catheters X

BAP, Blood agar plate; BCB, blood culture bottles; BBE, Bacteroides bile esculin agar; Campy, Campylobacter-selective medium; CBA, chocolate blood agar; CYE, charcoal yeast
extract, for Legionella or Nocardia requests; EB, enrichment broth, such as GN or Selenite broth, same for rectal swabs, minus the Campylobacter-selective culture; EMB,
eosin methylene blue; GBS, group B streptococcus; GC, gonococcus; HE, Hektoen enteric agar; Lim broth, enrichment broth for group B streptococcus; MAC, MacConkey’s
agar; PEA, phenylethyl alcohol; XLD, xylose-lysine-deoxycholate agar.
*Specific guidelines for individual organisms will be included where they are described in text.
†
Supplemented thioglycollate is the usual broth; when P. acnes is a suspected pathogen, broth should be incubated for at least 10 days; however, for aerobes, a brain-heart
infusion may be adequate.
‡
Consider a CDC BAP or a Brucella blood agar, or another “enriched” BAP for anaerobic recovery; a laked blood agar plate with antibiotics may also be appropriate.
§
Not recommended for anaerobic cultures.
#
If specific organisms, or situations, other media may be added.
¶
If a protected bronchoscope is used for collection of the specimen.
**Thayer-Martin or Martin-Lewis or other media enriched for recovery of N. gonorrhoeae.
††
If Propionibacterium acnes is suspected in cases of endophthalmitis, a thioglycollate broth and/or anaerobic BAP may be used.

Tests useful for distinguishing staphylococci from Micrococcus and

MEDICALLY IMPORTANT BACTERIA
GRAM-POSITIVE COCCI
Staphylococcus
Characteristics
Staphylococci are catalase-positive spherical cocci that often appear in
grape-like clusters in stained smears (Fig. 58-2). They grow well on any
peptone-containing nutrient medium under aerobic and anaerobic condi-
tions and may produce hemolysis of various species of animal blood cells
and yellow or orange pigment on certain types of agar. Growth of staphy-
lococci is readily detected on blood agar plates or in various types of
nutrient broth. A selective medium for the isolation of S. aureus is one
containing 7.5% to 10% sodium chloride (NaCl) with mannitol.
Kocuria spp. (generally considered nonpathogenic) are listed in Table 58-2
(Becker & von Eiff, 2011). S. aureus is differentiated from other species of
staphylococci principally by its production of coagulases, which are capable
of clotting plasma. Two antigenically distinct forms of coagulases have
been recognized: One bound to the cell wall is called clumping factor and
is detected with the slide coagulase test, and the other is free from the cell
wall and is detected with the tube coagulase test (often considered the
definitive test for the presence of coagulase enzyme). Commercial latex
agglutination products are available that detect clumping factor and
protein A in S. aureus with good sensitivity and specificity. These assays
may be appropriate in situations in which reproducibility of the test is in
question because of the inexperience of technologists performing the assay.
A FISH product (S. aureus peptide nucleic acid FISH) is also available for
the differentiation of S. aureus from coagulase-negative staphylococci in a

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Figure 58-2  Cytocentrifuge preparation of cerebrospinal fluid stained with
Gram stain shows many neutrophils, smooth amorphous material, and gram-
positive cocci in pairs, short chains, and clusters, suggestive of staphylococcal
infection (oil immersion).
TABLE 58-2
Tests Differentiating Staphylococci from Micrococci and
Kocuria spp.
Staphylococcus Micrococcus
spp. spp.
Lysostaphin susceptibility S R
Aerobic acid production + –
from glycerol
Anaerobic acid production +/– – Delayed +
from glucose
Furazolidone (100-µg disk) S R
Bacitracin susceptibility R S
(0.04 U)
Modified oxidase – +
Adapted from Becker K, von Eiff C: Staphylococcus, Micrococcus, and other catalase
positive cocci. In Versalovic J, Carroll KC, Funke G, et al, editors: Manual of clinical
microbiology, ed 10, Washington, DC, 2011, American Society for Microbiology,
p309.
+, Positive result; –, negative result; R, resistant; S, susceptible.
blood culture bottle found positive for gram-positive cocci in clusters. In
addition, many laboratories are now utilizing polymerase chain reaction
(PCR) assays for detection of S. aureus in nasal swabs and directly from
positive blood culture bottles. Many of these assays enable detection of
methicillin-resistant S. aureus (MRSA) versus methicillin-susceptible
S. aureus.
Clinical Manifestations and Pathogenesis
S. aureus may be present among the indigenous flora of the skin, eye, upper
respiratory tract, gastrointestinal tract, urethra, and, infrequently, vagina.
Therefore, infection may arise from an endogenous or an exogenous
source. Factors of importance in the development of infection due to S.
aureus include breaks in the continuity and integrity of mucosal and cuta-
neous surfaces, the presence of foreign bodies or implants, prior viral
diseases, antecedent antimicrobial therapy, and underlying diseases with
defects in cellular or humoral immunity.
Infections caused by S. aureus may affect multiple organ systems.
Among the most common are those involving the skin and its appendages,
such as impetigo, folliculitis, mastitis, and infection of surgical wounds. S.
aureus is among the leading causes of bacteremia in hospitalized patients,
and it may cause endocarditis, particularly in persons with left-sided val-
vular heart disease and in intravenous drug users. S. aureus is the most
common cause of spinal epidural abscess and suppurative intracranial phle-
bitis, and it may be recovered from brain abscesses, typically following
trauma. Meningitis caused by S. aureus is uncommon and generally follows
head trauma or a neurosurgical procedure.
S. aureus is responsible for many cases of osteomyelitis, is the most
common cause of septic arthritis in prepubertal children, and is occasion-
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ally responsible for septic arthritis in adults. S. aureus is an infrequent cause
of community-acquired pneumonia but a common cause of nosocomial
pneumonia, which usually follows aspiration of endogenous nasopharyn-
geal organisms. Predisposing factors include infection with measles or
influenza A virus, cystic fibrosis, and immune deficiency. Urinary tract
infections caused by S. aureus are rare, but cases of pyelonephritis and
intrarenal and perirenal abscesses can be found.
Several factors play a role in the virulence of S. aureus. The capsule, if
present, has antiphagocytic properties. Cell wall peptidoglycans have
endotoxin-like activity, stimulating the release of cytokines by macro-
phages, activation of complement, and aggregation of platelets. Protein A,
an immunologically active substance in the cell wall, has antiphagocytic
properties that are based on its ability to bind the Fc fragment of immu-
noglobulin IgG. Other surface proteins, designated as microbial surface
components recognizing adhesive matrix molecules, may play an impor-
tant role in the ability of staphylococci to colonize host tissues (Speziale
et al, 2009). Recently, the significant involvement of superantigens of S.
aureus in sepsis, endocarditis, and acute kidney injury (abscess) has been
elucidated (Salgado-Pabón et al, 2013).
S. aureus produces numerous toxins. The exotoxin TSST-1 is respon-
sible for toxic shock syndrome, and enterotoxins A to E are responsible
for staphylococcal food poisoning. The exfoliative toxins—epidermolytic
toxins A and B—cause skin erythema and separation, as seen in scalded
skin syndrome. Various enzymes are also produced, including protease,
lipase, and hyaluronidase, all of which destroy tissue and probably function
to facilitate the spread of the infection.
Toxin-mediated diseases caused by S. aureus include scalded skin
syndrome, food poisoning, and toxic shock syndrome. Scalded skin syn-
Kocuria drome occurs in infants infected with a strain of S. aureus producing
spp. exfoliative toxin. The illness begins abruptly with erythema, followed in
2 to 3 days by the formation of flaccid bullae, which slough, leaving
R denuded areas that eventually resolve completely. Staphylococcal food
PART 7
+ poisoning, characterized by nausea, vomiting, abdominal cramps, and
diarrhea, occurs 1 to 6 hours after ingestion of foods contaminated with
preformed staphylococcal enterotoxin, which is usually introduced into
the food product by food handlers who prepare and/or serve the food
R (Baumgartner et al, 2014).
S
Toxic shock syndrome is a multisystem disease affecting individuals
who have no antibodies to TSST-1 and are colonized or infected with
strains of S. aureus producing TSST-1 or rarely enterotoxin B or C. Toxic
+
shock syndrome (TSS) is primarily the result of a superantigen-mediated
cytokine storm and M protein-mediated neutrophil activation, resulting in
the release of mediators leading to respiratory failure, vascular leakage, and
shock (Low, 2013). The illness is most common in women 15 to 25 years
of age who use tampons during menstruation, but it also may occur in
nonmenstruating individuals, including women in the postpartum period,
male or female patients with a surgical wound or other focal infection, and
individuals who have had a surgical procedure in the nose or sinuses. Toxic
shock syndrome begins abruptly with fever, myalgias, vomiting, and diar-
rhea, followed by hypotension, hypovolemic shock, and an erythematous
rash that frequently involves the palms and soles and desquamates in 1 to
2 weeks. The diagnosis is clinical; isolation of S. aureus from any site is not
required. Full recovery is the rule, although repeated episodes may occur
(Becker & von Eiff, 2011).
Over the past 10 to 15 years, cases of community-acquired infection
with S. aureus that are oxacillin resistant (CA-MRSA) have become more
common. In these isolates, a toxin referred to as Panton-Valentine leuko-
cidin toxin (PVL), which has rarely been associated with hospital-acquired
strains of S. aureus (Becker & von Eiff, 2011), has been found. PVL has
been shown to be responsible for necrotizing skin and soft tissue infections
and has been infrequently demonstrated to cause a necrotizing and occa-
sionally fatal pneumonia (Hageman et al, 2006; MMWR, 2007a; Moskow-
itz & Wiener-Kronish, 2009, Toro et al, 2014). Individuals who were
initially felt to be at greatest risk are children involved in contact sports
and individuals in institutions such as prisons (MMWR, 2003; Pan et al,
2003). These CA-MRSA strains, unlike hospital-acquired strains of MRSA
(HA-MRSA), are often susceptible to most non–β-lactam classes of anti-
biotics. HA-MRSA strains are usually resistant to all antibiotics except the
glycopeptides, such as vancomycin. The mechanism of oxacillin resistance
is the same in CA-MRSA and HA-MRSA—the presence of a mecA gene
that is responsible for production of a new penicillin-binding protein
(PBP-2a or PBP-2′). However, the chromosomal cassette that houses the
CA-MRSA mecA gene is different from and much smaller than that con-
taining the mecA gene of HA-MRSA. Many experts believe that, in time,
blending of CA-MRSA and HA-MRSA strains will occur, and without
molecular typing of any isolate, it will be difficult to distinguish them. The
1117
 predominant strain of CA-MRSA in the United States is type USA 300
58  Medical Bacteriology
(Becker & von Eiff, 2011).
S. aureus bacteremia can be caused by community and nosocomial
strains of S. aureus. A recent study compared the risk factors, morbidity,
and mortality due to bacteremia related to health care–associated MRSA
(HCA-MRSA) bacteremia versus CA-MRSA bacteremia. Patients with
CA-MRSA bacteremia were likely to have diabetes mellitus, chronic liver
disease, and HIV infection, perhaps reflecting the community nature of
acquisition of the SA; in those patients with bacteremia due to HCA-
MRSA occurring with 48 hours or more of hospitalization, the risk factors
were found to include the presence of a central venous catheter, solid
tumor, chronic renal failure, and prior hospitalizations and previous anti-
biotic therapy. Those with bacteremia caused by HCA-MRSA that
occurred with less than 48 hours of hospitalization were more prone to
have had previous hospitalizations, been in long-term care facilities, and
have had received corticosteroid therapy (Bassetti et al, 2012).
Infections caused by coagulase-negative species (CoNS) of Staphylococ­
cus usually occur in association with foreign bodies, especially implanted
prosthetic valves, joints, and shunts. Isolates of CoNS are usually consid-
ered less pathogenic than S. aureus, although that varies among the species
and strains (Becker et al, 2014). The presence of biofilms and antibiotic
resistance of the species appears to be associated with bacteremia, whereas
specific adhesion capabilities of the CNS were associated with prosthetic
joint infections (Giormezis et al, 2014). CoNS are one of the most
common organisms associated with CSF shunt infections; they are rarely
involved in urinary tract infections, pneumonia, or skin and soft tissue
infections. More than 20 species of CoNS are known, of which S. epider­
midis is the species most frequently involved in such infections. S. sapro­
phyticus is an important cause of bacteriuria, particularly among sexually
active young women. S. hemolyticus, reported to rank second in frequency
to S. epidermidis in clinical specimens, can be resistant to vancomycin, an
agent to which most CoNS are susceptible (Giormezis et al, 2014). S.
lugdunensis can appear morphologically similar to S. aureus (i.e., in produc-
tion of a narrow zone of β-hemolysis on blood agar plates) and on occa-
sion will test positive in some assays for coagulase. However, it is usually
classified as a coagulase-negative staphylococcus. Clinically, it will act
more aggressively than most other CoNS and in this way mimics S. aureus
infection, including its role as an agent of endocarditis, osteomyelitis, and
other more severe staphylococcal infections (Sabe et al, 2014). It is impor-
tant to distinguish S. lugdunensis from other CoNS because the break-
points one uses (according to the Clinical Laboratory and Standards
Institute [CLSI]) for the interpretation of susceptibility results versus oxa-
cillin (or cefoxitin) should be those that are used to interpret S. aureus and
not those used to interpret breakpoints for oxacillin (or cefoxitin) versus
CoNS (CLSI, 2014).
Laboratory Diagnosis
The observation microscopically of typical rounded, gram-positive cocci
in clusters in smears of material taken from previously unopened or und-
rained lesions, or in smears of broth from a positive blood culture, is
indicative of staphylococcal infection.
S. aureus produces coagulase, an enzyme that binds plasma fibrinogen,
causing the organisms to agglutinate or plasma to clot; only rare strains
of other staphylococci are coagulase positive. More than 95% of isolates
of S. aureus are identified by the slide coagulase test, which detects
cell-bound enzyme (clumping factor); nearly 100% of all isolates are
identified by tube coagulase tests, which detect free coagulase (Becker &
von Eiff, 2011).
The slide coagulase test is performed by mixing a dense emulsion of
the organism with plasma on a glass slide. The test is positive if clumping
occurs within 30 seconds. Staphylococcus lugdunensis and Staphylococcus
schleiferi are two other staphylococci that may give a positive result with
this slide coagulase test. A control that consists of emulsifying the suspect
colony in saline should be run with each slide test to ensure that autoag-
glutination does not occur. If autoagglutination is present, slide test results
should be considered insufficient for determination of the coagulase nature
of the isolate.
For the tube coagulase test, several colonies are transferred into a tube
containing plasma that is incubated at 35° C for 4 hours and then is exam-
ined for clot formation. If no clot has formed, the tube is reincubated at
room temperature and reexamined after a total of 24 hours of incubation.
The test should be examined after 4 hours because most isolates of S.
aureus produce a clot within this interval, but some strains produce a
fibrinolysin that can lyse the clot, thus producing a false-negative reaction
if the test is observed only after 24 hours. Staphylococcus intermedius and
Staphylococcus hyicus will also be positive with the tube coagulase test, but
1118
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they are primarily pathogens of animals and are encountered only rarely
in human specimens.
Several commercial latex agglutination assays are available for
rapid identification of S. aureus. These assays detect protein A and clump-
ing factor; some also detect capsular polysaccharide, which may improve
the ability to detect methicillin (oxacillin)-resistant S. aureus. S. sapro­
phyticus and Staphylococcus sciuri are two other staphylococcus species that
may be latex agglutination positive, along with the rare Micrococcus
spp.; however, these should all be slide coagulase negative (Becker & von
Eiff, 2011).
Many species of CoNS have been recognized; however, with the excep-
tion of S. epidermidis, S. lugdenensis, and S. saprophyticus, which is resistant
to novobiocin, identification of these isolates to the species level is not
practical or clinically indicated in every culture. Identification to species
may be needed if isolates are found repeatedly in sterile sites, if S. lugdu­
nensis is being ruled out, and/or if correlation between isolates in a patient
is being sought to increase the likelihood that the two isolates are the same
and therefore are potentially clinically relevant. If necessary, attempts to
identify these isolates to the species level may be made using commercially
available identification kits or with use of Maldi-Tof (Peel et al, 2014).
Alternatively, isolates may be sent to a reference laboratory capable of
performing standard biochemical assays or molecular assays such as 16S
ribosomal RNA (rRNA) analysis (Loonen et al, 2012). Staphylococci may
be classified into strains for epidemiologic purposes in attempting to iden-
tify common sources of infection on the basis of their susceptibility to
different bacteriophages, plasmid profiles, cellular fatty acids, electropho-
resis of multilocus enzymes, or chromosomal molecular typing (pulsed
field gel electrophoresis and repetitive PCR). These tests are generally
available only through reference laboratories.
Antimicrobial Susceptibility
More than 90% of staphylococci are resistant to penicillin due to inducible
plasmid-encoded β-lactamase. A chromogenic β-lactamase test (i.e., nitro-
cefin disk test) or a penicillin disk diffusion zone edge test may be per-
formed. The latter is considered more sensitive than the nitrocefin assay,
but either can be used. When β-lactamase is positive by either, isolates
should be reported as resistant to penicillin, but some resistant isolates
could be missed by the nitrocefin β-lactamase test. If penicillin is being
considered for treatment of serious S. aureus infections, a zone edge disk
diffusion test or an MIC should be performed for confirmation; MICs to
penicillin of 0.12 or less are interpreted as susceptible (CLSI, 2014). Resis-
tance to the penicillinase-resistant penicillins (methicillin, oxacillin, nafcil-
lin) occurs in up to 80% of coagulase-negative staphylococci and in more
than 50% of isolates of hospital-acquired S. aureus. Resistance to this group
of antimicrobial agents is mediated by the mecA gene, which encodes an
altered penicillin-binding protein, PBP-2a. Resistance typically is hetero-
geneous, meaning that only rare cells may (1 in 104 to 108) express the
resistance trait. Because of this, specific guidelines must be followed to
ensure detection. An oxacillin disk should no longer be used to detect
resistance, but rather CLSI has suggested that if disk diffusion is used as
the method of detection of MRSA, a 30-µg cefoxitin disk test is the better
indicator of resistance than oxacillin. To predict the presence of mecA-
mediated resistance in S. aureus (and S. lugdunensis), the CLSI recommends
that isolates with zone sizes 22 mm or larger can be reported as susceptible
(S), and those with zone sizes 21 mm or smaller to cefoxitin can be reported
as oxacillin resistant (R) (Velasco et al, 2005; CLSI, 2014). Likewise, for
coagulase-negative staphylococci (except S. lugdunensis), 25 mm or larger
can be reported as susceptible, and 24 mm or smaller can be reported as
oxacillin resistant with cefoxitin disks (CLSI, 2014). Oxacillin in cation-
supplemented Mueller-Hinton broth containing 2% NaCl should be used
for microdilution testing; microtiter trays should be incubated a full 24
hours at 35° C. To screen isolates of S. aureus for oxacillin resistance,
Mueller-Hinton agar supplemented with 4% NaCl and containing 6 µg/
mL of oxacillin is spot inoculated with a cotton swab, and plates are incu-
bated for 24 hours at 35° C. All reports should list the results for oxacillin
and not for cefoxitin. Any growth on or in screening medium suggests an
MRSA, and further testing should be done for confirmation. This could
include either detection of mecA by molecular methods or detection of
PBP2a. There is a newly described mec A, the mec C gene, which cannot
be detected by the molecular mecA assays; mecC may be detected in tests
for PBP2a (Paterson et al, 2014).
For coagulase negative staphylococci (CNS) other than S. lugdenensis,
a broth microdilution using oxacillin (≤0.25 µg/ml = S) can be used, or if
using disk diffusion, with cefoxitin (≥25 mm zone diameter = S). If any
species of CoNS other than S. epidermidis has an MIC to oxacillin between
0.5 µg/ml and 2.0 µg/ml, a mecA or PBP2a assay should be used for
confirmation because the broth microdilution may overcome resistance in
these strains (CLSI, 2014).
Several assays have been developed for rapid detection of oxacillin
resistance. These include nucleic acid amplification, nucleic acid probe
assays for mecA, and latex agglutination assays for PBP-2a (the MRSA
Screen Test, Denka-Seiken Co., Tokyo, Japan) and PBP-2′ (Oxoid Ltd.,
Basingstoke, U.K.), Mastalex test (Mast Diagnostics, Bootle, U.K.), and
the Slidex MRSA detection assay (bioMerieux, Raleigh-Durham, N.C.)
and Clearview (Inverness Medical Innovations, Scarborough, Mass.)
(Bowers et al, 2003; Chediac-Tannoury & Araj, 2003; Chapin & Musgnug,
2004; Nonhoff et al, 2012). Not all of these assays are FDA cleared in
the United States; some have been used for direct detection of oxacillin
resistance in blood cultures positive for S. aureus. For direct detection of
MRSA in nasal swabs, two approaches can be used. Newer chromogenic
media specific for the detection of MRSA require overnight incubation
but allow easy detection of specific-colored colonies that are positive for
the presence of mecA. Those commercially available are MRSASelect
(bioRad Laboratories, Redmond, Wash.), CHROMagar MRSA (BD,
Sparks, Md.), Brilliance MRSA (Oxoid, Thermofisher Scientific, Lenexa,
Kan.), and MRSA-ID (bioMerieux, Raleigh-Durham, N.C.) (Perry et al,
2004; Manickam et al, 2013; Veenemans et al, 2013). Not all are FDA
cleared in the United States; most studies indicate they work well in detect-
ing MRSA but are less sensitive than the available molecular assays. They
are, however, less expensive than the molecular assays.
Three molecular assays that can detect MRSA directly in clinical speci-
mens (nasal swabs, blood cultures, and other) within 90 minutes are Gene-
Ohm (Becton Dickinson Microbiology Systems, Sparks, Md.), the Xpert
MRSA (Cepheid, Sunnyvale, Calif.), and Light Cycler MRSA Advanced
Test (Roche, Basel, Switzerland) (Warren et al, 2004; Al-Haj-Hussein
et al, 2005; Frey et al, 2011; Buchan et al, 2014). Both Gene-OHM and
Cepheid have assays for the detection of S. aureus and MRSA in clinical
specimens and from positive blood cultures. Although oxacillin-resistant
staphylococci may appear to be susceptible to cephalosporins, they should
be considered resistant to all β-lactam agents (penicillins, cephalosporins,
and carbapenems). Hospital-acquired strains usually are resistant to many
non–β-lactam antibiotics as well. Many of the CA-MRSA strains still
remain susceptible to most non–β-lactam antibiotics (Daum et al, 2002;
David et al, 2014).
Clindamycin may be used to treat staphylococcal infection. Inducible
resistance, due to mechanisms involving a class of enzyme-inactivating
genes referred to as erm genes, may not be detected in routine susceptibil-
ity testing. This erm gene also confers cross-resistance to the macrolides
(e.g., erythromycin) and streptogramins (quinupristin-dalfopristin). An
isolate of S. aureus that is resistant to erythromycin but susceptible to
clindamycin in a minimum inhibitory concentration (MIC) test (broth or
agar dilution or E-test) should be evaluated for inducible resistance to
clindamycin by the “D-zone” test as follows. A 15-µg erythromycin disk
and a 2-µg clindamycin disk are placed 15 mm apart on the surface of a
blood agar plate inoculated with the isolate in question. After overnight
incubation, if there is inducible resistance to clindamycin, blunting of the
clindamycin zone of inhibition will be seen on the side near the erythro-
mycin disk, giving the appearance of a D zone (CLSI, 2014).
Vancomycin resistance, although rarely seen in S. aureus, is a serious
issue that laboratories need to be aware of and should screen for. Isolates
of vancomycin intermediately susceptible S. aureus (VISA) have MICs in
the intermediate range (4-8 µg/mL) (Cosgrove et al, 2004). A number of
cases of vancomycin-resistant S. aureus with vancomycin MICs as high as
1024 µg/mL have been reported (MMWR, 2004). Because the latter have
not been uniformly detected in automated systems for susceptibility
testing, the Centers for Disease Control and Prevention (CDC) recom-
mends that all S. aureus isolates tested on an automated instrument should
also be tested by a vancomycin screen assay to ensure that the correct MIC
is determined. This is usually done by inoculating 100 µL of a 0.5 McFar-
land suspension of S. aureus to a Brain Heart Infusion Agar (BHIA) plate
containing 6 µg/mL of vancomycin, and incubating overnight at 35 °C. If
more than one colony is seen on the plate, this is evidence of presumptive
reduced susceptibility to vancomycin; confirmation with an MIC should
be done (CLSI, 2014). Most automated susceptibility testing systems have
been adjusted to detect vancomycin-resistant S. aureus (VRSA), if present;
however, detection of VISA is still variable among systems—both auto-
mated and manual. Isolates with an MIC of 4 µg/ml to vancomycin should
be considered representative of a possible VISA, and further testing with
microbroth dilution should be performed for confirmation. Screen-positive
isolates that have elevated vancomycin MIC values (≥8 organisms/µg/mL)
should be sent to a reference laboratory for confirmation, and confirmed
cases should be referred to your state health department and the CDC.
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The entity of hVISA (strains of S. aureus that are variable within the popu-
lation or colony for vancomycin nonsusceptibility) is difficult for a routine
clinical microbiology laboratory to detect. If a strain with an MIC of 2 µg/
mL to vancomycin is being clinically considered nonresponsive to vanco-
mycin, a macro E-test can be performed, employing a McFarland of 2.0
instead of the usual 0.5, and inner colonies can be looked for. Exposure of
suspected isolates to increasing concentrations of vancomycin in agar
plates, however, is the recommended approach for detection of hVISA,
although these methods are often beyond what a routine laboratory can
do (Bae et al, 2009).
Newer antimicrobial agents have good activity against susceptible and
resistant staphylococci. These include quinupristin/dalfopristin, a strepto-
gramin; the lipopeptide daptomycin; linezolid; televancin; and ceftaroline.
The last is the only cephalosporin to which MRSA isolates are susceptible.
For isolates of VISA or VRSA, or when clinicians request agents other
than vancomycin for MRSA strains, laboratories should consider testing
these agents or sending the isolates to reference laboratories that can test
for their susceptibility.
Streptococcus and Enterococcus
Characteristics
Streptococci are catalase-negative, gram-positive, spherical, ovoid, or
lancet-shaped cocci, often seen in pairs or chains. They are facultatively
anaerobic. Some strains require added CO2 for their initial isolation but
may lose this requirement in subcultures. Streptococci can be broadly
classified according to the hemolytic reaction on blood agar (Table 58-3).
Those strains that completely hemolyze the red cells around their colonies
are called β-hemolytic and can be further categorized into the Lancefield
groups based on serologically reactive carbohydrates. Important members
of this group include Streptococcus pyogenes (group A) and Streptococcus aga­
lactiae (group B). Figure 58-3 is a Gram stain of S. pyogenes (group A
streptococcus) in a specimen from abscess material on the arm of a patient
PART 7
with cellulitis. Those gram-positive cocci in chains that produce partial
hemolysis (cause “greening” of the agar) are α-hemolytic. An important
TABLE 58-3
Classification of Streptococci and Enterococci
Hemolysis Lancefield Group Species
β A Streptococcus pyogenes
B Streptococcus agalactiae
C Streptococcus dysgalactiae
D Enterococcus spp.
α or γ D Enterococcus spp.
D Streptococcus bovis complex (reclassified
into many new species as described
in text)
None Viridans group*
α None Streptococcus pneumoniae
*Small colony variants of Lancefield group A, C, F, or G, or nongroupable strains,
can be any hemolysis.
Figure 58-3  Gram stain of Streptococcus pyogenes (group A streptococcus)
from a case of cellulitis.
1119
 member of this group is S. pneumoniae. Streptococci that do not hemolyze
58  Medical Bacteriology
blood are γ-hemolytic. An important member of this group is Streptococcus
bovis complex. Some S. agalactiae may also be γ-hemolytic. Most of the
remainder of the α- and γ-hemolytic streptococci are collectively called
viridans streptococci, including Streptococcus mutans, Streptococcus sanguis,
Streptococcus mitis, Streptococcus salivarius, and Streptococcus anginosus. The
group of organisms previously referred to as nutritionally variant (pyri-
doxal or thiol dependent, satelliting) streptococci have now been assigned
to the genus Abiotrophia or Granulicatella.
Members of the genus Enterococcus, previously designated as group D
streptococci because their cell wall antigens reacted with group D antisera,
are different molecularly as well as metabolically from the other members
of the genus Streptococcus to be considered a separate genus. These organ-
isms are gram-positive cocci that occur singly, in pairs, and in short chains.
They are facultatively anaerobic. Most enterococci are α- or γ-hemolytic
on blood agar, but some may exhibit β-hemolysis. The most common
species are Enterococcus faecium and Enterococcus faecalis; yellow motile
strains of the enterococci, usually nonpathogenic, include Enterococcus cas­
silflavus and Enterococcus gallinarum. These latter two species are usually
intrinsically vancomycin resistant, and it is important to differentiate them
from the vancomycin-resistant E. faecium or E. faecalis strains.
Other genera of catalase-negative gram-positive cocci that may be
isolated from clinical specimens include Leuconostoc, Pediococcus, Stomatococ­
cus (Rothia), Gemella, Aerococcus, Lactococcus, and other less rarely isolated
species. These organisms are considered to have low virulence potential
and generally are pathogenic only in the compromised host, with the
exception of some species of Aerococcus (A. urinae and A. sanguinicola),
which are known urinary tract pathogens. However, some of these isolates
may be confused with viridans streptococci, in particular, and their dif-
ferentiation is important because of their lower virulence and their poten-
tial for vancomycin resistance. Further differentiation should be considered
if a vancomycin-resistant viridans streptococcus is thought to be clinically
relevant.
Clinical Manifestations and Pathogenesis
One of the most common clinical manifestations of group A streptococci
is pharyngitis. This may be accompanied by scarlet fever, a punctate exan-
them overlying diffuse erythema that usually first appears on the neck or
upper chest, becomes generalized, and then desquamates. Skin infections
caused by group A streptococcus include cellulitis, erysipelas, and pyo-
derma. Acute rheumatic fever, characterized by carditis, polyarthritis, ery-
thema marginatum, chorea, and subcutaneous nodules, may occur 1 to 5
weeks after group A streptococcal pharyngitis. Acute glomerulonephritis
may develop 10 days to 3 weeks after group A streptococcal pharyngitis or
pyoderma.
Beginning in the late 1980s, serious group A streptococcal clinical
syndromes, including necrotizing fasciitis, myositis, malignant scarlet
fever, bacteremia, and toxic shock–like syndrome, began to be seen with
increasing frequency. These have been associated with high morbidity
rates and mortality rates of up to 30% or more. The reason for this increase
is not completely understood but appears to be related to changes in the
prevalence of organisms having an enhanced virulence potential (Kaplan,
2005; Vucicevic et al, 2008; Lappin & Ferguson, 2009, Reglinski & Sris-
kandan, 2014).
S. pyogenes produces numerous virulence factors. One of the most
important is the antiphagocytic cell wall M protein. Antibodies against the
specific M protein confer lifelong type-specific immunity; however,
because more than 60 M protein types exist, infection with a group A
streptococcus possessing a different M protein may occur. Another impor-
tant cell wall component is lipoteichoic acid, which permits bacterial
adherence to the respiratory epithelium. S. pyogenes also elaborates about
20 extracellular products, including enzymes (streptolysins, hyaluronidase,
streptokinase, deoxyribonucleases [DNases], and nicotinamide adenine
dinucleotidase [NADase]) and erythrogenic toxins. Streptolysin O, an anti-
genic, oxygen-labile enzyme, produces subsurface hemolysis on blood agar
plates; streptolysin S, a nonantigenic, oxygen-stable enzyme, produces
surface hemolysis. Neither streptolysin has a proven role in the pathogen-
esis of human disease. Streptokinase promotes fibrinolytic activity by con-
verting plasminogen to plasmin, and hyaluronidase may enhance the
spread of the organism through connective tissue. The pathogenic signifi-
cance of the DNases and of NADase is unknown. Pyrogenic (erythrogenic)
toxins (serotypes A, B, C) are produced by isolates of S. pyogenes infected
with a specific temperate bacteriophage. Their pyrogenicity is caused by
a direct action on the hypothalamus. Streptococcus group A has also been
found to possess superantigens with high mitogenic capabilities; these have
been associated with cases of more severe streptococcal infection, such as
1120
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necrotizing fasciitis or toxic shock syndrome (Kotb et al, 2002; Reglinski
& Sriskandan, 2014).
The pathogenesis of acute rheumatic fever is not fully understood.
Certain M protein types of S. pyogenes may be rheumatogenic. The pres-
ence of complexes of immunoglobulin and the C3 component of comple-
ment along the sarcolemmal sheaths of cardiac myofibers from individuals
with rheumatic carditis suggests that myocarditis results from the produc-
tion of antibodies directed against a streptococcal cell wall M protein that
cross-reacts with myocardial tissue. Moreover, a heart or tissue cross-
reactive antigen of S. pyogenes that shares immunologic epitopes with, but
is distinct from, the M protein has been identified (Barnett & Cunning-
ham, 1990). Renal damage in acute glomerulonephritis is caused by depos-
its of circulating streptococcal–antistreptococcal immune complexes in the
glomeruli and subsequent activation of complement. Cell-mediated reac-
tions to an altered glomerular basement membrane or activation of the
alternate complement pathway also may be involved. Isolates of S. pyogenes
have been linked to toxic shock syndrome, with a clinical picture very
similar to S. aureus; streptococcal superantigens (Sags) are thought to be
responsible for the toxic shock–like syndrome caused by strains of S. pyo­
genes (Reglinski & Sriskandan, 2014).
The most common infections caused by Group B streptococci (GBS)
are neonatal sepsis, pneumonia, and meningitis. Colonization of the mater-
nal genital tract is associated with colonization of infants and risk of
neonatal disease, with early-onset infections occurring within the first few
days after delivery and late-onset infections appearing after 1 week of age.
To reduce the incidence of neonatal disease, the CDC published specific
guidelines to facilitate early identification and treatment of women colo-
nized with GBS and identification and treatment of neonates at risk for
developing disease (MMWR, 2010; Verani et al, 2010). All pregnant
women at 35 to 37 weeks of gestation should have vaginal/rectal specimens
collected and processed for detection of GBS. Results of this test should
be available during labor, so appropriate prophylaxis can be given to the
mother before delivery to prevent infection to the newborn. Isolation of
GBS from the urine of a pregnant female can also be used as a marker of
group B streptococcal vaginal carriage, and this information should be used
to direct prophylaxis to mothers found to be positive. If urine is positive,
screening of vaginal/rectal cultures may not be necessary. Molecular tests
that can rapidly identify the presence of GBS are available and are used in
some laboratories at the time of labor and delivery (Gray et al, 2012).
Group B streptococcal infections in adults include postpartum endometri-
tis, urinary tract infection, bacteremia, skin and soft tissue infections,
pneumonia, endocarditis, meningitis, arthritis, and osteomyelitis.
Group C and G streptococci are similar to S. pyogenes in that they cause
a wide range of infections, including bacteremia, endocarditis, meningitis,
arthritis, and respiratory and skin infections (Rantala, 2014). The pharyn-
geal infection caused by these streptococci is similar to that of group A
streptococci, except that the nonsuppurative sequelae of rheumatic fever
do not occur.
Infections caused by S. pneumoniae include pneumonia, meningitis
(especially in infants and the elderly), spontaneous bacteremia (in persons
who do not have a spleen), otitis, sinusitis, and spontaneous peritonitis. S.
pneumoniae is seen in the normal flora of the upper respiratory tract of 25%
to 50% of preschool children, 36% of primary school-age children, and
nearly 20% of adults, termed carriers (Lopez et al, 1999). Its spread is
enhanced by upper respiratory tract infections and crowding. Pneumonia
may develop when the host immune defenses are impaired. Most cases are
endogenous, following aspiration of oral secretions containing normal
flora that includes S. pneumoniae. Person-to-person transmission during
epidemics occurs by droplet aerosols. The major virulence factor of S.
pneumoniae is its antiphagocytic polysaccharide capsule, and strains with a
thick, mucoid capsule are especially virulent. Vaccines designed to protect
against infection by pneumococci of many of the predominant capsular
polysaccharide types are available. The CDC recommends that infants
receive the 13-valent conjugated vaccine starting at age 2 months. It also
is recommended that adults 65 years of age and older receive both the
13-valent conjugated vaccine and the 23-valent polysaccharide vaccine.
Immunosuppressed patients of any age should receive both vaccines (Mir-
saeidi & Schraufnagel, 2014). There are other conjugated vaccines being
investigated to further enhance the protection in various populations
(Feldman & Anderson, 2014).
Bacterial endocarditis is the most common infection caused by viridans
streptococci; others include abscesses in the brain or liver, bacteremia, and
dental caries. The milleri streptococci complex (S. constellatus, S. interme­
dius, and S. anginosus) consists of the most common viridans streptococci
responsible for liver, spleen, and brain abscesses; they often are more
susceptible to antibiotics than other strains of viridans streptococci,
although resistance to penicillin is increasingly being recognized. S. bovis
bacteremia has been associated with malignancies of the gastrointestinal
tract. S. bovis is now recognized as a complex of seven strains and/or sub-
species. Biochemical differentiation remains difficult within the species.
Presently, there are seven subspecies broken down into four branches: S.
gallolyticus (includes the subspecies gallolyticus, pasteurianus, and macedoni­
cus); S. equinus; S. infantarius (includes subspecies infantarius and lutetiensis);
and S. alactolyticus. S. gallolyticus subspecies gallolyticus and subspecies pas­
teurianus are isolated from blood cultures of patients with colonic cancer
more often than is S. infantarius or S. lutetiensis. S. gallolyticus subspecies
gallolyticus is associated with prosthetic joint infections and infective endo-
carditis; S. gallolyticus subspecies pasturianus and S. infantarius subspecies
infantarius are associated with hepatobiliary infections (including UTI) and
cases of meningitis. S. alactolyticus, S. equinus, and S. infantarius subspecies
lutetiensis are not often found in human disease (Jans et al, 2014; Vaska &
Faoagali, 2009). In many laboratories, isolates are still reported as S. bovis
because phenotypic identification may not be adequate for differentiation;
as use of molecular methods or MALDI-TOF for bacterial identification
increases, more of these differences may be appreciated (Vaska & Faoagali,
2009).
Enterococci are not highly pathogenic; however, they are a common
cause of urinary tract infection in hospitalized persons. They may also
cause endocarditis, bacteremia, and wound infection. Vancomycin-resistant
enterococci offer a greater potential for infection, especially in immuno-
compromised patients and patients with implanted foreign devices (Han
et al, 2009; McBride et al, 2009).
More and more clinically relevant cases of Aerococcus urinae (and other
species of aerococci) are being reported. Urinary tract infections are most
common, but rare cases of more serious infection, including bacteremia,
are seen (Ruoff, 2011; Shelton-Dodge et al, 2011). A. viridans remains
relatively uncommon as a pathogen when isolated from clinical samples;
A. sanguinicola isolation is not always associated with clinical disease, but
case numbers are increasing (Ibler et al, 2008; Shelton-Dodge et al, 2011).
In addition, higher MICs have been demonstrated to levofloxacin by A.
sanguinicola than by other species of aerococci (Ruoff, 2011; Shelton-
Dodge et al, 2011).
Laboratory Diagnosis
Streptococci grow well on blood or chocolate agar. Blood agar is preferred
because the hemolytic properties of the organism can be assessed. When
culturing vaginal/rectal swabs from pregnant women for group B strepto-
cocci, specimens should first be inoculated to a selective broth, such as
Lim or carrot broth, or on to selective agar, such as Granada agar, to
enrich for this organism (Church et al, 2008; Carvalho et al, 2009; Spell-
erberg & Brandt, 2011). Tests that may be used in the clinical microbiol-
ogy laboratory to presumptively name the β-hemolytic species of
Streptococcus are shown in Figure 58-4. More than 99% of isolates of group
A streptococcus are susceptible to bacitracin, but a very small percentage
of isolates of group B streptococcus and 10% to 20% of isolates of groups
C and G streptococcus are also susceptible. Therefore, results of the baci-
tracin susceptibility test provide a presumptive identification. An isolate
may be called group A streptococcus presumptively, based on hydrolysis
of the l-pyrrolidonyl-β-naphthylamide (PYRase) test (Spellerberg &
Brandt, 2011). All isolates of group A streptococcus and more than 99%
of isolates of Enterococcus are PYRase positive. Identification of group A
streptococcus is confirmed by serotyping, using latex agglutination or a
nucleic acid probe. A nucleic acid probe (Gen-Probe, San Diego) is also
Figure 58-4  Decision tree of tests to presumptively name the β-hemolytic
species of Streptococcus. +, Positive result; –, negative result; R, resistant; S, suscep-
tible. (With permission from Woods GL, Gutierez Y: Diagnostic pathology of infectious
diseases, Philadelphia, 1993, Lea & Febiger.)
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available for direct detection of group A streptococcus on throat swabs
(Chapin et al, 2002).
Group A streptococcal antigen may be detected directly in throat swab
specimens by using commercial kits designed to generate a rapid result.
These tests are highly specific but, given their low sensitivity, which varies
among studies from 31% to 95% (Carroll & Reimer, 1996; Lean et al,
2014), a negative antigen test in children should be followed by culture or
probe. More recently, clinical guidelines recommended that negative
antigen assays in adults need not be followed up with culture, but this is
controversial (Dingle et al, 2014; Lean et al, 2014). Serologic tests to detect
streptolysin O and DNase B antibodies in acute and convalescent serum
samples are used primarily to diagnose acute rheumatic fever and acute
glomerulonephritis following infection with group A streptococcus.
Catalase-negative colonies that are β-hemolytic and hippurate hydro-
lysis positive and/or that have a positive CAMP test (named for researchers
Christie, Atkins, and Munch-Petersen) reaction presumptively can be
called group B streptococcus (GBS). Isolates of presumed GBS from sterile
body sites should be identified by serotyping (using latex agglutination or
coagglutination tests) or by using a chemiluminescent DNA probe. The
DNA probe can also be used to identify GBS growing in Lim broth or
other selective broth cultures (Daly et al, 1991; Williams-Bouyer et al,
2000). This probe, however, is not sensitive enough to use directly on
clinical specimens for the detection of GBS. For culture of vaginal/rectal
swab specimens from pregnant women during weeks 35 through 37 of
gestation, it is recommended that a broth enrichment be used along with
or as a replacement for agar-based media. Selective broth media, including
Lim broth, selective Todd Hewitt broth, or a commercially available Trans
Vag broth supplemented with 5% sheep blood (Remel, Lenexa, Kans.),
can be used as enrichment media (Heelan et al, 2005). Chromogenic broth,
including carrot broth media, can be used as enrichment broth; colonies
of β-hemolytic GBS will convert the color of the tube from clear to yellow
or orange. However, nonhemolytic GBS will not change the tube color,
PART 7
and when used, a negative broth would still need to be planted onto solid
media for recovery of these strains.
In addition, a selective nonchromogenic enrichment broth can be sub-
cultured to Granada agar, on which colonies of GBS will appear yellow to
orange for ease in detection. Isolates of β-hemolytic groups C, D, F, and
G Streptococcus are identified by serotyping with latex agglutination
reagents. The molecular assays mentioned earlier can be used to detect
GBS directly in vaginal-rectal specimens or can be used to detect GBS in
Lim or carrot broth cultures (Picard & Bergeron, 2004; Block et al, 2008).
The Cepheid GeneXpert GBS assay can be performed directly on clinical
vaginal/rectal samples and has the potential to provide results intrapartum
(Gray et al, 2012). More nucleic acid amplification assays for the detection
of GBS in vaginal/rectal samples are rapidly becoming FDA cleared for
use in clinical laboratories.
Latex agglutination assays are available for direct detection of group B
streptococcus (as well as S. pneumoniae, some serotypes of N. meningitidis,
E. coli, and H. influenzae type b) in CSF, serum, and urine. These assays
have been shown to have sensitivities equivalent to or lower than a Gram
stain and may give false-positive results; hence, they do not in general
provide additional useful information above that provided by the CSF
Gram stain. The rapid bacterial antigen tests are much more expensive
and labor intensive than Gram stain; most laboratories no longer offer
these tests or strictly limit their use (Thomas, 1994).
Tests used to presumptively identify α- and γ-hemolytic streptococci
and enterococci are shown in Figure 58-5. α-Hemolytic colonies that are
mucoid or flattened with a depressed center are suggestive of S. pneu­
moniae; they should be tested for susceptibility to ethylhydroxycupreine
hydrochloride, more commonly called optochin (P disk), and for bile solu-
bility. S. pneumoniae is susceptible to both; other α-hemolytic streptococci
are resistant to optochin and are variable in response to bile. A urinary
antigen assay for detection of S. pneumoniae has been shown in some
studies to be the nonculture diagnostic method of choice for patients with
severe pneumococcal infection, for diagnosis of pneumococcal exacerba-
tion in chronic obstructive pulmonary disease patients, and as a tool for
diagnosis of otitis media. As with any antigen assay, caution needs to be
taken in interpreting results in cases where prior infection with S. pneu­
moniae may have occurred and the antigen may merely be reflecting this
(Gisselsson-Solen et al, 2007; Smith et al, 2009; Couturier et al, 2014;
Harris et al, 2014).
α-Hemolytic colonies that are not S. pneumoniae and γ-hemolytic colo-
nies are tested for PYRase hydrolysis; enterococci are PYRase positive, and
viridans streptococci are negative. Moreover, all enterococci grow in the
presence of 6.5% NaCl, but viridans streptococci do not. Enterococci
hydrolyze esculin in the presence of bile (causing visible growth and
1121
 blackening of the agar), but up to 10% of viridans streptococci are also
58  Medical Bacteriology
bile–esculin positive. Additional biochemical assays are required to identify
enterococci to the species level. A majority of vancomycin-resistant entero-
cocci (VRE) are E. faecium.
α-Hemolytic streptococci that are optochin resistant and PYRase nega-
tive and γ-hemolytic streptococci that are PYRase negative and do not
grow in 6.5% NaCl are grouped as nonhemolytic (viridans) streptococci.
Identification of individual species of viridans streptococci requires con-
ventional biochemical testing, molecular methods, or potentially MALDI-
TOF (Fang et al, 2012; Moon et al, 2013). Kit systems to identify these
organisms are commercially available. Full identification of species
members of viridans streptococci, however, is usually not necessary.
Members of viridans streptococci belonging to the milleri streptococci,
because they are usually recognized by their characteristic “caramel” odor,
can be reported, if present, to alert clinicians about this group of viridans
because of their propensity for abscess formation and their uniform sus-
ceptibility to penicillin. Figure 58-6 is an example of a Gram stain of a
member of the milleri group of viridans streptococci from a brain abscess.
There are no vancomycin-resistant streptococci, but occasionally a
“viridans”-like isolate is reported as vancomycin resistant. Usually this is
an enterococcus, but it could also be a member of some more uncommon
genera such as Leuconostoc or Pediococcus. If vancomycin resistance has been
demonstrated, it would be important to differentiate the intrinsically
vancomycin-resistant Leuconostoc and Pediococcus from enterococci that have
acquired vancomycin resistance. Characteristics that might be used to
accomplish this are listed in Table 58-4 (Facklam et al, 1989). Included in
this table is Aerococcus sp., because of their morphologic similarity to
Enterococcus sp. The clue that an isolate (especially from a urine culture)
might be an Aerococcus sp. is the finding on Gram stain that a catalase-
negative colony consists of gram-positive cocci in tetrads and clusters, not
in pairs and chains. Some species of Aerococcus are l-pyrrolidonyl-β-
naphthylamide (PYR) positive, which leads to further confusion with
Enterococcus sp.
Figure 58-5  Decision tree of tests to presumptively name the α-hemolytic
species of Streptococcus and Enterococcus. +, Positive result; –, negative result; R,
resistant; S, susceptible. (With permission from Woods GL, Gutierez Y: Diagnostic
pathology of infectious diseases, Philadelphia, 1993, Lea & Febiger.)
TABLE 58-4
Characteristics Differentiating Enterococcus, Leuconostoc, Pediococcus, and Aerococcus
Enterococcus Aerococcus
Gram stain Pairs and short chains Tetrads
Hemolysis β or α or γ α or γ
Bile esculin + V
Growth in 6.5% NaCl + +
PYR + *
LAP + *
Vancomycin susceptibility S/R S R
+, Positive result; –, negative result; LAP, leucine aminopeptidase; PYR, l-pyrrolidonyl-β-naphthylamide; R, resistant; S, susceptible; V, variable reactions.
*Aerococcus urinae is PYR and LAP positive; Aerococcus viridans is PYR positive and LAP negative.
1122
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Members of the genus Abiotrophia will not grow in the absence of pyri-
doxal. Often they are first recognized as satellite colonies growing around
a colony of S. aureus. Differential characteristics of Abiotrophia defectiva and
Abiotrophia adiacens are reviewed elsewhere (Ruoff, 2011; Giuliano et al,
2012). Methods of performing susceptibility tests for strains of Abiotrophia
and Granulicatella can be found in CLSI document M45-M2 (CLSI, 2010).
Antimicrobial Susceptibility
The antibiograms of groups A, B, C, and G streptococcus are predictable
(all are susceptible to penicillin); therefore, routine antimicrobial suscep-
tibility testing of these organisms is unnecessary unless penicillin cannot
be used, as in the case of a penicillin allergy. In the latter situations, testing
for resistance to macrolides, clindamycin, and the tetracyclines may be
warranted. Inducible resistance to clindamycin may occur with Streptococcus
(i.e., group B streptococcus), and a D zone test as described earlier for S.
aureus may be warranted if the streptococcal isolate is found resistant to
erythromycin. Specific guidelines for streptococcal D–zone testing recom-
mend that the clindamycin and erythromycin disks be separated by 12 mm
instead of the 15 to 16 mm recommended for testing S. aureus (CLSI,
2014). Because S. pneumoniae organisms with intermediate- or high-level
resistance to penicillin are found worldwide, isolates should be tested for
susceptibility to penicillin. A screening test using disk diffusion with a 1-µg
oxacillin disk (≥20 mm = susceptible) may be performed; however, isolates
that are not susceptible by this method must be further evaluated by mac-
rodilution or microdilution testing, using Mueller-Hinton broth supple-
mented with lysed horse blood or the E test to determine the penicillin
MIC. There are breakpoints for penicillin and the cephalosporins for
isolates from cases of meningitis versus those isolated from nonmeningitis
sites (CLSI, 2014). Resistance to third-generation cephalosporins also
occurs; therefore, isolates should be tested for susceptibility to these anti-
microbial agents as well.
Susceptibility testing should be performed on isolates of nonhemolytic
(viridans) streptococci from sterile body sites, because resistance to
Figure 58-6  Gram stain of a viridans Streptococcus, specifically a member of
the milleri group, from a brain abscess.
Leuconostoc Pediococcus
Cocci, coccobacilli, and rods; Tetrads and pairs;
pairs and chains spherical cells
α or γ α or γ
V +
V V
– –
– +
R
penicillin does occur. Enterococcus spp. should also be tested, primarily to
identify high-level resistance to penicillin or ampicillin, high-level resis-
tance to streptomycin and gentamicin, and resistance to vancomycin.
Enterococci are resistant to vancomycin (MIC >32 µg/mL) because of the
presence of resistance genes, referred to as the van genes (CLSI, 2014).
Although many of these genes have been described, the most common are
vanA, vanB, and vanC. The vanA and vanB genes, conferring high-level
resistance and predominantly found in E. faecium and much less frequently
in E. faecalis, are acquired, plasmid-borne genes that can create infection
control problems involving transmission of this resistance. This vancomy-
cin resistance can be differentiated from intrinsic and lower-level resis-
tance (vanC genes) in the yellow, motile species of Enterococcus, and this
should be done in laboratories and reported as such to the infection control
team (Teixeira et al, 2011).
Gemella and Aerococcus
Other nonstreptococcal gram-positive, catalase-negative cocci of increas-
ing importance are those that belong to the genera Gemella and Aerococcus.
Gemella spp. (Gemella haemolysans and Gemella morbillorum) resemble viri-
dans streptococci, although they usually produce smaller colonies. G. hae­
molysans has been associated with endocarditis and meningitis. Gram stain
usually demonstrates diplococci with adjacent sides flattened that can be
confused with a Neisseria sp. because cells can easily become decolorized.
G. haemolysans is aerobic, and G. morbillorum is anaerobic. The latter
usually appears as cocci in pairs or short chains. Both are PYR positive,
6% NaCl, and esculin–hydrolysis negative (to differentiate them from
enterococci). G. morbillorum is leucine-aminopeptidase (LAP) positive as
well. Both are usually susceptible to penicillin. As with other gram-positive
cocci, if there is doubt about the morphology of the organism (i.e., whether
it is a short rod or a coccus), Gram stain performed from a broth culture
will usually resolve the difficulty. Maldi-Tof has been shown to be a useful
tool in the correct identification of Gemella spp. (Schultness et al, 2013).
Two major species of Aerococcus may be clinically relevant and/or iso-
lated from clinical specimens: Aerococcus urinae and Aerococcus viridans. Both
resemble viridans streptococci or enterococci on agar plates; however, in
Gram stains, they usually appear in tetrads. A. urinae is a recognized
pathogen in urinary tract infection; in addition, it has been isolated from
the blood in cases of endocarditis. A. urinae is PYR negative and LAP
positive, in contrast to A. viridans, which is PYR positive and LAP negative
(see Table 58-4). Both will grow in the presence of 6.5% NaCl. Neither
are anaerobes, and A. viridans usually will not grow under anaerobic condi-
tions. A. urinae is usually susceptible to penicillin and nitrofurantoin but
may be resistant to sulfonamides. Variability in its response to trime-
thoprim has been noted (Ruoff, 2011; Senneby et al, 2015). A newer
member of the genus Aerococcus, A. sanguinicola, which is rarely recovered
from clinical specimens, can be both LAP and PYR positive, although this
is not a confirmatory identification. Use of Maldi-TOF can provide more
reliable results. It can be responsible for UTI as well as bacteremia and
infective endocarditis. Like A. urinae, isolates of A. sanguinicola are usually
susceptible to penicillin and nitrofurantoin, but are variable in their
response to sulfonamides and quinolones (Ibler et al, 2008; Rasmussen,
2013; Senneby et al, 2015).
GRAM-POSITIVE RODS
Corynebacterium and Arcanobacterium
Characteristics
The corynebacteria—or diphtheroids, as they are sometimes called—
appear in the Gram-stained smear as slightly curved, gram-positive rods
with nonparallel sides and sometimes wider ends, giving a clubbed appear-
ance (Fig. 58-7). These organisms are catalase positive. More than 46
species of Corynebacterium are known. Most are rarely pathogenic in
humans; notable exceptions are Corynebacterium diphtheriae and its closely
related species or varieties Corynebacterium ulcerans and Corynebacterium
pseudotuberculosis. C. pseudodiphtheriticum has been implicated in respiratory
tract infections, including pneumonia (Camello et al, 2009). Medically
relevant Arcanobacterium spp. include Arcanobacterium haemolyticum,
Arcanobacterium pyogenes, and Arcanobacterium bernardiae. Arcanobacterium
species also appear as irregular gram-positive rods on Gram stain but can
be easily differentiated from the corynebacteria by their negative catalase
reaction.
Clinical Manifestations and Pathogenesis
At the initial site of infection on the epithelial cells of the tonsils and
oropharynx, C. diphtheriae elaborates an exotoxin that causes local cell
necrosis and subsequent inflammation. The exotoxin produced by strains
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Figure 58-7  Sputum stained with Gram stain shows many neutrophils, amor-
phous debris, and coryneform gram-positive bacilli (oil immersion).
of C. diphtheriae infected with a specific bacteriophage is absorbed into the
circulation. Distribution of exotoxin through the bloodstream can produce
degenerative changes in the heart, nervous system, and kidneys. The toxin
molecule consists of two fragments: A, containing the enzymatically active
site, and B, comprising the receptor binding site. Once in the cell, protein
synthesis is disrupted. The bacteria and exotoxin produce a serum exudate
and cellular infiltrate of the mucous membrane in the pharynx. Exudative
lesions coalesce, forming a grayish-black adherent pseudomembrane,
which is characteristic of diphtheria. Although toxin production and
PART 7
pathogenicity are often considered to be synonymous, pseudomembranes
may form in persons infected with nontoxigenic strains. Extension of the
pseudomembrane superiorly into the nasopharynx or inferiorly into the
larynx may be so marked as to produce respiratory obstruction. Although
C. diphtheriae infections of other parts of the body do occur, those observed
most frequently in the United States today are infections of the skin.
Transmission of C. diphtheriae occurs by droplet nuclei from the respiratory
tract or by contact from cutaneous foci of infection (Byard, 2013; Mattos-
Guaraldi et al, 2003).
Because they are part of the normal flora of the skin and mucous
membranes, it is difficult to establish the etiologic role of the other cory-
nebacteria. Clinical significance is generally increased if the organism is
observed in the Gram-stained smear in association with leukocytes, is
isolated from a sterile site, and is isolated from multiple samples. Coryne­
bacterium jeikeium has been clearly associated with infections of implanted
prosthetic materials (e.g., heart valves, CSF, joints), has caused subacute
bacterial endocarditis, and has been involved in a variety of opportunistic
infections. Corynebacterium urealyticum has been associated with urinary
tract infection, as well as with bacteremia, endocarditis, and wound infec-
tion (Nebreda-Mayoral et al, 1994). When identified, Corynebacterium
striatum and Corynebacterium amycolatum are the most common normal
flora skin coryneforms. They may become pathogenic, especially in cases
of prosthetic joint infections and, of note, are often resistant to β-lactam
antibiotics—a characteristic usually attributed only to C. jeikeium (Crab-
tree & Garcia, 2003; Cazanave et al, 2012).
A. haemolyticum has been associated with pharyngitis and wound and
soft tissue infections (Fernandez-Suarez et al, 2009). A. pyogenes and A.
bernardiae are associated with abscess formation. (Funke & Bernard, 2011)
Laboratory Diagnosis
Because of the relative rarity of diphtheria in the United States today, the
diagnosis may be overlooked clinically, and the laboratory may easily fail
to recognize it in cultures. When the diagnosis of diphtheria is suspected,
the laboratory should be informed so that the specimen can be handled
appropriately. Specimens should be obtained with a cotton- or polyester-
tipped swab from inflamed regions of the nasopharynx and, if possible,
beneath the pseudomembrane. If skin lesions are suspected of being posi-
tive for C. diphtheriae, the most appropriate specimen would be an aspirate
of the lesion. Corynebacteria will grow on routine blood-containing agar;
however, cystine-tellurite (CT) blood agar or Tinsdale medium is pre-
ferred. On CT medium, colonies of C. diphtheriae are gray or black after
48 hours of incubation. Colonies may be large or small, and flat or convex.
Colonies of species other than C. diphtheriae may produce black colonies
on CT or Tinsdale media, although these will usually be smaller. If a
1123
 TABLE 58-5
58  Medical Bacteriology
Differential Characteristics of Some Species within the Genus Corynebacterium and Related Organisms
Test C. diphtheriae C. ulcerans
Catalase + +
Hemolysis v +
Gelatinase – +
Urease – +
Nitrate reduction v –
Sucrose fermentation – –
+, Positive; –, negative; v, variable.
laboratory does not have CT or Tinsdale medium and a request for C.
diphtheriae is made, CNA can be used, although it will be more difficult to
recognize possible C. diphtheriae strains (Funke & Bernard, 2011).
Classification of oral and skin corynebacteria or diphtheroids is difficult
and confusing. The differential characteristics of some species are shown
in Table 58-5. Commercial identification systems are available to identify
many of the members of this group of organisms (Funke & Bernard, 2011).
Isolates of suspected C. diphtheriae must be tested for production of exo-
toxin. The elaboration of toxin may be detected in vitro with the Elek
immunodiffusion test; however, this generally is not done in a routine
clinical laboratory. Isolates should be sent to a state health laboratory or a
reference laboratory where this can be performed. Alternatively, PCR-
based tests have been described that may be used for detection of the toxin
gene (Mancini et al, 2012). C. jeikeium often produces a characteristic
metallic sheen on the surface of blood agar plates. C. striatum and C.
amycolatum are common skin florae that can be responsible for infection;
whether they need to be specifically identified is controversial, and iden-
tification can be difficult. Species identification of Corynebacterium often
requires a combination of commercial kit systems, cellular fatty acid analy-
sis, and/or sequencing (Van den Velde et al, 2006). C. striatum and C.
amycolatum organisms are often resistant to many antibiotic agents—a
characteristic that often is more typically associated with hospital-acquired
strains, in particular C. jeikeium. Maldi-Tof has been reported to be very
useful in the identification of Cornebacterium spp. (Bernard, 2012) and may
become helpful in correlating the significance of species of coryneforms
in the future.
Arcanobacterium spp. are β-hemolytic on sheep blood agar. Colonies on
sheep blood agar are small after 48 hours of incubation, and the hemolysis
may go unnoticed. Adequate growth and noticeable hemolysis are best
demonstrated in a CO2-enhanced environment. Arcanobacterium spp. are
catalase negative. Biochemical reactions that are used to determine the
species of corynebacteria are also useful in identifying the Arcanobacterium
spp. (Funke & Bernard, 2011). A. haemolyticum produces phospholipase D,
which is responsible for the reverse CAMP reaction with S. aureus. This
organism inhibits hemolysis around the S. aureus streak, producing an
inverted triangle of no hemolysis.
Antimicrobial Susceptibility
Although antitoxin remains the only specific method of treatment of diph-
theria, antibiotics are administered to patients with disease and to asymp-
tomatic carriers of toxigenic strains. C. diphtheriae is usually inhibited by
penicillins and the macrolides. The antimicrobial susceptibilities of other
species of corynebacteria or diphtheroids are far less predictable. C. jeikeium
is usually resistant to the penicillins and cephalosporins, is variably suscep-
tible to most other antibiotics, and is almost uniformly susceptible to
vancomycin. Other species of Corynebacterium, however, may be similarly
resistant to β-lactam antibiotics. Treatment of infection caused by these
organisms is often complicated by the presence of compromised host
defenses and implanted prosthetic materials. Arcanobacteria are sensitive
to penicillin and other β-lactams, rifampin, tetracycline, and the macrolides.
Growth of the organisms may be inhibited by fluoroquinolones and ami-
noglycosides (Funke & Bernard, 2011). Methods used for performing
susceptibility tests on Corynebacterium spp. and interpretive criteria can be
found in CLSI document M45-M2 (CLSI, 2010). Bernard recently reviewed
the antimicrobial susceptibilities of gram-positive rods (Bernard, 2012).
Prevention
Methods of prevention of diphtheria are almost exclusively active and
passive immunization programs with supplemental antibiotics to eliminate
the carrier state of toxigenic strains during epidemics.
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Arcanobacterium Arcanobacterium
C. pseudotuberculosis C. jeikeium haemolyticum pyogenes
+ + – –
+ – + +
– – – +
+ – – –
v – – –
v – v v
Figure 58-8  Gram stain of a cerebrospinal fluid sample that grew Listeria
monocytogenes. The short bacilli are seen inside white blood cells.
Listeria
Characteristics
Listeria spp. are nonbranching, non–spore-forming gram-positive rods.
Listeria monocytogenes (Fig. 58-8) is the only species of Listeria that is
pathogenic for humans, and L. ivanovii is the only species of the other five
Listeria spp. that is pathogenic for animals. Optimal growth of L. monocy­
togenes is observed between 30° C and 37° C; however, growth may occur
as low as 4° C. Colonies are small after 24 hours and exhibit a narrow zone
of β-hemolysis on blood agar. A characteristic tumbling motility of saline
suspensions of the colonies occurs at room temperature but rarely at 35° C.
This same temperature-dependent motility is also noted in semisolid
media, in which growth appears as an umbrella shape at the top of the
medium (aerobic conditions), preferentially at lower temperatures.
Clinical Manifestations and Pathogenesis
L. monocytogenes is found in soil, dust, water, silage, sewage, and raw unpas-
teurized milk and in asymptomatic human and animal carriers. Transmis-
sion of the organism by foods such as coleslaw, pasteurized milk, soft
cheeses, and, more recently, cantaloupe has resulted in several major epi-
demics in North America and Europe (MacDonald et al, 2005; Cartwright
et al, 2013; McCollum et al, 2013). According to data from microbiologi-
cal surveys of food, L. monocytogenes has been detected in 2% to 3% of
dairy products, 20% of soft cheeses and processed meats, 30% of certain
vegetables (cabbages, radishes), and up to 50% of raw meat and poultry
(Wellinghausen, 2011; Simmons et al, 2014). About 1% to 10% of humans
are fecal carriers.
Listeriosis is mainly a disease of industrialized countries, occurring
sporadically or in epidemics. The primary mode of transmission is con-
taminated food products, although occasional non–food-related outbreaks
have occurred in health care settings, primarily in nurseries, as the result
of cross-infection; contaminated mineral oil for bathing was implicated in
one such outbreak (Schuchat et al, 1991). A meta-analysis of over 11,700
literature references pertaining to cases of listeriosis globally resulted in
23,150 illnesses and 5463 deaths in 2010. The proportion of perinatal cases
was 20.7% (de Noordhout et al, 2014). The incidence rate of listeriosis in
the United States was 0.27 cases per 100,000 between 2004 and 2009 and
0.29 per 100,000 from 2009 to 2011; in adults aged over 65 years, the
incidence increased 1.3 cases per 100,000 inhabitants (Hernandez-Milian
& Payeras-Cifre, 2014).
 Clinical manifestations of listeriosis differ among pregnant women,
neonates, and immunocompromised individuals, which constitute the
high-risk groups. Listeriosis during pregnancy, most common in the third
trimester, presents as a flu-like illness. Bacteremia occurs concomitantly,
during which time the uterine contents are infected. Progression to amnio-
nitis may induce premature labor or septic abortion in 3 to 7 days. Infec-
tion in the mother is self-limited because the source of infection is removed
with delivery of the infected fetus and uterine contents. Neonatal listeriosis
may have an early or late onset. Early-onset disease, manifested at birth
or a few days thereafter, results from in utero infection. Infants present
with temperature instability, hemodynamic compromise, and respiratory
distress; widely disseminated granulomas, particularly involving the pla-
centa, posterior pharynx, and skin, are characteristic of the illness but are
not always present. Late-onset disease, affecting full-term infants of
mothers with uncomplicated pregnancies, is assumed to be acquired post-
partum, but in most cases the source is unknown. Clinical manifestations
of meningitis become apparent several days to weeks after birth.
Nonperinatal listeriosis usually occurs in immunosuppressed individu-
als, but in about one-third of cases, no risk factor is identified. Tropism
for L. monocytogenes for the central nervous system (CNS) is manifested
predominantly as meningitis; other forms of CNS listeriosis include cere-
britis and brainstem and spinal cord abscesses. Severe disease with high
mortality rates (20% to 50%) and neurologic sequelae among survivors
are common. Primary bacteremia or focal infections outside the CNS are
uncommon. Primary cutaneous listeriosis has occurred occupationally in
veterinarians and abattoir workers after exposure to infected animal tissues.
Endocarditis, osteomyelitis, arthritis, endophthalmitis, and other focal
infections have been reported rarely. Febrile gastrointestinal disease due
to L. monocytogenes has been reported in nonimmunocompromised patients;
implicated foods include chocolate milk, rice salad, and delicatessen meats
and cheeses (Wellinghausen, 2011; Hernandez-Milian & Payeras-Cifre,
2014). Immunocompromised patients, such as those with leukemias or
bone marrow transplants and patients on immunosuppressive therapies,
are cautioned against eating uncooked dairy meats for fear of infection
with this organism.
The pathogenesis of listeriosis infection has been well elaborated in
recent years. Host susceptibility, gastric acidity, inoculum size, and viru-
lence properties of the organism, along with specific food products, are
the most common factors that determine progression from infection to
disease and the severity of that disease in the infected individual. L. mono­
cytogenes can penetrate the epithelial cells of the gastrointestinal tract and
grow within hepatic and splenic macrophages; from there, the organism
can spread to the CNS or the pregnant uterus. Virulence factors such as
internalin and E-cadherin, a placental receptor, interact to result in infec-
tion in the pregnant female/fetus. Immunity to listeriosis relies on T
cell–mediated activation of macrophages by lymphokines (Wellinghausen,
2011; Hernandez-Milian & Payeras-Cifre, 2014).
Laboratory Diagnosis
Colonies are small and grayish-blue, growing in 24 to 48 hrs, and are sur-
rounded by a narrow zone of β-hemolysis on blood agar. A positive catalase
reaction differentiates L. monocytogenes from similarly appearing group B
streptococci. Organisms are motile at room temperature and produce acid
from glucose, trehalose, and salicin and hydrolyze esculin. Other bio-
chemical characteristics of L. monocytogenes and differences between Liste­
ria and Erysipelothrix are listed in Table 58-6. Successful use of Maldi-Tof
for the identification of L. monocytogenes has been reported (Farfour et al,
2012).
Antimicrobial Susceptibility
L. monocytogenes is usually susceptible to penicillin, ampicillin, erythromy-
cin, chloramphenicol, tetracycline, and gentamicin. Isolates usually are
only moderately susceptible to the quinolones. Cephalosporins are inef-
fective against Listeria spp.; isolates should not be tested against cepha­
losporins because they are ineffective in vivo regardless of the in vitro
result. Methods of performing susceptibility tests for Listeria and inter­
pretive criteria can be found in CLSI document M45-A (CLSI, 2010).
Resistance to chloramphenicol, macrolides, and tetracyclines has been
found in several clinical isolates (Wellinghausen, 2011; Hernandez-Milian
& Payeras-Cifre, 2014). Ampicillin, alone or in combination with an ami-
noglycoside, has been used successfully in the treatment of infections
caused by L. monocytogenes. Trimethoprim-sulfamethoxazole may be used
as alternative therapy in penicillin-allergic patients. Newer gram-positive
antibiotics such as daptomycin, linezolid, and tigecycline appear sus­
ceptible in vitro, but limited clinical data document their in vivo
effectiveness.
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TABLE 58-6
Differential Characteristics of Listeria monocytogenes and
Erysipelothrix rhusiopathiae
Test L. monocytogenes E. rhusiopathiae
β-Hemolysis + –
Growth at 4° C + –
Catalase + –
Motility + –
Esculin hydrolysis + –
Gluconate utilization + –
Voges-Proskauer + –
H2S in triple sugar iron agar – +
+, Positive; –, negative.
Erysipelothrix
Characteristics
Erysipelothrix rhusiopathiae is a catalase-negative, non–spore-forming, non-
motile, facultatively anaerobic gram-positive bacillus that has a worldwide
distribution. Microscopically, they appear as short rods with rounded ends,
occurring singly, in short chains, or in nonbranching filaments. Two
species have been identified: E. rhusiopathiae and E. tonsillarum. A third
species, E. inipinata, has been proposed recently. E. rhusiopathiae is a rec-
ognized pathogen in humans, occasionally causing erysipeloid, a localized
cutaneous infection of hands and fingers, obtained after exposure to
animals or animal products. E. tonsillarum has not been isolated from
PART 7
human specimens (Wellinghausen, 2011).
Clinical Manifestations and Pathogenesis
E. rhusiopathiae is usually transmitted to humans from animals by means
of skin wounds produced by contaminated objects or in contact with blood,
flesh, viscera, or feces of infected animals. E. rhusiopathiae is widespread in
nature in wild and domestic animals, birds, fish, and decaying organic
matter and causes infection in swine, sheep, rabbits, cattle, birds, and fowl.
At risk of infection with this organism are butchers, abattoir workers,
fishermen, fish handlers, poultry processors, and veterinarians. The most
common form of erysipeloid is a local cutaneous infection manifested by
pain, swelling, and a cutaneous eruption characterized by a slowly progres-
sive, slightly elevated, violaceous zone around the site of inoculation. The
swelling and erythema migrate peripherally, and the lesion involutes
without desquamation. Systemic disease is rare, but numerous case reports
describe septicemia and endocarditis. Also rarely reported have been cases
of arthritis and brain abscess. Virulence factors include a hyaluronidase, a
neuraminidase, and a heat-labile capsule (Wang et al, 2010).
Laboratory Diagnosis
Biopsy and tissue aspirates from erysipeloid lesions are the best specimens
for culture. The organisms are located deep in the subcutaneous layer of
the leading edge of the lesion; therefore, swabs of the surface of the skin
are not useful. The organism will grow on blood or chocolate blood agar,
but may require up to 7 days for growth. Conventional blood culture media
are suitable for its isolation from blood. They are considered nonhemo-
lytic, although greenish discoloration of the media beneath the colonies is
often observed after 2 days of incubation.
E. rhusiopathiae is oxidase and catalase negative. Characteristically, it
produces hydrogen sulfide (H2S) in triple-sugar iron agar (TSIA) and fer-
ments glucose and lactose. It is nonmotile, does not reduce nitrates to
nitrites, and is negative for urease, gelatin, and esculin hydrolysis. A trait
highly characteristic of E. rhusiopathiae is the “pipe cleaner” pattern of
growth in gelatin stab cultures incubated at 22° C. This organism can be
readily distinguished from Listeria spp. (see Table 58-6).
Antimicrobial Susceptibility
Erysipelothrix is susceptible to the penicillins, the cephalosporins, imipe-
nem, erythromycin, clindamycin, chloramphenicol, the tetracyclines, and
the fluoroquinolones but is resistant to sulfonamides, aminoglycosides, and
vancomycin. Penicillin is the treatment of choice for localized and systemic
infection (Wellinghausen, 2011). Methods used for performing susceptibil-
ity tests for Erysipelothrix rhusiopathiae and interpretive criteria can be
found in CLSI document M45-M2 (CLSI, 2010).
1125

58  Medical Bacteriology
Figure 58-9  Gram stain of Bacillus cereus in pleural fluid.
Prevention
Prevention of human disease is recommended by control of animal disease
through sound husbandry, herd management, good sanitation, and immu-
nization procedures (Wang et al, 2010).
Bacillus
Characteristics
Members of this genus are strictly aerobic or facultatively anaerobic, rod-
shaped, spore-forming, gram-positive, and catalase-positive organisms.
Figure 58-9 shows a Gram stain of a Bacillus sp. seen in pleural fluid. With
the notable exception of Bacillus anthracis, they are usually motile by means
of lateral or peritrichous flagella. Some strains stain gram-negatively and,
because of their variable oxidase reactions, can be confused with gram-
negative bacilli. The most reliable diagnostic characteristic of the genus is
spore formation, which occurs optimally and on a variety of media under
aerobic conditions at 25° to 30° C. In Gram-stained smears, endospores
are detectable by the presence of unstained defects or holes within the cell.
The spores themselves can be stained by any of several methods.
Clinical Manifestations and Pathogenesis
Of the numerous species of Bacillus, B. anthracis is the only one that is
uniformly and highly pathogenic. Great care must be exercised when
handling material suspected of harboring this species. Work should be
performed in biological safety cabinets by gloved, gowned, masked, and
immunized personnel; work surfaces must be disinfected with 5% hypo-
chlorite or 5% phenol; and all supplies, materials, and equipment must be
decontaminated. Because B. anthracis spores have been used as a means of
bioterrorism, cultures containing suspect B. anthracis should be handled
only by reference or public health laboratories.
Three forms of anthrax are recognized: cutaneous, inhalation, and
intestinal. In its cutaneous form, anthrax produces a small, red, macular
lesion that progresses to a vesicle and finally to necrosis with formation of
a characteristic black eschar. Regional lymphadenopathy and septicemia
may occur. Mortality in untreated cases with this form of disease is approxi-
mately 20%. Inhalation of anthrax spores can lead to acute bronchopneu-
monia, mediastinitis, and septicemia (“woolsorter’s disease”). The mortality
in recognized cases of this form of disease is nearly 100%. Intestinal
anthrax follows the ingestion of contaminated food and is manifested by
nausea, vomiting, and diarrhea. In some cases, gastrointestinal bleeding is
followed by prostration, shock, and death. Septicemia can occur in all three
forms of anthrax and may lead to a fatal, purulent meningitis. More
recently, a fourth type of anthrax, injectional anthrax, has been recognized
in heroin addicts, resulting in severe soft tissue infections with an increased
risk of shock and higher level of mortality than is associated with cutaneous
infections (Sweeney et al, 2011).
A major factor in the organism’s pathogenic capabilities is its glutamyl
polypeptide capsule, which inhibits phagocytosis; anticapsular antibodies
do not protect against the disease. A complex toxin with three components
(edema factor, protective antigen, and lethal factor) is responsible for the
signs and symptoms of anthrax. The cell wall peptidoglycan is believed to
produce a robust intravascular inflammatory response that can lead to
hemodynamic and organ dysfunction, resulting in shock (Logan, 2011;
Sweeney et al, 2011).
1126
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Humans become infected with anthrax by contact with and inhalation
or ingestion of infected animals, their carcasses, or their by-products.
Cattle, sheep, horses, and goats are the animals most frequently infected
and provide a ready source of vegetative organisms that sporulate and
perpetuate the environmental contamination.
Although usually saprophytic, other species of Bacillus can cause disease.
Bacillus cereus has been associated with ear infection, pneumonia, post-
traumatic ocular wound infection, septicemia, endocarditis, and meningi-
tis. Patients with pneumonia and septicemia are often immunosuppressed.
Bacteremia is frequently associated with intravenous drug use and with
contaminated intravascular devices (Stevens et al, 2012).
Two forms of gastroenteritis are associated with Bacillus species. Food
poisoning caused by Bacillus may occur within 1 to 6 hours following
ingestion of food contaminated by B. cereus that has produced a preformed
heat-stable toxin. Major manifestations of Bacillus food poisoning include
nausea, vomiting, cramps, and occasionally diarrhea, but no fever. Typi-
cally, this form of Bacillus gastroenteritis results from the bulk preparation
of foods that are not reheated before they are served. B. cereus type 1 grows
particularly well in fried rice and is more heat resistant than other types,
so this form of gastroenteritis is frequently seen in association with con-
sumption of cooked rice in Chinese restaurants. The second form of
gastroenteritis caused by Bacillus spp. results from contamination of poultry
and vegetable dishes and is characterized by the onset of cramps and diar-
rhea 8 to 16 hours following ingestion of contaminated food. In this
instance, the major manifestations of B. cereus infection are caused by the
production of a heat-labile enterotoxin. From 1998 to 2008, 1229 food-
borne outbreaks caused by B. cereus, (rice dishes were implicated in 50%
of the cases), Clostridium perfringens, and Staphylococcus aureus were reported
in the United States. In 2008, there were 17 to 18 suspected outbreaks of
gastroenteritis associated with B. cereus as compared to over 40 outbreaks
due to S. aureus and 10 or 11 outbreaks due to C. perfringens (Bennett et al,
2013).
The genus Bacillus contains more than 100 species; other than B.
anthracis and B. cereus, common species include B. subtilis, B. licheniformis,
B. megaterium, B. pumilus, and B. thuringiensis. Many species have been
renamed, however, and more than 25 new genera of gram-positive spore-
producing aerobic bacilli have been named. One of those genera, Paeniba­
cillus, contains species that have been associated with clinical disease,
including meningitis and endophthalmitis. Species of Paenibacillus include
P. alvei, P. polymyxa, P. popilliae, P. sanguinis, P. massiliensis, P. timonensis, and
P. thiaminolyticus (Anikpeh et al, 2010; Logan, 2011). P. macerans has been
found in contaminated blood culture bottles in a neonatal intensive care
unit, and P. pasadenensis has been isolated in spacecraft facilities and also as
a cause of mediastinitis following heart surgery (Noskin et al, 2001;
Anikpeh et al, 2010).
Laboratory Diagnosis
In cases of suspected cutaneous anthrax, vesicle fluid and material under
the edge of the eschar should be collected with a swab for smear and
culture. For suspected inhalation anthrax, sputum should be collected for
smear and culture. Cultures of stool should be considered in the intestinal
form. Smears and cultures consisting of CSF should be requested in sus-
pected meningitis. In the septicemic stage, blood cultures should be
performed.
Finding large, boxcar-shaped gram-positive cells in smears of any of
these specimens should raise suspicion for the diagnosis. Fluorescent
microscopy, available in some state health laboratories and at the CDC,
can provide a rapid presumptive diagnosis. As mentioned earlier, because
of the use of B. anthracis in terrorist attacks, sentinel laboratories (level A)
should send any suspicious isolates of this organism to their state health
laboratory or the CDC.
Species of Bacillus grow well on sheep blood agar. Colonies of B.
anthracis are usually flat, with an irregular margin (“Medusa head”), appear
off-white with a ground glass surface, and are usually nonhemolytic. Figure
58-10 demonstrates the colonial morphology of B. anthracis on blood agar.
When touched with an inoculating loop, the colonies are tenacious and
will stand up like beaten egg white. The Medusa head colony may be seen
with B. cereus and certain other Bacillus species. Anthrax bacilli are non-
motile in a hanging drop test or in semisolid media, whereas most other
species of Bacillus are motile. Motility by the hanging drop method is a
useful differential test between B. anthracis and B. cereus but must be carried
out with a fresh broth culture of the organism. Additional characteristics
and biochemical reactions that may be used to identify isolates to the
species level are summarized by Logan (2011). A commercial system has
been evaluated recently for identification of aerobic endospore-formers;
93% of the strains were correctly identified to species level in the genera

Figure 58-10  Colonies of Bacillus anthracis on blood agar. Note the irregular
edges of the colonies.
Bacillus, Paenibacillus, Aneurinibacillus, Brevibacillus, Geobacillus, and Virgi­
bacillus (Halket et al, 2010). Bacillus spp. have been correctly identified
using Maldi-Tof (Farfour et al, 2012).
The diagnosis of B. cereus gastroenteritis cannot be accurately made by
culture of stool because the organisms may be a component of the indig-
enous gut flora. Diagnosis, therefore, depends on quantitative culture of
the suspected contaminated food. The presence of greater than or equal
to 105 colony-forming units (CFUs)/g in the suspected food constitutes
presumptive evidence of B. cereus food poisoning (Logan, 2011). This
testing is usually not performed in a routine clinical microbiology
laboratory.
Antimicrobial Susceptibility
Although susceptible to a variety of agents, antibiotic therapy of anthrax
has centered on the use of fluoroquinolones. These agents are highly active
against B. anthracis, strains of which are typically susceptible to β-lactam
antibiotics. However, many strains of Bacillus spp. elaborate β-lactamases
in nature, so these agents usually are not considered as first-line drugs of
choice until the specific susceptibility of the isolate is known. Most strains
of Bacillus spp. are inhibited by fluoroquinolones, tetracyclines, aminogly-
cosides, and chloramphenicol at low concentrations (Logan, 2011), and
most are susceptible to vancomycin, but reports have described vancomy-
cin resistance in B. circulans and P. thiaminolyticus. Bacillus strains have been
demonstrated to carry genes similar to the vanA gene of the enterococci
(Patel et al, 2000). Methods of performing susceptibility tests and of inter-
preting their results can be found in the CLSI document M45-M2 (CLSI,
2010).
Prevention
Prevention of anthrax in humans ideally depends on its control in animals.
Prompt diagnosis of sick animals, their isolation and therapy, and crema-
tion of carcasses are indicated when sporadic outbreaks occur. In enzootic
areas, vaccination with nonencapsulated spore preparations is used. Occu-
pationally exposed persons should also be immunized. Acute diarrheal
disease caused by B. cereus may be prevented by proper cooking and refrig-
eration of foods prepared in bulk to prevent proliferation of vegetative
forms of the bacteria and formation of the enterotoxin.
Nocardia
Characteristics.  In Gram-stained smears of clinical specimens,
Nocardia spp. appear as long, thin, beaded, branching gram-positive bacilli
(Fig. 58-11). The most distinguishing quality of the nocardiae is their
partial acid fastness; cells stain positively with a modified acid-fast (Ziehl-
Neelsen or Kinyoun) stain, differentiating them from Actinomyces, which
may have a similar Gram-stain appearance. Partial acid fastness may be
difficult to demonstrate and can be enhanced by growing the organism for
about 4 days on Middlebrook 7H10 agar or in litmus milk broth. Most
clinical infections have been caused by members of the Nocardia asteroides
complex, most commonly Nocardia cyriageorgica, Nocardia farcinica, and N.
nova, followed by Nocardia brasiliensis and rarely Nocardia otitidis caviarum
(Cloud et al, 2004).
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Figure 58-11  Sputum smear stained with Gram stain shows neutrophils,
amorphous debris, and filamentous, beaded, branched gram-positive bacilli
(oil immersion).
Clinical Manifestations and Pathogenesis
Nocardiae are found in soil and organic material worldwide and cause
disease in many animals and in fish. Human infection is slightly more
common in males than in females. It is usually acquired via inhalation of
the organism but may occur following trauma and contact with contami-
nated soil, or the organism may enter the body via the gastrointestinal tract
when contaminated material contacts an area of mucosal ulceration.
In the lungs, Nocardia spp. are phagocytosed by alveolar macrophages
PART 7
and grow intracellularly, eliciting a mixed inflammatory response (neutro-
phils, lymphocytes, and macrophages), eventually resulting in abscess or,
occasionally, granuloma formation. In vitro studies of the host defenses
against Nocardia spp. suggest that neutrophils, activated macrophages, and
cytotoxic T cells are involved. Although neutrophils do not kill virulent
Nocardia spp., they inhibit their growth, possibly suppressing the infection
until macrophages are fully activated. If the infection is not contained
within the lung, organisms spread to other tissues by advancing growth,
thus producing empyema, chest wall involvement, and draining sinuses,
or by hematogenous dissemination, resulting in abscess formation, espe-
cially in the brain; subcutaneous tissues; and kidneys. The primary host
factor associated with increased risk of nocardiosis is cellular immune
dysfunction, although many persons infected with Nocardia spp. have
no recognized cellular or humoral immune defect (Budzik et al, 2012;
Wilson, 2012).
Pulmonary disease, the most frequent manifestation of nocardiosis, is
characterized by fever, anorexia, weight loss, cough, dyspnea, and pleuritic
chest pain. Skin and subcutaneous disease may present as pyoderma, cel-
lulitis, single or multiple abscesses, lymphocutaneous disease resembling
sporotrichosis, or nodules. In Central and South America, primary infec-
tions of the skin with N. brasiliensis may produce an actinomycetoma (a
localized indurated granulomatous mass with sinus tracts draining pus and
“sulfur” granules), typically on the lower extremities. Disseminated disease
is usually caused by members of the N. asteroides complex. It originates in
the lung in most cases and typically is manifested as single or multiple
abscesses involving the CNS (Anagnostou et al, 2014). The skin is the
second most common site of dissemination, followed by kidney, liver, and
lymph nodes (Conville & Witebsky, 2011).
Laboratory Diagnosis
Nocardia spp. grow aerobically on most nonselective media such as sheep
blood and chocolate agars, potato dextrose agar, Sabouraud’s dextrose agar,
and Löwenstein-Jensen or Middlebrook media and in 7H9 broth used for
mycobacterial culture. These organisms will grow on buffered charcoal-
yeast extract (BCYE) used for the isolation of Legionella spp. as well. It is
important to note that Nocardia spp. may not always survive the decon-
tamination procedures used for recovery of mycobacteria. Incubation in
the presence of 10% CO2 enhances growth. Nocardia spp. may grow in 48
hours, but colonies typically appear in 5 to 10 days as waxy, bumpy, or
velvety rugose forms, often with yellow to orange pigment, depending on
the species. Observing branching filaments that stain only with a modified
acid-fast stain distinguishes Nocardia spp. from mycobacteria. Nocardia spp.
are differentiated from most other aerobic actinomycetes by testing resis-
tant to the action of lysozyme (Nocardia spp. are resistant; Streptomyces spp.,
1127
 TABLE 58-7
58  Medical Bacteriology
Differentiation of Nocardia spp. Based on Antimicrobial Susceptibility Pattern*
Amox/
Amik Clav Cefotax Ceftriax Ciproflox Clari Gent Imi Kana Linez Mino Sulfa Tobra Amp Eryth Carb

Nocardia cyracigeorgica S R S S R R S S S S/I S R R

(N. asteroides drug
pattern VI)/N.
asteroides VII
Nocardia farcinica S S R R S R R S R S S/I R/S R R R
(drug pattern V)
Nocardia nova complex† S R S S R S S S S/I S S S R
(drug pattern III)
Nocardia abscessus S S S S R R R/S S S/I S S R R
(drug pattern 1)
Nocardia brasiliensis S S S/R S/R R R S R R S S/I S S R R S
Nocardia S S S/R S/R S S S R R S R S S R S
pseudobrasiliensis
Nocardia S R R R S S R S‡ S S/I S R R
otitidiscaviarum
(often R to all
β-lactams)
Nocardia transvalensis R S S S S R R S R S S/I S R R R
complex (drug
pattern IV)
Nocardia brevicatana/ S S S R S S S S/I S S S S
paucivorans

Reproduced with permission of Dr. Richard Wallace and Ms. Barbara Brown-Elliott.
Amik, Amikacin; Amox/clav, amoxicillin/clavulanate; Amp, ampicillin; Carb, carbenicillin; Cefotax, cefotaxime; Ceftriax, ceftriaxone; Ciproflox, ciprofloxacin; Clari, clarithromycin;
Eryth, erythromycin; Gent, gentamicin; I, intermediate; Imi, imipenem; Kana, kanamycin; Linez, linezolid; Mino, minocycline; R, resistant; S, susceptible; Sulfa, sulfamethoxa-
zole; Tobra, tobramycin.
*Table based on majority of isolates tested.
†
Includes N. nova/veterana/africana.
‡
Kanamycin zone ≥ amikacin zone.

Rhodococcus spp., Gordona spp., and Actinomadura spp. are susceptible) and
examining their morphology on tap water agar. For example, the latter can
help in differentiating the branching Nocardia from nonbranching Rhodo­
coccus spp. Because many new species of Nocardia have been recognized
over the past several years, the use of biochemical tests (e.g., casein, hypo-
xanthine, tyrosine, xanthine) is not sufficient for identification. Molecular
tests (e.g., 16S rDNA sequencing, PCR-restriction enzyme pattern analy-
sis [PRA]) of MALDI-TOF (Farfour et al, 2012) are required for accurate
species identification (Cloud et al, 2004; Patel et al, 2004). Identification
to the species level is important because of the variability in antibiotic
susceptibility patterns noted among species (Table 58-7) (Brown-Elliott
et al, 2006; Schlaberg et al, 2014).
Antimicrobial Susceptibility
Sulfonamides (alone or in combination with trimethoprim—e.g.,
trimethoprim-sulfamethoxazole) are usually the antimicrobial agents of
choice; however, optimal antimicrobial therapy depends on the species of
Nocardia present, the susceptibility pattern of the individual strain, and the
type of infection. Other potential antimicrobial agents include amikacin,
clarithromycin, imipenem, or a quinolone, depending on the species iso-
lated. The CLSI has information about methods used for susceptibility
testing and interpretation of results for Nocardia and other aerobic actino-
mycetes (CLSI, 2011).
Other Aerobic Actinomycetes
Other genera of actinomycetes that are medically relevant to humans
include Rhodococcus, Gordona, Tsukamurella, Actinomadura, and Streptomyces.
One other member, Tropheryma whippelii, is nonculturable and is the puta-
tive agent of Whipple’s disease.
Clinical Manifestations and Pathogenesis
Members of this group of organisms are ubiquitous in the environment
and have been isolated from soil, fresh water, marine water, and organic
matter. Rhodococcus equi is the most common Rhodococcus sp. pathogenic to
humans. It is an opportunistic pathogen in severely immunocompromised
hosts, causing a slowly progressive granulomatous pneumonia. It may be
isolated from the blood of infected patients. The organism is likely
acquired via the respiratory route, possibly as a result of exposure to
infected animals. The organism’s ability to persist in and ultimately destroy
macrophages probably accounts for its ability to cause disease (Weinstock
& Brown, 2002). Infections caused by Gordona spp. and Tsukamurella spp.
are increasingly being reported (Savini et al, 2012; Ramanan et al, 2013).
Tsukamurella spp. appear to be pathogenic only when certain clinical condi-
tions are present (e.g., immunosuppression, presence of foreign body,
active chronic infection such as tuberculosis) (Woo et al, 2003). Actino­
madura spp. are a frequent cause of actinomycotic mycetomas, most of
which are seen in tropical and subtropical countries, where walking bare-
foot increases the chance of exposure through repeated puncture wounds.
Streptomyces spp. have traditionally been considered of little medical sig-
nificance; however, one species, Streptomyces somaliensis, has been identified
as an etiologic agent of actinomycotic mycetoma. Other Streptomyces spp.
have only occasionally been reported to be of medical importance (Mossad
et al, 1995).
Laboratory Diagnosis
Aerobic actinomycetes are slow growing and may require 2 to 3 weeks of
incubation. These microorganisms grow on most of the nonselective
media used to isolate bacteria, mycobacteria, and fungi. Species of Rhodo­
coccus grow as coccobacilli in a zigzag fashion. Rudimentary branched fila-
ments have been observed from liquid media. Colonies may be rough,
smooth, or mucoid and have pigments ranging from buff to coral to orange
to deep rose after several days of incubation. R. equi is usually pale pink,
pale yellow, or coral and may appear slimy. Gordona spp. range from
smooth, mucoid colonies that are adherent to the media to dry, raised
colonies. The pigment produced may be beige to salmon-colored. Tsuka­
murella spp. are slightly acid fast by the modified Kinyoun. They appear
as long rods that fragment into three parts. No aerial hyphae are produced.
The colonies are circular with entire or rhizoid edges. They may be dry
or creamy, with a white to orange pigment. Rough colonies may be pro-
duced after 7 days of incubation. Species of Actinomadura form waxy,
cerebriform, tough, membranous white, yellow, pink, or red colonies.
Colonies of Streptomyces are dry to chalky, heaped or folded, gray-white to
yellow, and have the odor of a musty basement. A wide variety of pigments
are produced that color the substrate and hyphae. Some species do not

1128
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Figure 58-12  Sputum smear stained with Gram stain shows many neutrophils
and intracellular gram-negative diplococci, suggestive of Neisseria meningitidis
infection (oil immersion).
produce aerial hyphae. As with the Nocardia spp., complete identification
of these members of the aerobic actinomycetes often requires molecular
sequencing methods (Conville et al, 2011).
GRAM-NEGATIVE BACTERIA—COCCI
Neisseria
Characteristics
This genus is composed of species that are nonmotile and catalase and
oxidase positive; these aerobic, gram-negative cocci are often arranged in
pairs, with flattened adjacent surfaces, giving the appearance of kidney
beans or coffee beans (Fig. 58-12). The organisms are somewhat fastidious,
in some instances requiring the addition of blood, serum, cholesterol, or
oleic acid to the medium to counteract growth inhibitors, such as fatty
acids. N. gonorrhoeae and N. meningitidis generally require prompt incuba-
tion in CO2 for growth; however, this is strain dependent, varies with the
phase of the organism’s growth curve, and is often lost in subcultures. N.
meningitidis and most N. gonorrhoeae are not inhibited by the presence of
vancomycin or lincomycin, colistin, and nystatin—a characteristic that is
particularly useful in their selective isolation from specimens contaminated
by other bacteria. Rarely, isolates of N. gonorrhoeae (especially AUH strains,
which require arginine, uracil, and hypoxanthine) are susceptible to van-
comycin (Elias et al, 2011).
Clinical Manifestations and Pathogenesis
Although opportunistic infections caused by species of Neisseria other than
N. gonorrhoeae and N. meningitidis have occasionally been reported in
compromised hosts, these species are generally nonpathogenic. N. menin­
gitidis may colonize the mucous membranes of the upper respiratory
tract—an event that is usually followed in 7 to 10 days by the formation
of bactericidal and hemagglutinating antibodies, which may not eliminate
the carrier stage but convey group-specific immunity. In a few cases,
disease results shortly after colonization, most frequently in the form of
meningococcemia and meningitis. The organism also has a tendency to
invade serous membranes and joint tissues, with the development of pleu-
ritis, pericarditis, and arthritis. Carriage of N. meningitidis in the nasophar-
ynx occurs in 5% to 15% of healthy individuals, and this rate may be higher
in confined groups such as military recruits. A direct correlation between
carrier rates and incidence of disease has not been established, with the
possible exception of members of large households or households with an
infant or childhood case during epidemics of disease. N. meningitidis has
also been isolated from genital sources, where its clinical significance is
uncertain. When cultured from these sources, bacteria may be misidenti-
fied as N. gonorrhoeae unless appropriate tests for distinguishing these
species are performed.
The principal virulence factor of N. meningitidis is a lipopolysaccharide–
endotoxin complex, which in experimental animals activates the clotting
cascade, depositing fibrin in small vessels, producing hemorrhage in the
adrenals and other organs, altering peripheral vascular resistance, and
leading to shock and death.
The pathogenesis and clinical manifestations of N. gonorrhoeae infec-
tions differ somewhat from those of N. meningitidis. Pathogenic types of
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N. gonorrhoeae adhere by means of pili, which are not produced by non-
pathogenic types, to various human cells. These antigenically heteroge-
neous pili, which represent one of the principal virulence factors of N.
gonorrhoeae, may inhibit phagocytosis and stimulate strain-specific anti-
body formation. Other possible virulence factors of N. gonorrhoeae are less
clearly defined. Both N. gonorrhoeae and N. meningitidis produce an IgA
protease, which may be important in their pathogenesis because IgA is the
antibody class that predominates in secretions on mucous membranes
(Elias et al, 2011).
Laboratory Diagnosis
The single most important element in the laboratory diagnosis of
infection caused by N. meningitidis or N. gonorrhoeae is the specimen,
including proper selection, collection, and transport to the laboratory (see
Chapter 64). The pathogenic species are sensitive to drying and extremes
of temperature, and material must be cultured promptly to enhance recov-
ery. They are mesophilic and grow poorly, if at all, at room temperature.
Many require prompt incubation in CO2 (2% to 8%) for primary isolation.
Media containing chocolatized blood are commonly used for cultures and
should contain antibiotics (i.e., vancomycin or lincomycin, as well as colis-
tin, nystatin or anisomycin, and trimethoprim) if the specimen is prone to
be contaminated by indigenous flora. Vancomycin-susceptible gonococci
will grow on media containing lincomycin; however, because of the syn-
ergistic interaction of lincomycin and trimethoprim, the latter must be
omitted from media containing lincomycin. Direct inoculation of speci-
mens at the bedside followed by prompt incubation at 35° C in CO2 is
optimal. This can be accomplished in several ways: placing the medium
into a candle jar; placing the medium in a sealed bag containing a citric
acid bicarbonate tablet; or using a medium contained within a bottle
having a CO2 atmosphere. If any of these culture systems must be mailed
to a reference laboratory for processing, they must first be incubated
overnight to ensure growth of the organisms.
PART 7
An isolate from a urogenital specimen showing the appropriate colony
appearance on a selective medium presumptively may be called N. gonor­
rhoeae based on results of Gram stain and oxidase and catalase tests. Gram-
stained smears prepared from colonies of N. gonorrhoeae should show
typical gram-negative diplococci, but organisms may occur in tetrads,
especially from young cultures. All species of Neisseria are oxidase positive,
and all species except N. elongata are catalase positive. Because Neisseria
other than N. gonorrhoeae may be recovered from urogenital sites, confir-
matory testing is strongly recommended and is required for all isolates
from extragenital sites, and when sexual abuse is suspected (preferably by
more than one method).
Confirmation of N. gonorrhoeae and identification of the other Neisseria
spp. are based on growth and biochemical characteristics (Table 58-8)
(Elias et al, 2011). The standard method of identification consists of detec-
tion of acid production from carbohydrates in a cystine trypticase acid
(CTA) base medium and other conventional biochemical tests. However,
given the drawbacks of conventional methods, more rapid identification
tests are used in most clinical laboratories. Tests for direct detection of N.
gonorrhoeae and N. meningitidis in clinical specimens are also available.
Typing of isolates of N. gonorrhoeae and N. meningitidis is done primarily
for epidemiologic studies.
With the standard method of identification, acid production from
glucose, maltose, lactose, sucrose, and fructose in a CTA base medium and
a carbohydrate-free control are tested. Tubes are inoculated, incubated at
35° to 37° C in ambient air, and examined at 24-hour intervals until reac-
tions are interpretable, or for 72 hours. Expected results for the species of
Neisseria are shown in Table 58-8. Occasionally, however, an isolate of N.
meningitidis yields aberrant carbohydrate reactions: glucose-negative,
maltose-negative, or asaccharolytic. If N. meningitidis is strongly suspected
in these cases, identification can be confirmed by slide agglutination, using
pooled polyvalent grouping antisera or sera specific for individual sero-
groups. In addition to conventional carbohydrate degradation tests, reduc-
tion of nitrates and nitrites and production of DNase should be evaluated.
The latter is especially useful for identification of Moraxella catarrhalis,
which is DNase positive (Neisseria spp. are DNase negative). Drawbacks
to conventional tests are the requirement for a heavy inoculum, the need
to work with pure cultures, long turnaround time, and failure of some
fastidious strains of N. gonorrhoeae to grow.
Several commercial systems detect acid production from carbohy-
drates, usually in 1 to 4 hours (Elias et al, 2011). The inoculum must be
prepared from a pure culture of the isolate, so identification is generally
available 24 hours after isolation. Acid reactions of some N. gonorrhoeae
and, to a lesser extent, N. meningitidis may be difficult to interpret or may
be aberrant with some kits, and strains of N. gonorrhoeae that are weak
1129
 TABLE 58-8
58  Medical Bacteriology
Differentiation of Species of Neisseria and Moraxella catarrhalis
N. gonorrhoeae N. meningitidis N. cinerea N. lactamica N. sicca N. subflava N. flavescens N. mucosa M. catarrhalis

Growth
Thayer-Martin +* + – + – – + – †

medium
Nutrient agar, – – – – + + – + +
25° C
Oxidase + + + + + + + + +
β-Galactosidase – – – + – – – – –
Reduction of – – – – – – – + +
nitrate
DNase – – – – – – – – +
Production of Acid from
Glucose + + –‡ + + + – + –
Maltose – + – + + + – + –
Lactose – – – + – – – – –
Sucrose – – – – + D§ – + –
Fructose – – – – + – – – –

+, ≥90% of strains positive; –, ≥90% of strains negative; D, variable; DNase, deoxyribonuclease.

*Most vancomycin-susceptible strains will not grow on Thayer-Martin medium.
†
Some strains positive and others negative.
‡
Weak reaction may occur in rapid carbohydrate utilization tests.
§
Biovar. perflava, +; biovar. flava, –.

producers of acid from glucose may appear to be glucose negative. Some
strains of Neisseria cinerea, which does not typically produce acid from
glucose, can appear glucose positive in certain systems.
Enzyme substrate tests provide rapid identification (1 to 4 hours) only
of isolates of oxidase-positive, gram-negative diplococci recovered on a
selective medium (Elias et al, 2011). They are valuable for differentiating
maltose-negative strains of N. meningitidis from N. gonorrhoeae, but color
changes may be subtle and if misinterpreted could cause isolates of N.
meningitidis and other Neisseria spp. to be incorrectly called N. gonorrhoeae.
Moreover, strains of N. cinerea and Kingella denitrificans that grow on
gonococcus-selective media could be misidentified as N. gonorrhoeae if not
confirmed by other procedures. Commercial products that combine
enzyme substrate tests with modified conventional tests provide accurate
identification of species of Neisseria and Haemophilus. Immunologic tests
for N. gonorrhoeae—in particular coagglutination—can be used to confirm
the biochemical identification of N. gonorrhoeae. Three tests are available
for this: the Phadebact Monoclonal GC Test (Boule Diagnostics AB, Hud-
dinge, Sweden), the GonoGen I (New Horizons Diagnostics, Columbia,
Md.), and the GonoGen II (New Horizons Diagnostics, Columbia, Md.).
Both false-positive and false-negative results have been reported with these
reagents A chemiluminescent nucleic acid probe for detection of N. gonor­
rhoeae can be used for culture confirmation (Limberger et al, 1992; Hale
et al, 1993). Nucleic acid amplification assays (NAAT) for use on endocer-
vical or urethral swab specimens and urine (see below) are also available
and may increase sensitivity when compared with nucleic acid probe and
culture techniques, largely because the problem with organism viability is
not an issue.
Cultural detection of Neisseria gonorrhoeae from urogenital sites has
been largely replaced by NAATs. Many NAATs are available that simulta-
neously detect N. gonorrhoeae and Chlamydia trachomatis in vaginal, cervical,
urethral, and urine samples with greater sensitivity than culture, and for
which loss of viability of N. gonorrhoeae in particular is an issue. These tests
are recommended by the CDC as the preferred test in symptomatic and
asymptomatic individuals, although there are also recommendations that
culture be available for both N. gonorrhoeae and C. trachomatis at least in
some laboratories in situations of potential antimicrobial resistance and in
some sexual assault situations (MMWR, 2014). These molecular tech-
niques are described in other sections of this text.
Antimicrobial Susceptibility
Despite the occasional recovery of N. meningitidis strains with decreased
susceptibility to penicillin, penicillin G remains the drug of choice for
treatment of meningococcal meningitis (Elias et al, 2011). Decreased sus-
ceptibility of N. meningitidis to penicillin is thought to be due to decreased
binding of penicillin by altered meningococcal cell wall penicillin-binding
proteins, PBP-2 and PBP-3. Other species of Neisseria have also demon-
strated this lowered affinity to penicillin. Other agents that have good
activity against N. meningitidis include the extended-spectrum cephalospo-
rins and chloramphenicol. Rifampin, minocycline, and the fluoroquino-
lones may be used for prophylaxis among household contacts. Standardized
methods of susceptibility testing and breakpoints in the CLSI documents
are now available (CLSI, 2014). The CLSI recommends broth microdilu-
tion or agar dilution with cation-supplemented Mueller-Hinton Broth
(with 2% to 5% laked horse blood) or Mueller-Hinton Agar (with 5% [vol/
vol] sheep blood) if testing is done. Enrichments such as IsoVitaleX (1%)
may also be needed. In laboratories where susceptibility testing for N.
meningitidis is not available, β-lactamase testing can be performed by using
the chromogenic cephalosporin test, the cefinase nitrocefin disk test, if
lowered susceptibility or clinical failure on penicillin is suspected. If posi-
tive, isolates can be shipped to a reference laboratory for further testing.
Because of widespread resistance of N. gonorrhoeae to penicillin and
tetracycline, current recommendations for treatment include extended-
spectrum cephalosporins but no longer include the newer fluoroquino-
lones. Although no resistance to the cephalosporins is apparent, resistance
to the fluoroquinolones has been documented (Fox et al, 1997; MMWR,
2007b). Therefore, susceptibility testing should be performed if symptoms
persist after treatment. β-Lactamase production can be detected using a
chromogenic cephalosporin. Disk diffusion using GC agar with 1% growth
supplement is recommended to determine the susceptibility of N. gonor­
rhoeae to the cephalosporins, quinolones, and spectinomycin. The CLSI
document recommends the agar dilution method or a disk diffusion
method for testing of N. gonorrhoeae (CLSI, 2014). In addition, an E-test
can be performed. For some agents, only a susceptible breakpoint is avail-
able because no resistant strains have yet been documented. If an isolate
with a nonsusceptible result to a third-generation cephalosporin is identi-
fied, for example, confirmation tests and referral to a reference laboratory
should be strongly considered (CLSI, 2014).
Prevention
A polysaccharide vaccine against N. meningitidis serogroups A, C, Y, and
W135 is licensed in the United States and is recommended for military
personnel for persons living in epidemic areas of developing countries, for
individuals with a nonfunctional or absent spleen, and for college students.
Two meningococcal conjugate vaccines have been developed for infants as
well (American Academy of Pediatrics, 2014), and there now is a separate
vaccine against serotype B. Antibiotic prophylaxis should be limited to
household contacts and those who have had contact with patients’ oral
secretions. Rifampin is currently the drug of choice. Laboratory safety is
very important when handling specimens and cultures positive for N.
meningitidis. Borrow and colleagues (2014) wrote an article on the

1130
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Figure 58-13  Gram stain of Moraxella catarrhalis in a sputum specimen. Note
the intracellular gram-negative diplococci that resemble Neisseria spp.
Figure 58-14  Gram stain of urine positive for Escherichia coli. The short, plump
gram-negative rods are typical of any member of the Enterobacteriaceae.

appropriate precautions for handling specimens and steps to take if acci-

dents do occur.
The use of preexposure antibiotics to prevent gonococcal disease is
discouraged because of the potential risks of sensitization and the emer-
gence of resistant strains. The sole exception to this rule is the application
of erythromycin ointment to the eyes of newborns to prevent gonococcal
(and chlamydial) ophthalmia (U.S. Preventive Services Task Force, 2012).
Moraxella catarrhalis

PART 7
M. catarrhalis may be carried in the oropharynx of healthy children and
adults. It is an encapsulated organism, and extending from its outer mem-
brane are pili that serve as adhesins. The most common infections caused
by this organism are bronchitis, otitis, sinusitis, and pneumonia (especially
in persons with underlying chronic lung disease) (Murphy & Parameswaran,
2009; Vaneechouette et al, 2011). More recently, molecular mechanisms
have been described that demonstrate the virulence factors possessed by
strains of M. catarrhalis responsible for otitis media (Hassan, 2013). M.
catarrhalis is an infrequent cause of bacteremia, endocarditis, meningitis,
urogenital infection, and ophthalmia neonatorum. Figure 58-13 shows the
Gram stain of sputum in which M. catarrhalis is seen in large quantities
inside and outside the polymorphonuclear leukocytes. Moraxella catarrhalis Figure 58-15  Gram stain of a cerebrospinal fluid specimen from a neonate
initially was part of the Neisseria genera and later was transferred to the containing gram-negative bacilli that grew Escherichia coli.
Branhamella genus for a short time. M. catarrhalis is a coccus that morpho-
logically resembles Neisseria spp., unlike other members of the genus
Moraxella (e.g., Moraxella lacunata, Moraxella osloensis, Moraxella atlantae),
which appear as rods. M. catarrhalis bacteria are oxidase and catalase posi- gram-negative bacilli that produce acid fermentatively from glucose and
tive but can be differentiated from Neisseria spp. in their ability to grow reduce nitrates to nitrites. Figure 58-14 shows a Gram stain of Escherichia
readily on blood agar, their lack of oxidative metabolism (sugars will be coli, but it could represent any member of the Enterobacteriaceae. Genera
negative), and their production of DNase. Nearly all isolates of M. catarrh­ included in this group are Budvicia, Buttiauxella, Cedecea, Citrobacter,
alis produce β-lactamase, which can be detected using nitrocefin. Although Edwardsiella, Enterobacter, Escherichia, Ewingella, Hafnia, Klebsiella, Kluyvera,
they should be assumed to be resistant to penicillin because of this, these Leclercia, Leminorella, Moellerella, Morganella, Obesumbacterium, Pragia,
isolates generally remain susceptible to cephalosporins, trimethoprim- Pantoea, Photorhabdus, Proteus, Providencia, Rahnella, Salmonella, Serratia,
sulfamethoxazole, and β-lactamase inhibitor combinations (Vaneechouette Shigella, Tatumella, Trabulsiella, Xenorhabdus, Yersinia, and Yokenella. Only a
et al, 2011). few of these are discussed here (Nataro et al, 2011).

Clinical Manifestations and Pathogenesis

GRAM-NEGATIVE BACTERIA—BACILLI
The gram-negative bacilli make up a complex group. They are broken
down into Enterobacteriaceae, which are those found normally in the
gastrointestinal (GI) tract or that colonize and cause infection there pri-
marily; nonfermentative gram-negative bacilli, which usually are not found
as part of the normal flora of humans but rather are environmental bacte-
ria; non-Enterobacteriaceae, which can cause GI infection, such as Vibrio
or Campylobacter; agents of infections with specific epidemiologic charac-
teristics, such as Legionella and Francisella; other gram-negative bacilli,
including Haemophilus spp.; and miscellaneous genera. A good overview of
how to approach the identification of the aerobic gram-negative rods can
be found in Wauters and Vaneechouette (2011).
Enterobacteriaceae
Characteristics
The Enterobacteriaceae are aerobic and facultatively anaerobic, non–
spore-forming, nonmotile or peritrichously flagellated, oxidase-negative,
Enterobacteriaceae are found on plants, in soil, in water, and in the intes-
tines of humans and animals. They have been associated with many clinical
infections, including abscesses, pneumonia, meningitis, septicemia, and
urinary tract infections. Those commonly associated with human infection
include E. coli, Klebsiella spp., Proteus spp., Enterobacter spp., Salmonella spp.,
Shigella spp., Serratia spp., Citrobacter spp., and Providencia spp. Figure
58-15 shows a Gram stain of CSF from a newborn child who was culture
positive for E. coli. In the urinary tract, those frequently isolated are E. coli,
Proteus mirabilis, Klebsiella pneumonia, and K. oxytoca. Gram-negative pneu-
monias associated with the Enterobacteriaceae are frequently caused by
K. pneumoniae. Gram-negative bacteremias related to the Enterobacteria-
ceae are frequently caused by E. coli, K. pneumoniae, Enterobacter spp., and
P. mirabilis. Infections acquired in the hospital are likely to be caused by
members of antibiotic-resistant genera, such as Citrobacter, Enterobacter,
and Serratia. Enterobacteriaceae associated with diarrhea include Shigella
spp., Salmonella spp., E. coli (enterohemorrhagic [Shiga toxin producing],
enterotoxigenic, enteroinvasive, enteropathogenic, enteroadherent), and

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 Yersinia spp. Shigellas are rarely isolated from sources other than the GI
58  Medical Bacteriology
tract, whereas salmonellas are more likely to be isolated from other sources,
such as urine or blood. Plesiomonas sp. have recently been added to the
Enterobacteriaceae and remain as the only oxidase-positive member of this
family. They also can be associated with infections of the GI tract. Calym­
matobacterium granulomatis, an agent of the sexually transmitted disease
donovanosis, has been renamed Klebsiella granulomatis and is now part of
the Enterobacteriaceae, even though the organism cannot be cultured on
bacteriologic media (Abbott, 2011; Lagergård et al, 2011). This topic is
discussed later in this chapter.
Endotoxins that are present within the cell walls of the Enterobacteria-
ceae, as well as other gram-negative bacilli, are responsible for much of
the morbidity and mortality resulting from infections associated with these
bacteria. Endotoxins consist of lipid and polysaccharide moieties with
small quantities of amino acids and are often referred to as lipopolysac-
charides. Lipopolysaccharides may elicit fever, chills, hypotension, granu-
locytosis, thrombocytopenia, disseminated intravascular coagulation, and
activation of both classic and alternate complement pathways. Endotoxic
shock is the result of gram-negative septicemia, with endotoxin reacting
with macrophages, leukocytes, platelets, complement, and other serum
proteins to increase the blood levels of proteolytic enzymes and vasoactive
substances, resulting in pooling of blood, increased peripheral vasocon-
striction, and diminution in cardiac output. It has become clear that the
lethal effects of endotoxin are dependent on macrophage activation and
responsiveness and that the production of cachectin from the activated
macrophage plays a major role in causing profound shock and multiple
organ injury (Munford, 2006).
Other pathogenetic factors of the Enterobacteriaceae include the K1
antigen, which is associated with a high percentage of strains of E. coli
causing neonatal meningitis; the capsule of K. pneumoniae, which, like that
of the pneumococcus, inhibits phagocytosis; the Vi antigen of Salmonella
serotype typhi, which may interfere with intracellular killing of this organ-
ism; and various surface antigens, such as fimbriae, that mediate adherence
of the organism to mucosal surfaces.
Plasmid-mediated factors appear to play an important role in the inva-
sive properties of Salmonella, Shigella, and enteroinvasive strains of E. coli.
Moreover, the heat-labile enterotoxins (LT) and the heat-stable enterotox-
ins of E. coli are plasmid mediated. LT stimulates adenylate cyclase in
mucosal cells of the small intestine, which, in turn, activates cyclic adenos-
ine monophosphate (cAMP); this causes secretion of fluid and electrolytes
into the intestinal lumen and produces watery diarrhea. In contrast, heat-
stable (ST) enterotoxins appear to activate guanylate cyclase.
Laboratory Diagnosis
The isolation of gram-negative bacilli from contaminated specimens is
greatly facilitated by the use of differential and selective media (Table
58-9). Eosin methylene blue (EMB) and MacConkey’s agar can be
used interchangeably as minimally selective and differential media to ini-
tially select for and differentiate lactose-fermenting from non–lactose-
fermenting gram-negative bacilli. XLD and HE agars are more selective
differential media that are especially useful in selecting for Salmonella spp.
and Shigella spp. in heavily contaminated specimens such as stool. Bismuth
sulfite is a highly selective medium that is especially useful for the detection
of salmonellae in endemics or epidemics. Salmonella spp. produce H2S and
are easily recognized by the production of colonies with black centers on
XLD, HE, and bismuth sulfite agars. An enrichment medium, such as
selenite-F or gram-negative (GN) broth, should be used to allow detection
of low numbers of Salmonella spp. and Shigella spp. in stool. Cefsulodin-
irgasan-novobiocin (CIN) agar incubated at room temperature is selective
and differential for the recovery of Yersinia enterocolitica. Colonies will
appear as bull’s-eyes with red centers and transparent borders. MacCon-
key’s agar with sorbitol as the fermentable sugar is a differential medium
that is capable of differentiating the sorbitol-fermentation–negative E. coli
0157:H7, which is associated with hemolytic-uremic syndrome from most
other E. coli (Nataro et al, 2011).
For some of the Enterobacteriaceae, a few simple colonial character-
istics or biochemical reactions can be used to presumptively identify an
isolate. For example, Proteus spp. swarm on blood agar, Klebsiella spp.
form lactose-positive mucoid colonies, Serratia marcescens may produce a
red pigment, Salmonella spp. produce H2S, and E. coli is indole positive.
Definitive identification of these and other species requires additional
biochemical testing and/or molecular methods. Innumerable schemes
based on the use of conventional biochemical media have been described
for identification of the Enterobacteriaceae. Differential characteristics
of the Enterobacteriaceae most commonly encountered in the clinical
laboratory are shown in Table 58-10. Commercially prepared kits and
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automated devices are available and offer convenience and accuracy in
identifying the vast majority of isolates belonging to the Enterobacteria-
ceae. In some instances, identification is accurately made in a few hours.
Semiautomated systems may combine identification and antimicrobial
susceptibility testing in a single disposable unit. In general, accuracy of
identification among these systems is very high and comparable. Maldi-
Tof has been shown to be an excellent tool for the identification of
members of the Enterobacteriaceae and can usually provide results more
quickly than traditional or semiautomated biochemical methods (Richter
et  al, 2013).
Classification of the Enterobacteriaceae has undergone considerable
revision in recent years as the result of DNA hybridization and relatedness
studies. Because phenotypic groupings on the basis of biochemical reac-
tions are not always consistent with their DNA relatedness, the use of
tribes (e.g., Klebsielleae, Proteae) for grouping species within the Entero-
bacteriaceae has been discontinued.
Historically, the genus Salmonella has been divided into the following
species: S. typhi, Salmonella paratyphi A and B, Salmonella choleraesuis, Sal­
monella typhimurium, and Salmonella enteritidis. Because all groups have
been found to be genetically very closely related, current terminology now
recognizes only two species: S. enterica and S. bongori (rarely isolated from
humans), each of which contains multiple subspecies. Six subspecies of S.
enterica are known, with subspecies I (S. enterica subsp. enterica) as the usual
human isolate. More than 2000 serotypes of Salmonella have been identi-
fied, most belonging in the subspecies enterica. Serotyping is based on
immunologic reactivity of the heat-stable somatic “O” antigens, which are
predominantly lipopolysaccharide in content, and the heat-labile flagellar
“H” antigens. In the United States, Salmonella serotypes typhimurium and
enteritidis are the most common. Salmonella serotype typhi also produces a
heat-labile capsular polysaccharide Vi antigen. In practice, most clinical
laboratories identify isolates as Salmonella spp. based on biochemical reac-
tions and use group-specific immunologic reagents to assign isolates to a
specific serogroup. Commercial slide agglutination tests to differentiate
large serogroups—A, B, C, and D—are useful in differentiating typhoidal
salmonella from nontyphoidal strains. S. serotype typhi carries the D sero-
group and Vi antigen. Further identification of the specific serotype is
generally performed only by State Health Departments or other reference
laboratories (Nataro et al, 2011).
Isolates biochemically resembling Shigella are also classified by the
reactivity of the “O” antigen, as are isolates of E. coli that are identified as
potential causes of diarrhea and hemolytic-uremic syndrome. Such E. coli
are classified by the type of toxin produced as well. Commercial kits are
available to identify E. coli O157 and to detect some types of toxins in
culture or stool specimens; however, this type of testing is often sent out
to referral laboratories or the State Health Department. The CDC has
recommended that all laboratories consider testing stool samples for the
presence of shiga toxins I and II produced by certain strains of hemorrhagic
E. coli (O157:H7 and other serotypes). Shiga toxin is also produced by
Shigella, but the species is S. dysenteriae, which is not often seen in the
United States. Serologic antigen tests are available and are widely used in
clinical laboratories for this toxin (Nataro et al, 2011).
Antimicrobial Susceptibility
The susceptibility of Enterobacteriaceae to various antimicrobial agents is
highly variable. Susceptibility to ampicillin was common among strains
of E. coli and P. mirabilis, for example (although resistance in both has
increased greatly over the past 10 to 20 years), but was not expected among
most other clinically significant members of the Enterobacteriaceae. Resis-
tance to first-generation cephalosporins (cefazolin, cephalothin) is expected
for Enterobacter spp., Serratia spp., Citrobacter spp., Proteus vulgaris, Provi­
dencia spp., Morganella spp., and Yersinia spp.; susceptibility to third-
generation cephalosporins (e.g., ceftriaxone, cefotaxime, ceftazidime)
continues for many members of the Enterobacteriaceae. However, the
presence of extended-spectrum β-lactamases and Amp-C genes, which are
recognized by resistance of the bacteria to third-generation cephalosporins
and all β-lactam antibiotics, except carbapenems, is increasing in selected
strains of E. coli, K. pneumoniae, P. mirabilis, and others. Most Enterobac-
teriaceae are susceptible to aminoglycosides and fluoroquinolones. It had
been very unusual for a member of the Enterobacteriaceae to be resistant
to carbapenems (e.g., imipenem, meropenem, ertapenem, doripenem);
however, in the late 1990s, in a few U.S. states, isolates of K. pneumoniae
were first recognized that produced a blaKPC gene, resulting in production
of carbapenemases that inactivate all carbapenems. These strains are often
referred to as “KPCs” or K. pneumoniae–producing carbapenemases. These
isolates are resistant to most classes of antibiotics, except for an aminogly-
coside, colistin, and tigecycline. The spread of these KPC genes has
 TABLE 58-9
Differentiation of Aerobic Gram-Negative Bacilli
Salmonella Salmonella Salmonella
Escherichia Klebsiella Klebsiella Proteus Proteus Shigella Citrobacter Yersinia Enterobacter Serratia Morganella Providencia Serotype Serotype Serotype
Test coli pneumoniae oxytoca mirabilis vulgaris spp. freundii enterocolitica cloacae marcescens morganii alcalifaciens choleraesuis typhi paratyphi A

Indole + – + – + D – D – – + + – – –
Methyl red + – or + D + + + + + – + or – + + + + +
Voges-Proskauer – + + – or + – – – + (25° C)/ + + – – – – –
– (37° C)
Citrate – + + D D – + – (25° C) + + – + (+) – –
(Simmons)
H2S (in triple – – – + + – + – – – – – D + – or +
sugar iron
agar)
Urease – + + + + – D + D – or + + – – – –
Phenylalanine – – – + + – – – – – + – – – –
deaminase
Lysine + or – + + – – – – – – – – – + + –
decarboxylase
Arginine – or + – – – – – D – + – – – (+) – or (+) – or +
dihydrolase
Ornithine D – – + – D – or + + + + + – + –
decarboxylase
Motility + or – – – +* +* – + + + (25° C)/ + + + or – + + + +
– (37° C)

Downloaded from ClinicalKey.com at Universidad de Monterrey August 12, 2016.

Acid produced + + + – – – + or (+) D D – – – – – –
from lactose

+, ≥90% positive reactions within 2 days; –, ≥90% negative reactions; (+), positive reactions in 3 to 7 days; + or –, reactions of most strains positive; – or +, reactions of most strains negative; D, different reactions [+, (+), or –].

For personal use only. No other uses without permission. Copyright ©2016. Elsevier Inc. All rights reserved.
*Swarm on blood and chocolate agar.

1133
PART 7
 TABLE 58-10
58  Medical Bacteriology
Enteric Differential and Selective Media
Gram-positive
Bacteriostatic Fermentable Colony Color
Medium Agent Carbohydrate Indicator Fermenter Nonfermenter Category

Eosin methylene blue (EMB) Eosin Y Lactose* Eosin Y Red or black Colorless S, D
Methylene blue Methylene blue with sheen
MacConkey’s Crystal violet Lactose Neutral red Red Colorless S, D
Bile salts
Xylose-lysine-deoxycholate Bile salts Xylose Phenol red Yellow Red S, D
(XLD) Lactose
Sucrose
Hektoen enteric (HE) Bile salts Salicin Bromthymol blue Yellow-orange Green, blue-green S, D
Lactose
Sucrose
Salmonella shigella (SS) Bile salts Lactose Neutral red Red Colorless S
† †
Bismuth sulfite (BS) Brilliant green Glucose Bismuth sulfite S
Thiosulfate citrate bile salts Bile salts Sucrose Thymol blue Yellow Colorless S, D
sucrose (TCBS)‡ pH 8.6 Bromthymol blue

D, Differential; S, selective.
*Levine’s formulation.
†
H2S-producing salmonellae have black colonies.
‡
Used for isolation of vibrios.

continued, and it is feared that these will be introduced into E. coli, for
example, which accounts for significant nosocomial infections (Peirano
et al, 2014).
Detection of carbapenem resistance may be difficult with some strains
possessing this resistance, so the modified Hodge test may be used for
confirmation. A potential KPC is inoculated near a carbapenem antibiotic
disk onto an agar plate, onto which a lawn of carbapenem-susceptible E.
coli has first been placed. Inactivation of the carbapenem will result in a
reduction in the zone size of E. coli near the disk in close proximity to the
inoculated KPC if the test is positive. Ertapenem is a better marker for
carbapenem resistance than is meropenem or imipenem, but resistance to
one of these leads to resistance to all carbapenems (Endimiani et al, 2009a;
2009b; Kitchel et al, 2009; Abbott, 2011; Nataro et al, 2011; CLSI, 2014).
Use of Maldi-Tof and molecular methods for the detection of carbapenem
resistance has been reported (Johansson et al, 2014). Infection control
procedures are essential to prevent the spread of these highly resistant
strains.
Because the susceptibility pattern of the Enterobacteriaceae is unpredict-
able, as a general rule, susceptibility testing should be performed if anti-
microbial therapy is being considered. No susceptibility testing need be
performed if antimicrobial therapy is not instituted, as is the case for
uncomplicated enteric infection due to salmonella, for which therapy may
actually prolong the carrier state, or when a mixed flora infection is present
and individual susceptibilities may not be appropriate. Methods for per-
forming susceptibility tests, including the modified Hodge test and con-
firmatory extended-spectrum ESBL tests, and interpretive criteria can be
found in CLSI documents (CLSI, 2014).
Plesiomonas
Characteristics
Plesiomonas shigelloides, the only species in the genus Plesiomonas, is a facul-
tatively anaerobic, oxidase- and catalase-positive, glucose-fermenting,
gram-negative rod. Recent molecular genetic evidence demonstrates that
the genus Plesiomonas is most closely related to the genus Proteus. There-
fore, it has been placed into the Enterobacteriaceae family (Abbott, 2011)
as the only oxidase-positive member of this group of gram-negative bacilli.
Clinical Manifestations and Pathogenesis
P. shigelloides is found in aquatic environments that are limited geographi-
cally by its minimum growth temperature of 8° C. It may be found in fresh
and estuarine water, usually in tropical countries. It has been implicated as
a cause of gastroenteritis, especially following the ingestion of uncooked
shellfish. The diarrheal stool specimen frequently contains polymorpho-
nuclear leukocytes and red blood cells, although a cholera-like illness may
occur. Gastroenteritis may occur in sporadic cases, as well as in outbreaks.
Extraintestinal manifestations of infection with P. shigelloides include men-
ingitis, septicemia, cellulitis, arthritis, and endophthalmitis (Ampofo et al,
2001; Ozdemir et al, 2010). Virulence factors of P. shigelloides include
hemolysins, cytotoxins, production of exoenzymes associated with patho-
genicity, adhesive ability, and vacuolation of cell lines in vitro (Salerno
et al, 2010).
Laboratory Diagnosis
P. shigelloides can be isolated on a variety of nonselective and enteric-
selective media, including HE agar. Acid production from lactose is vari-
able, but on enteric media the organism usually appears to be a non–lactose
fermenter. It is indole positive; reduces nitrates to nitrites; produces cata-
lase; is methyl red positive; and ferments glucose, maltose, and trehalose
(Abbott, 2011).
Antimicrobial Susceptibility
P. shigelloides is susceptible to a variety of antimicrobial agents, including
cephalosporins, trimethoprim-sulfamethoxazole, imipenem, and the qui-
nolones (Abbott, 2011). Susceptibility to the penicillins is variable because
of the presence of a β-lactamase similar to that of Aeromonas spp.
GRAM-NEGATIVE BACTERIA—
NONFERMENTATIVE BACILLI
A group of gram-negative bacilli that do not ferment glucose and other
sugars are often lumped together under the heading of “nonfermenters.”
They account for about 15% of the gram-negative bacilli isolated from
hospitalized patients. Although many genera of nonfermenters are known,
75% of the clinically relevant ones are Pseudomonas aeruginosa, and most
of the remaining 25% are Acinetobacter spp., Stenotrophomonas maltophilia,
or Burkholderia cepacia. As a group, they are environmental bacteria and
are not usually found as part of the normal flora of the human body, except
as colonizers in hospitalized patients. They can be readily isolated from
water, soil, vegetables, plants, and hospital surfaces. Although no uniform
biochemical characteristics have been noted, they are often oxidase-
positive (except for Acinetobacter spp.), lactose-negative colonies on selec-
tive media such as MacConkey’s agar (although some of the species do not
grow on this media), and they are frequently resistant to many of the
antibiotics that are effective against members of the Enterobacteriaceae.
The four main species mentioned previously, as well as a few others, are
discussed in this chapter.
Pseudomonas
Characteristics
The genus Pseudomonas has undergone extensive revision, and now many
of the species that were previously classified in this genus have been
reclassified into the genera Burkholderia, Stenotrophomonas, Comamonas,
Shewanella, Ralstonia, Methylobacterium, Sphingomonas, Acidovorax, and Bre­
vundimonas. Of the species that remain, P. aeruginosa is the most significant
human pathogen. Figure 58-16 shows the Gram stain of P. aeruginosa in a
sputum specimen.
Pseudomonads are strictly aerobic, catalase-positive, oxidase-positive,
gram-negative bacilli. Their metabolism is respiratory and never

1134
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fermentative, with oxygen as the terminal electron acceptor. Some pseu-
domonads are motile by means of polar flagella.
Clinical Manifestations and Pathogenesis
Pseudomonads are found in moist environments. Some of the more
unusual habitats for these organisms include cosmetics, swimming pools,
hot tubs, and the inner soles of sneakers. The latter can lead to puncture
wounds that are infected with P. aeruginosa. The species causing the great-
est morbidity and mortality today is P. aeruginosa. Other species of Pseu­
domonas, although often isolated from clinical specimens, are only
occasionally involved in disease.
P. aeruginosa is ubiquitous in the hospital environment, existing almost
anywhere there is moisture, including medical equipment and disinfectant
solutions and soaps. It is only rarely found as part of the normal flora of
healthy people, but in hospitalized patients, the rates of colonization
increase with the length of hospitalization. P. aeruginosa may produce
serious infection in patients with burns and traumatic and operative
wounds; following urinary tract manipulation; in patients with diseases of
the hematopoietic, reticuloendothelial, and lymphoid systems; and in those
with impaired cellular or humoral defenses. Pulmonary infection occurs
commonly in patients with cystic fibrosis. The mortality rate is highest in
severely leukopenic patients.
P. aeruginosa produces a slime polysaccharide, an endotoxin, and pro-
teases that inactivate components of complement, thereby inhibiting to
some degree opsonization and the inflammatory response, and perhaps
contributing to its invasiveness. Exotoxin A promotes cellular damage and
tissue invasion and is toxic for macrophages.
Laboratory Diagnosis
The presence of P. aeruginosa in cultures can often be suspected because
of its musty grape-like (or corn tortilla) odor, the rough or ground glass
appearance of its colonies on sheep blood agar, and the presence of one or
and/or a metallic sheen caused by pyoverdin pigment. It can be identified
easily with a positive oxidase reaction, an alkaline slant/neutral butt reac-
tion in TSIA, growth at 42° C, and the formation of sheen and/or pigment
on the slants of TSIA and Pseudomonas P agar. Additional tests are shown
in Table 58-11. Tests of carbohydrate utilization should be carried out in
O-F basal medium, which contains a minimal quantity of peptone and a
relatively large quantity of carbohydrate and so can allow detection of very
small quantities of acid formed by this group of bacteria. Reactions are
usually complete within 48 hours but may require as long as 7 days.
MALDI-TOF can also be used for the identification of P. aeruginosa (Desai
et al, 2012; Manji et al, 2014).
Antimicrobial Susceptibility
As a general rule, susceptibility testing should be performed for all clini-
cally significant isolates of P. aeruginosa. Hospital strains of P. aeruginosa
may be resistant to many classes of antibiotic. Isolates are often susceptible
to the aminoglycosides, the carboxypenicillins and ureidopenicillins, cef-
tazidime or cefepime, carbapenems, and the quinolones. P. aeruginosa is
always resistant to sulfamethoxazole-trimethoprim (SXT) and tetracy-
clines, including the newer broad-spectrum tigecycline, ertapenem, and
nitrofurantoin. Multiple resistance to drugs to which P. aeruginosa was once
susceptible is increasing, especially in intensive care units and among
patients who have long-standing Pseudomonas infections, such as patients
with cystic fibrosis and other chronic syndromes (Friedland et al, 2004;
Hauser & Siriam, 2005; Tai et al, 2015). Laboratories are being asked to
test additional antibiotics, especially colistin or polymyxin B, when these
resistant isolates are encountered. The CLSI has recently provided break-
points for testing of polymyxin B that can be interpreted for polymyxin B
or colistin (CLSI, 2014).

PART 7
both of two pigments: a “blue-green” fluorescent pigment (Fig. 58-17)

Figure 58-16  Gram stain of Pseudomonas aeruginosa in a sputum specimen.

Note the longer, gram-negative bacilli compared with Figure 58-13. Figure 58-17  Extracted pyocyanin pigment from Pseudomonas aeruginosa.

TABLE 58-11
Differential Characteristics of Nonfermentative Gram-Negative Bacilli Isolated from Clinical Material
Pseudomonas Pseudomonas Pseudomonas Burkholderi Stenotrophomonas Acinetobacter
aeruginosa fluorescens putida cepacia maltophilia baumanii

Oxidase + + + + – –
Pyocyanin + – – – – –
Fluorescein + – + – – –
Glucose oxidation + + + + +/– +
42° C + – – +/– +/– +
DNase – – – – + –/+
Growth on MacConkey’s agar + + + + + +*
Motility + + + + + –

+, Positive; –, negative; +/–, variable results, most strains positive; –/+, variable results, most strains negative; DNase, deoxyribonuclease.
*Purplish color on MacConkey’s agar.

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58  Medical Bacteriology
Acinetobacter
Characteristics
Organisms in this genus are short, rod shaped to spherical, nonmotile,
oxidase negative, strictly aerobic, and gram negative. In a Gram-stained
smear, they often appear in pairs and may be difficult to decolorize.
Clinical Manifestations and Pathogenesis
Acinetobacter spp. are found commonly in soil and water and uncommonly
on the skin and mucous membranes of healthy people. Current research
has begun to elucidate the virulence factors in this group of organisms, as
they are being more recognized as serious pathogens in some patients.
Although usually considered nonpathogenic in the past, they have been
increasingly associated with nosocomial septicemia, pneumonia, bacteri-
uria, and wound infection, especially due to the increased antimicrobial
resistance that has developed in many strains of A. baumanii. The mortality
rate associated with Acinetobacter baumanii infections can reach 35%
(Antunes et al, 2014; Spellberg & Bonomo, 2014).
Laboratory Diagnosis
Acinetobacter spp. can be distinguished readily from pseudomonads on the
basis of their lack of motility, inability to reduce nitrates, and negative
oxidase reaction. They may produce characteristic purplish colonies on
MacConkey’s agar. More than 25 species are known, but their differentia-
tion biochemically is difficult, and they are often lumped together in the
Acinetobacter calcoaceticus–Acinetobacter baumanii complex. The glucose-
oxidizing (saccharolytic strains), nonhemolytic clinical strains are usually
referred to as A. baumanii; nonsaccharolytic strains (non–glucose oxidizers)
may be Acinetobacter lwoffi if nonhemolytic, or Acinetobacter haemolyticus if
hemolytic (Vaneechouette et al, 2011). The most clinically relevant are
members of the A. baumanii complex, and these are the most resistant to
antimicrobials as well.
Antimicrobial Susceptibility
Members of the Acinetobacter baumanii complex are resistant to most
available β-lactam and aminoglycoside antibiotics. Resistance to the ami-
noglycosides is caused by plasmid-mediated acetyl-, adenylyl-, and phos-
photransferases. Acinetobacter spp. may be susceptible to doxycycline,
trimethoprim-sulfamethoxazole, quinolones, ureidopenicillins, imipenem,
ampicillin-sulbactam, and ceftazidime. The carbapenems (excluding ertap-
enem) are considered the most active, but resistance rates up to 11% have
been described in nosocomial strains throughout the United States; in
some hospitals in the United States, Acinetobacter has become a predomi-
nant nosocomial pathogen (Gales et al, 2001; Chopra et al, 2013). These
isolates of carbapenem-resistant A. baumanii are often referred to as
carbapenem-resistant A. baumanii (CRAB); they are often resistant to all
classes of antibiotics, except colistin and tigecycline (Perez et al, 2007;
Chopra et al, 2013). Appropriate infection control procedures are neces-
sary to prevent their transmission (Rodríguez-Baño et al, 2009). Suscepti-
bility testing should be performed for clinically significant isolates or upon
request of the clinician.
Burkholderia
Burkholderia spp. are aerobic, non–spore-forming, gram-negative rods;
except for Burkholderia mallei, all are motile because they have polar fla-
gella. These organisms are catalase positive, and most are oxidase positive.
On MacConkey’s agar, they produce lactose-negative colonies.
Clinical Significance and Pathogenesis
These organisms are found in the environment in water, in soil, and on
plants. Because of their predilection for watery environments, some can
be found in the hospital environment and have the potential to cause noso-
comially acquired infection.
Two important human pathogens in the genus Burkholderia are Burk­
holderia pseudomallei and Burkholderia cepacia complex. B. pseudomallei,
which is acquired via inhalation or contact through cut or abraded skin,
causes melioidosis. The infection can be asymptomatic, can become
chronic, or can cause a fulminant sepsis. Melioidosis is most prevalent in
Southeast Asia and Australia but may occur in other tropical and subtropi-
cal environments.
B. cepacia, a nosocomial pathogen that is associated with contaminated
equipment, medications, and disinfectants, can cause bacteremia, urinary
tract infection, septic arthritis, and respiratory tract infection. It is an
important pathogen in patients with cystic fibrosis (CF) and in those with
chronic granulomatous disease. Patients with CF who become chronically
infected with this organism have a decreased rate of survival. B. cepacia
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complex comprises at least nine species, and all species have been recov-
ered from patients with CF. In the United States, however, approximately
85% of strains are B. multivorans or B. cenocepacia. B. cenocepacia has been
shown in many studies to possess potent virulence factors that lead to
increased mortality in CF patients who are infected with it versus other
strains of Burkholderia (LiPuma et al, 2011).
Laboratory Diagnosis
Burkholderia species grow well on standard laboratory media, including
blood and chocolate agar. Isolation of B. cepacia from contaminated speci-
mens such as sputa may be made easier through the use of selective
media—for example, PC, Pseudomonas cepacia selective agar; OFBL,
oxidation-fermentative base-polymyxin B, bacitracin-lactose-agar; and
BCSA, B. cepacia selective agar (LiPuma et al, 2011). No good biochemical
methods are available to differentiate among the B. cepacia complex or
between the complex and related species such as B. gladioli and Ralstonia,
Cupriavidus, and Pandoraea spp. Molecular techniques are often required
for confirmatory identification and should be pursued when a B. cepacia
complex organism is suspected. The use of Maldi-Tof may also assist
in further speciation in the future (Desai et al, 2012). The CF Founda-
tion (http://www.cff.org) has established a B. cepacia reference laboratory
to confirm the identity of possible isolates in CF patients (LiPuma et al,
2011).
Antimicrobial Susceptibility
The susceptibility of these organisms to antimicrobial agents varies
considerably. B. cepacia is highly resistant to many antimicrobials but
is usually susceptible to piperacillin, ceftazidime, chloramphenicol, and
trimethoprim-sulfamethoxazole. Strains from CF patients who have been
on repeated courses of antibiotics are, however, likely to become resistant
to these agents. The CLSI recommends reporting only ceftazidime,
meropenem, minocycline, and trimethoprim-sulfamethoxazole for B.
cepacia (CLSI, 2014). All B. cepacia organisms are intrinsically resistant to
polymyxins (colistin).
Stenotrophomonas maltophilia
Characteristics
Stenotrophomonas maltophilia are becoming significant nosocomial patho-
gens. Risk factors for colonization or infection with this organism are
mechanical ventilation, use of broad-spectrum antibiotics, catheterization,
and neutropenia (Brooke, 2012; Behnia et al, 2014).
Laboratory Diagnosis
Important distinguishing biochemical reactions of S. maltophilia are its
negative oxidase reaction and positive DNase activity. Colonies grow on
blood agar (lavender green colonies) and MacConkey’s agar; the bacteria
are nonmotile and nonfermentative.
Antimicrobial Susceptibility
S. maltophilia is inherently resistant to many antibiotics, especially the
carbapenems. Trimethoprim-sulfamethoxazole is the antibiotic of choice,
although some strains have become resistant (Gales et al, 2001; Brooke,
2012). CLSI recommends reporting only levofloxacin, trimethoprim-
sulfamethoxazole, and minocycline. Breakpoints have been put forth for
interpretation of these agents for MIC and disk diffusion testing (CLSI,
2014).
Other nonfermentative gram-negative bacilli include members of the
genera Alcaligenes, Achromobacter, Flavobacterium, Flavimonas, Chryseomonas,
Acidovorax, Brevundimonas, Comamonas, and Ralstonia, among others. These
are infrequently isolated from clinical specimens, often when they may
represent contamination or colonization. They can be considered as
significant, especially when isolated from sterile sites, on multiple occa-
sions in immunosuppressed patients, or patients with foreign devices in
place.
Vibrio
Characteristics
Vibrio spp. are facultatively anaerobic, oxidase-positive, short, curved, or
straight gram-negative bacilli that are usually motile by means of polar
flagella; they ferment carbohydrates and reduce nitrates to nitrites. Several
species are medically important (Table 58-12).
Clinical Manifestations and Pathogenesis
Among the vibrios, Vibrio vulnificus causes the most severe disease. Wound
infections and septicemia with this organism are often fatal. Disease is
usually associated with consumption of raw oysters or oyster-related injury.
TABLE 58-12
Differential Characteristics of Vibrio Species*
V. V. V. V. V. V. V. V. V.
Test cholerae mimicus damsela parahaemolyticus alginolyticus vulnificus fluvialis metschnikovii hollisae

Indole + + – + or – D + – or + D +
Voges-Proskauer – or + – + – + – – + –
Lysine + + + + + + – D –
decarboxylase
Ornithine + + – + or – D D – – –
decarboxylase
Arginine – – + – – – + D –
dihydrolase
Lactose (+) – or + – – – + – D –
Sucrose + + – + + D + + –
Mannitol + + – + + D + + –
Maltose + + + + + + + + –
Arabinose – – – + or – – – + – +
Salicin – – – – – + – – or + –
Cellobiose – – – – – + D – or + –
NO3→NO2 + + + + + + + – +
Oxidase + + + + + + + – +
Growth in Nutrient Broth Plus NaCl, %
0 + + – – – – W+ or – – –
1 + + + + + + + + +
6 – or (+) – or (+) + + + + + + (+)
8 – – – + + – – D –

PART 7
10 – – – – + – – D –
12 – – – – – – – – –

W+, Weakly positive.

*For key to symbols, see Table 58-9.

Preexisting hepatic disease is almost always present in serious illness.
Decreased liver function results in increased available iron and appears to
facilitate the growth of the organism.
Cholera toxin–producing Vibrio cholerae O1 is a well-known cause of
epidemic cholera, which manifests itself by massive intestinal fluid loss and
dehydration. The cholera toxin mediates this effect by binding to and
activating the adenylate cyclase of cells in the small intestine, resulting in
hypersecretion of electrolytes and water (Abbott et al, 2011). Non-O1
strains of V. cholerae cause a self-limited gastroenteritis but are not respon-
sible for epidemics of disease. Nearly all non-O1 strains of V. cholerae do
not produce cholera toxin but do produce two types of hemolysins and a
heat-stable enterotoxin.
Vibrio mimicus and Vibrio parahaemolyticus primarily cause gastroenteri-
tis. The mechanism of pathogenicity of V. parahaemolyticus appears to be
related to invasiveness rather than to enterotoxin production. More than
95% of isolates of V. parahaemolyticus isolated from patients with gastro-
enteritis produce a cell-free hemolysin that is lethal to mice when injected
in high doses and is described as the Kanagawa phenomenon. This halo-
philic organism is widely distributed in marine environments and has been
found to contaminate fish and shellfish. Outbreaks of acute diarrheal
disease following ingestion of contaminated food have been especially
common in Japan but have also occurred in the United States and other
countries (Abbott et al, 2011).
Laboratory Diagnosis
Although formerly of concern only to U.S. travelers to endemic areas,
cases of V. cholerae disease have been described in the United States in
association with ingestion of contaminated shellfish. In 2012, CDC
reported 22 cases, 4 patients with V. cholera serogroup 01 or 0141 (cdc.gov/
cholera/index.html). In addition, gastroenteritis caused by V. parahaemo­
lyticus and by other halophilic vibrios in contaminated shellfish has been
described in many parts of the country, particularly in coastal areas. In
2012, there were 944 cases of Vibrio infection in the United States, approxi-
mately 50% by V. parahaemolyticus, followed by 32% either V. alginolyticus
or V. vulnificus; 16% of the Vibrio infections were from noncoastal states
(cdc.gov/vibrio/index.html). Thus it is important for clinical laboratories
to have the capability to culture stool for Vibrio spp. when indicated on
the basis of travel and dietary history.
With the exception of V. cholerae and V. mimicus, growth of this group
of organisms requires media containing NaCl. Most solid and liquid media
used for bacterial culture contain enough sodium that the use of special
media in such instances is not necessary. Selective media containing
sucrose, such as thiosulfate citrate bile salts medium, are very useful for
culturing stool specimens for Vibrio spp. Certain species (V. cholerae and V.
alginolyticus) ferment the sucrose and appear as yellow colonies on this
medium. An enrichment medium, such as alkaline peptone water, may be
used prior to subculture to solid medium to enhance recovery of Vibrio
spp. from stool. Vibrio spp. can be differentiated among themselves and
from other enteric gram-negative bacilli according to the reactions listed
in Table 58-12. It may be necessary to carry out biochemical testing of the
halophilic vibrios in media supplemented with 1% to 3% NaCl. If TSIA
and lysine iron agar are inoculated for screening purposes, their reactions
will be acid slant/acid butt with no gas (A/A) or H2S and alkaline slant/
alkaline butt (K/K), respectively (Abbott et al, 2011). Not all commercially
available gram-negative identification systems are reliable for the identifi-
cation of Vibrio spp.; they should be used only if they have proved accurate.
Use of Maldi-Tof and molecular methods for identification have become
more useful if full identification is needed (Erler et al, 2014).
Antimicrobial Susceptibility
Antimicrobial susceptibility testing can be performed using the disk diffu-
sion method with Mueller-Hinton agar and broth microdilution using
cation-adjusted Mueller-Hinton broth and incubation at 35° C for 16 to
18 hours. CLSI has established interpretive standards for V. cholerae tested
with ampicillin, tetracycline, doxycycline, trimethoprim-sulfamethoxazole,
chloramphenicol, and sulfonamides (CLSI, 2010). Interpretive standards
have been put forth for the other Vibrio spp. in CLSI document M45-2A
(CLSI, 2010).
Aeromonas
Characteristics
Members of this genus are facultatively anaerobic, oxidase- and catalase-
positive, rod-shaped, gram-negative bacilli. They are usually motile by
means of polar flagella, although some species may be nonmotile. They
form acids from carbohydrates by respiratory and fermentative metabolism
and reduce nitrates to nitrite.

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58  Medical Bacteriology
Clinical Manifestations and Pathogenesis
Aeromonas spp. are mainly found in aquatic environments. They have been
isolated from tap water, rivers, soil, and various foods, and are only rarely
found in marine environments. These organisms have been associated with
both intestinal and extraintestinal disease. Although no definitive evidence
has demonstrated the role of Aeromonas spp. in GI disease, the presence
of Aeromonas spp. in stool is more often associated with diarrhea than with
an asymptomatic carrier state. Its role in producing diarrheal disease is
possibly related to the production of an enterotoxin by some strains. A
hemolysin and a cytopathic factor have also been described. Aeromonas spp.
may cause infection of traumatically acquired wounds or septicemia in
patients who are immunocompromised (Janda & Abbott, 2010). An
unusual association has also been reported between the use of leeches
(which harbor A. hydrophila in their digestive tract) to decrease vascular
congestion and human skin or bloodstream infections (Maetz et al, 2012).
Laboratory Diagnosis
Isolation of a fermenting, oxidase-positive, gram-negative bacillus from an
appropriate specimen should suggest the possibility of Aeromonas spp.
Organisms grow readily on conventional laboratory media and produce
colonies that resemble those of Pseudomonas spp.; have a greenish, ground
glass appearance; and have a fruity odor. Most species are β-hemolytic on
blood agar. Isolation of Aeromonas spp. from stool specimens may be
enhanced by inoculation of blood agar containing ampicillin or CIN
medium. More than 18 species of Aeromonas are now recognized. Aeromo­
nas hydrophila complex, Aeromonas caviae complex, and Aeromonas veronii
complex are the most common isolates from human specimens. Esculin,
Voges-Proskauer, gas from glucose fermentation, and l-arabinose are four
biochemicals that can be used to separate Aeromonas spp. into one of these
three complexes, but a more definitive identification would require con-
ventional biochemicals in conjunction with molecular sequencing methods
or Maldi-TOF identification (Horneman & Ali, 2011).
Antimicrobial Susceptibility
Aeromonas spp. are susceptible to the quinolones, aminoglycosides, carbap-
enems, and trimethoprim-sulfamethoxazole but produce a β-lactamase
that mediates resistance to the penicillins and first-generation cephalospo-
rins (Horneman & Ali, 2011). Carbapenemases, although rare, may be
difficult to detect by conventional susceptibility testing methods, including
automated systems (Horneman & Ali, 2011). Aeromonas spp. have been
found to maintain resistance plasmids of both the Enterobacteriaceae and
Pseudomonas spp.
Campylobacter
Characteristics
Campylobacter spp. are small (0.5-8 µm long × 0.2-0.5 µm wide), motile,
non–spore-forming, curved (comma-shaped) or S-shaped gram-negative
bacilli that grow optimally in an atmosphere containing 5% to 10% oxygen
and, therefore, are considered to be microaerophilic. Figure 58-18 shows
the Gram stain of Campylobacter jejuni from culture. C. jejuni is among the
Figure 58-18  Campylobacter jejuni Gram stain: Note the comma-shaped
appearance of the bacilli.
1138
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most common cause of bacterial enteritis in the United States. Other
Campylobacter spp. associated with enteritis are Campylobacter coli, Campy­
lobacter lari, and Campylobacter upsaliensis. Campylobacter fetus subsp. fetus is
a cause of septic thrombophlebitis, arthritis, peritonitis, abscess, and peri-
carditis (Fitzgerald & Nachamkin, 2011), especially in persons with an
underlying chronic disease. Arcobacter are aerotolerant, Campylobacter-like,
spiral-shaped bacteria that are frequently isolated from bovine and porcine
products of abortion and feces of animals with enteritis (Fitzgerald &
Nachamkin, 2011). A. butzleri has been isolated from patients with bacte-
remia, endocarditis, diarrhea, and peritonitis. A. cryerophilus has been iso-
lated from patients with bacteremia and diarrhea. A. skirrowii has been
isolated from stool of patient with chronic diarrhea, although clinical
relevance was unclear (Fitzgerald & Nachamkin, 2011).
Clinical Manifestations and Pathogenesis
C. jejuni is found worldwide as a commensal of the GI tract of wild or
domesticated cattle, sheep, swine, goats, dogs, cats, and fowl, especially
turkeys and chickens. It is the most common cause of bacterial enteritis in
some areas of the United States, with an estimated occurrence of 1000
cases per 100,000 individuals. The incidence of infection in the United
Kingdom and in other developed nations is similar to that in the United
States, and the incidence in underdeveloped countries is probably even
higher. Infections generally occur in the summer and fall and are com-
monly the result of ingestion of improperly cooked foods, usually poultry.
In addition, several outbreaks of C. jejuni enteritis have been linked to
unpasteurized milk and to defects in municipal water systems. The spec-
trum of illness ranges from asymptomatic to severely ill. The stool from
patients with diarrhea may contain blood or leukocytes. Symptoms can last
up to 1 week and generally are self-limited. Extraintestinal infections,
including bacteremia, reactive arthritis, urinary tract infection, and men-
ingitis, may also occur. C. jejuni is the most recognized antecedent cause
of Guillain-Barré syndrome. The pathogenesis of this organism is not
completely understood; it appears to first colonize the intestinal mucous
layer and then is able to translocate through the epithelial surface to the
underlying tissue (Fitzgerald & Nachamkin, 2011).
The major habitat of C. fetus subsp. fetus is the intestine of sheep and
cattle; it also may be found in the genital tract of these animals, their
placentas, and the gastric contents of their aborted fetuses and, less fre-
quently, in other animals and birds. The mechanisms of transmission of
infection to humans are not understood completely. Direct contact with
an infected animal is possible, but less than one-third of infected individu-
als have a history of environmental or occupational exposure. Contami-
nated food or water may be a vehicle for infection, or infection may
originate from an endogenous source.
Laboratory Diagnosis
A single stool specimen is generally adequate to detect enteric pathogens,
including Campylobacter spp. Examination for fecal leukocytes is not recom-
mended because they may be present in as few as 25% of cases. An enzyme
immunoassay (EIA) is available for direct detection of Campylobacter jejuni
and Campylobacter coli antigens in stool specimens (Granato et al, 2010;
Fitzgerald & Nachamkin, 2011). Several media can be used for the selective
isolation of Campylobacter spp., including charcoal-cefoperazone-deoxy-
cholate agar, charcoal-based selective medium, semisolid blood-free motil-
ity medium, Skirrow’s medium, and Campylobacter agar with 5% sheep
blood and five antimicrobials (cephalothin, trimethoprim, vancomycin,
polymyxin B, and amphotericin B). Most Campylobacter spp. require a
microaerobic environment (5% O2, 10% CO2, and 85% N2), which can
be produced using commercially available gas generator packs. The amount
of oxygen in a candle jar is too little to support the growth of Campylobacter
spp. and should not be used. Incubation of the plates at 42° C increases
selectivity for C. jejuni. Commercial multiplex PCR assays that detect
multiple potential pathogens in stool also are available.
If Campylobacter spp. are suspected in a blood culture based on clinical
history or appearance of an organism on Gram stain, the broth should be
subcultured to a nonselective blood agar plate and incubated at 37° C in a
microaerobic environment.
In general, Campylobacter spp. produce gray, flat, irregular, spready
colonies, which may become round, convex, and glistening as the moisture
content in the media is reduced. A typical Gram stain appearance and a
positive oxidase reaction from a colony growing on selective media at 42° C
can be reported as Campylobacter spp. C. jejuni is able to hydrolyze hip-
purate and is susceptible to nalidixic acid and resistant to cephalothin. C.
coli is hippuricase negative.
Strains of C. fetus subsp. fetus are resistant to nalidixic acid, fail to
hydrolyze hippurate, and do not ordinarily grow at 42° C.
Antimicrobial Susceptibility
C. jejuni is variably susceptible to antimicrobial agents. Most are not sus-
ceptible to penicillins or cephalosporins. For intestinal infection, erythro-
mycin is the drug of choice, with quinolones used as alternative therapy.
Treatment often is not warranted, however, for simple gastroenteritis with
Campylobacter sp. in an otherwise healthy individual. Resistance to both
agents has been encountered. Currently, no standardized methods are used
for susceptibility testing of this group of organisms; however, CLSI docu-
ment M45-A2 provides guidelines for testing (CLSI, 2010).
Helicobacter
Characteristics
Helicobacter spp. are spiral-shaped or curved, gram-negative, non–spore-
forming bacilli, measuring 0.3 to 1.0 µm wide and 1.5 to 10 µm long. They
are motile by multiple bipolar or monopolar flagella, are microaerobic, and
have a respiratory metabolism.
Clinical Manifestations and Pathogenesis
Helicobacter spp. are found in the GI tracts of mammals and birds. Trans-
mission from one host to another occurs through both oral–oral and
fecal–oral routes. Helicobacter pylori is considered to be one of the “gastric”
helicobacters. In the stomach, it lives within or beneath the mucous layer
adjacent to the epithelium. It is also found transiently in the duodenum,
saliva, and feces.
Infection with H. pylori may result in acute gastritis symptoms. Most
infected patients develop chronic active gastritis, which may lead to non-
ulcer dyspepsia or duodenal ulcers. H. pylori has been associated with 90%
of duodenal ulcers and nearly all gastric ulcers. Infection with H. pylori has
also been associated with gastric carcinoma and gastric lymphoma (Tester-
man & Morris, 2014). The prevalence of gastritis associated with H. pylori
increases with age, suggesting that the organism is acquired as people
become older.
The “enteric” helicobacters, such as Helicobacter (formerly Campylo­
bacter) cinaedi and Helicobacter fennelliae, have been implicated in cases of
gastroenteritis. Rarely, these organisms may invade the bloodstream and
be isolated from cultures of blood.
Laboratory Diagnosis
Typically, nonculture methods have been utilized to diagnose H. pylori
infection. One such test is the urea breath test. This is a noninvasive test
that detects urease activity of H. pylori by measuring 14C- and 13C-labeled
CO2 in the patient’s expelled air after ingestion of labeled urea. Serologic
assays are also widely used in symptomatic patients to detect antibodies
against H. pylori; however, because most adults will have been exposed to
H. pylori, detection of IgG antibodies is not helpful diagnostically but can
be used as an epidemiologic or surveillance tool. In some cases, biopsies
of the affected tissues are obtained and are examined histologically using
the hematoxylin and eosin or immunohistochemical stain for the presence
of organisms with morphology typical of H. pylori. Because the organism
hydrolyzes urea very rapidly, a portion of the gastric biopsy may be placed
directly into urea broth or onto urea-containing agar to detect urea hydro-
lysis in 1 to 24 hours (CLO test). A commercially available EIA for detec-
tion of H. pylori antigen in stool is a noninvasive alternative for diagnosis
of H. pylori infection (Premier Platinum HpSA, Meridian Bioscience,
Cincinnati, Ohio). Sensitivity of this assay is as high as 89%, with specifici-
ties up to 95% (Masoero et al, 2000; Montiero et al, 2001). PCR has also
been reported as a sensitive method for detection of H. pylori (Montiero
et al, 2009).
If culture is requested, tissue specimens should be maintained at 4° C
and processed within 2 hours of collection. Transport media include Bru­
cella broth with 20% glycerol, cysteine Albemi broth with 20% glycerol,
isotonic saline with 4% glucose, and Stuart’s transport media. Processed
specimens may be inoculated to one of several media, including brain-
heart infusion, Brucella, Trypticase soy agar, Columbia agar base with 10%
defibrinated horse blood, or Wilkens Chalgren agar. Inoculated media
should be incubated in a microaerobic atmosphere (5% to 10% CO2, 80%
to 90% N2, and 5% to 10% O2) under high humidity at 35° C for 5 to 10
days. Addition of 5% to 8% H2 in the atmosphere enhances growth of H.
pylori, which generally produces small, gray, translucent colonies on these
media; has the characteristic gram-negative spiral appearance on stained
smears; and is oxidase, catalase, and urease positive. Feces generally are
not cultured for the enteric helicobacters. Helicobacter spp. will grow and
will be detected by the automated blood culture systems used in many
laboratories, but may require incubation for longer than the standard 5
days (Lawson, 2011).
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Figure 58-19  Gram stain of a Haemophilus influenzae coccobacillus in a brain
abscess.
Antimicrobial Susceptibility
Multidrug regimens are used to treat H. pylori infection. These usually
include two antibiotics (metronidazole, clarithromycin, tetracycline, or
amoxicillin) and an “antiacid” drug. Strains resistant to metronidazole and
clarithromycin have been reported. For susceptibility testing, the CLSI
recommends agar dilution using Mueller-Hinton agar plus 5% sheep
blood (CLSI, 2010). Interpretive breakpoints are given only for clarithro-
mycin. Updated information about the treatment of H. pylori gastritis can
be found in two review articles (Papastergiou et al, 2014; Testerman &
PART 7
Morris, 2014).
Haemophilus
Characteristics
Members of the genus are oxidase-positive, facultatively anaerobic, small,
gram-negative, pleomorphic rods or coccobacilli with a potential require-
ment for X (hemin) and/or V (NAD) factor. Figure 58-19 shows an H.
influenzae bacterium in a brain abscess specimen.
Clinical Manifestations and Pathogenesis
Most Haemophilus spp. are normal inhabitants of the upper respiratory
tract. Some may reside in the gastrointestinal or urogenital tract. Person-
to-person spread occurs by respiratory droplets. Infections caused by Hae­
mophilus spp. range from conjunctivitis and otitis media to meningitis and
endocarditis. Those that are generally considered human pathogens are H.
influenzae, Haemophilus parainfluenzae, Haemophilus ducreyi, and Aggregati­
bacter (Haemophilus) aphrophilus (Ledeboer & Doern, 2011).
The major virulence factor of H. influenzae is the polysaccharide
capsule, of which there are six serotypes (a to f). Strains that do not possess
a capsule are referred to as nontypeable. Endotoxin is not produced by H.
influenzae, and this species is rapidly killed once ingested by macrophages
unless antibody, complement, or the phagocytes are deficient. The role of
antibodies in immunity is also poorly understood. Antibodies develop with
age, presumably following natural infection with H. influenzae or with
cross-reacting antigenic organisms, so most persons older than 15 years
have antibodies. Which antibodies are present and what level of those
antibodies is protective remain unknown.
Since the introduction of a vaccine for H. influenzae type b in the mid-
1980s, there has been a sharp drop in the incidence of invasive infection
such as meningitis and epiglottitis due to this organism. Figure 58-20
demonstrates the pleomorphic nature of the H. influenzae seen in a CSF
specimen. Nontypeable strains of H. influenzae are most frequently associ-
ated with acute otitis media and acute exacerbations of chronic bronchitis.
H. parainfluenzae is usually a commensal in the upper respiratory tract but
may also cause serious illness, such as endocarditis. Aggregatibacter (Hae­
mophilus) aphrophilus, another upper respiratory tract commensal, can cause
endocarditis, brain abscess, pneumonia, meningitis, and bacteriuria. H.
ducreyi is responsible for the sexually transmitted disease chancroid (Lede-
boer & Doern, 2011).
Laboratory Diagnosis
Isolation of Haemophilus spp. usually requires the presence of X and/or V
factor in the culture medium. The former is most frequently supplied by
the incorporation of heat-lysed (“chocolatized”) blood cells in agar,
1139
 although it may also be provided by whole human, horse, or rabbit blood
58  Medical Bacteriology
cells. NAD is commonly supplied by the incorporation of yeast extract or
other appropriate supplements in the medium or by a suspension of staph-
ylococci, which is streaked across the agar surface and about which satellite
colonies of dependent strains of Haemophilus spp. grow. The differential
characteristics of members of this genus are listed in Table 58-13.
Requirements for X and V factors are determined by absence or pres-
ence of growth on media containing these factors. An alternative method
to test for X factor dependence is the porphyrin test described by Kilian,
which determines the ability of dependent species to use δ-aminolevulinic
acid in the biosynthesis of porphobilinogen and porphyrins. The forma-
tion of porphobilinogen can be detected by adding Kovac’s reagent to the
reaction mixture and observing the development of a red color in the
aqueous phase. Alternatively, the formation of porphyrins in the reaction
mixture can be demonstrated by red fluorescence under a Wood’s lamp.
Hemolytic properties of Haemophilus spp. can be determined on rabbit or
horse blood agar.
Aggregatibacter (Haemophilus) aphrophilus must often be distinguished
from species such as Aggregatibacter (Actinobacillus) actinomycetemcomitans,
Cardiobacterium hominis, and Eikenella corrodens (Table 58-14), all of which
have been associated with subacute bacterial endocarditis.
Cultivation of H. ducreyi from chancroid lesions is problematic. A
Gram-stained smear of material from the lesion may be helpful if gram-
negative bacilli in pairs or in rows (“schools of fish”) are seen. Figure 58-21
shows a “typical” Gram stain of H. ducreyi from clinical material. Material
may be inoculated onto GC medium base plus 1% hemoglobin, 5% to
10% fetal calf serum, 1% IsoVitaleX (BBL Microbiology Systems), and
3 µg/mL of vancomycin or Mueller-Hinton agar plus 5% horse blood, 1%
cofactor-vitamin-amino acid enrichment, and 3 µg/mL vancomycin.

Figure 58-20  Note the pleomorphic nature of the Haemophilus influenzae
seen in this Gram stain of cerebrospinal fluid.
Antimicrobial Susceptibility
Currently, the CLSI recommends testing H. influenzae isolated from blood
or CSF against ampicillin, chloramphenicol, a third-generation cephalo-
sporin, and meropenem (CLSI, 2014). Resistance to ampicillin may be as
high as 60% in the United States, varying geographically. Resistance to
ampicillin is usually mediated by the production of β-lactamase; however,
rare isolates are resistant on the basis of alterations in outer membrane
permeability or affinity to penicillin-binding proteins. Resistance to
second- or third-generation cephalosporins has not been documented in
the United States. Susceptibility testing of H. influenzae requires the use
of Haemophilus test medium (HTM). The recommended treatment for H.
ducreyi infection is erythromycin; alternative agents include azithromycin,

TABLE 58-13
Differential Characteristics of Medically Important Haemophilus Species
X Factor V Factor Fermentation
Porphyrin Dependent Dependent of Sucrose Hemolysis* Catalase

Haemophilus influenzae – + + – – +
Haemophilus haemolyticus – + + – + +
Haemophilus parahaemolyticus + – + + + V
Haemophilus ducreyi – + – – – –
Haemophilus parainfluenzae + – + + V
Haemophilus pittmaniae + – + + + w
Haemophilus paraphrohaemolyticus + – + + + +

Adapted from Ledeboer NA, Doern GV: Haemophilus. In Versalovic J, Carroll KC, Funke G, et al, editors: Manual of clinical microbiology, ed 10, Washington, DC, 2011,
American Society for Microbiology, p 589.
*On horse and rabbit blood.

TABLE 58-14
Differential Characteristics of Aggregitabacter (Haemophilus) aphrophilus, Aggregitabacter (Actinobacillus) actinomycetemcomitans,
Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae*
Test A. aphrophilus A. actinomycetemcomitans C. hominis E. corrodens K. kingae

Oxidase +/– –/W + + +

Catalase – + – – –
δ-ALA utilization + + + + +
V requirement – – – – –
Indole – – + – –
Urease – – – – –
Lysine decarboxylase – – – + –
Acid from glucose + + + – (+)
Sucrose + – + – –
Lactose + – – – –
Mannitol – + D – –
Xylose – D – – –

*For key to symbols, see Table 58-9.

δ-ALA, δ-Aminolevulinic acid; W, weak.

1140
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Figure 58-21  Haemophilus ducreyi bacilli from a genital lesion.
ciprofloxacin, ceftriaxone, amoxicillin-clavulanate, and trimethoprim-
sulfamethoxazole. Although few data are available on the susceptibility of
other Haemophilus spp. to antibiotics, resistance is assumed higher than
among H. influenzae strains (Ledeboer & Doern, 2011).
GRAM-NEGATIVE BACTERIA—THE   C. hominis can cause subacute bacterial endocarditis, similar to other
HACEK BACTERIA
Five small gram-negative coccobacilli are part of the normal oral flora and
are associated occasionally with bacterial endocarditis and rarely with other
infections. They are opportunists that enter the bloodstream, settle on
damaged heart valves, and cause a relatively slowly progressive, indolent
form of endocarditis. They typically require an additional 1 to 2 days
before they are isolated from blood cultures, and they are uniformly sus-
ceptible to many antimicrobial agents (Yew et al, 2014). The word HACEK
is an acronym for the bacteria responsible for this disease: Haemophilus spp.
(influenzae, parainfluenzae), Aggregatibacter (Haemophilus) aphrophilus (most
commonly, Aggregatibacter [Actinobacillus] actinomycetemcomitans), Cardio­
bacterium hominis, Eikenella corrodens, and Kingella spp. Some taxonomic
changes have been noted more recently, and some of the HACEK members
have been reassigned to the genus Aggregatibacter (Norskov-Lauritsen &
Kilian, 2006). Differential characteristics of the members of this group are
listed in Table 58-14.
Haemophilus
H. influenzae and H. parainfluenzae were discussed earlier in this chapter.
Aggregatibacter (Haemophilus) aphrophilus is also part of the HACEK group.
It does not require X or V factor, so it can easily grow on blood and
chocolate agars, or CO2 for growth. Along with causing endocarditis, A.
aphrophilus has been reported in cases of endophthalmitis (following oph-
thalmic procedures), bacteremia, meningitis, brain abscess, cervical lymph-
adenitis, empyema, and a few other infectious syndromes. In one study,
39% of patients reported undergoing prior dental procedures before devel-
oping their A. aphrophilus infection (Huang et al, 2005). It is usually sus-
ceptible to β-lactam agents; successful treatment may require combination
therapy with a β-lactam and an aminoglycoside.
Aggregatibacter (Actinobacillus)
Characteristics
A. actinomycetemcomitans is a gram-negative, non–spore-forming coccoba-
cillus or short rod. It grows both aerobically and anaerobically. The addi-
tion of 5% to 10% CO2 enhances growth. Colonies on blood agar appear
slowly and remain small. A description of “star-shaped” colonies has been
given to their appearance on agar media.
Clinical Manifestations and Pathogenesis
Aggregatibacter organisms are found in the mucous membranes of the
respiratory and genitourinary tracts of humans and animals. They gener-
ally cause disease only in immunocompromised individuals, or when they
are accidentally introduced into healthy surrounding tissue—for example,
by trauma. A. actinomycetemcomitans has a low level of pathogenicity. It
derives its species name from its frequent association with actinomycotic
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lesions. In recent years, however, it has most frequently been reported as
a cause of subacute bacterial endocarditis, periodontitis, and brain abscess.
Two virulence factors are known: a leukotoxin and a collagenase (Zbinden
& von Graevenitz, 2011).
Laboratory Diagnosis
A. actinomycetemcomitans grows on blood and chocolate agar. After 24–72
hours, colonies are 1–3 mm in diameter with a central wrinkling. The
organism is catalase positive, oxidase negative or weakly positive, and
urease negative (Zbinden & von Graevenitz, 2011). Additional biochemical
assays must be used to differentiate it from other slowly growing, some-
what fastidious gram-negative bacilli (see Table 58-14).
Antimicrobial Susceptibility
This organism is resistant to penicillin but is usually susceptible to many
other antibiotics, including the cephalosporins, β-lactam–β-lactamase
inhibitor combinations, fluoroquinolones, and tetracycline. Methods for
performing susceptibilities and interpretation of results can be found in
the CLSI document M45-2A (CLSI, 2010).
Cardiobacterium hominis
Characteristics
Cardiobacterium hominis is a gram-negative, non–spore-forming bacillus
that is part of the normal oral flora. It is a facultative anaerobe that does
not require CO2, although growth is enhanced in microaerophilic condi-
tions. Growth occurs on blood and chocolate agar but not on MacConkey’s
agar and is better at longer than 48 hours.
Clinical Manifestations and Pathogenesis
HACEK members; it may also be responsible for cases of periodontitis
and peritonitis (Bhan et al, 2006). The usual habitat of C. hominis is the
PART 7
upper respiratory tract, but it may also be found in the gastrointestinal and
genitourinary tracts (Zbinden & von Graevenitz, 2011).
Laboratory Diagnosis
Colonies at 48 hours’ incubation are small and may have a yellow pigment,
although most are white. The organism is generally oxidase and indole
positive but negative for catalase, urease, esculin, and nitrate reduction.
Acid may be produced from glucose, maltose, and sucrose (Zbinden & von
Graevenitz, 2011).
Antimicrobial Susceptibility
Isolates are usually susceptible to penicillins and cephalosporins, amino-
glycosides, and tetracyclines. Resistance to clindamycin is common.
β-Lactamases have been rarely reported (Lu et al, 2000). Methods for
performing susceptibilities and interpretation of results can be found in
the CLSI document M45-2A (CLSI, 2010).
Eikenella
Characteristics
Formerly classified as Bacteroides corrodens, the “corroding bacilli” that are
facultatively anaerobic have been assigned to the species Eikenella corrodens.
E. corrodens organisms are oxidase-positive, catalase-negative, nonfermen-
tative, gram-negative bacilli, colonies of which may corrode or pit agar.
Growth is enhanced by 5% to 10% CO2 and/or the presence of hemin (X
factor) in the medium.
Clinical Manifestations and Pathogenesis
Little is known about factors contributing to the organism’s virulence in
human disease; it has a low level of pathogenicity for animals. E. corrodens
resides predominantly in the oral cavity and is isolated frequently from
the upper respiratory tract. Similar to other HACEK bacteria, it is
responsible for subacute bacterial endocarditis. It has been recovered
from abscesses, cellulitis, and wound infections, often following human
bites. Infections are usually mixed with other organisms (Zbinden & von
Graevenitz, 2011).
Laboratory Diagnosis
Growth is observed on blood or chocolate agar but not on MacConkey’s
agar. The most striking features of E. corrodens in culture are the distinctive
odor of bleach and the characteristic pitting of the agar; however, pitting
does not occur with all strains. Colonies appear slowly (2 to 4 days) and
are generally small (0.5-1.0 mm in diameter). E. corrodens must usually be
distinguished from other fastidious, slowly growing gram-negative bacilli
(see Table 58-14).
1141
58  Medical Bacteriology
Antimicrobial Susceptibility
E. corrodens is susceptible to the penicillins, quinolones, and tetracycline;
variably susceptible to aminoglycosides; and resistant to clindamycin and
metronidazole. β-Lactamases have been described in clinical strains, but
resistance can be overcome with the use of β-lactamase inhibitors in com-
bination with β-lactam antibiotics (Zbinden & von Graevenitz, 2011).
Kingella
Characteristics
Kingella has three recognized species: K. kingae (the HACEK species),
Kingella oralis, and Kingella denitrificans. They are gram-negative rods to
coccobacilli, requiring increased CO2 for optimum growth. Colonies will
grow on blood (β-hemolytic) and chocolate, but not on MacConkey’s agar,
after 2 days.
Clinical Manifestations and Pathogenesis
K. kingae is the most pathogenic of the three species. It is a member of the
HACEK group, causing an indolent, slowly progressive endocarditis. In
addition, it is associated with septic arthritis/osteomyelitis (usually in chil-
dren younger than 4 years of age) (Sena et al, 2009; Yagupsky, 2015) and
septicemia. K. oralis has been isolated from patients with periodontitis, but
with unclear clinical relevance. K. denitrificans is a rare clinical isolate that
can be associated with endocarditis (Zbinden & von Graevenitz, 2011).
Laboratory Diagnosis
K. kingae is oxidase positive and nonmotile and produces acid from glucose,
although in delayed fashion. Indole and catalase are negative.
Antimicrobial Susceptibility
K. kingae is susceptible to penicillin and most antibiotics to which other
members of the HACEK group are susceptible. β-Lactamases have been
described in clinical strains, but resistance can be overcome with the use
of β-lactamase inhibitors in combination with β-lactam antibiotics
(Zbinden & von Graevenitz, 2011). Methods for performing susceptibili-
ties and interpretation of results can be found in the CLSI document
M45-2A (CLSI, 2010).
Clinical Manifestations and Pathogenesis
Legionella spp. are found in the environment in association with water.
Growth within environmental protozoa is thought to be an important
factor for survival of the organism in the environment. Transmission to
humans occurs through exposure to contaminated water (e.g., faucets,
shower heads, public fountains). Human-to-human infection and laboratory-
acquired infections are not known to occur.
Infections can be subclinical, pulmonary, or extrapulmonary. Infection
is usually manifested as an acute, fibrinopurulent pneumonia with lobular
distribution. Histologically, there is an alveolar infiltrate of neutrophils and
macrophages, accompanied by fibrin and red blood cell extravasation.
Legionella spp. may be found within alveolar macrophages. Figure 58-23
shows the Gram stain of sputum of a patient with L. pneumophila pneumo-
nia. L. pneumophila has also been isolated from cultures of blood.
Laboratory Diagnosis
Legionella spp. may be isolated on BCYE agar supplemented with growth
factors, including l-cystine, ferric salt, and α-ketoglutarate. This medium
may be made selective for culture of nonsterile body sites by the addition
of cefamandole, polymyxin B, and anisomycin or polymyxin B, anisomycin,
and vancomycin (Edelstein, 2011). Chocolate agar may also support the
growth of legionellae. Treatment of sputum with a weak acid (0.2M HCl/
KCl pH 2.2) for 4 minutes or with heat (60° C) for 2 minutes may help to
reduce contamination from other organisms but may reduce the number
of legionellas as well.
Inoculated media should be incubated for at least 5 days in a humid
atmosphere containing no more than 2% to 5% CO2. Colonies often
appear iridescent and have a sticky consistency. Isolates with typical Gram
stain morphology should be subcultured to blood agar, where no growth
will be observed. These organisms may be weakly oxidase and catalase

MISCELLANEOUS GRAM-NEGATIVE BACILLI

Legionella
Characteristics
Legionella spp. are non–spore-forming, faintly staining, thin, gram-negative
bacilli. Legionella spp. were first recognized to cause human disease during
an epidemic of pneumonia that occurred among members of the Pennsyl-
vania American Legion who had gathered in Philadelphia to celebrate the
1976 bicentennial. There are now more than 52 named species and a
number of unnamed species. Most clinical cases have been due to Legionella
pneumophila, serogroup 1. Figure 58-22 demonstrates excellent staining of
Legionella spp. with a Dieterle silver stain.

Figure 58-23  Legionella pneumophila faintly staining negative with Gram

stain of pulmonary infiltrate.

Figure 58-22  Legionella pneumophila in a clinical specimen stained with a

Dieterle silver stain. Figure 58-24  Gram stain of culture of Legionella pneumophila.

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positive, are gelatinase positive, and often are motile. Identification of this
organism is most accurately achieved using type-specific antibody assays
or sequencing with appropriate primers. Figure 58-24 shows a Gram stain
of L. pneumophila from culture.
Legionella spp. can be detected or identified by direct fluorescent anti-
body (DFA) staining of specimens or colonies in cultures, but the sensitiv-
ity of direct DFA examination of respiratory specimens is very low
compared to newer methods of detection and is not commonly utilized
today. The urine antigen test for L. pneumophila serogroup 1 has a reported
sensitivity of 80% to 90%, although it should be noted that antigenuria
may persist for many months following infection. Use of the urinary
antigen test within and outside of the intensive care unit has been shown
to have a positive impact on patient cases (Edelstein, 2011; Couturier
et al, 2014).
The diagnosis of legionellosis can also be established serologically by
detecting a fourfold or greater rise in antibody titer to at least 1 : 128. A
single antibody titer of 1 : 256 is presumptive evidence of past infection.
The sensitivity of serologic diagnosis for disease caused by species other
than L. pneumophila serogroup 1 is not known, and the specificity of anti-
body tests for disease caused by other species is less than that of L. pneu­
mophila serogroup 1. PCR assays for direct detection of Legionella spp. in
clinical specimens and for sequencing identification of cultured isolates
have been developed. Sensitivities of 64% to 100% and specificities of 88%
to 100% have been reported (Edelstein, 2011). Use of these assays can
provide more rapid results, especially when used in direct specimen testing;
in addition, identification of species other than L. pneumophila, which is
not possible with DFA and serology, could increase the demand for such
assays. PCR for Legionella spp. directly in clinical samples will be per-
formed most often in the future as assays become cleared for clinical labo-
ratory testing to enhance recovery or in assays in which other respiratory
pathogens can be simultaneously searched for.
Antimicrobial Susceptibility
Because of the intracellular nature of Legionella spp. in clinical infection,
in vitro susceptibility test results do not predict the clinical response of
antibiotics. Susceptibility testing should not be performed. Therapy gener-
ally consists of a macrolide (clarithromycin and azithromycin are as effica-
cious and result in fewer side effects than erythromycin) or a fluoroquinolone
alone or in combination. Other agents that have been used include
trimethoprim-sulfamethoxazole, rifampin, or a tetracycline (Valve et al,
2009; Edelstein, 2011). No β-lactam antibiotic has acceptable intracellular
activity against L. pneumophila. Macrolides and fluoroquinolones should
always be efficacious against infection with Legionella micdadei, longbeacheae,
bozemanae, or dumoffi (Muder & Yu, 2002).
Bordetella
Characteristics
Bordetella spp. are strictly aerobic, nonfermentative, catalase-positive,
minute coccobacilli that can oxidize amino acids but do not ferment sugars.
Members of the genus Bordetella include B. pertussis and B. parapertussis,
which are responsible for pertussis or a pertussis-like disease, respectively,
in humans; B. bronchiseptica and B. avium, which have been responsible for
respiratory disease in humans; and B. hinzii, B. trematum, B. holmseii, B.
petri, and B. ansorpii, which have been found in a wide variety of human
nonrespiratory infections, primarily in immunocompromised hosts
(Wirsing et al, 2011).
Clinical Manifestations and Pathogenesis
Bordetella spp. are found in the respiratory tracts of warm-blooded animals.
Bordetella bronchiseptica primarily causes kennel cough in dogs, although it
may rarely cause pertussis-like symptoms in immunocompromised human
hosts. Bordetella parapertussis, which infects both humans and lambs, is an
uncommon human pathogen. Infection may be asymptomatic or may cause
a pertussis-like illness, most frequently bronchitis. B. pertussis, the etiologic
agent of whooping cough, causes disease only in humans. In 2012, more
than 48,000 cases of whooping cough were reported to the CDC, the
highest number seen in the United States since 1955. In California alone
in 2014, an outbreak with nearly 10,000 cases was reported (cdc.gov/
pertussis). Most of the increased cases have been associated in nonvacci-
nated individuals. Whooping cough is less easily diagnosed in adults, but
there are indications that the number of cases is increasing in adults as well
as children (McGuiness et al, 2013).
B. pertussis and B. parapertussis can cause pertussis or whooping cough,
although cases due to the latter are usually milder and of shorter duration.
B. pertussis is transmitted via droplets from other infected individuals;
in nonvaccinated individuals, the transmission rate can be as high as
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90%, whereas lower rates are seen in vaccinated populations. Protection
against B. pertussis wanes over the years post infection or vaccination, so
currently it is recommended that adults be revaccinated with the acellular
pertussis vaccine (Tdap) in order to reduce the burden of circulating
B. pertussis, especially to newborns and infants (Cortese et al, 2007;
Spector & Maziarz, 2013).
B. pertussis, B. parapertussis, and B. bronchiseptica produce a number of
virulence factors, including adhesions; autotransporters (filamentous hem-
agglutinin, FHA, fimbriae or FIM, and pertactin, or PRN, which is highly
immunogenic); and toxins (adenylate cyclase and lipopolysaccharide, or
LPS). In addition, B. pertussis produces pertussis toxin (PT), an exotoxin
that is involved in colonization of the respiratory tract and establishment
of the infection with B. pertussis, and a virulence factor that has ADP-
ribosyltransferase activity and ribosylates G proteins. PT also induces
lymphocytosis and suppresses chemotaxis oxidative responses and the
overall activity of neutrophils and macrophages (Wirsing et al, 2011). It is
thought that in a pertussis infection, the organism first attaches to the cili-
ated epithelium of the respiratory tract and immune effector cells by means
of fimbriae, FHA, PT, and pertactin. PT and adenylate cyclase toxin work
together to inhibit the host’s immune system, and tracheal cytotoxin
damages the respiratory epithelium. Results may include inflammation and
epithelial necrosis; leukocytosis and lymphocytosis; accumulation of secre-
tions; cough; and ultimately bronchopneumonia, hypoxic episodes, and
encephalopathy (Xu et al, 2009). B. pertussis contains a protective antigen
that when combined with antibody abolishes its infectivity. It appears,
however, that both cellular immunity and humoral immunity are needed
to eradicate the organism.
Laboratory Diagnosis
The rate of isolation of B. pertussis from patients declines with the duration
of illness. The most commonly recommended specimen is the nasopha-
ryngeal aspirate, especially in infants and young children. In adults, older
PART 7
children, and adolescents, a nasopharyngeal swab may be adequate if taken
by trained professions, although aspirates are still considered more sensi-
tive for recovery overall (Wirsing et al, 2011). B. pertussis and B. parapertus­
sis in particular are sensitive to transport and should be cultured as quickly
as possible after collection or placed into special transport devices. The
other species of Bordetella are not sensitive to transport conditions. In
general, swabs or aspirates should be inoculated onto suitable media, such
as Regan-Lowe agar, as quickly as possible after collection. The medium
used is usually supplemented with an antibiotic such as cephalexin to sup-
press contaminating bacteria. Incubate at least 7 days at ambient atmo-
sphere, 35° to 37° C. For shipment to reference laboratories, the inoculated
medium should be incubated for at least 24 hours in ambient air at 35° C
prior to transport to allow some initial growth of the organism. Isolation
of B. pertussis from culture is very specific, so sensitivity will depend on the
patient’s age, the duration of the illness, and the patient’s vaccination status.
Direct examination of smears stained with fluorescein-conjugated B.
pertussis monoclonal or polyclonal antiserum may provide a rapid diagno-
sis; however, DFA assays suffer from low sensitivity (30% to 71%) and low
specificity. Results of DFA should be considered presumptive only and
should be used as an adjunct to culture or PCR (Wirsing et al, 2011).
Direct detection of B. pertussis in clinical specimens by means of PCR is
the best method for detection of B. pertussis. Specimen collection for PCR
is the same as that for culture, but transport is less of an issue when PCR
is being used for detection. A study examining a few transport devices
demonstrated equal performance among them, and transport times are not
as critical as they are when culture is the method of detection (Arbefeville
et al, 2014). As more commercial products become available, the reproduc-
ibility of results between laboratories have improved (Leber, 2014). A
survey of many public health laboratories using different PCR methods
revealed comparable results that were better than had been reported in a
previous survey (Williams et al, 2015). PCR test results, however, may
remain positive longer than results obtained with culture or DFA, even
with appropriate antimicrobial therapy. PCR methods for the direct detec-
tion of B. parapertussis in clinical specimens are also available (Arbefeville
et al, 2014), and consideration should be given to use of a PCR that can
detect both organisms, because in some outbreaks, a significant cause of
whooping cough is actually B. parapertussis (Wirsing et al, 2011).
Culture for B. pertussis provides the most specific diagnosis of whoop-
ing cough and enables a laboratory to perform susceptibility testing
and/or genotypic analysis if required. Colonies of B. pertussis are small,
smooth, round, and shiny and may have the appearance of a drop of
mercury. Organisms with typical colony morphology and Gram stain
appearance should be subcultured to blood agar to verify the absence
of growth on this medium. Positive catalase and oxidase reactions and
1143
 negative urease can be used for presumptive identification of an organism
58  Medical Bacteriology
as B. pertussis. B. parapertussis grows more rapidly and will grow on blood
agar and occasionally on MacConkey’s agar. Colonies are oxidase negative
and catalase and urease positive. B. bronchiseptica grows well on both blood
and MacConkey’s agars and biochemically is the most active of the three.
It is catalase, oxidase, urease, and nitrate reduction positive. B. holmseii has
been described more recently in the genus, not as a cause of whooping
cough, but rather in association with bacteremia, endocarditis, and respi-
ratory illness in immunocompromised patients, especially in asplenic
patients (Shepard et al, 2004). B. holmseii will grow well on blood agar, and
its appearance on MacConkey’s agar may be delayed. It is negative for
oxidase, nitrate reduction, and urease. Other Bordetella spp., including
B. hinzii, B. trematum, and B. avium, grow on both blood agar and Mac-
Conkey’s agar and are motile, unlike all other members of the genus
(Wirsing et al, 2011).
Serologic methods for B. pertussis and B. parapertussis may be used to
make the diagnosis of whooping in nonvaccinated older children, adoles-
cents, and adults; EIA is the usual method of choice. Demonstration of
seroconversion or a significant rise in concentration of IgG against PT is
thought to be the most sensitive and specific test. Serology, however,
should not be used for 1 year after a person is vaccinated with the acellular
pertussis vaccine. In a study comparing culture, PCR, and serology for
diagnosis of pertussis, if a minimum of two antigens to B. pertussis (IgG,
IgA, or IgM) were obtained in both acute and convalescent sera, serology
was found to be as sensitive as PCR, and both were more sensitive than
culture (Cengiz et al, 2009).
Antimicrobial Susceptibility
Antimicrobial agents probably play no role in the therapy of pertussis, but
nasopharyngeal cultures become negative after 1 to 2 days of treatment,
which may prevent bacterial complications in patients with the disease
and may be effective in preventing spread of the disease to nonimmune
contacts. Susceptibility testing is not indicated for B. pertussis, and
methods are not standardized. A macrolide (erythromycin, azithromycin,
or clarithromycin) is the drug of choice for treatment and prophylaxis;
trimethoprim-sulfamethoxazole (SXT) is an acceptable alternative in
patients with macrolide intolerance and in those in whom the isolate is
resistant to the macrolides, which occurs very rarely (Wirsing et al, 2011).
Prevention
Several formulations of vaccines can be used to prevent diphtheria, tetanus,
and pertussis. Some are combined with vaccines to prevent other diseases
and reduce the total number of injections that someone receives at one
office visit. In the United States, DTaP, Tdap, and Td vaccines are most
commonly used. DTaP is given to children younger than 7 years of age,
and Tdap and Td are given to older children and adults (cdc.gov/vaccines/
vpd-vac/pertussis/default.htm).
Brucella
Characteristics
Brucella spp. are small, gram-negative coccobacillary organisms (0.5-
0.7 µm × 0.6-1.5 µm). In smears, they occur predominantly as single coc-
cobacilli, but they may occur in pairs or in short chains. They have been
described as having the appearance of sand. They are nonmotile, strictly
aerobic, catalase- and usually oxidase-positive organisms. They are non-
fermenters. They can grow on a variety of laboratory media, but growth
is often enhanced by the presence of 5% to 10% CO2 and the addition of
blood or serum. Of the recognized species, Brucella melitensis, Brucella
abortus, Brucella suis, and Brucella canis are human pathogens, although B.
canis has reduced virulence for humans as compared with other species. B.
ovis is a pathogen of sheep, and B. neotomae has been isolated from the
desert wood rat. Three species have been recovered from marine animals:
B. delphini, B. pinnipediae, and B. cetaceae. All species hydrolyze urea, except
B. ovis, and this remains a significant characteristic of the genus (Petersen
et al, 2011).
Clinical Manifestations and Pathogenesis
Brucella spp. are intracellular bacteria that infect a wide range of animal
species (including humans) and have been found in some insects and ticks.
They are important veterinary and human pathogens. Preferential hosts
are sheep and goats for B. melitensis, cattle for B. abortus, swine for B. suis,
and dogs for B. canis; however, each species may occasionally infect other
animals. Humans become infected by inhalation of the organism; by
direct contact with infected material, including animal carcasses, fetal
membranes, vaginal discharge, fetuses, skin, or mucous membranes; or
by ingestion of unpasteurized milk or milk products from infected
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animals. To prevent infection with Brucella spp., CDC recommends not
consuming undercooked meats or unpasteurized milk, cheese, or ice
cream and related dairy products. If animals are handled, use of rubber
gloves, goggles, and protective gowns/aprons is essential to prevent infec-
tion. Approximately 100 cases of human brucellosis are reported per year
in the United States; in 2010, there were 105 reported cases, with half of
these in California or Texas. A common risk factor for brucellosis in
the United States is consumption of imported cheese made from unpas-
teurized goat’s milk. In addition, brucellosis is the most often reported
disease associated with laboratory accidents, so care must always be taken
when working with specimens and/or cultures from suspected cases
(cdc.gov/brucella).
Local lymphadenopathy often occurs with dissemination and secondary
localization in the reticuloendothelial system and formation of granulomas
in the liver, spleen, bone, genitourinary tract, lungs, and soft tissues.
Organisms may be seen within phagocytes. Signs and symptoms are often
variable and nonspecific, with chills, fever, sweats, and anorexia occurring
frequently. The fever is characteristically diurnal (“undulant”). The incu-
bation period is generally considered to be 1 to 4 weeks. Although specific
virulence factors have not been demonstrated, the intracellular nature of
the organisms and their survival intracellularly contribute to the virulence
and pathogenicity of the organism. In addition, the urease produced by
most species enables passage through the stomach when infection occurs
through ingestion. Brucella-containing vacuoles enable escape from the
host immune system and provide an acidic environment to avoid antibiotic
killing (Petersen et al, 2011).
Laboratory Diagnosis
Brucella spp. are recovered most often from blood and bone marrow
and less often from material obtained from spleen and liver abscesses.
The organism grows on standard laboratory media, including Brucella,
blood, chocolate, and trypticase soy agar. Some strains will grow on
MacConkey’s agar. Brucella spp. will grow in media used for culturing
blood specimens and should be held 10 to 14 days, although bottles may
become positive in as little as 5 to 7 days (Petersen et al, 2011). Brucella
spp. are recognized as Class A bioterrorism organisms, and as such should
be handled only in public health laboratories and/or by the CDC. Identi-
fication and testing of this organism are described here for completeness
but should not be performed in sentinel laboratories, which include most
hospital laboratories.
Solid media should be incubated in an atmosphere containing 5% to
10% CO2. These organisms grow slowly, and even after 48 hours of incu-
bation, colonies may be difficult to see. Organisms can be presumptively
identified as Brucella spp. based on a characteristic Gram stain appearance
and may be positive for catalase, oxidase, and urease. Urease activity is
manifested rapidly (about 15 minutes) by B. suis and more slowly (2 to 24
hours) by B. melitensis and B. abortus. Because brucellosis can be laboratory
acquired, laboratory personnel should be notified if this organism is sus-
pected, and all manipulations of possible Brucella spp. should be conducted
in a biological safety cabinet (Petersen et al, 2011).
Identification of Brucella spp. to the species level requires tests for CO2
requirement, H2S production, urea hydrolysis, dye sensitivity, and phage
sensitivity. Most hospital laboratories refer this testing to State Health
Departments or other reference laboratories. Molecular tools such as PCR
have become more effective means of identifying cases of brucellosis
(Queipo-Ortuno et al, 2005).
There are many serologic tests employed in the laboratory diagnosis
of brucellosis. The serum agglutination test (SAT) is among the most
widely used; however, it has limitations, including false-negative results in
chronic or complicated cases, cross-reactivity with Francisella tularensis,
and a 24-hour turnaround time. The slide agglutination test is simpler and
faster (10 minutes) and is relatively good in acute cases, although it is also
prone to false-negative and false-positive reactions. The Indirect Coombs
test is more useful in complicated cases, but it does take up to 48 hours
for a result. ELISA is the method of choice in most cases of chronic,
complicated cases and is very sensitive and specific. Brucella agglutination
titers of greater than 1 : 160 are usually considered positive, although lower
titers with SAT have been reported. It is recommended that two of the
above tests be used in combination for diagnosis to limit misdiagnosis
(Petersen et al, 2011).
Antimicrobial Susceptibility
Several agents are effective against species of Brucella, including doxycy-
cline, aminoglycosides, rifampin, trimethoprim-sulfamethoxazole, some
quinolones, and cephalosporins. Combination therapy is recommended
because studies have shown more failure and relapses when monotherapy
is used (Skalsky et al, 2008). For uncomplicated cases, the WHO recom-
mends treatment with doxycycline and rifampin for 6 to 8 weeks, and
longer treatment periods are recommended for cases of neurobrucellosis
and patients with endocarditis (Petersen et al, 2011. Although treatment
failures do occur, they are not due to antimicrobial resistance, and suscep-
tibility testing is not recommended, although the CLSI describes a method
using a Mueller Hinton broth dilution to determine the MICs of tetracy-
cline and doxycycline. Such testing, if needed, would be done at a public
health laboratory or CDC because sentinel laboratories should not be
working with these possible bioterrorism organisms (CLSI, 2010; Petersen
et al, 2011).
Pasteurella
Characteristics
Pasteurellae are facultatively anaerobic, oxidase- and catalase-positive,
nonmotile, gram-negative bacteria that range morphologically from coc-
cobacilli to long filamentous rods. Of the eight species known to infect
humans (Pasteurella multocida, Pasteurella bettyae, Pasteurella canis, Pasteu­
rella dagmatis, Pasteurella stomatis, Pasteurella pneumotropica, Pasteurella hae­
molytica, Pasteurella aerogenes), P. multocida is the most important human
pathogen. Pasteurella spp. are phenotypically similar to the Actinobacillus
spp., and DNA–DNA hybridization studies and comparisons of 16S rRNA
have shown that P. pneumotropica, P. haemolytica, and P. aerogenes are more
closely related to the genus Actinobacillus than to the genus Pasteurella.
Clinical Manifestations and Pathogenesis
Pasteurella spp., especially P. multocida, may be found as commensals in the
upper respiratory tracts of fowl and mammals and are frequently isolated
from animal bite or scratch wounds. Cat bites more often become infected
than dog bites. Local infections can become systemic, and a number of
reports have described septicemia, osteomyelitis, and meningitis. Pasteu-
rellae have also been associated with respiratory tract infections, including
sinusitis, peritonsillar abscess, mastoiditis, pulmonary abscess, pneumonia,
empyema, bronchitis, and bronchiectasis, usually in patients with underly-
ing chronic pulmonary disease (Zbinden & von Graevenitz, 2011; Wilkie
et al, 2012). Little is known about the virulence factors, but a dermone-
crotic toxin that targets G proteins, similar to that found in Bordetella spp.,
E. coli, and Yersinia, has been recently found in P. multocida; further studies
may elucidate how this toxin plays a role in the pathogenicity of Pasturella
spp. (Wilson & Ho, 2011).
Laboratory Diagnosis
Pasteurellae grow well on blood agar and are only rarely able to grow on
gram-negative differential media, such as EMB or MacConkey’s agar. The
finding of a gram-negative bacillus that grows on blood agar only and is
oxidase and indole positive and ortho-Nitrophenyl-β-galactoside (ONPG)
negative provides strong presumptive evidence for the isolation of P. mul­
tocida. In addition, susceptibility to penicillin, as evidenced by a wide zone
of inhibition around a penicillin disk on a blood agar plate, is good evi-
dence that the isolate is P. multocida.
Antimicrobial Susceptibility
Pasteurellae are usually susceptible to penicillin, broad-spectrum cephalo-
sporins, tetracyclines, and quinolones, but they are resistant to macrolides,
amikacin, and narrow-spectrum cephalosproins. Penicillin is the usual
therapeutic drug of choice. Rare strains of P. multocida have produced
β-lactamase, but the combination of a β-lactam with a β-lactamase inhibi-
tor drug should be effective (Zbinden & von Graevenitz, 2011). Although
susceptibility testing is not usually required, a method is available from the
CLSI (CLSI, 2010).
Francisella tularensis
Characteristics
F. tularensis is a very small, strictly aerobic, coccobacillary to pleomorphic
rod-shaped, gram-negative bacillus that requires cystine or cysteine for
growth. Faint bipolar staining occurs with aniline dyes.
Clinical Manifestations and Pathogenesis
F. tularensis is found in both wild and domesticated animals, birds, arthro-
pods, water, mud, and animal feces. The primary reservoir for this organ-
ism is the cottontail rabbit. Transmission to humans occurs through bite
of tick or deerfly, direct cutaneous inoculation from the handling of an
infected or dead animal, conjunctival inoculation, inhalation, or ingestion
of undercooked infected animal meat or ingestion of contaminated water.
Several forms of the disease occur, including ulceroglandular, glandular,
oculoglandular, oropharyngeal, intestinal, pneumonic, and typhoidal.
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There were 203 cases of tularemia reported to the CDC in the United
States between 2004 and 2013 (cdc.gov/francisella); disease has been
reported from every state except Hawaii. F. tularensis is considered to be
one of the class A agents of bioterrorism; because of this, workup of sus-
pected cases is limited to approved laboratories. Most clinical laboratories
are considered sentinel laboratories, and if F. tularensis is suspected, isolates
should be sent to State Health Departments or other selected laboratories
(Petersen et al, 2011).
Tularemia manifests in various forms after an incubation period of 1 to
10 days. Headache, fever, chills, vomiting, and myalgias characteristically
occur at the onset. In ulceroglandular disease, lymphadenitis and lymph-
adenopathy occur in the region draining the primary lesion. The lesion is
initially papular and is later ulcerative. Oculoglandular disease is character-
ized by inflammation of the conjunctiva and usually a papule of the lower
lid with lymphadenitis of the preauricular, parotid, submaxillary, and ante-
rior cervical nodes. The intestinal form of tularemia is characterized by
ulcerative lesions of the mouth, throat, and upper gastrointestinal tract.
The CDC recommends the following protective measures for prevention
of tularemia: Use insect repellent when outdoors in tick- and deerfly-
infested areas, use gloves when handling potentially infected animals, and
do not mow over dead animals (cdc.gov/tularemia). There is no safe and
efficacious vaccine available as yet to protect against tularemia.
Virulence appears to be related to the ability of this pathogen for
intracellular replication, especially within macrophages, and its ability to
evade host recognition. In addition, its ability to control reactive oxygen
and nitrogen species provides mechanisms for its intracellular survival,
adding to its pathogenicity (Steiner et al, 2014). Tularemia should be
suspected in anyone who has been in an endemic area, has had contact
with wild animals or livestock, has a history of tick bite, has been engaged
in farming operations, has drunk impure water, or has been exposed to
cultures or infected animals in the laboratory. Trappers, hunters, fur and
meat industry workers, agricultural workers, and laboratory personnel are
PART 7
at greatest risk. Because of its protean manifestations, tularemia is readily
confused with many other diseases, such as brucellosis, anthrax, sporotri-
chosis, typhoid fever, tuberculosis, histoplasmosis, and syphilis.
Laboratory Diagnosis
Material suitable for examination includes fluid or curettings from the
primary lesion, aspirates of enlarged regional nodes, sputum, pharyngeal
washes, and gastric aspirates. Due to the low dose of organisms required
for infection, care must be taken by all laboratory personnel when handling
suspicious specimens. Bacterial isolation is difficult because the organism
has special growth requirements and grows slowly, allowing for over-
growth of other organisms present in the specimen. The organism grows
on glucose-cysteine agar supplemented with 5% defibrinated rabbit blood,
chocolate agar with IsoVitalX, or BCYE agar. Some isolates may even grow
on blood agar or trypticase soy agar. If clinical material is contaminated
by other organisms, penicillin, polymyxin B, and cycloheximide can be
added to inhibit their growth. Special care must be exercised in handling
infected material to prevent aerosolization or direct contact with the skin.
Clinicians should always notify laboratory personnel if F. tularensis is sus-
pected so proper precautions can be taken to prevent exposure to this
organism (www.bt.cdc.gov) (Petersen et al, 2011).
Cultures are incubated at 35° C with or without added CO2. Colonies
usually appear within 2 to 4 days and are blue-gray to white, round,
smooth, and slightly mucoid. On blood-containing agar, a small zone of
α-hemolysis may be seen. Isolates are weakly catalase positive, nonmotile,
and non–spore-forming, and they react with only a few carbohydrates.
Because working with the organism in the laboratory is dangerous, sus-
pected isolates should be sent to the local public health laboratory or the
CDC for confirmation. MALDI-Tof has also been shown to identify some
of the agents of bioterrorism, including F. tularensis (Murray, 2010).
The diagnosis of F. tularensis can be established serologically. Aggluti-
nation titers determined by tube agglutination (TA) or microagglutination
(MA) are the standard methods utilized for detection of antibodies to
Francisella. In the United States, a single TA titer of 1 : 160 or greater or
an MA titer of 1 : 128 or greater is considered positive (Petersen et al,
2011). As with other serological assays, demonstration of a fourfold change
in titers between an acute and convalescent serum would also be consid-
ered positive. Brucella agglutinins may also rise nonspecifically, but usually
to a considerably lower level (Petersen et al, 2011)).
Antimicrobial Susceptibility
All isolates are β-lactamase positive, so penicillins and cephalosporins are
not effective. The antibiotics recommended for treatment and prophylaxis
include chloramphenicol, ciprofloxacin, gentamicin, streptomycin, and
1145
 tetracycline; resistance to these agents has not been reported as yet
58  Medical Bacteriology
(Petersen et al, 2011). CLSI has published interpretative criteria and a
method utilizing Mueller Hinton media supplemented with 2% IsoVitalex
for broth microdilution. However, most clinical laboratories should not
perform susceptibility testing because all suspected isolates should be sub-
mitted to a public health laboratory or CDC for such testing (CLSI, 2010).
Gardnerella
Characteristics
Gardnerella vaginalis is a thin, gram-variable rod or coccobacillus. Over the
years, this organism, in its association with bacterial vaginosis, has been
called Haemophilus vaginalis and Corynebacterium vaginale, further demon-
strating its gram-variable appearance. Catalase is not produced, and cells
are nonmotile. Growth is best observed after 48 hours of incubation in a
5% CO2-enriched atmosphere. Colonies are small and exhibit β-hemolysis
on media containing rabbit or human blood.
Clinical Manifestations and Pathogenesis
This organism is associated with bacterial vaginosis but is not the cause.
It has been found in the blood of patients with postpartum fever and can Figure 58-25  Blood smear positive for Capnocytophaga canimorsus in patient
cause infection in newborns. G. vaginalis is a part of the anorectal flora of with septicemia (Wright’s stain).
healthy adults of both sexes, as well as of children. It is part of the endog-
enous vaginal flora of women of reproductive age.
Laboratory Diagnosis
Diagnosis of bacterial vaginosis (BV) does not require culture. The diag-
nosis is made by direct examination of vaginal secretions for the presence
of clue cells (epithelial cells covered with bacteria on the cell margins) and
small gram-negative rods and coccobacilli, the absence of lactobacilli
(gram-positive thin rods), a pH greater than 4.5, and a fishy amine odor
after addition of 10% potassium hydroxide (KOH) to the secretions. A
scored Gram stain is the laboratory test that should be performed when
vaginal discharge is submitted for the diagnosis of BV (Nugent et al, 1991).
Alternatively, a nucleic acid probe (Affirm, Becton Dickinson Microbiol-
ogy Systems, Sparks, Md.) is available that tests for a high concentration
of G. vaginalis as a marker for bacterial vaginosis. In comparison to PAP
smears, the AFFIRM was more sensitive and specific in the diagnosis of
BV (Briselden & Hillier, 1994; Levi et al, 2011). When observed in culture,
the organism is presumptively identified based on the presence of diffuse
beta-hemolysis around small colonies (<0.5 mm) after 24 to 48 hours on
medium containing human or rabbit blood and a consistent Gram stain
morphology for G. vaginalis (Funke & Bernard, 2011). Figure 58-26  Gram stain of Capnocytophaga ochraceus. Note the fusiform
bacilli.
Antimicrobial Susceptibility
Susceptibility testing of G. vaginalis is not recommended, and no guide-
lines exist for performing this testing. Metronidazole is the drug of choice canimorsus is oxidase and catalase positive. Capnocytophaga spp. are usually
for bacterial vaginosis. Systemic infection due to G. vaginalis may be susceptible to cephalosporins, macrolides, tetracycline, clindamycin, and
treated with ampicillin because this organism has not been found to quinolones, but are resistant to colistin and the aminoglycosides (Zbinden
produce β-lactamase (Funke & Bernard, 2011). & von Graevenitz, 2011).
Capnocytophaga
Calymmatobacterium granulomatis
Capnocytophaga is a genus of facultatively anaerobic, gram-negative rods to
filamentous bacteria. Species include Capnocytophaga ochraceus, Capnocyto­ (Klebsiella granulomatis)
phaga canimorsus (formerly DF-2), Capnocytophaga gingivalis, and Capnocy­ Characteristics
tophaga sputigena, among others. C. canimorsus is part of the oral flora of Calymmatobacterium granulomatis is a gram-negative, nonmotile, encapsu-
dogs and cats; the others can be found as part of normal human oral flora. lated, pleomorphic rod that may be cultured in yolk sacs or on fresh egg
yolk medium. The organism possesses antigenic and molecular determi-
Clinical Manifestations and Pathogenesis nants similar to those of Klebsiella spp., leading taxonomists to now classify
C. ochraceus can cause transient bacteremia or endocarditis in both immu- it among the Enterobacteriaceae as Klebsiella granulomatis (Abbott, 2011).
nocompromised and immunocompetent patients (Zbinden & von Graeve-
nitz, 2011). It also has been associated with periodontitis. C. canimorsus is Clinical Manifestations and Pathogenesis
a cause of a fatal septicemia following wound infection subsequent to the The organism does not produce disease in animals. In humans, it causes
bite of dogs or cats. Patients with this fatal septicemia have predisposing granuloma inguinale (donovanosis), a rare sexually transmitted disease seen
factors of a prior splenectomy or alcoholism (Oehler et al, 2009; Gaastra predominantly in tropical countries, characterized by ulcerogranuloma-
& Lipman, 2010). Meningitis, endocarditis, arthritis, and eye infection tous lesions of the skin and mucosa of the genital and inguinal areas
have also been documented with C. canimorsus (Gasch et al, 2009). Figure (O’Farrell & Moi, 2010; Abbott, 2011).
58-25 shows a Wright’s stain of C. canimorsus seen on the blood film of a
patient with C. canimorsus septicemia. Note the intracellular nature of the Laboratory Diagnosis
bacilli. A fragment of tissue removed from the margin of an ulcer is pressed and
Isolation of Capnocytophaga spp. requires inoculation of blood or choco- rubbed against a glass slide and stained with Wright’s or Giemsa stain. The
late agar and incubation in a 5% to 10% CO2 environment, generally for finding of small, straight or curved, pleomorphic rods with rounded ends
longer than 24 hours. Colonies are usually slightly pigmented yellow or and characteristic polar granules, giving a “safety pin” appearance within
orange and may spread because of a “sliding” motility. Gram stains often mononuclear cells, is the most effective way of establishing the diagnosis.
show long, thin, spindle-shaped cells, almost fusiform in appearance. K. granulomatis does not grow on routine culture media (Abbott, 2011).
Figure 58-26 shows the Gram stain of C. ochraceus, demonstrating the PCR methods for detection of this organism along with other agents of
fusiform bacilli. C. ochraceus is oxidase and catalase negative, and C. genital ulcers are available (Bialasiewicz et al, 2012).

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Figure 58-27  Gram stain of Streptobacillus moniliformis from culture. (Cour-
tesy Dr. Nancy Cornish.)
Antimicrobial Susceptibility
The tetracyclines, macrolides, ampicillin, and chloramphenicol are active
against K. granulomatis. Azithromycin is the standard treatment.
Streptobacillus
Characteristics
Streptobacillus is a facultatively anaerobic, fermentative, nonencapsulated,
and nonmotile pleomorphic gram-negative rod, frequently occurring in
chains or long filaments, and often with a series of oval to elongated bulbous
swellings, giving a string-of-beads appearance. The microscopic morphol-
ogy of the organism in culture varies with time, being more homogeneously
filamentous in young cultures and becoming fragmented into irregular
coccobacilli with age. L-phase organisms having a “fried egg” appearance
may occur spontaneously on agar and may become stabilized if penicillin
is incorporated in the medium. Figure 58-27 is a smear of Streptobacillus
moniliformis from the culture of a patient with rat-bite fever.
Clinical Manifestations and Pathogenesis
S. moniliformis occurs as indigenous flora in the upper respiratory tract of
wild and laboratory rodents. Infection (rat-bite fever or Haverhill disease)
in humans follows rodent bites, ingestion of contaminated food, or trau-
matic injury. Local lymphangitis and lymphadenitis may develop up to 3
weeks later, followed by fever, chills, malaise, and, later, a general morbil-
liform maculopapular or petechial rash. Some patients develop a migratory
polyarthritis. Endocarditis has been reported, as have cases of myocarditis,
pericarditis, meningitis, pneumonia, and abscess development due to S.
moniliformis (Madhubashini et al, 2013). The histopathology is nonspecific
chronic inflammation (Zbinden & von Graevenitz, 2011).
Laboratory Diagnosis
Specimens for recovery of S. moniliformis include blood, joint fluid, and
abscess material. The organism grows on media enriched with 15% sheep
or rabbit blood, serum, or ascitic fluid. Inoculated media should be incu-
bated at 35° C in a humid environment containing an atmosphere of 5%
to 10% CO2. Colonies on solid media are small and slightly translucent
to opaque, with slightly irregular edges. Coccal forms and gram variability
may occur, but usually colonies will demonstrate long filaments with gran-
ules and bulbs or banding forms on Gram stain. L-forms may be present
in some cultures, along with the usual bacterial forms.
Colonies in broth form as fluff balls, usually at the bottom of the tube.
In cultures, the organism dies quickly unless subcultured often. Because
this organism is relatively inert, identification of S. moniliformis is difficult.
Biochemical tests must be performed in heart infusion agar or broth supple-
mented with yeast extract and horse serum. 16S rRNA sequencing methods
or fatty acid analysis is required for complete identification (Zbinden &
von Graevenitz, 2011). In addition, direct specimen testing with molecular
methods may prove more sensitive than culture. (Mackey et al, 2014)
Antimicrobial Susceptibility
S. moniliformis is susceptible to penicillins and tetracyclines, which are the
recommended drugs for therapy. It is also susceptible to cephalosporins,
clindamycin, macrolides, and aztreonam; intermediately susceptible to the
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aminoglycosides and fluoroquinolones; and resistant to colistin, nalidixic
acid, and SXT.
Prevention and Control
Because 10% to 65% of rats are infected with the organism, their control
and precautions against bites represent the only effective methods of
control of the disease.
ANAEROBIC BACTERIA
It is important to reemphasize that anaerobes represent a major component
of the indigenous flora of the skin and mucous membranes. Their isolation
and identification are contingent on the proper selection and collection of
specimens, as well as on their proper transport to the laboratory. Anaerobic
infections are frequently mixed, consisting of several species of anaerobes,
or of anaerobes mixed with facultatively anaerobic bacteria or aerobes;
therefore, the first task in examining an anaerobic culture is to separate
facultatively anaerobic from obligate anaerobic bacteria. With experience,
the more commonly isolated anaerobes can often be recognized on the
basis of their colonial and microscopic morphologies and presumptively
identified on the basis of a few additional tests. Definitive identification is
based on biochemical reactions, physiologic and molecular characteristics,
and occasionally pathogenicity and toxin neutralization tests.
The extent to which anaerobes are identified varies according to the
facilities and expertise available, the interest of laboratory personnel and
clinical staff, and the clinical relevance of the information available from
the laboratory. With the advent of Maldi-Tof, identification of anaerobes
has become something that many laboratories will be able to do success-
fully, although it would still be prudent to continue to use clinical relevance
as a guide to when full identification is necessary. Anaerobes have been
fairly consistent in their response, at least in vitro, to antibiotics, but as
with other groups of bacteria, resistance is becoming an issue with many
PART 7
anaerobes; this often prompts additional identification and susceptibility
testing (Hecht, 2006).
Definitions and Characteristics
An anaerobe requires an atmosphere with reduced oxygen tension for its
growth and fails to grow on the surface of solid media in an atmosphere
of room air with 10% CO2. A facultatively anaerobic bacterium will grow
in the presence or absence of room air. The term microaerophile has not
been strictly defined but is commonly applied to bacteria—usually campy-
lobacters and streptococci—that grow only or preferentially in an atmo-
sphere with reduced O2 and with increased CO2.
Pathogenesis and Virulence Factors
Little is known about the factors responsible for the pathogenic and viru-
lence properties of most anaerobes, other than the clostridia and some
Bacteroides spp. Endotoxic, proteolytic, and heparinase activities have been
identified among the Bacteroidaceae. The polysaccharide capsule of B.
fragilis promotes abscess formation. Clostridia, on the other hand, elabo-
rate potent exotoxins, including lethal and necrotizing toxins, hemolysins,
lecithinases, gelatinases, and hyaluronidases.
Although clostridial infection may be exogenous or endogenous in
origin, disease caused by other anaerobes usually originates endogenously
from the normal indigenous anaerobic flora of a contiguous mucous mem-
brane, the integrity of which has been disrupted by surgery, instrumenta-
tion, trauma, or malignancy. Essential to the establishment of anaerobes
in the infectious process is a decrease in the oxidation-reduction potential
(Eh) of the area, which may result from failure of its blood supply or from
the multiplication of other bacteria at the site.
Although clostridial infections and intoxications are unquestionably of
major medical importance, the role of other anaerobes in causing cellulitis
and myonecrosis has been recognized more recently. Most isolates of
Clostridium perfringens are the result of simple contamination of a wound.
In such instances, the clostridia may multiply in cellular debris, a hema-
toma, or necrotic tissue without observable clinical symptoms. Anaerobic
cellulitis is a necrotizing process of the soft tissues. Its onset is gradual, but
it can progress rapidly and extensively. Gas is produced; however, the
process typically does not involve muscle. In addition to or instead of
clostridia, the bacteriology of anaerobic cellulitis may involve anaerobic
cocci and anaerobic gram-negative bacilli.
In contrast to anaerobic cellulitis, gas gangrene or clostridial myone-
crosis is an acute and rapidly progressive invasive process that produces
marked changes in muscle. Distinguishing between anaerobic cellulitis and
gas gangrene is critical to avoid performing unnecessarily aggressive and
mutilating surgery in the former condition.
1147
 Histotoxic clostridia associated with gas gangrene include C. per­
58  Medical Bacteriology
fringens, Clostridium novyi, Clostridium septicum, Clostridium histolyticum,
Clostridium sporogenes, and Clostridium bifermentans. C. perfringens has been
the species most frequently involved in gas gangrene; the prevalence of
other species in this process has varied widely.
Tetanus and botulism, caused by C. tetani and C. botulinum, respectively,
are described as intoxications rather than as infections, because their mani-
festations are related to the elaboration of potent neurotoxins. Antibiotic-
associated diarrhea is a disease caused by the toxins of C. difficile. Botulism
is most frequently due to the ingestion of home-processed foods that have
been improperly preserved or canned; sporadic outbreaks of the disease
have been associated with commercially processed food and with wounds
infected by the organism. The incubation period for botulism is short.
Signs and symptoms usually occur between 18 and 36 hours following
ingestion of contaminated food. Of the seven antigenic types of botulinum
toxin known, type A is the most common in cases of food poisoning in
North America, followed by types B, E, and F. The toxin is absorbed from
the intestinal tract and, rarely, from an infected wound and ultimately
attaches to motor nerve terminals, thereby preventing acetylcholine release
at the nerve endings.
Tetanus typically occurs in nonimmunized persons within the first 2
weeks following a traumatically acquired puncture, laceration, or abrasion.
Cases have been reported to occur postoperatively; following dental work,
childbirth, and abortion; or in association with stasis and decubitus ulcers.
The toxin, tetanospasmin, is transported to gangliosides in the CNS via
the lymphatics and bloodstream and by migration through the perineural
spaces of peripheral nerves.
Clostridium difficile is the major cause of nosocomial diarrhea and the
primary pathogen responsible for pseudomembranous colitis. It is a rare
cause of abscesses, wound infections, osteomyelitis, pleuritis, peritonitis,
septicemia, and urogenital tract infections. Carriage rates of C. difficile
and its toxins are high (50% or more) in neonates, but disease is rare
(Bartlett, 1997). Colonization, with or without toxin production, may be
maintained for several months, but when the adult flora becomes estab-
lished at 6 to 12 months of age, colonization rates fall, and only about
3% of normal, healthy adults are colonized with the organism. C. difficile
almost always is acquired in the hospital by persons receiving antimi­
crobial agents via direct or indirect exposure to human or inanimate
reservoirs. Although the penicillins and the cephalosporins are implicated
most frequently, any antimicrobial agent may trigger C. difficile–associated
disease. Disease rarely occurs without antibiotic exposure, but cases have
been reported following therapy with antineoplastic agents that have anti-
bacterial activity. Pseudomembranous colitis, the most severe form of
C. difficile disease, is a toxin-mediated illness in which microbial invasion
of the mucosa is not known to occur. C. difficile produces two toxins.
Toxin A, a weakly cytopathic toxin, is predominantly responsible for the
enterotoxic activity of the organism. Toxin B, a potent cytotoxin, appears
to play a minor role in human disease, although toxin A–negative,
B-positive strains have been isolated from symptomatic patients. Use of
a PCR assay in the laboratory for detection of both toxin A and toxin B
have increased sensitivity of detection of the disease (Eastwood et  al,
2009; Kvach et  al, 2009).
As previously mentioned, other anaerobic bacteria, particularly anaero-
bic cocci and gram-negative bacilli, have been associated with anaerobic
cellulitis. These organisms are part of the indigenous flora of the mucous
membranes of the oral cavity and of the gastrointestinal and genitouri-
nary tracts. As such, they are encountered in aspiration pneumonias, lung
abscesses, empyemas, intraabdominal infections and abscesses, pelvic
abscesses, brain abscesses, and bacteremias. Anaerobic intraabdominal
infections commonly follow abdominal and especially colon surgery, and
are most frequently associated with the B. fragilis complex. Clinically
significant anaerobic bacteremias are also most frequently caused by this
species. Propionibacterium acnes have been increasing in isolation and in
association with clinical disease.
Significant taxonomic changes have occurred among the anaerobes.
Some of these are delineated in Tables 58-15 through 58-19.

TABLE 58-15
Most Frequently Isolated Gram-Positive Non-Clostridium spp. Anaerobic Bacteria
Species Gram Stain Colony Indole PYR

Gram-Positive Cocci
Peptostreptococcus anaerobius Large cocci, often in chains Nonhemolytic; gray with Negative Negative
raised center; sweet odor
P. micros Clusters or short chains; Small; dull color; “halo” Negative Positive
<0.6 µm around colony
Finegoldia marna Pairs, tetrads, and clusters; Small, white, convex Negative Positive
>0.6 µm
Peptoniphilus asaccharolyticus “Clumps,” pairs, or tetrads Small; slight yellow pigment Usually positive Negative
Anaerococcus tetradius Clumps and tetrads Nonglistening; gray; convex Negative Weak
A. prevotii Clumps and tetrads Nonglistening; gray; convex Negative Positive
Staphylococcus saccharolyticus Clusters and tetrads Catalase and coagulase ND ND
negative
Gram-positive non–spore- Gram stain Colony Aerotolerance/ Other characteristic
forming bacilli Biochemical clues
Actinomyces israelii Short rods; branching or White; opaque; “molar + Most common cause of actinomycosis:
beading tooth” genital; pulmonary; abdominal
A. naeslundii Short rods; branching or Gray-white; translucent +++ Oral flora; rarely pathogenic
beading
A. odontolyticus Short rods; branching or May produce pink to red +++ Oral flora; rarely pathogenic
beading colonies in ambient air
A. turicensis ++ Genital sites
Propionibacterium acnes Short rods; may appear White round colonies ++++ Normal flora on skin; associated with
“spidery” Catalase positive; endophthalmitis (post cataract
indole positive surgery); ventricular shunt infections;
infrequent cause of endocarditis,
osteomyelitis
Propionimicrobium Coccoid; singly, pairs, or White, glistening Nonaerotolerant Isolation: lymph nodes; uncertain
lymphophilum short chains relevance
Bifidobacterium spp. Diphtheroidal; bifurcated Nonaerotolerant Normal flora of GI and GU tract; rarely
or forked ends clinically significant
Eggerthella lenta (formerly Straight rod with rounded Nonaerotolerant Grow in 20% bile; saccharolytic; have
Eubacterium lentum) ends Catalase positive; been isolated in anaerobic
indole negative bacteremia

+, ++, +++, ++++, Positive with increasing strength; ND, not done; PYR, l-pyrrolidonyl-β-naphthylamide.

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 TABLE 58-16
Most Frequently Isolated Gram-Negative Anaerobic Bacteria (All Should Grow in Presence of 20% Bile)
Species Colony Biochemicals Other Characteristics

Bacteroides fragilis complex* White, round; requires 24-48 hours

B. fragilis Negative for indole Most common and most pathogenic
Positive for catalase and indole of the complex
B. distasonis** Negative for indole
Usually positive for catalase and esculin
B. thetaiotaomicron Positive for indole, catalase, and esculin
B. vulgatus Negative for indole; usually negative for
catalase and esculin
B. ovatus Positive for indole, catalase, and esculin
B. goldsteinii** Negative for indole; variable for catalase
and positive esculin
Bacteroides urealyticus Colonies often “pit” the agar No growth in 20% bile; indole, esculin, Part of normal anaerobic oral flora
and catalase usually negative; urease
positive
Bilophila wadsworthia Will grow in 20% bile; catalase positive;
negative for indole and esculin
Porphyromonas asaccharolyticus Colonies fluoresce brick red (ultraviolet Bile sensitive; indole positive and Normal oral flora; pulmonary disease
light exposure); black pigment with age catalase negative
Prevotella disiens and P. bivia Bile sensitive; indole and esculin Normal genitourinary flora; can be
negative; saccharolytic and involved in pelvic abscesses
nonpigmented
Fusobacterium nucleatum Breadcrumb-like and speckled; Gram Bile sensitive; indole positive and Oral flora; can be involved in
stain: long thin, pointed bacilli esculin negative bacteremia, pulmonary infection
F. necrophorum Umbonate colonies; greening of agar; Usually sensitive to bile; indole positive, Associated with Lemierre’s syndrome
Gram stain: round, not tapered ends; lipase positive
often “bizarre” forms seen

PART 7
F. mortiferum “Fried egg” appearance of colonies; Grows on 20% bile; indole negative Infrequent isolate
nontapered rods and bizarre round and esculin positive
“bodies” on Gram stain
Gram-Negative Cocci
Veillonella spp. Small white colonies; Gram stain: well Catalase variable; nitrate reduction Common isolate, but rarely involved
staining, round cocci, singly or in pairs positive in clinical disease

*In addition to those listed, B. fragilis complex includes B. caccae, B. cellulosilyticus, B. coprocola, B. coprophius, B. dorei, B. eggerthii, B. faecis, B. finegoldia, B. intestinalis, B.
massiliensis, B. nordii, B. plebius, B. salyersiae, B. stercoris, B. uniformis, B. xylanisolvens, and a few additional species for which testing is based on only one strain. The new
B. fragilis complex species can be found in Table 6 of Kononen E, Wade WG, Citron DM: Bacteroides, Porphyromonas, Prevotella, Fusobacterium, and other anaerobic gram-
negative rods. In Versalovic J, Carroll KC, Funke G, et al, editors: Manual of clinical microbiology, ed 10, Washington, DC, 2011, American Society for Microbiology, p 870.
**These organisms have been placed in the Parabacteroides genus along with P. gordonii, P. johnsonii, and P. merdae.

TABLE 58-17
Most Common Species of Clostridium (Spore-Former) in Clinical Samples
Species Colony Gram Stain Characteristics

C. perfringens Large, white, with double-zone “Boxcar” (short and fat)-shaped rods; short Lipase negative; lecithinase positive; indole
hemolysis chains may be seen; rare spores seen negative; reverse CAMP positive
Most common isolate; skin and soft tissue
infections (including gangrene); bacteremia;
also found in gastrointestinal tract as part of
normal flora
C. ramosum Large, spready white colony Often stain gram negative or variable; Commonly isolated from clinical samples
cells are more slender and longer than
C. perfringens; spores not commonly seen
C. sordelii White Large, gram-positive rods with subterminal Less frequently isolated than C. perfringens, C.
spores seen; lecithinase positive; lipase ramosum, and C. septicum
negative; indole and urease positive
C. tetani Thin bacilli with terminal spores: “snowshoe” May be part of gastrointestinal flora; cause of
or “tennis racquet” appearance; lecithinase tetanus
negative; lipase weak positive; indole
variable and urease negative
C. septicum White, swarming colonies Long, filamentous bacilli with rare When isolated from blood cultures, indicates a
subterminal spores seen; lecithinase, lipase, possible gastrointestinal malignancy
indole, and urease negative
C. difficile White colonies with distinctive Thin, long bacilli with spores seen Cause of pseudomembranous colitis and
“horse-barn” odor; selective antibiotic-associated diarrhea; rarely found
CCFA media: yellow, ground outside of the gastrointestinal tract
glass appearing colonies

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 TABLE 58-18
58  Medical Bacteriology
New Taxonomic Designations of Anaerobic
Laboratory Diagnosis
Gram-Positive Cocci Identifying anaerobic bacteria to the species level can be a complex task;
however, the extent to which isolates of anaerobic bacteria are identified
New Designation Older Designation varies. It may be limited to basic information that is of clinical relevance
and/or that might be needed to predict antimicrobial susceptibilities. For
Finegoldia magna Peptostreptococcus magnus example, the mixed anaerobic flora from a site such as a decubitus ulcer,
Parvimonas micra Micromonas micros perirectal fistula, or intraabdominal abscess may simply be reported as
Anaerococcus prevotii Peptostreptococcus prevotii mixed fecal flora (mentioning specifically whether there are aerobes and
Anaerococcus tetradius Peptostreptococcus tetradius anaerobes or only anaerobes present) without specific identification of its
Anaerococcus vaginalis Peptostreptococcus vaginalis components. Determining the species of an isolate may be limited to those
Anaerococcus lactolyticus Peptostreptococcus present in pure culture from a normally sterile body fluid or site, and can
lactolyticus be readily accomplished with the use of any of several commercially avail-
Anaerococcus hydrogenalis Peptostreptococcus able biochemical kits, in conjunction with microscopic and colonial mor-
hydrogenalis phology and the use of select antibiotic disk sensitivity results. Some
Anaerococcus octavius Peptostreptococcus laboratories, however, have begun to use sequencing as a means of identify-
octavius ing anaerobic isolates. Results are often available rapidly, and increasing
Peptoniphilus indolicus Peptostreptococcus numbers of databases are available for the identification of many more
indolicus species than can be identified with phenotypic kit systems (Simmon et al,
Peptoniphilus asaccharolyticus 2008). MALDI-TOF-MS (matrix-assisted laser desorption/ionization
Peptostreptococcus asacchaolyticus time-of-flight mass spectrometry) is a recently developed method for the
identification of a wide variety of bacteria, including anaerobes (Stingu
Peptoniphilus harei Peptostreptococcus harei
et al, 2008). One should understand that the clinical value of any report
Peptoniphilus lacrimalis Peptostreptococcus of the presence of anaerobic bacteria is directly related to the speed of
lacrimalis
reporting such results from the laboratory. Identification procedures that
Peptostreptococcus anaerobius No name change require 1 to 2 weeks to complete are generally of academic interest only;
Peptoniphilus ivorii Peptostreptococcus ivorii identification of every anaerobe isolated in a culture is often not appropri-
Peptoniphilus gorbachii New species ate; clinical relevance should still be used to guide any workup of anaerobic
Anaerococcus murdochii New species cultures, as it should be used for all cultures.
Gallicola harnesae Peptostreptococcus harnesae Because of their rapid progression and considerable morbidity and
Atopobium parvulum Streptococcus parvulus mortality, the initial diagnosis and management of diseases caused by the
Blautia wexlerae New species clostridia must be based on their clinical presentation and manifestations.
Blautia producta Peptostreptococcus productus In some patients with tetanus, no primary wound is evident. When a
Staphylococcus saccharolyticus wound is present, organisms typical of C. tetani are seldom seen in stained
Peptostreptococcus saccharolyticus smears, even though they may be recovered from cultures. Moreover,
because of this organism’s widespread distribution in nature, its isolation
Adapted from Song Y, Finegold SM: Peptostreptococcus, Finegoldia, Anaerococcus,
Peptoniphilus, Veillonella, and other anaerobic cocci. In Versalovic J, Carroll KC,
from a wound is not necessarily indicative of the diagnosis of tetanus.
Funke G, et al, editors: Manual of clinical microbiology, ed 10, Washington, DC, Laboratory confirmation of botulism requires detection of the toxin in
2011, American Society for Microbiology, p 804. serum, wounds, gastric contents, feces, or the food suspected of causing
the disease. Procedures for extracting the toxin and for performing mouse
neutralization tests are complex; therefore, it is suggested that the appro-
priate materials be referred to the CDC for examination. Telephone con-
sultation should be made in such instances to ensure that the requisite
specimens are properly collected and transported and that the appropriate
TABLE 58-19 authorities are alerted (Stevens et al, 2011).
Newer Taxonomy of Less Frequently Encountered Anaerobic In cases of suspected anaerobic cellulitis or gas gangrene, the laboratory
Gram-Negative Bacilli can be helpful by examining exudate or tissue microscopically. The finding
of numerous, large, “boxcar”-shaped, gram-positive bacilli (Fig. 58-28)
New Designation Older Designation provides presumptive confirmation of the diagnosis. Stained smears may
also be diagnostic of anaerobic streptococcal myositis. Cultures of exudate,
Alistipes putredinis Bacteroides putredinis
tissue, and blood should be performed. Once again, the level or extent of
Alistipes finegoldii New species identification varies considerably among laboratories; however, C. perfrin­
Allistipes indistinctus (new species)
gens may be easily identified by its Gram stain morphology, the production
Allistipes onderdonkii (new species)
of double zones of hemolysis on blood agar, and a positive Nagler’s reac-
Allistipes shahii (new species)
tion on egg yolk agar. For many years, laboratory diagnosis of C. difficile
Dialister micraerophilus New diarrhea was based on detecting one or both toxins directly in stool using
Dialister propionicifaciens New an EIA or cell culture assay. Culture of stool for the organism and, when
Barnesiella is a new genus with species positive, determination of whether toxin is being produced are primarily
B. intestinihominis reserved for epidemiologic and surveillance studies; many believe it is the
Faecalibacterium prausnitzii Fusobacterium prausnitzii “gold standard” for detection. Assays that detect a “common” antigen—
Porphyromonas uenonis Porphyromonas endodontalis (nonoral) glutamate dehydrogenase—which is found in virtually all C. difficile iso-
Sneathia sanguinegens Leptotrichia sanguinegens lates, are now being used as part of a two- or three-step procedure for
Tannerella forsythensis Bacteroides forsythus laboratory diagnosis of C. difficile to increase sensitivity. Unfortunately, the
antigen can be found in other bacteria, so a positive antigen must be con-
Sutterella wadsworthensis Campylobacter (Bacteroides) gracilis
firmed before it is reported as C. difficile positive. Confirmation is carried
(some strains)
out in the two-step assay with PCR or cytotoxicity studies. In the three-
Odoribacter (new genus) O. laneus step assay, a positive antigen is followed by an EIA, and any antigen-
O. splanchnicus
positive/EIA toxin–negative samples would have a PCR or a cytotoxicity
Paraprevotella (new genus) P. clara test conducted for confirmation. The prevalence of C. difficile usually is
P. xylaniphila not greater than 20% in any single facility, and the two- or three-step
Phocaeicola (new genus) P. abscessus approach allows rapid results to be obtained from negative samples. PCR
Porphyromonas P. benonis (new species) assays for initial detection of the toxins of C. difficile are now commercially
available, and many laboratories use PCR as a replacement for EIA or
Adapted from Kononen E, Wade WG, Citron DM: Bacteroides, Porphyromonas, Pre-
votella, Fusobacterium, and other anaerobic gram-negative rods. In Versalovic J, cytotoxicity (Eastwood et al, 2009; Kvach et al, 2009; Stevens et al, 2011).
Carroll KC, Funke G, et al, editors: Manual of clinical microbiology, ed 10, Wash- One of the most common groups of clinically relevant and frequently
ington, DC, 2011, American Society for Microbiology, p 859. isolated anaerobic bacteria is the B. fragilis complex. Isolates from this

1150
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Figure 58-28  Gram stain of a smear of exudate from a wound that had gas
bubbles shows large, boxcar-shaped, gram-positive bacilli, suggestive of clos-
tridial disease (oil immersion).
Figure 58-29  Gram stain of Bacteroides fragilis from broth culture.
complex will grow selectively on Bacteroides bile esculin medium, and they
have characteristic Gram stain morphology, as seen in Figure 58-29. These
gram-negative bacilli are found as normal flora throughout much of the
gastrointestinal and genitourinary tracts. Infections caused by the complex
include abdominal abscess, pelvic abscess, bacteremia, and brain abscess.
They may rarely be involved in pulmonary disease. Although most anaero-
bic infections are polymicrobial, B. fragilis alone may be responsible for
infection. Members of the B. fragilis complex include those organisms
listed in Table 58-16. Also noted in Table 58-16 is a newly named genus,
Parabacteroides, into which some of the Bacteroides have been placed. With
rare exception, members of the complex will grow in the presence of 20%
bile and will hydrolyze esculin. Inclusion of a Bacteroides bile esculin agar
(BBE) plate when anaerobic specimens are processed will allow more rapid
detection and identification of these organisms (Kononen et al, 2011).
Another important group of anaerobic gram-negative bacilli are the
Fusobacterium spp. They often appear as thin and spindly (or pointed)
gram-negative bacilli on Gram stain or produce rather bizarre forms, as is
seen in Figure 58-30. Table 58-16 lists some of the more common Fuso­
bacterium spp. Fusobacterium nucleatum is isolated from many clinical
samples and can be the cause of bacteremia and other syndromes, but it
can also be isolated as part of the normal oral or genital flora. F. necropho­
rum is associated with Lemiere’s syndrome. Table 58-16 provides a list of
other gram-negative anaerobic bacilli, not members of the Bacteroides or
Fusobacterium. Some have been linked to disease, and others have been
isolated from clinical specimens, but their clinical relevance is yet to be
determined.
Anaerobic gram-positive cocci were at one time referred to for the most
part as Peptostreptococcus spp. Many taxonomic changes have occurred with
this group, as well as with the ones described above, and these changes are
shown in Table 58-15. Finegoldia magnus and Parvimonas micra are the most
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For personal use only. No other uses without permission. Copyright ©2016. Elsevier Inc. All rights reserved.
Figure 58-30  Gram stain of Fusobacterium nucleatum.
PART 7
Figure 58-31  Gram stain of Propionibacterium acnes from vitreous fluid.
commonly isolated anaerobic gram-positive cocci isolated from clinical
specimens and the ones most often associated with disease. Anaerobic
gram-negative cocci are members of the genera Veillonella (the most com-
monly isolated), Acidaminococcus, Negativicoccus, Megasphaera, and Anaero­
globus, and although not infrequently isolated from clinical samples, they
are rarely involved in disease (Song & Finegold, 2011).
An anaerobic gram-positive bacillus, Propionibacterium acnes, has been
associated with cases of endophthalmitis that occur after cataract surgery.
In addition, P. acnes may be associated with ventricular shunt infection in
patients with hydrocephalus; rarely, P. acnes can cause endocarditis, septic
arthritis, or other infections. Isolation of this organism may require
extended incubation, often up to 10 days. Figure 58-31 shows a Gram stain
of P. acnes in vitreous fluid obtained from a patient months after cataract
surgery. Actinomyces israelii and other species of Actinomyces can be involved
in lung abscesses, brain abscesses, skin and soft tissue infections, and
mycetoma, and may be involved in genital infections involving use of an
intrauterine device (IUD), although this is a controversial topic (Westhoff,
2007). Other genera of anaerobic gram-positive rods are listed in Table
58-15; many of these anaerobes are isolated from clinical samples, but their
relevance in any disease process is not always clear. As with other anaer-
obes, it is always important to consider whether the anaerobe has been
isolated as the only organism in a sterile site, such as body fluids and tissues;
how often the organism has been isolated from these sites; and whether
other pathogens are present that can account for the patient’s disease
(Wade & Kononen, 2011).
Antimicrobial Susceptibility
Susceptibility testing of anaerobic bacteria in the past was considered
unnecessary, but as more resistance is detected, this is not the case today.
In addition, there are certain clinical settings in which decisions regarding
the selection of agents are critical: failure of usual therapeutic regimens
and persistence of infection; the key role of antimicrobial agents in
1151
 determining the outcome of infection; or the difficulty involved in making
58  Medical Bacteriology
empirical therapeutic decisions based on precedent. Infections from which
antibiotic susceptibility testing of isolates should be considered include
brain abscess, endocarditis, osteomyelitis, joint infections, infections of
prosthetic devices or vascular grafts, and refractory or recurrent bactere-
mia. Isolates to test under these circumstances should include members of
the B. fragilis complex, certain Fusobacterium spp., C. perfringens, and Clos­
tridium ramosum. Agents such as metronidazole, the carbapenems, and
ampicillin-sulbactam are usually active against anaerobic bacteria, with
rare exceptions such as P. acnes, Actinomyces spp., and a few others. Agents
with more unpredictable activity include penicillin, clindamycin, and
cefoxitin. Although an agar dilution method has been recommended for
reference purposes, testing in the clinical laboratory is generally performed
by broth microdilution (CLSI, 2012) or the Etest method. Batch testing
of saved isolates on a yearly basis is recommended to develop antibiograms
for empirical use by clinicians and to monitor for possible development of
resistance (Boyanova et al, 2007).
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