MICROBIOLOGY LECTURE 12 – Other Spirochaetales • Diffuse chronic inflammation devastating destruction of

Notes from Lecture virtually any organ (arthritis, dementia, blindness

USTMED ’07 Sec C - AsM • Gummas-granulomatous lesions

Order Spirochaetales (2 Families, 7 Genera)

• Treponema
• Borrelia D. Congenital
• Leptospira  In-utero can lead to fetal death organ malformations, or
latent infections (desquamation maculopapular rash
TREPONEMA body destruction, cardiovascular syphilis common in
• T. pallidum ssp. Pallidum - Syphilis untreated infants
• T. pallidum ssp. Endemicron - Bejel
• T. pallidum spp. Pertenue - Yaws Laboratory Diagnosis
• T. carateum - Pinta Diagnostic Test Method or Examination
Microscopy * Darkfield
Structure * Direct fluorescent antibody staining
• spirochete 0.1 X 5 – 15 um, coiled Culture Not used
• too thin to be seen by light microscopy even with grams Serology Nontreponemal tests
- VDRL
or giemsa stain
- RPR
• motile forms – darkfield illumination
Treponemal tests
• appreciated thru staining with specific antibodies - FTA-ABS
labeled with flourescent dyes - MHA-TP
Physiology Darkfield examination. Illumination for
• Grown in cell cultures (rabbit epithelium) darkfield microscopy is obtained when
light rays strike the objection in the field
• Doubling time is 30 hours at an oblique angle so that no light rays
• Maintained only for a few generations pass directly from the condenser to the
• Can utilize glucose oxidatively objective of the microscope, but only the
light rays that are reflected from the
object will enter. A treponema is seen in
 Natural syphilis is not found in any other species the center field, distinguished by its
corkscrew, spiral shape
Virulence Factors
• Outer membrane proteins – adherence to surface of
Direct fluorescent antibody for Treponema pallidum (DFA-TP). A tissue
host’s cells impression smear from a brain biopsy specimen shows fluorescent
• Hyaluronidase – perivascular infiltration treponemes. In the DFA-TP test, a
• Fibronectin coating from host cells antiphagocytic monoclonal antibody labeled with
fluorescein isothiocyanate specific
Pathogenesis for T. pallidum binds to the
treponemes in either smears or
 Tissue destruction and lesions are primarily the result of tissue sections prepared from
the patient’s immune response to infection paraffin-embedded tissue blocks.
Because a monoclonal antibody is
Epidemiology used in the DFA-TP test, motility
1. Incidence of the organism is not required for
• worldwide; no seasonal incidence identification.
• true incidence underestimated
• in US, next only to Neisseria and Chlamydia VDRL TEST
• high incidence among blacks and Hispanic population

2. Transmission
• Man is the only natural host
• Sexually transmitted
• Congenital transmission thru infected birth canal
• Transfusion with contaminated blood

3. Who are at risk Reacive

Weakly Reactive
• People with multiple sexual partners
• Newborns of infected mothers
• Patient with AIDS increased risk for neurosyphilis

Clinical Syndromes
A. Primary Syphilis
• Chancre (at site of
inoculation) painless,
indurated ulcer, with
Non-reactive
regional
lymphadenopathy RPR TEST
• Represents local focus for
spirochete multiplication
• Heals spontaneously after
2 months

B. Secondary Syphilis
• Disseminated disease
• Flu-like syndrome (fever,
sorethroat, myalgia
• Generalized mucocutaneous rash
(highly infectious)
• Resolve spontaneously

C. Late Syphilis, Tertiary Non-treponemal Tests

• Measures reagin antibodies developed against lipids
released from damaged cells and also on surface cells
of treponemes
 • Antigen used is cardiolipin – derived from beef heart persist for years and result in hypopigmented lesions and
scarring
VDRL – Venereal Disease Research Laboratory • (Mexico, Central and South America)
RPR – Rapid Plasma Reagin • spread by direct contact with infected lesions
 for suspected neurosyphilis – only VDRL should be used
 can be used to monitor therapy of patients
 specificity = 98%
Treponemal Tests
• Specific antibody tests – to confirm positive VDRL and Diagnosis
RPR  Microscopy
• They are positive even before non-treponemal tests  Darkfield microscopy for Spirochetes
become positive
• Remain positive even when the non-treponemal tests Treatment
are negative  Penicillin
 Tetracyclines
FTA-ABS – Flourescent Treponemal Antibody Absorption  Chloramphenicol
MHA – TP – Microhemagglutination Test for T. pallidum

WESTERN BLOT ASSAY BORRELIA

 they remain generally positive for life
 a negative test is unreliable for AIDS patients  Relapsing fever (Borrelia recurrentis)
 epidemic or louseborne
Medical Conditions Associated with False-positive Treponemal
 endemic or tick born
and Non-treponemal Serology Tests
 Lyme disease – tick borne disease protean
1. Non treponemal Tests manifestations with initial skin lesion erythema migrans
• Viral infection
• Collagen-vascular disease Structure
• Acute or chronic illness • Gram negative bacilli resembling spirochetes
• Pregnancy • Weakly staining, 0.2 – 0.5 X 3 – 30 um
• Recent immunization • Stains well with Aniline dyes (Wright and Giemsa)
• Heroin addiction • With periplasmic flagella
• Leprosy
• Malaria Physiology
• Microaerophilic with complex nutritional requirements
2. Treponemal Tests • Recovery in culture difficult
• Pyoderma • Generation time – 18 hours
• Skin neoplasm
• Acne vulgaris Pathogenesis and Immunity
• Mycoses • Able to spread in the blood stream and the clinical
manifestations of relapsing fever are in part a response
• Crural ulceration
to the release of endotoxins
• Rheumatoid arthritis
• Organism can undergo antigenic variation hence those
• Psoriasis residing in internal tissues can alter their serotype
• Systemic lupus erythematosus specific outer envelop proteins by gene rearrangement
• Pregnancy and emerge as antigenically novel organism
• Drug addiction
• Herpes genitalis

Treatment, Prevention and Control

• Penicillin drug of choice
• alternatives – tetracyclines, erythromycin,
chloramphenicol
• Safe sex techniques
• Adequate treatment of partners with documented
infections

Other Treponemes
1. T. pallidum ssp. Endemicum (bejel) or endemic syphilis
• Initial oral lesions
• Secondary lesions are
oral papules and
mucosal patches
• Late manifestation of
gummas of skin, bones
and nasopharynx Crowded unsanitary conditions required for maintenance of
• (Africa, Asia and disease.
Australia)

2. T. pertenue – Yaws
• Granulomatous disease
with early skin lesions
and late destructive
lesions of the skin,
lymph nodes and bones
• (South America,
Central Africa and SEA)
• Spread by direct
contact with infected skin lesions

3. T. carateum (Pinta)
• Start as small pruritus Epidemiology of Borrelia Infections
papules, enlarge and Clinical Syndromes
1. RELAPSING FEVER
 • 1 week incubation period Physiology
• abrupt onset of shaking cells, fever, myalgia, headache • obligate aerobes, motile (two peripasmic flagella)
• Splenomegaly and hepatomegaly frequent • Utilize alcohol and fatty acids
• corresponds to the bacteremia phase • Grown on enriched media – rabbit or bovine serum
albumin
• Relief after 3-7 days. Recurrence of manifestation after
• Fletchers, EMTH, Tween 80 – albumin 6-16 hrs incubation
1 week, with milder symptoms
• 2-3 relapses, but as many as 13 Pathogenesis and Immunity
• More severe with epidemic than endemic • Subclinical infection
• Mortality less than 5 in endemic; 4-40% in epidemic • Mild flu-like illness
• Severe systemc disease with renal, hepatic failure,
2. LYME DISEASE extensive vasculitis myocarditis
• Incubation period – 3 –30 days
• One or more skin lesion at bite site (erythema migrans
• Malaria, headache fever, chills, myalgia,
Epidemiology
lymphadenopathies ( 4 weeks)
• Dogs, cattle rodents and wild animals are reservoir
• Late manifestations (80% of untreated cases) one week • Transmission thru break in skin after exposure to urine
to 2 years later – 10 – 15% of cases contamination
o Phase 1  wadding in streams, fields, flood waters
 neurologic and cardiac  occupational exposure – farmers, meat-handlers,
o Phase 2 veterinarians
 arthritis and antralgia  worldwide distribution
 more common in warm months
Laboratory Diagnosis
Clinical Syndrome
A. Lyme Disease
• Isolation of Borrelia burgdorferi Stages of Icteric and Anicteric Leptospirosis
• Demonstration of diagnostic levels of IgM or IgG (ELISA,
Western Blot)
 B. burgdorferi is rarely seen in clinical
specimens or blood
 Serologic test should not be performed in the
absence of appropriate history and clinical
disease

B. Borrelia Recurrentis
• Microscopy – Giemsa or Wrights stain of blood during
febrile periods (70%)
o increased sensitivity with mouse inoculation
test
• Serologic test not useful because of antigenic phase
variation

• Organism can be demonstrated in the blood and CSF

Borreiia sp. Indirect immunofluorescent test for
Antibodies to Borrelia burgdorferi.
Prevention and Control
• Relapsing fever; tetracycline (drug of choice except for
pregnant women and young children), chloramphenicol
• Lyme disease; doxycline, amoxicillin, erythromycin for
early manifestations, ceftriaxone, penicillin,
doxycycline, amoxicillin for late manifestations
• Avoid ticks and their natural habitats, wear protective
clothing, and use insect repellants
• Control of rodents (epidemic relapsing fever)
• Delousing sprays and improved hygiene important to
control of epidemic louse-borne disease
early in disease and later in urine
• Clearance of organisms occurs when humoral immunity
develops
• Clinical manifestations may be related to immunologic
reactions with the organisms
 meningitis develop after organism have been
removed from CSF
 immune complexes have been detected in
renal lesions
• 10% mortality with icteric form of generalized disease
• Surviving patients with hepatic and renal damage usually
attains full recovery
• Congenital leptospirosis can also occur
Diagnostic Tests for Leptospirosis

LEPTOSPIRA

• Leptospira interrogans – pathogenic for many wild

domestic animals and humans
• Leptospira biflexa – free living saphrophyte

Structure
• Thin coiled bacilli 0.1 X 6-20 um with a hook at one both Mode of Control
ends • Antimicrobials (PCN, Tetracycline, Doxycyline
• Prophylaxis – Doxycycline
LEPTOSPIRA INTERROGANS
• Vaccination of livestock and pets
 Icterohemorrhagiae, Weil’s disease, pomona,
• Control of rodents
Swineherd’s disease, Autumnalis, Fort Bragg fever or
pre-tibial eruptions
CAMPYLOBACTER

Structure
• Gram negative bacillus, 0.3 – 0.6 um diameter
• Comma shaped, motile with polar flagella
Physiology
• Grows best at 5 – 7% O2, 5 – 10% CO2
• Oxidase and Catalase (+)
Pathogenesis and Immunity
Development of disease is associated with:
• Infectious dose of organisms
• Level of specific immunity of host
 Low gastric acids
 Antibodies (higher among people in endemic
areas – less severe
 Hypogamma globulinemia associated with
prolonged severe infection
• Development of destruction of mucosal
surface crypt abscess and infiltration into the
lamina propria. Enteroxins, cytopathic toxins
and endotoxic activity detected but precise
roles, poorly defined
• Campylobacter fetushas the propensity to go
systemic especially among immuno-
compromised patients. It is covered with S -
protein that prevents complement-mediated
killing
Epidemiology of Campylobacter
 Most common cause of bacterial gastroenteritis in US
 Transmission is associated with contaminated food
(especially poultry), milk and water
 Asymptomatic carriage in animals is an important
reservoid for human disease
 Fecal-oral transmission as wells as person to person is
also possible
 It has world wide distribution but more common in warm
months
Clinical Syndrome
 Acute enteritis with diarrhea, malaria, fever and
abdominal pain, blood stools may be present
 Disease self-limiting
Laboratory Diagnosis
1. Microscopy
 small curved bacilli, pairs resemble wings of sea gull –
cannot be easily seen on gram stain
 Darting motility by darkfield a phase contrast
microscopy
2. Culture
 Microaerophilic selective media containing blood or
charcoal and antibiotic slow growers (48-72 hours
incubation). Identification based on biochemical
reactions
Campylobacter sp.
Mode of Control
 Antibiotics – Erythromycin, Tetracyclines,
Aminoglycoside, Clindamycin
 Proper food prepration, Pasteurization of milk, potable
water
HELICOBACTER PYLORI
 Associated with gastritis, recently implicated in gastric
and duodenal ulcers and gastric cancer
 Sequence analysis of 16S rRNA – hence their
reclassification; previously classified as Campylobacter
Potential Virulence Factors
 urease production (protection from gastric acids
 Motility
 mucinase activity
 adherence factors – anchors bacteria at the
 intracellular junction of enteric cells
 heat-labile cytotoxin, hemolysin, lipopolysaccharide
Epidemiology
 infection low during childhood but increase to 50% in
older adults
 early infections among low socio-economic classes and
developing nations
 identified in 70 – 100% of patients with gastritis
 no animal reservoir identified
 infection via food or water not demonstrated
 ubiquatous and worldwide
 no seasonal incidence
Laboratory Diagnosis
 Detected by histological examination of gastric
biopsis
 Warthin-starry silver stain is most sensitive; also
seen by hemolytic, eosin and gram stain
 Urease test most rapid test, directly on clinical
specimen
 Culture may be done but not very sensitive due to
extraneous factors
 Serology cannot discriminate between past and
current infection. Titers do not correlate with
severity of disease or response to therapy
Control
• Antibiotics combined with bismuth nitroimidazole and
either Amoxycillin or Tetracycline
• Prevention is difficult because organism is ubiquitous
LEGIONELLA PNEUMOPHILA
• ubiquitous aquatic saprophyte, that causes human
respiratory infections
Structure
• 0.3 – 0.9 X 2-5 um long pleomorphic in cultures,
appreciated by Dieterle silver stain in tissues, motile
Physiology
• nutritionally fastidious, growth enhanced
• by iron solutions and L-cysteine
• Non-fermentative, catalase positive,
• liquify gelatin, do not reduce nitrate nor hydrolyze urea
Epidemiology
• worldwide distribution
• organism commonly present in natural bodies of water,
air conditioning power and water systems, survives in
moist environments at relatively high temperature
• most infections occur in late summer and fall
• Elderly and decreased cellular immunity and people
with compromised pulmonary function are at risk
• Transmission by inhalation of aerosols
Clinical Syndromes
Pontiac fever – flu-like illness, self-limited
Legionnaire’s Disease
 2-10 days incubation, with abrupt onset of fever, chills,
dry cough, primary manifestations of pneumonia, with
common multiple organ involvement, mortality of 15 –
20% in compromised patients
Laboratory Diagnosis
Microscopy
• direct flourescent antibody test (DFA) – sensitivity is low
because antibody present are species-specific, organism
is relatively small and predominantly intracellular
Culture – BCYE
• small colonies with ground glass appearance
Antigenic Detection and Serology
• limited usefulness – due to limited sensitivity
Legionella pneumophilia on
buffered charcoal-yeast
extract (BCYE) agar. BCYE
agar is a selective medium
for the recovery of Legionella
spp. This buffered medium is
the agar of choice for the
isolation of Legionella spp.
because it contains the
requirements for optimal
growth of microorganism: L-
cystein, iron salts and a pH
of 6.9. Antibiotics are added to inhibit the growth of other bacteria.
Growth appears in 2 to 3 days and the colonies are circular, glistening, Classification and Clinical Features of Periodontitis
entire and measure up to 4 mm as shown here.

Mode of Control
• Antibiotics - Erythromycin, Rifampicin and
Flouroquinolones
• Hyperchlorination of water supply

SPIRILUM MINOR
• agent of rat-bite fever
• gram negative bacillus, polar flagella
• has not been cultured in vitro
• found in nasopharynx of rats and small rodents
• produce ulcrations at bite site with lymphadenopathy
and recurrent febrile
• episodes in untreated cases
• diagnosed by darkfield examination of blood ulcer
exudates or lymph node aspirates or by intraperitoneal
inoculation of rats

TREATMENT
Penicillin Tetracycline

ORAL MICROBIOLOGY

Most diseases of the oral cavity are the result of bacterial

Microbial Profile for Periodontitis
colonization and infection. The predominant etiologic agents are
colonized microorganisms and their producets à an adherent
mass or the teeth called dental plaque.

Bacterial Plaque

 total bacterial count – 108 – 1011 per gram

 removed from teeth by mechanical means
 factors enhancing rate of colonization
 inadequate oral hygiene
 diet rich in fermentable carbohydrate
 malocclusions and malposed teeth
 reduced oral immune factors
 impaired saliva flow
 rough teeth surfaces
 early plaque development are formed by streptococci
and gram positive bacilli
 plaque that extends underneath the gingiva where there
is less O2 tensin will grow facultative and strict
anaerobic bacteria – mainly bacilli and filamentous
bacteria
 the mixed complex colonies have > 200 sp. of bacteria
 the types of bacterial vary among individuals, with age Laboratory Diagnosis
of dental plaque area of the oral cavity where it was  Made primarily by clinical means
collected  There are no laboratory diagnostic tests specifically for
oral bacteria associated with gingivitis
Clinical Syndromes
1. Dental Caries Treatment, Prevention and Control
• soft, leather like consistency of teeth
1. Dental Caries
• demineralization and decalcification accelerated by
acids hydrolysis of enamel phosphoprotein by  mechanical removal of carious lesion
phosphoprotein phosphatase  PCN, Erythromycin
• oral bacteria penetrate the enamel, mostly streptococci
2. Gingivitis
(streptococcus mutans)
 improved oral hygiene
2. Periodontal Disease
 Gingivitis 3. Periodentitis
 reversible  measures as for control of gingivitis
 plaque is the primary etiologic agent  surgical approaches if necessary
 organisms involved mostly gram + cocci, bacilli  systemic antibiotics in the presence of abscess
and filamentous forms  Palliative therapy for ANUG
 Antiplaque mouth rinse like Chlorhexidine
 Trench mouth or Vincent infection (ANUG – Acute
 Dietary considerations
Necrotizing Ulceration Gingival stomatitis)
 Reduction of acids from fermentable
 very painful, malodorous
carbohydrate
 bacteria involved are spirochetes, vibrios and
filamentous forms
 Periodontitis -fin-
 inflammation extends beyond soft tissues into
the alveolar bone ligament and cementum
with loss of gingival attachment irreversible audsmartinez@gmail.com
dental disease ustmedc3@yahoogroups.com

 generally associated with age of onset and

types of bacteria present in the pockets
surrounding the teeth
.