Bacillus anthracis
 B. anthracis is the causative agent of anthrax, has a world wide distribution. Anthrax is caused by
inhalation, skin exposure, or by gastrointestinal (GI) absorption.
 B. anthracis is a Gram-positive spore forming bacillus.
 It is a very large bacillus measuring 1-2µm in width and 3-5µm in length.
 In smears from infected tissues, the bacteria are found as single, in pairs and in short chains, the entire
chain being surrounded by a capsule.
 In culture, B anthracis grows as long chains and may appear similar to streptobacilli. In these chains, the
bacilli are arranged end-to-end and the end of the bacilli are truncated, not rounded, or often concave
and somewhat swollen. This gives the chain of bacilli a “bamboo-stick” appearance.
 It is a capsulated. The bacterium forms the capsule only when grown on nutrient agar containing 0.7%
sodium bicarbonate in the presence of 5-20% carbon dioxide. The capsule is polypeptide (polymer of D-
glutamic acidd) in nature. It protects the bacteria against leukocytic phagocytosis and lysis.
 It is non motile and nonacid fast.
 Anthrax spores: Anthrax spores are oval, central in position and are refractile.
Virulence Factors:
 Capsule: The capsule protects the organism against the bactericidal components of serum and
phagocytes and against phagocytic engulfment. It plays most important role during the establishment of
the infection.
 Anthrax toxin complex: Anthrax toxin is an exotoxin. It comprises of three components: (a) protective
antigen(PA), (b) edema factor(EF) and (c) lethal factor (LF).
 Protective antigen: It is called PA because antibodies against this antigen are protective against the
action of anthrax toxin. This PA is the binding(B) domain of anthrax toxin and is necessary for entry
of the bacteria into the host cell.
 Edema Factor: It is a component of the edema toxin. It is a type of adenylate cyclase, which acts by
converting adenosine triphosphate (ATTP) to cyclic adenosine monophosphate (cAMP). This causes
an increase in the cellular cAMP levels.
 Lethal Factor: It is a zinc metalloprotease that inactivates mitogen-activated protein kinase, leading
to the inhibition of intracellular signalling.
Pathogenesis of anthrax
 Spore is the infective stage of the bacilli. Lethal dose of inhalation anthrax in human is approximately by
8,000 to 10,000 spores.
 Spores initiate the disease process in the following ways:
 The spores are attached into the entry site and then they are ingested by macrophages at the site of entry.
Inside macrophage, the spores are germinated to form the vegetative forms of the bacteria. Then capsules
are formed and bacteria are released from macrophage. Capsulated bacteria produce anthrax toxins (PA, EF,
LF)
 Phagocytes migrate to the area of infection but the capsulated anthrax bacilli resist phagocytic engulfment;
or in engulfed, resist killing and digestion.
 The anthrax toxin causes further impairment of phagocytic activity.
 The PA of B. anthracis binds to specific receptors on the host cell surface, thereby creating a secondary
binding site for which LF and EF compete and bind, leading to formation of lethal toxin and edema toxin,
respectively. The lethal toxin or edema toxin is internalized (into the cell) by endocytosis. Edema toxin
produces the characteristics edema of anthrax. Subsequently, the bacteria and their toxins enter the
circulation, causing systemic morbidity.
 The bacteria multiply locally and may invade the bloodstream or other organs back into the bloodstream
may result in bacteremia.
Laboratory Diagnosis: It is essential to follow biosafety (Level 11) precautions while handling clinical specimen
suspected for anthrax in a microbiology laboratory.
Specimens
 Vesicular fluid, fluid from under the eschar (in cutaneous anthrax)
 Blood, splenic aspirates (inhalational anthrax)
 Blood, splenic aspirates(inhalational anthrax)
 CSF (in anthrax meningitis)
 Sputum and blood ( in inhalational anthrax)
Microscopy
 The smears are stained with Gram stain, polychrome Methylene blue (McFadyean’s stain) and Giemsa Stain.
 Gran-Stained smear of a skin lesion (vesicular fluid of eschar), CSF, or blood show encapsulated, broad, large,
Gram-positive bacilli. The bacteria are found as single in pairs and in short chains. The bacilli do not show
any spores. By Gram staining, a presumptive identification of anthrax can be done.
Culture
 It is an aerobe and facultative anaerobe. The bacteria grow at a temperature range of 12-450C, optimum
temperature being 370C.
 Nutrient agar: On nutrient agar after 24 hours of incubation, B anthracis produces grayish and granular
colonies measuring 2-3 mm in diameter.
 Blood agar: On horse or sheep blood agar, B anthracis colonies are gray or white, typically nonhemolytic,
with a dry, ground-glass appearance. The colonies are at least 3 mm in diameter and sometimes have
tails.
 Selective medium: Knisely’s polymyxin B-lysozyme-EDT Athallous acetate (PLET) agar medium is a
selective medium used for isolation of B. anthracis from mixtures containing other spore bearing bacilli.
The medium is composed of heart infusion agar, polymyxin, lysosome, ethylene diamine tetra acetic acid
(EDTA), and thallous acetate. Production of capsular material is associated with the formation of a
characteristics mucoid or “smooth”(S) colony type. Capsulated bacteria on serum or bicarbonate medium
produce smooth or mucoid colonies. Smooth variants that form capsule are virulent strains of B anthracis.
Biochemical Reactions
 It produces acid from glucose, maltose, sucrose, trehalose and dextrin, but not from lactose, arabinose,
D-xylose or D-mannitol.
 It reduces nitrate to nitrite.
Treponema pallidum
T. pallidum is the causative agent of syphilis, the most common sexually transmitted disease.
Properties of Bacteria
Morphology
T. pallidum shows the following morphological features:
 It is a thin, coiled spirochete. It measures 0.1µm in breadth and 5-15µm in length.
 It has six to ten sharp and angular coils, which are present at regular interval of 1µm.
 It is actively motile.
 It is too thin to be seen by microscopy in specimens stained by simple Gram or Giemsa staining. It is stained
by silver impregnation method, which makes the bacteria thickened by deposition of silver compounds
during the process of staining.
 Dark ground or phase contrast microscopy is useful for demonstrating the morphology and motility of live T.
pallidum
Virulence Factors
 T. pallidum is a strict human pathogen.
 Outer membrane protein: Outer membrane protein of T. pallidum appears to play an important role in
virulence of the bacteria. It promotes adherence of T. pallidum to the surface of host cells, thereby facilitating
the infections.
 Enzyme hyaluronidase: This enzyme, produced by only pathogenic treponemes, may break hyaluronic acid
of the connective tissue and help to spread the infection.
 Fibronectin: Host cell fibronectin forms a coating on the surface of pathogenic Treponema, thereby
preventing it from phagocytosis by macrophages.
Pathogenesis of syphilis
 On sexual contact T. pallidum from infected partner is passed to another partner through minor skin
abrasions. The organisms invade the skin at these lesions and multiply at the site of infection. Then bacterium
travels via the circulatory system (including the lymphatic system and regional lymph nodes) throughout the
body. Invasion of the central nervous system (CNS) can occur can occur during any stage of syphilis.
 1st step of invasion is attachment bacterium to epithelial, fibroblast like and endothelial cells. Corkscrew
motility of bacterium via periplasmic flagella (flagella not exposed to the surface) transverses (crosswise
direction) junctions between endothelial cells. There bacterium produces metalloproteinase 1 (MMP-1) in
dermal cells which breaks down collagen.
 Bacterium then enters lymphatic and bloodstream and disseminates. Invasion of the central nervous system
occurs in greater than 30-40% of patients with primary and secondary syphilis.
 Some organisms lodge at the entry site, proliferate, sensitive, lymphocytes and activate macrophages,
resulting in the primary lesion or chancre developing at the site of infection.
 The secondary lesion of syphilis generally occur 3-6 weeks (upto 6 months) after the appearance of the
chancre. Primary and secondary stages of syphilis therefore may overlap. Clinically, generalized or localized
rashes can occur along with mucosal lesions. The secondary stage results from multiplication of the bacteria
at multiple organ sites after hematogenous spread.
 Relapses of secondary symptoms can occur usually during the first year of infection
Laboratory Diagnosis
 Collection of specimens
 Specimens for microscopy include serous transudates from moist lesions.
 Serum is used for serodiagnosis and cerebrospinal fluid (CSF) is used for diagnosis of neurosyphills.
Microscopy
 Dark-field microscopy is useful for diagnosis of primary, secondary or congenital syphilis by demonstration
of treponemes in the clinical specimen. Dark field microscopy is particularly helpful for diagnosis early in the
disease before the appearance of serum antibodies. T. pallidum is identified by its slender spiral structure
and slow movement. Dark-field microscopy, although useful has many limitations. The method is of low
sensitivity because as high as 104 bacteria/ml of the exudates need to be present for demonstration by dark
ground microscopy.
Culture
Since T. pallidum cannot be cultured on artificial medium, culture is not used for diagnosis of syphilis.
RICKETTSIA
Rickettsiae are obligate, intracellular, very small (0.3-1-2µm), Gram negative bacilli that multiply within
cytoplasm of eukaryotic cells. They have very small genomic composed of 1-1.5 million base pairs. These
organisms because of their small size, were once thought to be viruses.
Properties of Bacteria
Morphology
 They are small, Gram-negative coccobacilli varying from 0.3-0.6 to 0-8.2µm in size.
 They are nonmotile and noncapsulated.
 They are stained poorly with Gram stain but are stained well with the following: deep red with Gimenez
stain and bluish purple with Giemsa and Castaneda stain.
Other properties
Susceptibility to physical and chemical agents
 Rickettsia are rapidly killed by heating at 560C and also room temperature.
 They are preserved at -700C or in a lyophilized state.
Disease
Spotted fever group or rickettsial disease include:
 Rocky Mountain spotted fever caused by Rickettsia rickettsiae.
 Rickettsial pox caused by R. akar.
 Boutonneuse fever (i.e, Kenya tick-bite fever, African tick typhus, Mediterranean spotted fever, Indian tick
typhus and Marseilles fever) caused by R. conori.
 Rickettsiae of this group possess a common soluble group antigen. They also multiply in the nucleus as
wall as in the cytoplasm of the infected cells. R. rickettsiae is the most common species belonging to the
spotted fever group and is responsible for Rocky Mountain spotted fever.
Pathogenesis and Immunity
 R. rickettsiae multiply within the endothelial cells of the small blood vessels and invade the blood streams.
Subsequently, they cause vasculitis and vascular lessions, which are found in almost all organs but are
commonly found in the skin and in the adrenal glands, liver, heart and CNS.
 The condition progresses to hypoalbunemia, hyponatremia and hypovolemia due to loss of plasma into
the tissues.
Laboratory Diagnosis
 Specimens
 These includes skin biopsy for antigen detection and serum for serodiagnosis.
 Culture
 R. rickettsiae can be isolated in embryonated egg or tissue cultures, but cultures are rarely attempted
because of associated risk of infection.
 They ususlly grow inside the cell, usually in the cytoplasm (most Rickettsia) or in the nucleus of the cell
(Rickettsia causing spotted fever). They grow well at optimum temperature of 32-350C. They grow in
various cell lines, in the developing chick embryo, and also in many laboratory animals.
 Cell Lines: They grow on HeLa, Hep2, Detroit-6, mouse fibroblasts and other continuous cells lines.
Cultures in the cell lines are used primarily for the maintenance of Ricekttsia but are not useful for
primary isolation of Rickettsia from clinical specimens.
 Chick embryo: In the developing chick embryo 5-6 days old , Rickettsia spp, grow well in the yolk sac.
The inoculated eggs are incubated at 350C for most Rickettsia spp. and at 330C for spotted group. The
yolk sac is widely used as a source of Rickettsia for preparation of rickettsial antigens and vaccines.
Rickettsia shows poor growth on chorioallantoic membrane.
 Laboratory animals: Guinea pigs and mice are the commonly used laboratory animals for isolation of
Rickettsia organisms from animal specimens.
MYCOPLASMA
Mycoplasma belongs to class Mollicutes (Mollis, soft; cutis, skin), order Mycoplasmatales. This order contains
four families. Most mycoplasma causing human infections belong to the family Mycoplasmataceae.
Morphology
 Mycoplasmas are very small bacteria measuring 150-250 mm in dimension. They do not have a cell wall and
typically their cell membranes contain sterols.
 Many mycoplasma can pass through 0.45µm filter, hence were once believed to be viruses.
 Mycoplasma species was also considered to be L form of bacteria they lack a wall.
 They multiply by binary fission.
 They donot possess flagella or pili, but some Mycoplasma species including M. pneumonia show gliding
motility on liquid-covered surfaces.
Virulence Factors
 P1 Antiegn: P1 antigen is a membrane associated protein, which helps in adhesion mycoplasmas to
epithelial cells. This protein or adhesion combines specifically with sialated glycoprotein receptors present
at the base if the cilia on epithelial surface. This sampe receptor is also present on the surface of the
erythrocytes. The P1 protein induces production of antibodies which not only react with P1 protein but
also react with antigenic determinants of RBCs, leading to lysis of erythrocytes in autoimmune disease
process.
Pathogenesis of Mycoplasma infections
 Mycoplasmas are primarily extracellular pathogens that adhere to surface of ciliated and nonciliated
epithelial cells.
 Following attachment, Mycoplasma causes direct damage to the epithelial cells in which first cilia and
then ciliated epithelial cells are destroyed.
 Loss of the cells interferes with normal functioning of the upper respiratory tract. This results in the lower
respiratory tract to become infected with microbes and mechanically irritated.
 The mechanical irritation causes persistent cough typically seen in patients with respiratory infection
caused by M. pneumoniae.
 M. pneumoniae behaves as a super antigen. This causes migration of inflammatory cells to the site of
infection and produces cytokines, such as tumor necrosis factor-alpha, interleukin-1, and interleukin-6.
Those cytokines help in clearing of the bacteria and of the disease.
 The bacteria usually do not cause invasion of the blood to produce systemic manifestation of the disease.
 Mycoplasma rarely penetrates the submucosa except in rare cases of immunosuppression or following
instrumentation. In these conditions, they may invade the blood stream and cause infection in different
organs of the body.
Laboratory Diagnosis: Initial treatment of M. pneumoniae infection is based on clinical diagnosis of the
condition. The definite diagnosis of the condition usually takes 3-4 weeks. Hence treatment is started without
waiting for the result of laboratory tests.
Specimens: Respiratory specimens include throat washings, bronchial washings, and expectorated sputum.
Tracheal washings are most useful than sputum specimens, because most patients with respiratory tract
infections do not produce any sputum as they have a dry and non-productive cough.
Microscopy: Microscopy is of no value in diagnosis of M. pneumoniae infections. Mycoplasmas are stained
poorly, because they lack cell wall.
Culture
 Mycoplasmas are aerobic and facultative anaerobes. M. pneumoniae is an exception which is a strict aerobe.
They grow at 370C and at pH range of 7.3-7.8.
 PPLO broth/PPLO agar: Pleuropneumonia-like organism (PPLO) broth is a medium widely used for isolation
of mycoplasma. This medium is supplemented with 20% horse serum, 10% yeast extraction and glucose.
Phenol red is used as a pH indicator. The high concentration of animal serum (horse serum) is used as a
source of exogenous sterols ( cholesterol and other lipids). Addition of agar makes the medium solid. The
medium is supplemented with penicillin, ampicillin, and polymyxin B to inhibit growth of pneumoniae does
not produce fried egg appearance colonies but instead produces a colony known as mulberryshaped
colonies. These colonies do not show any thin hallow unlike that of fried-egg appearance colonies. Colonies
may be seen with a hand lens, which is the best studied after staining by Diene’s method.
Biochemical Reactions: Mycoplasmas show the following biochemical reactions:
 M. pneumonia and other species (M. fermentans, M. genitalia and M. agalactiae) utilize glucose and other
carbohydrates as the major source of energy.
 M. salivarium and other species (M. orale, M. hominis, and M. fermentans) utilize agiginine as a major source
of energy.
 Mycoplasma are chemo-organotrophs, the metabolism being mainly fermentative.