critical. Immunologically activated monocytes and macrophages restrict intracellular bacterial growth. The role

of humoral immunity is unclear.

Epidemiology

Legionella species are widespread in nature. Interactions with other environmental organisms may facilitate

growth. Disease may be sporadic or epidemic and may occur in the community or in hospitals. People with

compromised host defenses are at increased risk.

The clinical manifestations of Legionella infections are primarily respiratory. The most common presentation

is acute pneumonia, which varies in severity from mild illness that does not require hospitalization (walking

pneumonia) to fatal multilobar pneumonia.

Typically, patients have high, unremitting fever and cough but do not produce much sputum. Extra

pulmonary symptoms, such as headache, confusion, muscle aches, and gastrointestinal disturbances, are

common. Most patients respond promptly to appropriate antimicrobial therapy, but convalescence is often

prolonged (lasting many weeks or even months).

Laboratory Diagnosis

There are no reliable distinguishing clinical features of Legionella pneumonia, so the diagnosis must come from

the laboratory.

Some clinical features suggest legionnaire's disease; however, and should prompt the selection of appropriate

laboratory tests. The diagnosis is confirmed in the laboratory by culture, demonstration of bacterial antigen in

body fluids, or a serologic response.

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The preferred diagnostic method is culturing, because it is both sensitive and specific; however, appropriate

specimens are not always available.

The laboratory must be alerted to the possibility of legionellosis, because specially designed media must be

used. The medium of choice is buffered charcoal-yeast extract - α-ketoglutarate medium.

This medium contains yeast extract, iron, L-cysteine, and α-ketoglutarate for bacterial growth; activated

charcoal to inactivate toxic peroxides that develop in the media; and buffer with a pH 6.9, the optimum for

growth of Legionella organisms.

Addition of albumin to the media may further facilitate growth of species other than L. pneumophila. For

contaminated specimens such as sputum, antibiotics should be added. Morphologically distinctive bacterial

colonies can usually be detected within 3 to 5 days and identified presumptively as Legionella species if the

isolated bacteria depend on cysteine for growth. The identification can be confirmed by specific immunologic

typing of the isolated bacteria or, in problematic cases, by molecular analysis.

Direct detection of bacterial antigen in clinical specimens is potentially much faster than culturing.

Unfortunately, direct immunofluorescence detection (DFA) of Legionella antigen in respiratory specimens is

neither sensitive nor specific enough to warrant general use. A commercially available radioimmunoassay for

bacterial antigen in urine is satisfactory, but is available only for serogroup 1 of L. pneumophila.

Serologic diagnosis is moderately sensitive and reasonably specific. It should be considered as an adjunct to

diagnosis by culture. Indirect immunofluorescence has been used most frequently.

It is important to use an assay that detects IgM and IgG. The advantages of serologic diagnosis are that it is

performed on easily obtained blood specimens and can detect mild or even asymptomatic infection.

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Lecture Ten

Bordetella

Bordetella pertussis and Bordetella parapertussis is the human pathogens of this genus. The former causes the disease

pertussis (also known as whooping cough), and the latter causes a mild pertussis-like illness. Whooping cough is a highly

contagious disease and a significant cause of morbidity and mortality worldwide (51 million cases and 600,000 deaths

each year). Members of the genus Bordetella are aerobic. They are small, encapsulated coccobacilli that grow singly or

in pairs. They can be serotyped on the basis of cell-surface molecules including adhesins and fimbriae.

Epidemiology

The major mode of transmission of Bordetella is via droplets spread by coughing, but the organism survives only briefly

outside the human respiratory tract. The incidence of whooping cough among different age groups can vary substantially,

depending on whether active immunization of young children is widespread in the community. In the absence of an

immunization program, disease is most common among young children (ages 1 to 5 years). Adolescent and adult

household members, whose pertussis immunity has disappeared, are an important reservoir of pertussis for young

children.

Pathogenesis

B. pertussis binds to ciliated epithelium in the upper respiratory tract. There, the bacteria produce a variety of toxins and

other virulence factors that interfere with ciliary activity, eventually causing death of these cells.

Clinical significance

The incubation period for pertussis generally ranges from 1 to 3 weeks. The disease can be divided into two phases:

catarrhal and paroxysmal.

1. Catarrhal phase: This phase begins with relatively nonspecific symptoms, such as rhinorrhea, mild conjunctival

infection (hyperemia, or bloodshot conjunctivae), malaise, and/or mild fever, and then progresses to include a dry,

nonproductive cough. Patients in this phase of disease are highly contagious.

2. Paroxysmal phase: With worsening of the cough, the paroxysmal phase begins. The term “whooping cough” derives

from the paroxysms of coughing followed by a “whoop” as the patient inspires rapidly. Large amounts of mucus may be

produced. Paroxysms may cause cyanosis and/or end with vomiting. Pertussis typically causes leukocytosis that can be

quite striking as the total white blood cell count sometimes exceeds 50,000 cells/μl (normal range = 4,500–11,000 white

blood cells/μl), with a striking predominance of lymphocytes. Following the paroxysmal phase, convalescence requires at

least an additional 3 to 4 weeks. During this period, secondary complications, such as infections (for example, otitis

media and pneumonia) and central nervous system (CNS) dysfunction (for example, encephalopathy or seizures), may

occur. Disease is generally most severe in infants.

Laboratory identification

Presumptive diagnosis may be made on clinical grounds once the paroxysmal phase of classic pertussis begins. Pertussis

may be suspected in an individual who has onset of catarrhal symptoms within 1 to 3 weeks of exposure to a diagnosed

case of pertussis.

Culture of B. pertussis on Bordet-Gengou or Regan-Lowe media (selective and enrichment media) from the nasopharynx

of a symptomatic patient supports the diagnosis. The organism produces pin- point colonies in 3 to 6 days on selective

agar medium (for example, one that contains blood and charcoal), which serves to absorb and/or neutralize inhibitory

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substances and is supplemented with antibiotics to inhibit growth of normal flora. More rapid diagnosis may be

accomplished using a direct fluorescent antibody test to detect B. pertussis in smears of nasopharyngeal specimens.

Serologic tests for antibodies to B. pertussis are primarily useful for epidemiologic surveys.

Treatment

Erythromycin is the drug of choice for infections with B. pertussis, both as chemotherapy (where it reduces both the

duration and severity of disease) and as chemoprophylaxis for household contacts. For erythromycin treatment failures,

trimethoprim-sulfamethoxazole is an alternative choice. Patients are most contagious during the catarrhal stage and

during the first 2 weeks after onset of coughing. Treatment of the infected individuals during this period limits the spread

of infection among household contacts.

Prevention

Pertussis vaccine is available and has had a significant effect on lowering the incidence of whooping cough. It contains

proteins purified from B. pertussis and is formulated in combination with diphtheria and tetanus toxoids. To protect

infants who are at greatest risk of life-threatening B. pertussis disease, immunization is generally initiated when the

infant is 2 months old. Until the middle of the first decade of the 21st century. However, because neither disease- nor

vaccine-induced immunity is durable, there has been resurgence, with reported cases in 2010 the highest since the

1950’s. A new vaccine, licensed for adolescents and adults, and vaccination of women even during the last trimester of

pregnancy.

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Lecture 11

(Part-one)

Anaerobic Bacteriology

Infections caused by anaerobic bacteria are common. The infections are often polymicrobial; that is, the anaerobic

bacteria are found in mixed infections with other anaerobes, facultative anaerobes

Anaerobic bacteria are found throughout the human body; on the skin, on mucosal surfaces, and in high concentrations

in the mouth and gastrointestinal tract, as part of the normal microbiota

Infection results when anaerobes and other bacteria of the normal microbiota contaminate normally sterile body sites.

Several important diseases are caused by anaerobic Clostridium species from the environment or from normal flora:

botulism, tetanus, gas gangrene, food poisoning, and

Pseudomembranous colitis

Aerobic bacteria: Bacteria that require oxygen as a terminal electron acceptor and will not grow under anaerobic

conditions (i.e., in the absence of O2). Some Bacillus species and Mycobacterium tuberculosis are obligate aerobes (i.e.,

they must have oxygen to survive).

Anaerobic bacteria: Bacteria that do not use oxygen for growth and metabolism but obtain their energy from

fermentation reactions. A functional definition of anaerobes is that they require reduced oxygen tension for growth and

fail to grow on the surface of

solid medium in 10% CO2 in ambient air. Bacteroides and Clostridium species are examples of anaerobes.

Facultative anaerobes: Bacteria that can grow either oxidative, using oxygen as a terminal electron acceptor, or

anaerobically, using fermentation reactions to obtain energy. Such bacteria are common pathogens. Streptococcus

species and the Enterobacteriaceae (e.g., Escherichia coli) are among the many facultative anaerobes that cause disease.

Often, bacteria that are facultative anaerobes are called “aerobes.”

Anaerobic bacteria do not grow in the presence of oxygen and are killed by oxygen or toxic oxygen radicals.

Aerobes and facultative anaerobes often have the metabolic systems listed below, but anaerobic bacteria

frequently do not.

1. Cytochrome systems for the metabolism of O2

2. Superoxide dismutase (SOD), which catalyzes the following reaction:

O2−

+O2−

+ 2H+ → H2O2 +O2

3. Catalase, which catalyzes the following reaction:

2H2O2 → 2H2O + O2 (gas bubbles)

Anaerobic bacteria do not have cytochrome systems for oxygen metabolism. Less fastidious anaerobes may

have low levels of SOD and may or may not have catalase. Most bacteria of the Bacteroides fragilis group have

small amounts of both catalase and SOD. There appear to be multiple mechanisms for oxygen toxicity.

Presumably, when anaerobes have SOD or catalase (or both), they are able to negate the toxic effects of oxygen

radicals and hydrogen peroxide and thus tolerate oxygen. Obligate anaerobes usually lack SOD and catalase

and are susceptible to the lethal effects of oxygen; such strict obligate anaerobes are infrequently isolated from

human infections, and most anaerobic infections of humans are caused by “moderately obligate anaerobes.”

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Facultative anaerobes grow as well or better under anaerobic conditions than they do under aerobic

conditions. Bacteria that are facultative anaerobes are often termed aerobes. When a facultative anaerobe such

as E coli is present at the site of an infection (e.g., abdominal abscess), it can rapidly consume all available

oxygen and change to anaerobic metabolism, producing an anaerobic environment, and thus allow the

anaerobic bacteria that are present to grow and produce disease.

Gram-Negative Anaerobes

A. Gram-Negative Bacilli

1. Bacteroides: The Bacteroides species are very important anaerobes that cause human infection. They are a

large group of bile-resistant, non–spore-forming, slender gram negative rods that may appear as coccobacilli.

Many species previously included in the genus Bacteroides have been reclassified into the genus Prevotella or

the genus Porphyromonas. Those species retained in the Bacteroides genus are members of the B fragilis

group. Bacteroides species are normal inhabitants of the bowel and other sites. Normal stools contain 1011 B

fragilis organisms per gram (compared with 108

/g for facultative anaerobes).

Other commonly isolated members of the B fragilis group include Bacteroides ovatus, Bacteroides distasonis,

Bacteroides vulgatus, and Bacteroides thetaiotaomicron. Bacteroides species are most often implicated in intraabdominal

infections, usually under circumstances of disruption of the intestinal wall as occurs in perforations related to

surgery or trauma, acute appendicitis, and diverticulitis.

These infections are often polymicrobial. Both B fragilis and B thetaiotaomicron

are implicated in serious intrapelvic infections such as pelvic inflammatory disease and ovarian abscesses.

B fragilis group species are the most common species recovered in some series of anaerobic bacteremia, and

these organisms are associated with a very high mortality rate. B fragilis is capable of elaborating numerous

virulence factors, which contribute to its pathogenicity and mortality in the host.

2. Prevotella—Prevotella species are gram-negative bacilli and may appear as slender rods or coccobacilli.

Most commonly isolated are P melaninogenica, Prevotella bivia, and Prevotella disiens. P melaninogenica and

similar species are

found in infections associated with the upper respiratory tract. P bivia and P disiens occur in the female genital

tract.

Prevotella species are found in brain and lung abscesses, in empyema, and in pelvic inflammatory disease and

tubo-ovarian abscesses. In these infections, the Prevotella are often associated with other anaerobic organisms

that are part of the normal microbiota; particularly Peptostreptococcus, anaerobic Gram-positive rods, and

Fusobacterium species as well as Gram-positive and Gram-negative facultative anaerobes that are part of the

normal microbiota.

3. Porphyromonas: The Porphyromonas species also are Gram-negative bacilli that are part of the normal oral

microbiota and occur at other anatomic sites as well. Porphyromonas species can be cultured from gingival and

periapical tooth infections and, more commonly, breast, axillary, perianal, and male genital infections.

4. Fusobacteria: There are approximately 13 Fusobacterium species, but most human infections are caused by

Fusobacterium necrophorum and Fusobacterium nucleatum. Both species differ in morphology and habitat as

well as the range of associated infections. F necrophorum is a very pleomorphic; long rod with round ends and

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tends to make bizarre forms. It is not a component of the healthy oral cavity. F necrophorum is quite virulent,

causing severe infections of the head and neck.

Bacterial Vaginosis

Bacterial vaginosis is a common vaginal condition of women of reproductive age. It is associated with

premature rupture of membranes and preterm labor and birth. Bacterial vaginosis has a complex microbiology;

one organism, Gardnerella vaginalis, has been most specifically associated with the disease process.

G vaginalis is a serologically distinct organism isolated from the normal female genitourinary tract and also

associated with vaginosis, so named because inflammatory cells are not present. In wet smears, this

“nonspecific” vaginitis, or bacterial vaginosis, yields “clue cells,” which are vaginal epithelial cells covered

with many Gram-variable bacilli (pleomorphic), and there is an absence of other common causes of vaginitis

such as trichomonads or yeasts. Vaginal discharge often has a distinct “fishy” odor and contains many

anaerobes in addition to G vaginalis. The pH of the vaginal secretions is greater than 4.5 (normal pH is <4.5).

The vaginosis attributed to this organism is suppressed by metronidazole, suggesting an association with

anaerobes. Oral metronidazole is generally curative.

Gram-Positive Anaerobes

A. Gram-Positive Bacilli

1. Actinomyces: The Actinomyces group includes several species that cause actinomycosis, of which

Actinomyces israelii and Actinomyces gerencseriae are the ones most commonly encountered. Several new,

recently described species that are not associated with actinomycosis have been associated with infections of

the groin, urogenital area, breast, and axilla and postoperative infections of the mandible, eye, and head and

neck.

On Gram stain, they vary considerably in length; they may be short and club shaped or long, thin, beaded

filaments. They may be branched or unbranched. Because they often grow slowly, prolonged incubation of the

culture may be necessary before laboratory confirmation of the clinical diagnosis of actinomycosis can be

made. Some strains produce colonies on agar that resemble molar teeth.

Figure shows colony of Actinomyces

Species after 72 hours growth on brain–heart infusion agar, which

usually yields colonies about 2 mm in diameter; they are often termed

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“Molar tooth” colonies

Some Actinomyces species are oxygen tolerant (aerotolerant) and grow in the presence of air; these strains may

be confused with Corynebacterium species. Actinomycosis is a chronic suppurative and granulomatous

infection that produces pyogenic lesions with interconnecting sinus tracts that contain granules composed of

microcolonies of the bacteria embedded in tissue elements.

Figure shows granule of Actinomyces species in tissue with Brown and

Breen stain. Filaments of the branching

bacilli are visible at the periphery of the granule. Such granules

are commonly called “sulfur granules” because of their unstained

gross yellow color

Infection is initiated by trauma that introduces these endogenous bacteria into the mucosa. The organisms grow

in an anaerobic niche, induce a mixed inflammatory response, and spread with the formation of sinuses, which

contain the granules and may drain to the surface.

The infection causes swelling and may spread to neighboring organs, including the bones. Based on the site of

involvement, the three common forms are cervicofacial, thoracic, and abdominal actinomycosis. Cervicofacial

disease presents as a swollen, erythematosus process in the jaw area (known as “lumpy jaw”). With

progression, the mass becomes fluctuant, producing draining fistulas. The disease will extend to contiguous

tissue, bone, and lymph nodes of the head and neck. The symptoms of thoracic actinomycosis resemble those of

a subacute pulmonary infection and include a mild fever, cough, and purulent sputum. Eventually, lung tissue is

destroyed, sinus tracts may erupt through to the chest wall, and invasion of the ribs may occur.

Abdominal actinomycosis often follows a ruptured appendix or an ulcer. In the peritoneal cavity, the pathology

is the same, but any of several organs may be involved. Genital actinomycosis is a rare occurrence in women

that results from colonization of an intrauterine device with subsequent invasion.

Diagnosis can be made by examining pus from draining sinuses, sputum, or specimens of tissue for the

presence of sulfur granules. The granules are hard, lobulated, and composed of tissue and bacterial filaments,

which are club shaped at the periphery. Specimens should be cultured anaerobically on appropriate media.

Treatment requires prolonged administration of penicillin (6-12 months). Clindamycin or erythromycin is

effective in penicillin-allergic patients. Surgical excision and drainage may be required.

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2. Propionibacterium species are members of the normal microbiota of the skin, oral cavity, large intestine,

conjunctiva, and external ear canal. Their metabolic products include propionic acid, from which the genus

name derives. On Gram stain, they are highly pleomorphic; showing curved, clubbed, or pointed ends; long

forms with beaded uneven staining; and occasionally coccoid or spherical forms. Propionibacterium acnes,

often considered an opportunistic pathogen, causes the disease acne vulgaris and is associated with a variety of

inflammatory conditions.

It causes acne by producing lipases that split free fatty acids off from skin lipids. These fatty acids can produce

tissue inflammation that contributes to acne formation.

P acnes is frequently a cause of postsurgical wound infections, particularly those that involve insertion of

devices, such as prosthetic joint infections, particularly of the shoulder, central nervous system shunt infections,

osteomyelitis, endocarditis,

and endophthalmitis.

3. Clostridia

(In lecture of spore-forming gram-positive Bacilli: Bacillus and Clostridium Species)

B. Gram-Positive Cocci

The group of anaerobic gram-positive cocci has undergone significant taxonomic expansion. Many species

within the genus Peptostreptococcus have been reassigned to new genera such as Anaerococcus, Finegoldia,

and Peptoniphilus. The species contained within these genera, as well as Peptococcus niger, are important

members or the normal microbiota of the skin, oral cavity, upper respiratory tract, gastrointestinal tract, and

female genitourinary system. The members of this group are opportunistic pathogens and are most frequently

found in mixed infections particularly from specimens that have not been carefully procured. However, these

organisms have been associated with serious infections such as brain abscesses, pleuropulmonary infections,

necrotizing fasciitis, and other deep skin and soft tissue infections, intra-abdominal infections, and infections of

the female genital tract.

The polymicrobial anaerobic infections

Most anaerobic infections are associated with contamination of tissue by normal microbiota of the mucosa of

the mouth, pharynx, gastrointestinal tract, or genital tract. Typically, multiple species (five or six species or

more when standard culture conditions are used) are found, including both anaerobes and facultative anaerobes.

Oropharyngeal, pleuropulmonary, abdominal, and female pelvic infections associated with contamination by

normal mucosal microbiota have a relatively equal distribution of anaerobes and facultative anaerobes as

causative agents; about 25% have anaerobes alone, about 25% have facultative anaerobes alone, and about

50% have both anaerobes and facultative anaerobes. Aerobic bacteria may also be present, but obligate aerobes

are much less common than anaerobes and facultative anaerobes.

Diagnosis of anaerobic infections

Clinical signs suggesting possible infection with anaerobes include the following:

1. Foul-smelling discharge (caused by short-chain fatty-acid products of anaerobic metabolism)

2. Infection in proximity to a mucosal surface (anaerobes are part of the normal microbiota)

3. Gas in tissues (production of CO2 and H2)

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4. Negative aerobic culture results

Diagnosis of anaerobic infection is made by anaerobic culture of properly obtained and transported specimens.

Anaerobes grow most readily on complex media such as trypticase soy agar base, blood agar, brucella agar,

brain–heart infusion agar, and each highly supplemented (e.g., with hemin, vitamin K1, blood). A selective

complex medium containing kanamycin is used in parallel. Kanamycin (similar to all aminoglycosides) does

not inhibit the growth of obligate anaerobes; thus, it permits them to proliferate without being overshadowed by

rapidly growing facultative anaerobes. Cultures are incubated at 35-37°C in an anaerobic atmosphere

containing CO2. Colony morphology, pigmentation, and fluorescence are helpful in identifying anaerobes.

Biochemical activities and production of short-chain fatty acids as measured by gas liquid chromatography are

used for laboratory confirmation.

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Lecture 11

(Part –Two)

Spore-forming gram-positive Bacilli: Bacillus and Clostridium Species

These bacilli are ubiquitous, and because they form spores, they can survive in the environment for many years.

Bacillus species are aerobes and the Clostridium species are anaerobes.

Figure shows the vegetative cells with spores

Many species of Bacillus and related genera, most do not cause disease and are not well characterized. There

are a few species, however, that cause important diseases in humans. Anthrax, a classical disease in the history

of microbiology, is caused by Bacillus anthracis.

Anthrax remains an important disease of animals and occasionally of humans. Because of its potent toxins, B

anthracis is a major potential agent of bioterrorism and biologic warfare.

Bacillus cereus and Bacillus thuringiensis cause food poisoning and occasionally eye or other localized

infections.

The genus Clostridium is extremely heterogeneous and more than 200 species have been described.

Clostridia cause several important toxin mediated diseases, including tetanus (Clostridium tetani), botulism

(Clostridium botulinum), and gas gangrene (Clostridium perfringens), and antibiotic-associated diarrhea and

pseudomembranous colitis (Clostridium difficile)

Bacillus species

The genus Bacillus includes large aerobic, gram-positive rods occurring in chains. The members of this genus

are closely related but differ both phenotypically and in terms of pathogenesis.

Pathogenic species possess virulence plasmids. Most members of this genus are saprophytic organisms

prevalent in soil, water, and air, and on vegetation (e.g., Bacillus subtilis).

Some are insect pathogens, such as B thuringiensis. This organism is also capable of causing disease in

humans. B cereus can grow in foods and cause food poisoning by producing either an enterotoxin (diarrhea) or

an emetic toxin (vomiting). Both B cereus and B thuringiensis may occasionally produce disease in

8

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immunocompromised humans (e.g., meningitis, endocarditis, endophthalmitis, conjunctivitis, or acute

gastroenteritis). B anthracis, which causes anthrax, is the principal pathogen of the genus.

The typical cells, measuring 3–4 μm, have square ends and are arranged in long chains; spores are located in

the center of the bacilli

Colonies of B anthracis are round and have a “cut glass” appearance in transmitted light. Hemolysis is

uncommon with B anthracis but common with B cereus and the saprophytic bacilli. Gelatin is liquefied, and

growth in gelatin stabs resembles an inverted fir tree.

Anthrax is primarily a disease of herbivores—goats, sheep, cattle, horses, and so on; other animals (e.g., rats)

are relatively resistant to the infection.

Humans become infected incidentally by contact with infected animals or their products. In animals, the portal

of entry is the mouth and the gastrointestinal tract. Spores from contaminated soil find easy access when

ingested with spiny or irritating vegetation. In humans, the infection is usually acquired by the entry of spores

through injured skin (cutaneous anthrax) or rarely the mucous membranes (gastrointestinal anthrax) or by

inhalation of spores into the lung (inhalation anthrax). A fourth category of the disease, injection anthrax, has

caused outbreaks among persons who inject heroin that has been contaminated with anthrax spores. The spores

germinate in the tissue at the site of entry, and growth of the vegetative organisms results in formation of a

gelatinous edema and congestion. Bacilli spread via lymphatics to the bloodstream, and they multiply freely in

the blood and tissues shortly before and after the animal’s death.

Anthrax toxins are made up of three proteins, protective antigen (PA), edema factor (EF), and lethal factor

(LF). PA is a protein that binds to specific cell receptors, and after proteolytic activation, it forms a membrane

channel that mediates entry of EF and LF into the cell. EF is an adenylate cyclase; with PA, it forms a toxin

known as edema toxin. Edema toxin is responsible for cell and tissue edema. LF plus PA form lethal toxin,

which is a major virulence factor and cause of death in infected animals and humans.

When injected into laboratory animals (e.g., rats), the lethal toxin can quickly kill the animals by impairing both

innate and adaptive immunity, allowing organism proliferation and cell death.

In inhalation anthrax (woolsorters’ disease), the spores from the dust of wool, hair, or hides are inhaled;

phagocytosed in the lungs; and transported by the lymphatic drainage to the mediastinal lymph nodes, where

germination occurs. This is followed by toxin production and the development of hemorrhagic mediastinitis and

sepsis.

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Figure shows Bacillus anthracis in broth culture

In humans, approximately 95% of cases are cutaneous anthrax, and 5% are inhalation. Gastrointestinal anthrax

is very rare; it has been reported from Africa, Asia, and the United States when people have eaten meat from

infected animals. The bioterrorism events in the fall of 2001 resulted in 22 cases of anthrax 11 inhalation and

11 cutaneous. Five of the patients with inhalation anthrax died.

Cutaneous anthrax generally occurs on exposed surfaces of the arms or hands followed in frequency by the face

and neck. A pruritic papule develops 1–7 days after entry of the organisms or spores through a scratch. Initially,

it resembles an insect bite. The papule rapidly changes into a vesicle or small ring of vesicles that coalesce, and

a necrotic ulcer develops. The lesions typically are 1–3 cm in diameter and have a characteristic central black

eschar. Marked edema occurs. Lymphangitis, lymphadenopathy, and systemic signs and symptoms of fever,

malaise, and headache may occur.

After 7–10 days, the eschar is fully developed. Eventually, it dries, loosens, and separates; healing is by

granulation and leaves a scar. It may take many weeks for the lesion to heal and the edema to subside.

Antibiotic therapy does not appear to change the natural progression of the disease but prevents dissemination.

In as many as 20% of patients, cutaneous anthrax can lead to sepsis, the consequences of systemic infection

including meningitis and death.

The incubation period in inhalation anthrax may be as long as 6 weeks. The early clinical manifestations are

associated with marked hemorrhagic necrosis and edema of the mediastinum. Substernal pain may be

prominent, and there is pronounced mediastinal widening visible on chest radiographs. Hemorrhagic pleural

effusions follow involvement of the pleura; cough is secondary to the effects on the trachea.

Sepsis occurs, and there may be hematogenous spread to the gastrointestinal tract, causing bowel ulceration, or

to the meninges, causing hemorrhagic meningitis. The fatality rate in inhalation anthrax is high in the setting of

known exposure; it is higher when the diagnosis is not initially suspected.

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Animals acquire anthrax through ingestion of spores and spread of the organisms from the intestinal tract. This

is rare in humans, and gastrointestinal anthrax is extremely uncommon. Abdominal pain, vomiting, and bloody

diarrhea are clinical signs.

Injection anthrax is characterized by extensive, painless, subcutaneous edema and the notable absence of the

eschar characteristic of cutaneous anthrax. Patients may progress to hemodynamic instability due to septicemia.

Laboratory Diagnosis

Specimens to be examined are fluid or pus from a local lesion, blood, pleural fluid, and cerebrospinal fluid in

inhalational anthrax associated with sepsis and stool or other intestinal contents in the case of gastrointestinal

anthrax. Stained smears from the local lesion or of blood from dead animals often show chains of large grampositive rods. Anthrax can be identified in dried smears by immunofluorescence staining techniques.

When grown on blood agar plates, the organisms produce nonhemolytic gray to white, tenacious colonies with

a rough texture and a ground-glass appearance. Comma-shaped outgrowths (Medusa head, “curled hair”) may

project from the colony. Demonstration of capsule requires growth on bicarbonate- containing medium in 5–7%

carbon dioxide. Gram stain shows large gram-positive rods. Carbohydrate fermentation is not useful. In

semisolid medium, anthrax bacilli are always nonmotile, but related organisms (e.g., B cereus) exhibit motility

by “swarming.”

Definitive identification requires lysis by a specific anthrax γ-bacteriophage, detection of the capsule by

fluorescent antibody, or identification of toxin genes by polymerase chain reaction (PCR). These tests are

available in most public health laboratories. A rapid enzyme-linked immunoassay (ELISA) that measures total

antibody to PA, but the test result is not positive early in disease.

Bacillus cereus

Food poisoning caused by B cereus has two distinct forms;

1. The emetic type; which is associated with fried rice, milk, and pasta

2. The diarrheal type; which is associated with meat dishes and sauces

B cereus produces toxins that cause disease that is more of intoxication than a food-borne infection. The emetic

form is manifested by nausea, vomiting, abdominal cramps, and occasionally diarrhea and is self-limiting, with

recovery occurring within 24 hours. It begins 1-5 hours after ingestion of a plasmid-encoded preformed cyclic

peptide (emetic toxin) in the contaminated food products.

B cereus is a soil organism that commonly contaminates rice. When large amounts of rice are cooked and

allowed to cool slowly, the B cereus spores germinate, and the vegetative cells produce the toxin during logphase growth or during sporulation. The diarrheal form has an incubation period of 1-24 hours and is

manifested by profuse diarrhea with abdominal pain and cramps; fever and vomiting are uncommon. In this

syndrome, ingested spores that develop into vegetative cells of B cereus secrete one of three possible

enterotoxin which induce fluid accumulation in the small intestine.

The presence of B cereus in a patient’s stool is not sufficient to make a diagnosis because the bacteria may

be present in normal stool specimens; a concentration of 105

bacteria or more / gram of food is considered

diagnostic.

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Clostridia species

The Clostridia are large anaerobic, gram-positive, motile rods. Many decompose proteins or form toxins, and

some do both.

Their natural habitat is the soil, marine sediments, sewage, or the intestinal tract of animals and humans,

where they live as saprophytes. Among the pathogens are the organisms causing botulism, tetanus, gas

gangrene, and pseudomembranous colitis.

Clostridia are usually wider than the diameter of the rods in which they are formed; the spore is placed

centrally, subterminally, or terminally.

Most species of Clostridia are motile and possess peritrichous flagella. They are anaerobes and grow under

anaerobic conditions ((grow well on the blood-enriched media)).

Clostridia produce large raised colonies (e.g., C perfringens); others produce smaller colonies (e.g., C tetani).

Some Clostridia form colonies that spread or swarm on the agar surface (Clostridium septicum). Many

Clostridia produce a zone of β-hemolysis on blood agar. C perfringens characteristically produces a double

zone of β-hemolysis around colonies.

Clostridia can ferment a variety of sugars (saccharolytic) and many can digest proteins (proteolytic); some

species do both. These metabolic characteristics are used to divide the Clostridia into groups. Milk is turned

acid by some and digested by others and undergoes “stormy fermentation” (clot torn by gas).

Clostridium botulinum

C botulinum which causes the disease botulism is worldwide in distribution; it is found in soil and occasionally

in animal feces. Types of C botulinum are distinguished by the antigenic type of toxin they produce. Spores of

the organism are highly resistant to heat, withstanding 100°C for several hours. Heat resistance is diminished at

acid pH or high salt concentration.

During the growth of C botulinum and during autolysis of the bacteria, toxin is liberated into the environment.

Seven antigenic varieties of toxin (serotypes A-G) are known. Types A, B, E, and F are the principal causes of

human illness.

Botulinum toxins have three domains:

1- Two of the domains facilitate binding to and entry of toxin into the nerve cell

2-The third domain is the toxin which is protein that is cleaved into a heavy chain and a light chain that are

linked by a disulfide bond.

Botulinum toxin is absorbed from the gut, enters the blood circulation, and binds to receptors of motor neurons

of the peripheral nervous system and cranial nerves. The toxin does not cross the blood brain barrier or affect

the central nervous system. It inhibits the release of acetylcholine at the neurons synapse, resulting in lack of

muscle contraction and paralysis.

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Figure shows C botulinum toxins action pattern

Pathogenesis

Most cases of botulism represent an intoxication resulting from the ingestion of food in which C botulinum has

grown and produced toxin. The most common offenders are spiced, smoked, vacuum packed or canned

alkaline foods that are eaten without cooking. In such foods, spores of C botulinum germinate; that is, under

anaerobic conditions, vegetative forms grow and produce toxin. In infant botulism, honey is the most frequent

vehicle of infection.

The infant ingests the spores of C botulinum, and the spores germinate within the intestinal tract. The vegetative

cells produce toxin as they multiply; the neurotoxin then gets absorbed into the bloodstream.

The toxin acts by blocking release of acetylcholine at synapses and neuromuscular junctions; the result is

flaccid paralysis.

Figure shows C botulinum gram stained film

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The clinical picture begin 18-24 hours after ingestion of the toxic food, with visual disturbances (incoordination

of eye muscles, double vision), inability to swallow, and speech difficulty, and death occurs from respiratory

paralysis or cardiac arrest.

The infants in the first months of life develop poor feeding, weakness, and signs of paralysis (floppy baby).

Infant botulism may be one of the causes of sudden infant death syndrome. C botulinum and botulinum toxin

are found in feces but not in serum.

Laboratory diagnosis carried out by detection of toxin and not the organism. Toxin can often be demonstrated

in serum, gastric secretions, or stool from the patient, and toxin may be found in leftover food.

Clinical swabs or other specimens obtained from patients should be transported using anaerobe containers.

Mice injected intraperitoneally with such specimens from these patients die rapidly. The antigenic type of toxin

is identified by neutralization with specific antitoxin in mice. This mouse bioassay is the test of choice for the

confirmation of botulism. In infant botulism, C botulinum and toxin can be demonstrated in bowel contents but

not in serum. Other methods used to detect toxin include Elisa and PCR.

Clostridium tetani

C tetani which causes tetanus is worldwide in distribution in the soil and in the feces of horses and other

animals. Several types of C tetani can be distinguished by specific flagellar antigens. All share a common O

(somatic) antigen, which may be masked, and all produce the same antigenic type of neurotoxin

((tetanospasmin)) which composed of two peptides linked by a disulfide bond. The larger peptide initially

binds to receptors on the presynaptic membranes of motor neurons; it then migrates by the retrograde axonal

transport system to the cell bodies of these neurons to the spinal cord and brainstem. The release of the

inhibitory glycine and GABA (γ-aminobutyric acid) is blocked, and the motor neurons are not inhibited; so

hyper-reflexia, muscle spasms, and spastic paralysis result.

Pathogenesis

C tetani is not an invasive organism, the infection remains strictly localized in the area of devitalized tissue

(wound, burn, injury, umbilical stump, surgical suture) into which the spores have been introduced.

Figure shows C tetani gram film (tennis rackets or drumsticks)

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The volume of infected tissue is small, and the disease is almost entirely a toxemia. Germination of the spore

and development of vegetative organisms that produce toxin are aided by:

1- Necrotic tissue

2- Calcium salts

3- Associated pyogenic infections all of which aid establishment of low oxidation-reduction potential

The toxin released from vegetative cells reaches the central nervous system and rapidly becomes fixed to

receptors in the spinal cord and brainstem and exerts the actions

The incubation period may range from 4-5 days up to 3 weeks. The disease is characterized by tonic contraction

of voluntary muscles. Muscular spasms often involve first the area of injury and infection and then the muscles

of the jaw (trismus, lockjaw), which contract so that the mouth cannot be opened. Gradually, other voluntary

muscles become involved, resulting in tonic spasms. The patient is fully conscious, and pain may be intense.

Death usually results from interference with the mechanics of respiration. The mortality rate in generalized

tetanus is very high.

Diagnosis and tetanus prevention

Anaerobic culture of tissues from contaminated wounds may yield C tetani, but neither preventive nor

therapeutic use of antitoxin should ever be withheld pending such demonstration. Proof of isolation of C tetani

must rest on production of toxin and its neutralization by specific antitoxin.

Prevention of tetanus depends on:

1- Active immunization with toxoids

2- Aggressive wound care

3- Prophylactic use of antitoxin

4- Administration of penicillin

Active immunization with tetanus toxoid should accompany antitoxin prophylaxis; they are given very large

doses of antitoxin (3000-10,000 units of tetanus immune globulin) intravenously in an effort to neutralize toxin

that has not yet been bound to nervous tissue. Surgical debridement is vitally important because it removes the

necrotic tissue that is essential for proliferation of the organisms. Penicillin strongly inhibits the growth of C

tetani and stops further toxin production.

Tetanus toxoid is produced by detoxifying the toxin with formalin and then concentrating it. Aluminum salt

adsorbed toxoids are used. Three injections comprise the initial course of immunization followed by another

dose about 1 year later. Initial immunization should be carried out in all children during the first year of life.

“booster” injection of toxoid is given upon entry into school. Thereafter, “boosters” can be spaced 10 years

apart to maintain serum levels In young children; tetanus toxoid is often combined with diphtheria toxoid and

a cellular pertussis vaccine (DPT).

Clostridia with tissue invasion

Many different toxin-producing Clostridia (C perfringens and related Clostridia) can produce invasive

infection (including myonecrosis and gas gangrene) if introduced into damaged tissue.

About 30 species of Clostridia may produce such an effect, but the most common in invasive disease is C

perfringens (90%). An enterotoxin of C perfringens is a common cause of food poisoning. The invasive

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Clostridia produce a large variety of toxins and enzymes that result in a spreading infection. Many of these

toxins have lethal, necrotizing, and hemolytic properties.

The alpha toxin of C perfringens type A is a lecithinase, and its lethal action is proportionate to the rate at

which it splits lecithin (an important constituent of cell membranes). Alpha toxin also aggregates platelets,

thereby leading to formation of thrombi in small blood vessels and adding to poor tissue profusion and

extending the consequences of anaerobiosis, namely, destruction of viable tissue (gas gangrene).

The theta toxin has similar hemolytic and necrotizing effects but is not a lecithinase that act by forming pores

in cell membranes.

Epsilon toxin is a protein that causes edema, and hemorrhage is very potent. DNase and hyaluronidase, a

collagenase that digests collagen of subcutaneous tissue and muscle, are also produced.

Some strains of C perfringens produce a powerful enterotoxin especially when grown in meat dishes. When

more than 108

vegetative cells are ingested and sporulate in the gut. It induces intense diarrhea in 7-30 hours.

The action of C perfringens enterotoxin involves marked hypersecretion in the jejunum and ileum, with loss of

fluids and electrolytes in diarrhea; this illness is similar to that produced by B cereus and tends to be selflimited.

Pathogenesis

In invasive Clostridia infections, spores reach tissue either by contamination of traumatized areas (soil, feces)

or from the intestinal tract. The spores germinate at low oxidation-reduction potential; vegetative cells multiply,

ferment carbohydrates present in tissue, and produce gas.

The distention of tissue and interference with blood supply, together with the secretion of necrotizing toxins

and hyaluronidase, favor the spread of infection. Tissue necrosis extends, providing an opportunity for

increased bacterial growth, hemolytic anemia, and,