CLINICAL BACTERIOLOGY BSMLS-3D

Prof. Keith Ivan Jemuel Luis, RMT

BACILLUS AND SIMILAR ORGANISMS

BACILLUS • The organism is normally found in the soil and

primarily causes disease in herbivores.
G E N E R A A N D S P E C I E S T O B E CONSIDERED • Humans acquire infections when inoculated with the
Bacillus cereus Group endospores, by either traumatic introduction,
• Bacillus anthracis injection, ingestion, or inhalation during exposure
• Bacillus cereus (type species)
to contaminated animal products, such as hides
• Bacillus thuringiensis
• Although rare, person-to-person transmission has
• Bacillus mycoides
been reported from mother to child, between siblings,
• Bacillus pseudomycoides
and nosocomial spread from an infected umbilical
• Bacillus megaterium
lesion.
• Bacillus cytotoxicus
• Bacillus toyonensis • B. anthracis produces endospores that may be
• Bacillus weihenstephanensis effectively used as an agent of biologic warfare.
Bacillus circulans Group
PATHOGENESIS AND SPECTRUM OF DISEASE
• Bacillus circulans (type species)
• Bacillus firmus • B. anthracis is the most highly virulent species and is
• Bacillus coagulans the causative agent of anthrax.
Bacillus subtilis Group • The four forms of disease are cutaneous,
• Bacillus licheniformis gastrointestinal (ingestion), inhalation or woolsorters’
• Bacillus amyloliquefaciens disease, and injectional anthrax
• Bacillus subtilis (type species)
• Bacillus pumilus CUTANEOUS ANTHRAX ESCHAR ON A 7-MONTH-OLD
Other Related Organisms CHILD.
Brevibacillus spp.
Lysinibacillus spp. • The cutaneous anthrax accounts for most human
Paenibacillus spp. infections and is associated with contact with infected
animal products.
• Infection results from close contact and inoculation
Bacillus anthracis
of endospores through a break in the skin.
• Even though this organism is rarely found, sentinel • After inoculation and an incubation period of
laboratory protocols require ruling out the possibility approximately 2 to 6 days in most cases, a small
of anthrax before reporting any blood, cerebrospinal papule appears that progresses to a ring of vesicles.
fluid (CSF), or wound cultures in which a large gram- The vesicles then develop into an ulceration.
positive aerobic rod is isolated • The typical presentation of the ulceration is a black,
• B. anthracis, along with the other species within the necrotic lesion known as an eschar. The eschar may
genus, are capable of forming spores within the become thick and surrounded by edema. The lesion,
mother cell or endospores. however, is usually painless.
• The endospores are produced during conditions of • The patient presents with no fever, and the lesion
environmental stress such as nutrient deprivation, lacks purulence. If any of these symptoms are
temperature extremes, and drying or desiccation. present, the patient may be experiencing a secondary
• The endospores are able to survive the harsh bacterial infection.
environmental conditions that also include resistance • Cutaneous anthrax infections can be effectively
to radiation and disinfectants. The highly resistant treated with antibiotics. Fatalities may occur when
nature of the endospore provides a mechanism for the lesions form on the face or neck and cause an
dissemination and survival of the organism in the obstructed airway after edema or progression to
environment. systemic disease.
• The mortality rate for untreated cutaneous anthrax is
EPIDEMIOLOGY low, at approximately 1%.

• B. anthracis remains the most widely recognized

bacillus in clinical microbiology laboratories.
• Anthrax is primarily a disease of wild and domestic
animals including sheep, goats, horses, and cattle.
The decline in animal and human infections is a result
of the development of veterinary and human vaccines
as well as improvements in industrial applications for
handling and importing animal products.

PATARAY 1
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

• Colonies of B. anthracis on sheep-blood agar • The disease appears flulike and includes symptoms
demonstrating the characteristic comet tail or Medusa- such as fever, chills, fatigue, a nonproductive cough,
head morphology. and nausea or vomiting that then progresses to
respiratory distress, edema, cyanosis, shock, and
GASTROINTESTINAL (INGESTION) ANTHRAX death.
• Patients typically demonstrate abnormal chest x-rays
• Gastrointestinal (ingestion) anthrax results from
with pleural effusion, infiltrates, and mediastinal
ingestion of endospores and presents in two forms:
widening.
oral or oropharyngeal, with the lesion appearing in
the buccal cavity or on the tongue, tonsils, or • Woolsorters’ disease and ragpickers’ disease are
pharyngeal mucosa. terms used to describe respiratory infections that result
from exposure to endospores during the handling of
• Gastrointestinal anthrax, with the lesions developing
animal hides, hair, fibers, and other animal products.
typically in the mucosa of the terminal ileum or cecum.
• Oropharyngeal symptoms may include sore throat,
lymphadenopathy, and edema of the throat (neck), and
chest.
• The initial symptoms of gastrointestinal anthrax may
be nonspecific with progression to abdominal pain,
nausea, vomiting, anorexia, fever, bloody diarrhea, and
hematemesis (vomiting of blood).
• The mortality rate of gastrointestinal anthrax is much
higher than that of cutaneous anthrax and associated
with toxemia and sepsis. This may be associated with
the delayed treatment of the disease because of the INJECTIONAL ANTHRAX
nonspecific nature of the symptoms resulting in the
failure of the patient to seek appropriate medical care. • Injectional anthrax is associated with injection of
contaminated drugs of abuse, frequently heroin.
• Animal skins are often used to move contraband, such
as illegal drugs, to avoid detection by custom
authorities.
• The skin around the injection site may appear
bruised, but the characteristic lesion or eschar is
absent. The infection typically presents as a severe soft
tissue infection leading to rapid systemic dissemination
and septic shock
• Although when properly identified anthrax is a
treatable disease, complications may occur.
Patients can develop meningitis within 6 days after
exposure. Successful recovery, however, results in
long-term immunity to subsequent infections.

Injection anthrax of the right upper arm

following heroin abuse, on referral to
dermatology unit

Thigh and popliteal space with severe

skin and soft-tissue involvement in
anthrax infection. Multiple intravascular
and perivascular injection sites in the
region

INHALATION ANTHRAX

• Inhalation anthrax, previously referred to as

pulmonary anthrax, is the result of the inhalation of
endospores.
• The endospores are then ingested by macrophages in
the lungs and transported to the lymph nodes, leading
to the development of systemic infection.

PATARAY 2
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

VIRULENCE FACTORS • As previously described for B. anthracis, infections with

B. cereus group organisms have also been linked to
Each of these toxins consists of two proteins: the contaminated drugs.
protective antigen (PA) and the functional enzyme,
• "food poisoning" is associated with this ingestion of a
lethal factor (LF) and edema factor (EF). The PA facilitates
wide variety of foods including meats, vegetables,
the transport of the other protein into the cell.
desserts, sauces, and milk.
A higher incidence is seen after the ingestion of rice
1. Lethal toxin dishes. After ingestion, patients present with one of
• responsible for death two types of symptoms: diarrhea and abdominal
2. Edema toxin pain within 8 to 16 hours, or nausea and vomiting
(emetic food poisoning) within 1 to 5 hours.
• responsible for edema
• B. cereus produces several toxins implicated in the
Loss of the toxin-encoding plasmid from a B. anthracis diarrheal symptoms, including hemolysin BL (HBL),
strain appears to attenuate or reduce the pathogenesis of nonhemolytic enterotoxin (Nhe), and cytotoxin K
the organism. In addition, acquisition of the virulence (CytK; also referred to as hemolysin IV).
plasmid also enhances the pathogenesis of other Bacillus The three toxins are believed to act synergistically,
spp. organisms with Nhe responsible for the major symptoms in the
diarrheal presentation of the infection.
• Emetic form: cereulide
Bacillus cereus The cereulide toxin is encoded on a plasmid-
borne gene cluster and has been identified in a
B. cereus, previously a single clinically relevant species,
subset of B. cereus and B. thuringiensis isolates
consists of a group of organisms that are classified as
individual species. • B. cereus from a patient’s eye can cause permanent
damage and should be reported to the primary care
These include a variety of species: crystal-forming B. provider immediately.
thuringiensis; a cold-tolerant B. weihenstephanensis;
heat tolerant B. cytotoxicus; a probiotic, B. toyonensis; B. thuringiensis
and morphologic variants, B. mycoides and B.
• identified as harboring the cereulide enterotoxin.
pseudomycoides.
• toxins have been commercialized for the control of
insects that cause agricultural damage such as
• causes food poisoning. moths, beetles, flies, and parasitic worms
• Transmission: Spores on grains such as rice survive (nematodes).
steaming and rapid frying.
B. subtilis, Brevibacillus and Paenibacillus spp.
• spores germinate when rice is kept warm for many
hours (e.g reheated fried rice) • B. subtilis (type species) has been identified in
• portal of entry is the gastrointestinal tract clinical specimens in a variety of cases, including
pneumonia, bacteremia, septicemia, surgical
EPIDEMIOLOGY
wounds, meningitis after head trauma, and other
• B. cereus group is found within the soil and widely surgical infections.
distributed in nature. The organisms are considered • Rare human infections have been associated with a
opportunistic pathogens and often associated with variety of Bacillus spp., including B. licheniformis, B.
foodborne illness. circulans, B. coagulans, B. megaterium, B. pumilus,
Paenibacillus polymyxa, Brevibacillus spp., and
PATHOGENESIS AND SPECTRUM OF DISEASE others

• are often associated with infections in EPIDEMIOLOGY

immunocompromised patients who have debilitating
disease such as cancer or diabetes. • Most Bacillus spp., other than B. anthracis, B. cereus,
• may be associated with localized skin infections or and B. thuringiensis, are generally considered to be
systemic conditions such as bacteremia, opportunistic pathogens of low virulence and are
endocarditis, and septicemia. associated with immunocompromised patients after
• Additional sites of infection include the urinary tract or exposure to contaminated materials.
respiratory tract. PATHOGENESIS AND SPECTRUM OF DISEASE
• Health care–associated infections have also been
identified and linked to contaminated material such as • The endospores of Bacillus spp. are ubiquitous in
gowns, gloves, linens, and dressings, as well as nature, and contamination of various clinical
medical devices including catheters, ventilators, and specimens may occur. The clinical significance of the
bronchoscopy equipment.

PATARAY 3
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

isolate should be carefully established during the isolation and identification of the organism. These
identification of the microorganism media take advantage of the phospholipase C–
positive reaction on egg yolk agar, lack of production
LABORATORY DIAGNOSIS of acid from mannitol, and incorporation of pyruvate or
polymyxin as the selective agents.
Specimen Processing
• chromogenic medium that uses chromogenic
• Specimens collected from patients suspected of having substrates in place of egg yolk for the identification of
anthrax should be placed in leak-proof containers and phospholipase C include B. cereus/B.thuringiensis
placed in secondary container. media and B. cereus group medium.
• Cutaneous anthrax specimens should be collected • Heat shock treatment can be used for the growth
from underneath the eschar. and enhancement of endospores from clinical
• Inhalation anthrax specimens should include blood specimens.
cultures, pleural fluid, and a serum specimen for → Heat treatment at 70°C for 30 minutes or
serology. 80°C for 10 minutes is effective for killing
• may contain endospores that pose an aerosolization vegetative cells and retaining spores for
and inhalation risk to the laboratory professional, most Bacillus spp.
requiring the use of personal → B. anthracis heat treatment is carried out at
• protective equipment, including a proper respiratory lower temperatures, 62°C to 65°C for 15 to
mask. 20 minutes.
• Specimen processing may include heat or alcohol → After heat treatment, samples are plated to
shock before culture medium along with a sample of
• plating on solid media. disinfection with formaldehyde, untreated specimen to ensure maximal
glutaraldehyde, or hydrogen recovery of the isolate.
• peroxide and peracetic acid should be performed
EPIDEMIOLOGY
before the
Bacillus anthracis
Bacillus anthracis demonstrating
serpentine form on a Gram stain when Virulence Factors: Capsule exotoxins (edema toxin and
grown in culture. Note the endospore, lethal toxin) swelling and tissue death
as designated by the arrow.
Spectrum of Diseases and Infections:

Causative agent of anthrax, of which there are three forms:

Gram stain of Bacillus cereus. The
arrows indicate endospores, the clear Cutaneous anthrax occurs at site of spore penetration 2–5
area inside the gram-positive days after exposure and is manifested by progressive stages
vegetative cell. from an erythematous papule to ulceration and finally to
formation of a black scar (i.e., eschar); may progress to
toxemia and death
CULTIVATION METHOD Pulmonary anthrax, also known as woolsorters’ disease,
follows inhalation of spores and progresses from malaise with
• grow well on 5% sheep blood agar, chocolate agar, mild fever and nonproductive cough to respiratory distress,
routine blood cult media, and nutrient broths. massive chest edema, cyanosis, and death
• Columbia agar with nalidixic acid and colistin (CAN)
• Phenylethyl alcohol agar (PEA) an additional Gastrointestinal anthrax may follow ingestion of spores and
selective agar for gram-positive organisms, is useful affects either the oropharyngeal or the abdominal area; most
for the removal of contaminating organisms and the patients die of toxemia and overwhelming sepsis
isolation of Bacillus spp
• Polymyxin-lysozyme-EDTA-thallous acetate Injectional anthrax after the intravenous injection of
(PLET) can be used for selection and isolation from contaminated drugs; soft tissue infections that lack the eschar
contaminated specimens. Colonies appear as creamy associated with cutaneous anthrax. May result in death of
white, domed, circular colonies. In addition, shock, coma, organ failure, and necrotizing fasciitis.
bicarbonate agar is used to induce B. anthracis Bacillus cereus
capsule formation, providing a means for presumptive
morphologic identification. Virulence Factors: Enterotoxins and pyogenic toxin
• B. cereus media referred to as mannitol, egg yolk, and
polymyxin B agar (MEYP or MYP); polymyxin B, egg Spectrum of Diseases and Infections: Food poisoning of
yolk, mannitol, bromothymol blue (PEMBA); B. cereus two types: diarrheal type, characterized by abdominal pain
medium (BCM) have been developed for the specific and watery diarrhea, and emetic type, which is manifested by

PATARAY 4
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

profuse vomiting; B. cereus–type species is the most Bacillus thuringiensis

commonly encountered of Bacillus in opportunistic infections,
including posttraumatic eye infections, endocarditis, and Virulence Factors: Cereulide enterotoxin
bacteremia; infections of other sites are rare and usually
Spectrum of Diseases and Infections: Food poisoning and
involve intravenous drug abusers or immunocompromised
other infections such as wound, burn, pulmonary, and ocular
patients.
infections
Bacillus circulans, B. firmus, Bacillus licheniformis, Bacillus
Bacillus cytotoxicus
subtilis, other Bacillus spp., Brevibacillus sp., and
Paenibacillus spp. Virulence Factors: Cytotoxin K, enterotoxin
Virulence Factors: Virulence factors unknown Spectrum of Diseases and Infections: Severe food
poisoning
Spectrum of Diseases and Infections: Food poisoning has
been associated with some species but is uncommon; these
organisms may also be involved in opportunistic infections
similar to those described for B. cereus

PATARAY 5
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

PATARAY 6
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

Lecithinase production by Bacillus

cereus on egg yolk agar. The
organism has been streaked down
the center of the plate. The positive
test for lecithinase is indicated by the
opaque zone of precipitation around
the bacterial growth (arrows).

ANTIMICROBIAL SUSCEPTIBILITY TESTING AND

THERAPY

Bacillus anthracis

Therapeutic Options: Ciprofloxacin or doxycycline plus one

or two other antibiotics; other agents that may demonstrate
in vitro activity include rifampin, vancomycin, penicillin,
ampicillin, chloramphenicol, imipenem, clindamycin, and
clarithromycin.

Resistance to Therapeutic Options: Beta-lactamases

Validated Testing Methods: See CLSI document M100:

Performed in Approved Reference Laboratories Only

Other Bacillus spp., Brevibacillus sp., Paenibacillus spp.

Therapeutic Options: No definitive guidelines; vancomycin,

ciprofloxacin, imipenem, and aminoglycosides may be
effective

Resistance to Therapeutic Options: B. cereus frequently

produces beta-lactamase

Validated Testing Methods: See CLSI document M45:

Methods for Antimicrobial Dilution and Disk Susceptibility
Testing of Infrequently Isolated or Fastidious Bacteria

Comments: Whenever isolated from clinical specimens, the

potential for the isolate to be a contaminant must be strongly
considered

PREVENTION

A cell-free inactivated vaccine (BioThrax, Emergent

Biodefense Operations, Lansing, MI) given in three primary
doses followed by two boosters thereafter (0 weeks, 1 month,
6 months, 12 months, and 18 months) is available for
immunizing high-risk adults (i.e., public health laboratory
workers, workers handling potentially contaminated industrial
raw materials, and military personnel) against anthrax.
Chemoprophylaxis with ciprofloxacin (or doxycycline) is
recommended after aerosol exposure to B. anthracis, such as
in a bioterrorist event.

PATARAY 7
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

CLOSTRIDIUM • Ampicillin and fluoroquinolones, cancer

chemotherapy also predisposes to
There are four medically important species: Clostridium tetani, pseudomembranous colitis
Clostridium botulinum, Clostridium perfringens (which causes
either gas gangrene or food poisoning), and Clostridium Note: C. difficile rarely invades the intestinal mucosa.
difficile. All clostridia are anaerobic, spore-forming, gram-
positive rods. CLINICAL FINDINGS

• C. difficile causes diarrhea associated with

pseudomembranes (yellow-white plaques) on the
colonic mucosa.
• The diarrhea is usually not bloody, and neutrophils are
found in the stool in about half of the cases. Fever and
abdominal pain often occur.
• The pseudomembranes are visualized by
sigmoidoscopy. Toxic megacolon can occur, and
surgical resection of the colon may be necessary.
• Pseudomembranous colitis can be distinguished
from the transient diarrhea that occurs as a side effect
of many oral antibiotics by testing for the presence of
the toxin in the stool.
• In 2005, a new, more virulent strain of C. difficile
Clostridium difficile
emerged. This hypervirulent strain causes more severe
disease, is more likely to cause recurrences, and
responds less well to metronidazole than the previous
DISEASE strain. The strain is also characterized by resistance to
quinolones. It is thought that the widespread use of
• C. difficile causes antibiotic-associated quinolones for diarrheal disease may have selected for
pseudomembranous colitis (Figure 17–6). C. difficile is this new strain.
the most common nosocomial (hospital-acquired)
cause of diarrhea. LABORATORY DIAGNOSIS

TRANSMISSION • The presence of exotoxins in the filtrate of a patient's

stool specimen is the basis of the laboratory diagnosis.
• The organism is carried in the gastrointestinal tract in It is insufficient to culture the stool for the presence of C.
approximately 3% of the general population and up to difficile because people can be colonized by the
30% of hospitalized patients. Most people are not organism and not have disease.
colonized, which explains why most people who take
antibiotics do not get pseudomembranous colitis. It is Note that isolation of C. difficile from the stool,
transmitted by the fecal–oral route. The hands of followed by evidence that the isolate is a toxin
hospital personnel are important intermediaries. producing one, can be used. However, this process
takes time and may not be able to be completed in
PATHOGENESIS a clinically relevant time frame.

• Antibiotics suppress drug-sensitive members of the

normal flora, allowing C. difficile to multiply and • There are two types of tests usually used to detect
produce exotoxin A and B. the exotoxins. One is an ELISA using known antibody
Both exotoxin A and exotoxin B are to the exotoxins. The ELISA tests are rapid but are less
glucosyltransferases (i.e., enzymes that glucosylate sensitive than the cytotoxicity test. In the cytotoxicity
[add glucose to] a G protein called Rho GTPase). test, human cells in culture are exposed to the exotoxin
The main effect of exotoxin B in particular is to cause in the stool filtrate and the death of the cells is observed.
depolymerization of actin, resulting in a loss of
This test is more sensitive and specific but requires 24
cytoskeletal integrity, apoptosis, and death of the
to 48 hours of incubation time. To distinguish between
enterocytes
cytotoxicity caused by the exotoxins and cytotoxicity
caused by a virus possibly present in the patient's stool,
• Clindamycin was the first antibiotic to be recognized antibody against the exotoxins is used to neutralize the
as a cause of pseudomembranous colitis, but many cytotoxic effect. In addition to tests that detect the toxin,
antibiotics are known to cause this disease. a PCR assay for the presence of the toxin gene DNA is
• At present, third-generation cephalosporins are the also used.
most common cause

PATARAY 8
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

TREATMENT PATHOGENESIS & CLINICAL FINDINGS

The causative antibiotic should be withdrawn. Oral

metronidazole or vancomycin should be given and fluids
replaced.

Metronidazole is preferred because using vancomycin may

select for vancomycin-resistant enterococci. However, in
lifethreatening cases, vancomycin should be used because
it is more effective than metronidazole.

Fidaxomicin (Dificid) is used both in the treatment of Tetanus toxin (tetanospasmin) is an exotoxin produced by
pseudomembranous colitis and in preventing relapses of this vegetative cells at the wound site. This polypeptide toxin is
disease. It is effective in life-threatening cases. carried intraaxonally (retrograde) to the central nervous
system, where it binds to ganglioside receptors and blocks
Fecal bacteriotherapy is another possible therapeutic release of inhibitory mediators (e.g., glycine and γ-
approach. It involves administering bowel flora from a normal aminobutyric acid [GABA]) at spinal synapses. Tetanus toxin
individual either by enema or by nasoduodenal tube to the and botulinum toxin (see later) are among the most toxic
patient with pseudomembranous colitis. This approach is substances known.
based on the concept of bacterial interference (i.e., to replace
the C. difficile with normal bowel flora). Very high cure rates Tetanus is characterized by strong muscle spasms (spastic
are claimed for this technique, but safety issues have limited paralysis, tetany). Specific clinical features include lockjaw
its acceptance. (trismus) due to rigid contraction of the jaw muscles, which
prevents the mouth from opening; a characteristic grimace
PREVENTION known as risus sardonicus; and exaggerated reflexes.
Opisthotonos, a pronounced arching of the back due to
• There are no preventive vaccines or drugs. Because spasm of the strong extensor muscles of the back, is often
antibiotics are an important predisposing factor for seen. Respiratory failure ensues.
pseudomembranous colitis, they should be prescribed
only when necessary. A high mortality rate is associated with this disease. Note
• In the hospital, strict infection control procedures, that in tetanus, spastic paralysis (strong muscle contractions)
including rigorous handwashing, are important. occurs, whereas in botulism, flaccid paralysis (weak or absent
Probiotics such as Lactobacillus, Bifidobacterium, or the muscle contractions) occurs.
yeast Saccharomyces may be useful to prevent
pseudomembranous colitis. LABORATORY DIAGNOSIS

Clostridium tetani

Tetanus. Note the marked There is no microbiologic or serologic diagnosis. Organisms

hyperextension of the back, a position are rarely isolated from the wound site. C. tetani produces a
called opisthotonos, caused by terminal spore (i.e., a spore at the end of the rod). This gives
tetanus toxin, an exotoxin that inhibits the organism the characteristic appearance of a “tennis
the release of mediators of the racket.”
inhibitory neurons in the spinal cord TREATMENT
TRANSMISSION Tetanus immune globulin (tetanus antitoxin) is used to
neutralize the toxin. The role of antibiotics is uncertain. If
• Spores are widespread in soil.
antibiotics are used, either metronidazole or penicillin G can
• The portal of entry is usually a wound site (e.g., where
be given. An adequate airway must be maintained and
a nail penetrates the foot), but the spores can also be
respiratory support given. Benzodiazepines (e.g., diazepam
introduced during “skin-popping,” a technique used
[Valium]) should be given to prevent spasms.
by drug addicts to inject drugs into the skin.
• Germination of spores is favored by necrotic tissue
and poor blood supply in the wound.
• Neonatal tetanus, in which the organism enters
through a contaminated umbilicus or circumcision
wound, is a major problem in some developing
countries

PATARAY 9
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

PREVENTION • risk from home-canned foods can be reduced if the

food is boiled for more than 20 minutes before
• Tetanus is prevented by immunization with tetanus consumption.
toxoid (formaldehyde-treated toxin) in childhood and
every 10 years thereafter. Pathogenesis
• Tetanus toxoid is usually given to children in Botulinum toxin is absorbed from the gut and carried via
combination with diphtheria toxoid and the acellular the blood to peripheral nerve synapses, where it blocks
pertussis vaccine (DTaP). release of acetylcholine. It is a protease that cleaves the
• When trauma occurs, the wound should be cleaned proteins involved in acetylcholine release. The toxin is a
polypeptide encoded by a lysogenic phage. Along with
and debrided, and tetanus toxoid booster should be
tetanus toxin, it is among the most toxic substances known.
given. If the wound is grossly contaminated, tetanus
immune globulin, as well as the toxoid booster, should There are eight immunologic types of toxin; types A, B,
be given and penicillin administered. Tetanus immune and E are the most common in human illness. Botox is
globulin (tetanus antitoxin) is made in humans to a commercial preparation of exotoxin A used to remove
avoid serum sickness reactions that occur when wrinkles on the face. Minute amounts of the toxin are
antitoxin made in horses is used. effective in the treatment of certain spasmodic muscle
• The administration of both immune globulins and disorders such as torticollis, “writer’s cramp,” and
tetanus toxoid (at different sites in the body) is an blepharospasm.
example of passive–active immunity

Clostridium botulinum Clinical Findings

• During the growth of C botulinum and during Descending weakness and paralysis, including
autolysis of the bacteria, toxin is liberated into the diplopia, dysphagia, and respiratory muscle failure, are
environment. seen. No fever is present. In contrast, Guillain-Barré
• Seven antigenic varieties of toxin (A-G) are known syndrome is an ascending paralysis.

TRANSMISSION Two special clinical forms occur:

Spores, widespread in soil, contaminate vegetables and (1) wound botulism, in which spores contaminate a
meats. When these foods are canned or vacuum-packed wound, germinate, and produce toxin at the site; and
without adequate sterilization, spores survive and germinate
in the anaerobic environment. Toxin is produced within the (2) infant botulism, in which the organisms grow in the gut
canned food and ingested preformed. and produce the toxin there. Ingestion of honey containing
the organism is implicated in transmission of infant
The highest-risk foods are (1) alkaline vegetables such as botulism. Affected infants develop weakness or paralysis
green beans, peppers, and mushrooms and (2) smoked fish. and may need respiratory support but usually recover
The toxin is relatively heat-labile; it is inactivated by boiling for spontaneously. In the United States, infant botulism
accounts for about half of the cases of botulism, and wound
several minutes. Thus, disease can be prevented by sufficient
botulism is associated with drug abuse, especially skin-
cooking.
popping with black tar heroin.
PATHOGENESIS AND CLINICAL FINDINGS
LABORATORY DIAGNOSIS

The organism is usually not cultured. Botulinum toxin is

demonstrable in uneaten food and the patient’s serum by
mouse protection tests. Mice are inoculated with a sample of
the clinical specimen and will die unless protected by antitoxin.

TREATMENT

Trivalent antitoxin (types A, B, and E) is given, along with

respiratory support. The antitoxin is made in horses, and
serum sickness occurs in about 15% of antiserum recipients.
PREVENTION AND CONTROL PREVENTION
• Proper sterilization and strict regulation of all canned Proper sterilization of all canned and vacuum-packed
and vacuum-packed foods is essential. foods is essential. Food must be adequately cooked to
• Toxic foods may be spoiled and rancid, and cans inactivate the toxin. Swollen cans must be discarded
may "swell," or the appearance may be innocuous. (clostridial proteolytic enzymes form gas, which swells cans).

PATARAY 10
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

Clostridium perfringens TREATMENT

• Penicillin G is the antibiotic of choice. Wounds

should be debrided
DISEASE: GAS GANGRENE
PREVENTION

• Wounds should be cleansed and debrided.

Gas gangrene (myonecrosis, necrotizing
• Penicillin may be given for prophylaxis.
fasciitis) is one of the two diseases caused
• There is no vaccine
by C. perfringens. Gas gangrene is also
caused by other histotoxic clostridia such DISEASE: FOOD POISONING
as Clostridium histolyticum, Clostridium
septicum, Clostridium novyi, and • Food poisoning is the second disease caused by C.
Clostridium sordellii. (C. sordellii also perfringens.
causes toxic shock syndrome in
postpartum and postabortion women.)

TRANSMISSION

Spores are located in the soil; vegetative cells are members

of the normal flora of the colon and vagina. Gas gangrene
is associated with war wounds, automobile and motorcycle
accidents, and septic abortions (endometritis).

PATHOGENESIS

Organisms grow in traumatized tissue (especially muscle) and

produce a variety of toxins. The most important is alpha toxin
(lecithinase), which damages cell membranes, including those
of erythrocytes, resulting in hemolysis. Degradative enzymes
produce gas in tissues. TRANSMISSION
CLINICAL FINDINGS Spores are located in soil and can contaminate food. The
heat-resistant spores survive cooking and germinate. The
Pain, edema, cellulitis, and gangrene (necrosis) occur in
organisms grow to large numbers in reheated foods,
the wound area
especially meat dishes.
Crepitation indicates the presence of gas in tissues.
PATHOGENESIS
Hemolysis and jaundice, blood- tinged exudates.
C. perfringens is a member of the normal flora in the colon
Shock and death can ensue. but not in the small bowel, where the enterotoxin acts to
cause diarrhea. The mode of action of the enterotoxin is the
Mortality rates are high. same as that of the enterotoxin of Staphylococcus aureus (i.e.,
it acts as a superantigen).
LABORATORY DIAGNOSIS
CLINICAL FINDINGS
Smears of tissue and exudate samples show large gram-
positive rods. The disease has an 8- to 16-hour incubation period and is
characterized by watery diarrhea with cramps and little
Spores are not usually seen because they are formed
vomiting. It resolves in 24 hours.
primarily under nutritionally deficient conditions.
LABORATORY DIAGNOSIS
The organisms are cultured anaerobically and then
identified by sugar fermentation reactions and organic acid This is not usually done. There is no assay for the toxin.
production. Large numbers of the organisms can be isolated from uneaten
food.
C. perfringens colonies exhibit a double zone of hemolysis
on blood agar. TREATMENT
Egg yolk agar is used to demonstrate the presence of the Symptomatic treatment is given; no antimicrobial drugs
lecithinase. are administered.
Serologic tests are not useful.

PATARAY 11
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

LISTERIA & CORYNEBACTERIUM • Colonizer: Human nasopharynx but only in carrier

state; not considered part of normal microbiota
G E N E R A A N D S P E C I E S T O B E CONSIDERED Isolation from healthy humans is not common
• Arcanobacterium haemolyticum
• Arthrobacter spp. Mode of Transmission
• Brevibacterium spp.
• Cellulomonas spp. • Direct contact: Person to person by exposure to
• Cellulosimicrobium spp. contaminated respiratory droplets Contact with
• Corynebacterium amycolatum exudate from cutaneous lesions Exposure to
• Corynebacterium aurimucosum contaminated objects
• Corynebacterium coyleae
• Corynebacterium diphtheriae (toxigenic and Corynebacterium glucuronolyticum
nontoxigenic)
Habitat (Reservoir)
• Corynebacterium glucuronolyticum
• Corynebacterium jeikeium • Colonizer of the male genitourinary tract Uncertain
• Corynebacterium kroppenstedtii
• Corynebacterium macginleyi Mode of Transmission
• Corynebacterium propinquum
• Corynebacterium pseudodiphtheriticum • Endogenous strain: Access to normally sterile site
• Corynebacterium pseudotuberculosis (toxigenic)
Corynebacterium jeikeium
• Corynebacterium resistens
• Corynebacterium simulans Habitat (Reservoir)
• Corynebacterium striatum
• Corynebacterium tuberculostearicum • Colonizer: Skin microbiota of hospitalized patients,
• Corynebacterium ulcerans (toxigenic) most commonly in the inguinal, axillary, and rectal
• Corynebacterium urealyticum sites
• Dermabacter spp.
• Exiguobacterium spp. Mode of Transmission
• Leifsonia aquatica
• Listeria monocytogenes • Uncertain
• Microbacterium spp. • Direct contact: May be person to person
• Oerskovia spp. • Endogenous strain: Selection during antimicrobial
• Other Corynebacterium spp. therapy
• Rothia spp. Introduction during placement or improper care of
• Turicella otitidis intravenous catheters

Corynebacterium ulcerans
EPIDEMIOLOGY
Habitat (Reservoir)
Listeria monocytogenes
• Normal microbiota: Humans and cattle
Habitat (Reservoir)
Mode of Transmission
• Colonizer: Animals, soil, and vegetable matter;
widespread in these environments • Uncertain
• Zoonosis: Close animal contact, especially during
Mode of Transmission summer

• Direct contact: Corynebacterium pseudotuberculosis

• Human gastrointestinal tract
Habitat (Reservoir)
• Ingestion of contaminated food, such as meat and dairy
products • Normal microbiota: Animals such as sheep, goats,
• Endogenous strain: Colonized mothers may pass and horses
organism to fetus. Portal of entry is probably from
gastrointestinal tract to blood and in some instances Mode of Transmission
from blood to meninges.
• Uncertain
Corynebacterium diphtheriae • Zoonosis: Close animal contact, but infections in
humans are rare
Habitat (Reservoir)

PATARAY 12
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

Corynebacterium pseudodiphtheriticum

Habitat (Reservoir) Mode of Transmission

• Normal microbiota: Human pharyngeal and • Uncertain Rarely implicated in human infections
occasionally skin microbiota
Dermabacter spp.
Mode of Transmission
Habitat (Reservoir)
• Uncertain
• Endogenous strain: Access to normally sterile site • Normal microbiota: Human skin

Corynebacterium urealyticum Mode of Transmission

Habitat (Reservoir) • Uncertain Rarely implicated in human infections

• Normal microbiota: Human skin Turicella otitidis

Mode of Transmission Habitat (Reservoir)

• Uncertain Endogenous strain: Access to normally • Uncertain: Probably part of normal human microbiota
sterile site Mode of Transmission
Leifsonia aquatica
• Uncertain Rarely implicated in human infections
Habitat (Reservoir)
Arthrobacter spp., Microbacterium spp., Cellulomonas
• Environment: Fresh water Uncertain spp., and Exiguobacterium sp.
Corynebacterium striatum Habitat (Reservoir)
Mode of Transmission
• Uncertain: Probably environmental
• Normal microbiota: Skin Uncertain Endogenous strain: Mode of Transmission
Access to normally sterile site
• Uncertain Rarely implicated in human infections
Corynebacterium amycolatum
PATHOGENESIS & SPECTRUM OF DISEASE
Habitat (Reservoir)
• host response to C. diphtheriae consists of the
• Normal microbiota: Human conjunctiva Skin
following:
Nasopharynx
→ A local inflammation in the throat, with a
Mode of Transmission fibrinous exudate that forms the tough,
adherent, gray pseudomembrane characteristic
• Uncertain Endogenous strain: Access to normally of the disease.
sterile site → Antibody that can neutralize exotoxin activity by
blocking the interaction of the binding domain
Corynebacterium coyleae with the receptors, thereby preventing entry into
Habitat (Reservoir) the cell.
• Listeria infections occur primarily in two clinical
• Uncertain: Probably part of normal human microbiota settings:
→ in the fetus or in a newborn as a result of
Mode of Transmission transmission across the placenta or vagina
during delivery (Granulomatosis infantiseptica)
• Uncertain Rarely implicated in human infections
→ in pregnant women and immunosuppressed
Brevibacterium spp. adults
• the organism produces listeriolysin, which allows
Habitat (Reservoir) it to escape from the phagosome into the
cytoplasm, thereby escaping destruction in the
• Normal microbiota: Human Various foods phagosome.

PATARAY 13
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

PATARAY 14
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

PATARAY 15
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

CLINICAL FINDINGS: LISTERIA MONOCYTOGENES Resistant strains are rare. Listeria gastroenteritis typically
does not require treatment.

PREVENTION
Infection during pregnancy can cause abortion, premature
delivery, or sepsis during the peripartum period. Prevention is difficult because there is no immunization.

Newborns infected at the time of delivery can have acute Limiting the exposure of pregnant women and
meningitis 1 to 4 weeks later. immunosuppressed patients to potential sources such as
farm animals, unpasteurized milk products, and raw
The bacteria reach the meninges via the bloodstream vegetables is recommended.
(bacteremia). The infected mother either is asymptomatic or
has an influenzalike illness. L. monocytogenes infections in Trimethoprim-sulfamethoxazole given to
immunocompromised adults can be either sepsis or immunocompromised patients to prevent Pneumocystis
meningitis. pneumonia can also prevent listeriosis

Gastroenteritis caused by L. monocytogenes is ANTIMICROBIAL SUSCEPTIBILITY TESTING AND

characterized by watery diarrhea, fever, headache, myalgias, THERAPY
and abdominal cramps but little vomiting. Outbreaks are
usually caused by contaminated dairy products, but Definitive guidelines are established for antimicrobial therapy
undercooked meats such as chicken and hot dogs and ready- for L. monocytogenes against some antimicrobial agents.
to-eat foods such as coleslaw have also been involved. Because there is no resistance to the therapeutic agents of
choice, antimicrobial susceptibility testing is not routinely
LABORATORY DIAGNOSIS necessary.

Laboratory diagnosis is made primarily by Gram stain and

culture. The appearance of gram-positive rods resembling
diphtheroids and the formation of small, gray colonies with
a narrow zone of β-hemolysis on a blood agar plate
suggest the presence of Listeria. The isolation of Listeria is
confirmed by the presence of motile organisms, which
differentiate them from the nonmotile corynebacteria.
Identification of the organism as L. monocytogenes is made
by sugar fermentation tests.

Clinical and Laboratory Standards Institute (CLSI)

• is a short, gram-positive rod that may occur singly or in document M45 provides some guidelines for testing of
short chains, resembling streptococci Corynebacterium spp.
• trimethoprim- sulfamethoxazole
• ampicillin and gentamicin or ampicillin and
trimethoprim- sulfamethoxazole. CLINICAL FINDINGS: CLOSTRIDIUM DIPHTTHERIAE

There are three prominent complications:

• Extension of the membrane into the larynx and

trachea, causing airway obstruction.
• Myocarditis accompanied by arrhythmias and
circulatory collapse.
• Nerve weakness or paralysis, especially of the
cranial nerves.

CULTIVATION: CULTURE MEDIA

TREATMENT Corynebacterium
urealyticum on blood agar
Treatment of invasive disease, such as meningitis and sepsis,
with Tween 80 (A) and blood
consists of trimethoprim-sulfamethoxazole.
agar (B) at 48 hours. This
Combinations, such as ampicillin and gentamicin or organism is lipophilic and
ampicillin and trimethoprim-sulfamethoxazole, can also grows much better on the lipid-containing medium
be used.
Colony of Corynebacterium diphtheriae on Tinsdale agar.
Note black colonies with brown halo.

PATARAY 16
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

PREVENTION completed. Because of hypersensitivity reactions to

antibodies (proteins) produced in horses, it is recommended
The only effective control of diphtheria is through to question the patient regarding potential allergies and in
immunization with a multidose diphtheria toxoid prepared by some instances complete a skin scratch test to the forearm.
inactivation of the toxin with formaldehyde. DAT is no longer licensed in the United States, but a product
is available from the Centers for Disease Control for use in
There are currently four combination vaccines for the
other countries.
prevention of diphtheria, tetanus , and pertussis.
A single dose of intramuscular penicillin or a course of
Two of these are given to children younger than 7 years
oral erythromycin is recommended for 14 days for all
of age (DTap and DT), and two are given to older children
individuals that present with symptoms of diphtheria. In
and adults (Tdap and Td).
addition, prophylaxis may be indicated for individuals exposed
Td boosters are recommended every 10 years to maintain to diphtheria.
active protection.
Immunized contacts should receive a booster dose of
TREATMENT diphtheria toxoid; nonimmunized contacts should begin the
primary series of immunizations.
Hyperimmune antiserum produced in horses, DAT, is a
preparation of antibodies capable of toxin neutralization
before its entry into the patient’s cells. It is critical that DAT be
administered as soon as a presumptive clinical diagnosis is

PATARAY 17
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

PATARAY 18
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

PATARAY 19
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

PATARAY 20
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

PATARAY 21
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

ERYSIPELOTHRIX, LACTOBACILLUS, AND SIMILAR Trueperella bernardiae has been identified in skin abscesses,
ORGANISMS but it is unclear whether the organism is normal microbiota of
the skin or gastrointestinal tract in cows.
G E N E R A A N D S P E C I E S T O B E CONSIDERED
• Arcanobacterium haemolyticum T. pyogenes is found on the mucous membranes of cattle,
• Corynebacterium lipophiloflavum sheep, and pigs.
• Erysipelothrix rhusiopathiae
• Gardnerella vaginalis PATHOGENESIS AND SPECTRUM OF DISEASE
• Lactobacillus spp.
G. vaginalis and Lactobacillus spp. are natural inhabitants
• Trueperella bernardiae
of the human vagina. Vaginal infections with G. vaginalis are
• Trueperella pyogenes
often found in association with a variety of mixed anaerobic
• Weissella confusa
flora. Extravaginal infections are uncommon but have been
associated with postpartum endometritis, septic abortion, and
E. rhusiopathiae consists of several serovars based on cesarean birth.
peptidoglycan structure. The serovars most commonly
Lactobacillus spp. are important for maintaining the proper
associated with human infection include serovars 1 and 2.
pH balance in vaginal secretions. The organisms metabolize
Arcanobacterium spp. consists of seven species; however, glucose to lactic acid, producing an acidic vaginal pH and
only A. haemolyticum has been recovered from clinical resulting in an environment that is not conducive to the growth
specimens. of pathogenic bacteria. Lactobacilli are frequently associated
with dental caries. The organism enters the bloodstream
Arcanobacterium spp. demonstrate irregular, gram-positive during chewing, brushing teeth, and dental procedures,
rods on Gram stain. resulting in bacteremia and endocarditis.

Trueperella spp. (T. pyogenes and T. bernardiae) W. confusa is a Lactobacillus-like organism recovered in
demonstrate the same Gram stain morphology; however, they blood cultures from patients with clinical symptoms of
are Christie, Atkins, Munch-Petersen (CAMP) test negative, endocarditis and is of particular concern because the
unlike Arcanobacterium spp. Gardnerella vaginalis is the organism is vancomycin resistant.
only species in the genus, and although it is a gram-positive
bacterium, it has a much thinner layer of peptidoglycan than Erysipelothrix infections are associated with individuals
other organisms. As a result, the organism appears as a thin, employed in occupations such as fish handlers, farmers,
gram-variable rod or coccobacilli. slaughterhouse workers, food preparation workers, and
veterinarians. Infections are typically a result of a puncture
Although Lactobacillus spp. may be beneficial to the human wound or skin abrasion.
host in immunocompetent individuals, numerous species
have been isolated from serious infections in Three categories of human disease have been
immunocompromised patients. The species most frequently characterized, including localized skin lesions or cellulitis
isolated from invasive human infections include L. (erysipeloid), diffuse cutaneous infection with systemic
acidophilus, L. casei, L. fermentum, L. plantarum, L. symptoms, and bacteremia. Bacteremia results in
rhamnosus, and L. paracasei. dissemination of the organism and can manifest as
endocarditis.
Weissella confusa it is easily confused on culture media with
the organisms included in this chapter, and in rare cases, it Arcanobacterium sp. and Trueperella spp. are primarily
has been isolated associated with bacteremia and animal pathogens, but they have been associated with
endocarditis pharyngitis, septicemia, tissue abscesses, and ulcers in
immunocompromised patients. Arcanobacterium
EPIDEMIOLOGY haemolyticum is primarily associated with mild to severe
pharyngitis.
Erysipelothrix spp. are found worldwide in a variety of
vertebrate and invertebrate animals, including mammals, T. bernardiae, in particular, can be recovered from the blood,
birds, and fish. Other domestic animals that may be infected abscesses, the urinary tract, joints, the eyes, and wounds. The
include sheep, rabbits, cattle, and turkeys. organism has also been implicated in necrotizing fasciitis.

The organism may be transmitted through direct contact or T. pyogenes are typically isolated from infections in patients
ingestion of contaminated water or meat. from rural environments and has been identified in abscesses,
wounds, and blood infections.
Arcanobacterium haemolyticum is a normal inhabitant of the
mucosal membranes of cattle, sheep, dogs, cats, and pigs. Often the primary challenge is to determine the clinical
relevance of these organisms when they are isolated in
specimens from normally sterile sites.

PATARAY 22
 CLINICAL BACTERIOLOGY BSMLS-3D
Prof. Keith Ivan Jemuel Luis, RMT

PATARAY 23