Gram-Positive Bacilli of Medical

Importance
 MLS 400 quiz 1
• Ahmed is a 43 year-old man who works in
the Tamale abattoir. His main job is removing
hide from the slaughtered cattle. Ahmed
reported to the Tamale Teaching Hospital
with a 2-cm lesion on his left arm. The lesion
began as a painless papule that enlarged
within a few days, ulcerated and formed a
black crust (eschar).
 Answer all questions in 20min on A4 sheets

1. State the bacteria likely to be the cause of Ahmed ‘s infection.

2. Briefly describe how you will obtain appropriate specimen for lab
diagnosis of the infection.
3. State the Gram staining characteristics of the likely causative agent.
4. State one selective medium and appropriate conditions you will
recommend for culturing the likely agent.
5. Briefly describe the colonial characteristics of the likely causative agent
on blood agar.
6. List the virulent factors associated with the causative agent and indicate
the role of the listed virulent factors in the pathogenesis of the infection.
 Gram positive bacilli
Differentiation base on
• Spore formation
• Staining characteristics
• Morphology
 Objectives
The objectives:
• List and describe the general characteristics of spore forming Gram positive bacilli
• Describe the sources, route of transmission, pathogenesis of Spore forming Gram
positive bacilli
• Identify the types of infections, signs and symptoms associated with Spore forming
Gram positive bacilli.
• Outline laboratory tests utilized for the diagnoses of Spore forming Gram positive
bacilli infections
– Appropriate specimen and collection
– Staining characteristics
– Appropriate media and culture conditions
– Colonial morphology
– Biochemical, serological, molecular methods of identification
 Spore-forming Gram Positive Bacilli
Two main genera:
• Genus – Bacillus
– form endospores aerobically

• Genus – Clostridium
– form endospores anaerobically

Read the structure and the composition of bacterial spores

Aerobic endospore forming bacilli

Bacillus
 Bacillus
General Characteristics Species of medical importance

• Endospore forming bacilli – Bacillus anthracis

• Motile rods – Bacillus cereus
• Aerobic and facultative anaerobes – Bacillus mycoides
• Mostly saprobic – Bacillus circulans
• Primary habitat is soil and water – Bacillus licheniformis
• Seen in medical laboratory as – Bacillus subtilis
airborne contaminants
– Bacillus megaterium

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Bacillus anthracis
 Bacillus anthracis
General characteristics
• Agent of anthrax
• Spores may remain viable for many years
in contaminated pastures, or in bones,
wool, hair, hides of livestock
• Produce extremely potent toxins
• Agent of biologic warfare Anthrax
• Highly infectious disease with High mortality
rate
• Enzootic disease (typically affects ruminants/
herbivores (such as cows, sheep, and goats)
• It is a zoonosis (transmissible from animals to
humans)

10
 Anthrax
Epidemiology
• Endemic in warmer regions of the world.
• Highest in Africa, central and southern Asia
• High in countries with poor veterinary supervision
• Higher occupational risk of exposure in men (occupational
hazard among wool sorters)
• Decline in animal and human infections (due to development of
veterinary and human vaccines)
• Where the disease is infrequent in livestock, it is rarely seen in
humans
Risk of Transmission:
Transmission
Disease does not spread from animal to animal or human to human.
In humans In livestock
Industrial anthrax
• occurring in individual employed in processing
bones, hides, wool and other animal products • Ingest of contaminated
Non-industrial anthrax • Vegetation
• in individuals who handle infected carcasses • Animal feed contaminated with bone
(At risk; Farmers, Butchers, Veterinarians) meal of infected animals
• laboratory accidents • Insects can transmit the bacterium between
• heroin-injecting drug-users (contaminate animals.
heroin)
• Deliberate release event
– Biological warfare and bioterrorism
 Transmission
Rout of transmission

Spores are inoculated through

• Traumatic (cuts/abrasions/insect
bites) skin
• Inhalation
• Ingestion
• Injection
 Transmission
The endospores of Bacillus anthracis are the infectious particles of anthrax.

In the host

• B. anthracis germination is induced by specific amino acids and nucleoside

combinations that are recognized by a family spore germination gene gerA-like
environmental sensor
• Activation of the germinant sensors is believed to be the initiating event of germination.
• Processes that follow involve
• hydration of the core
• expulsion of cations and dipicolinic acid
• breakup of the cortex
• onset of vegetative metabolism, including production of potent virulence factors
 Pathogenesis

Two large plasmids are essential for toxicity

Plasmid pXO2
Plasmid pXO1
• Contains genes that encodes bacteria
capsule • Contains the lef, pag, and cya toxin genes
• Poorly antigenic (aids B. anthracis in that encodes the three synergistically acting
evading host immune response to its
presence) proteins (Exotoxins)
• Prevents antibodies to deeper surface • Protective antigen(PA)
antigens from reaching those antigens • Eodema factor (EF)
• Resistant to hydrolysis by host proteolytic • Lethal (LF) factor) respectively
enzyme
• Capsule does not form under normal
aerobic culture and, in in vitro cultures
• Requires an atmosphere of elevated Co2
together with the presence in the medium of
serum
 Pathogenesis
Protective antigen (PA)
• It facilitates the transport and entry of EF and LF
• Effect of EF and LF is seen when either is combined with PA
• Eodema results from the combination of PA with EF (oedema toxin)
• Death occurs when PA and LF combine (lethal toxin)
• Use for producing protective anthrax vaccines

Eodema factor (EF) Lethal factor (LF)

• a calmodulin-dependent adenylate cyclase, • zinc-dependent protease
catalysis abnormal production of cyclic-AMP • cleaves the amino termini of six mitogen-
(adenosine monophosphate) (cAMP) activated protein kinase kinases (MAPKK)
• elevation of cAMP produces altered water and ion • disrupts the transduction of extracellular
movements in host - severe eodema regulatory signals (cell growth, maturation,
• impair neutrophil function and incapacitates cellular stress responses)
phagocytes and cytokine pathways • impairs host B and T cell immune responses
• causes endothelial cell injury with resulting
thrombosis and haemorrhage
 Diseases
The route of spore entry into the host dictates the specific pathology and severity of the disease

Anthrax – a zoonotic disease

4 forms of anthrax:
– Cutaneous - "malignant pustule”: “black sore- eschar” localized
infection (least dangerous)
– Pulmonary – "woolsorter's disease” “rag pickers” may result in
respiratory distress and death
– Gastrointestinal – ingested spores from contaminated raw meat
– Injection - direct injection of spores into tissue generally during drugs
administration
 Diseases
• Cutaneous
– accounts for about 99% of anthrax
cases worldwide
– infection results from inoculation of
endospores through a break in the skin.
– incubation; 2 to 6 days
– starts as a small papule that progresses to
a swollen vesicles.
– vesicles ulcerate to form a depressed
painless black necrotic central lesion
(Eschar)
– mortality rate is approximately 1%.
Ingestion
Diseases
• Presented in two forms:
• Oral/oropharyngeal
– lesion in the buccal cavity, on the tongue, tonsils,
or pharyngeal mucosa
– sore throat, lymphadenopathy, and oedema of the
throat and chest
• Gastrointestinal
– lesions develop anywhere in the gastrointestinal
tract
– abdominal pain, bloody diarrhoea, haematemesis,
toxaemia and sepsis
• Mortality rate is much higher than that of cutaneous
anthrax
 Diseases
• Pulmonary (woolsorter’s disease)
– The most severe, and rarest
– Inhalation of the spores.
– Resembles an upper respiratory tract
infection (colds and flu)
– Develops into a systemic infection and
progressive haemorrhagic lymphadenitis
– Develops from flulike to respiratory distress,
oedema, cyanosis, shock, and death.
– There is abnormal chest x-rays with pleural
effusion, infiltrates, and mediastinal
widening.
 Diseases
Injection .

• Results from the direct injection of the spores into tissue

• Characterized by soft tissue infection
• Associated with necrotizing fasciitis, organ failure, shock, coma, and
meningitis,
• Not associated with black eschar formation.
• Lack of eschar, severity of disease, and increased mortality rate make this
form clinically distinct from the cutaneous form
 Diseases
• Complications

– Approximately 5% of patients with anthrax develop meningitis

– The symptoms are typical of any bacterial meningitis and
develop rapidly
– Unconsciousness and death,
• Bacillus cereus
 General characteristics

• Grows in foods, spores survive cooking and reheating

• Associated with foodborne illnesses
• Cause food poisoning and opportunistic infections in
susceptible hosts
 Transmission

• Food poisoning
– ingestion of meat, poultry and vegetable soups,
– ingestion of fried rice

• Non-gastrointestinal infections
– Opportunistic infections of the eye
 General characteristics
Virulence factors

• Toxins
– Diarrhoeal
• Haemolysin BL (Hbl)
• Non haemolytic enterotoxin (Nhe) responsible for the major
symptoms
• Cytotoxin K (CytK)
– The emetic forms
• Cereulide – a heat-stable acid resistant toxin, proteolytic, produced
in food.
 Diseases
Food poisoning

Diarrhoeal syndrome Emetic syndrom

– Incubation period is 8 to16 hours – Characterized by nausea and
– Characterized by abdominal pain vomiting 1 to 5 hours after ingestion
and diarrhoea. of contaminated food
– About 25% of individuals have – Abdominal cramps and vomiting
vomiting – Diarrhoea is present in about one
– Not associated with fever third of affected individuals
– Resolves within 24 hours – Self limiting - resolves in 9 hours
 Diseases
Non-gastrointestinal infections

• Occur in immunosuppressed hosts and

postsurgical patients

• Infections include
– Opportunistic infections of the eye
(endophthalmitis, panophthalmitis, and
keratitis)
– Meningitis
– Septicemia
– Osteomyelitis
 Laboratory diagnosis
use adequate protective clothing and good laboratory practice
Specimen types
Type of infection and appropriate Specimen
• Categorized broadly into: • Cutaneous anthrax
– fresh specimens from untreated – vesicular fluid underneath of the Eschar
animals or humans • Inhalation anthrax
– specimens from old and • Blood, CSF and Nasal swab
decomposed animal carcasses • Ingestion anthrax
or from animal products
• Pleural, Ascitic, Peritoneal fluid
– environmental specimens
• Food poisoning
including those from suspected
deliberate release events • Suspected food, vomitus
 Lab diagnosis
Microscopy

• In blood, tissues or lesion smears, appear two to a

few cells in length

• In smears made from in vitro cultures, they appear

as endless strings of cells
– Spores are central/subterminal, ellipsoidal and
do not swell the vegetative cell
– In Gram stained, spores appear as unstained
area within the cell.
– Malachite green/safranin (or malachite
green/basic fuchsin)- spore appear green and
the vegetative forms pink Gram positive, blunt-ended bacilli, Spores
– Ziehl-neelsen- spore appear pink and the are oval and centrally located
vegetative forms are blue.
 Laboratory diagnosis
Culture

• Specimen processing (selection procedure)

heat (62° to 65°C for 15 to 20 minutes) or

alcohol shock prior to plating on solid media.
– removes contaminating organisms, and
only the spore forming bacilli survive.
– culture along with untreated specimen
– increases the chance for laboratory
isolation of the organisms
 Lab diagnosis
• Routine culture media for specimen
from sterile body sites
– 5% sheep blood agar
– Chocolate agar
– Nutrient agar

• Colonies on blood agar

– large, grayish, raised
– Non-haemolytic
– Fingerlike/irregular border “Medusa
head”/ Fringed edges “beaten egg
whites”/ground glass colonies
• Non-motile
 Lab diagnosis – culture

• Selective media for contaminated samples (stool)

– B. anthracic Columbia agar with nalidixic acid and colistin (CNA)
– Phenylethyl alcohol agar (PEA)
– Polymyxin-lysozyme-EDTAthallous acetate (PLET)
– B cereus mannitol, egg yolk, and polymyxin B agar (MEYP or MYP);
polymyxin B, egg yolk, mannitol, bromthymol blue (PEMBA)
– B. cereus medium (BCM)
 Laboratory Identification: Bacillus spp
Characteristics B. anthracis B.cereus

Hemolysis on = +
BAP
Motility = +

String of pearls + =

Growth on PEA = +

Gelatin hydrolysis = +

Susceptibility to Susceptible Resistant

Penicillin (10U/ml)
 Lab diagnosis
• Serodiagnosis
– Indirect hemagglutination and enzyme-linked immunosorbent assays (
ELISA)
– M‘Fadyean (capsule) test

– detection of
• B. cereus toxin in food and stool
• antibodies to B. anthracis

– Laboratory animals
– PCR detection systems
 Control and Treatment

• Chemotherapy
– Treat with penicillin, tetracycline, or ciprofloxacin
• Vaccines
– live spores and toxoid to protect livestock
– purified toxoid; for high risk occupational and military personnel

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 Prevention

– A cell-free vaccine is available for workers in high risk occupations

– Post exposure prophylaxis with ciprofloxacin or doxycycline is
recommended
– Autoclaving is the most reliable means of decontamination
Anaerobic endospore bacilli

Clostridium
 Clostridium
General characteristics

• Obligate anaerobes
• Large gram-positive rods
• Most species are motile
 Clostridium
Classification
Lesions and clinical manifestation
Species
• The genus Clostridium comprises nearly Histotoxic Clostridia
100 species sub-divided into C. chauvoei, C. novyi, C. bifermentans, C.
– non-pathogenic septicum, C. hemolyticum,
– minor pathogenic C. perfringens .
major pathogenic species. Neurotoxic Clostridia
C. botulinum and C. tetani
Pathogenic spp of medical importance Enterotoxic Clostridia
– C. perfringens C. perfringens, C. ramosum, C. difficile, C.
septicum, C. sordellii)
– C. botulinum
– C. tetani
– C. difficile
 Clostridium
Habitat and Transmission
• Found in soil, sewage, and freshwater and saltwater sediments
• Part of the intestinal flora in humans and other mammals

acquired through
• exogenous anaerobic infections
– spores enter through open wounds and
germinate in vivo (tetanus, gas gangrene, and
wound botulism)
exogenous intoxications
– consumption of contaminated food (foodborne
disease in C. perfringens)
• endogenous origin
– In antibiotic-associated pseudomembranous
colitis caused by C. difficile
• Clostridium perfringens
 General characteristics

General characteristics
• large, straight rods with blunt ends
• non motile
• occur singly or in pairs
• spores are large oval and central to
subterminal, swell cell; large boxcar
shapes.
 Habitat and Transmission
Transmission
Habitat
ingestion of enterotoxin-producing
• found worldwide strains in
• ubiquitous in nature
• vegetative form are part of the – contaminated improperly
normal flora of the vagina and prepared and handled foods
gastrointestinal (GI) tract – contamination of wounds,
• spores are found in soil through trauma or surgery
– inhalation of Clostridium
perfringens bacilli
 Pathogenesis
• Virulence factors
– Capsules
– Toxins
• Exotoxins
• Enterotoxins
• Hydrolytic enzymes

The disease processes initiated by C. perfringens result from a

combination of infection with the production of exotoxins and/or
enterotoxins and degradative enzymes
 Pathogenesis
• C. perfringens strains are grouped, A
through E on the basis of their spectrum of
exotoxins
• Type A strains, which produce both alpha
toxin and enterotoxin, are responsible for
most human clostridial infections
• Two types of food poisoning strains
– type A, a relatively mild and self-
limited GI illness,
– type C, (enteritis necroticans) a more
severe but rarely seen disease. produce
β-toxin but less, α-toxin.
 Pathogenesis
• Exotoxins (alpha toxin)
• Alpha toxin is a lecithinase (phospholipase C) that degrades lecithin in
mammalian cell membranes, causing lysis of endothelial cells,
erythrocytes, leukocytes, and platelets. Hemolytic or other cytotoxic and
necrotic effects, either locally or when dispersed in the bloodstream
• Enterotoxin
• (heat-labile protein) acts in the lower portion of the small intestine. The
molecule binds to receptors on the epithelial cell surface disrupting ion
transport and leading to loss of fluid and intracellular proteins
• Degradative enzymes
• (hydrolytic enzymes) proteases, DNases, hyaluronidase, and collagenases,
liquefy tissue and promote the spread of infection
• Myonecrosis (gas gangrene)
Diseases
– Production of enzymes that break down ground substance facilitates the spread of
infection. Fermentation of tissue carbohydrates yields gas, and an accumulation of
gas bubbles in the subcutaneous spaces
• Anaerobic cellulitis:
– infection of connective tissue in which the spread of bacterial grow along fascial planes
(fasciitis) does not involve invasion of muscle tissue
• Food poisoning
– nausea, abdominal cramps, and diarrhea occurs eight to eighteen hours after eating
contaminated food. Fever is absent . self-limited, with recovery within one to two days
• Enteritis necroticans
associated with severe abdominal cramps. Necrotic inflammation of the small intestines
(necrotizing bowel disease) bloody diarrhea, high mortality (>50%)
• Endometritis
• complication of incomplete abortion, or the use of inadequately sterilized instruments.
Gangrenous infection of uterine tissue is followed by toxemia and bacteremia.
 Diseases

Anaerobic cellulitis and Myonecrosis

Bullae (fluid-filled blisters), serous discharge, discolouration, and tissue necrosis
 Laboratory diagnosis
• Specimen
– Tissue, food and stool

• Gram stain
– usually vegetative forms, no
spores
– short and fat Gram-positive
rods in clinical specimen
 Laboratory diagnosis
• Culture
– anaerobically on blood agar
– grows rapidly, producing
colonies with a unique
double zone of haemolysis

Read
Clostridium botulinum
 General characteristics

– causes botulism (flaccid paralysis) (a Neurotoxins exist as macromolecular

food poisoning condition ) complexes known as progenitor toxins
– occurs in several clinical forms • Consist of a neurotoxin subunit
– caused by the action of a (derivative toxin) and one or more
neurotoxin nontoxic subunits
– toxin is produced during vegetative • Seven antigenic botulinum toxins; A –
growth within the canned food and G (human disease is almost always
ingested preformed caused by types A, B, or E)
Epidemiology
– found worldwide
Clostridium spp. that produce botulinal
neurotoxin
– types A and B are predominant in the
soils of the continental United States
– type E most common in soils of northern
Europe
– type F from marine sediments from the
Pacific Coast
– type G from soil samples in Argentina

– outbreaks frequently occur in families or

other eating groups
 Pathogenesis
Mode of action of toxins
– toxin absorbed from the gut are carried via the blood/lymph
– toxin molecule are recognized by receptors on the nerve ending and
bind to the nerve ending
– the portion of the toxin molecule is internalized into the nerve cell
– the internalized toxin fragment act to prevent
acetylcholine release from the nerve ending
– this then blocks muscle response to a nerve impulse leading to
paralysis
 Diseases
• Regardless of the manner in which toxin gains access to the body system
(ingestion of preformed toxin or elaboration of toxin by the organism in a
wound or the gastrointestinal tract), the mechanism of paralysis is identical
and the manifestations of the disease are generally the same.

• five clinical categories of botulism

– foodborne botulism
– wound botulism
– infant botulism
– adult infectious botulism
– accidental following botulinum toxin injection
 Diseases
Food borne

• occurs 18 to 36 hours after exposure

• nausea, vomiting, abdominal cramps
or diarrhoea
• Dry mouth, blurred vision, and
diplopia
• dysphonia, dysarthria, dysphagia, and
peripheral muscle weakness
• Symmetric descending paralysis
 Diseases
• Infant botulism:
– often associated with ingestion of
contaminated honey
– organism colonizes the large bowel of
infants 3 to 24 weeks of age; the toxin
produced is slowly absorbed
– the first clinical sign is usually diminished
gastro-intestinal motility (constipation)
– cause floppy baby syndrome
– weakness is symmetrical and descending
– sudden infant death syndrome
 Diseases
Wound botulism
– occurs 4 to 14 days after
exposure
– a rare form of botulism
– occurs when a wound becomes
contaminated with the organism,
and toxin is absorbed from that
site
 Diagnosis
Clinical • confirmed by detecting toxin in
• patient's signs and symptoms patient serum or stool or in food
• food history sample
• epidemiological evidence. • isolation of organism from a
wound or faecal specimen
• the mouse bioassay
(Read)
 Laboratory diagnosis
Specimen
– serum, stool, and food.
Direct identification
• Gram
– Gram-positive straight rods
– occur singly or in pairs
– spores usually subterminal
and resemble a tennis racket
• Identification of toxins in
– serum, stool, and food
Lab diagnosis
 Treatment and prevention

– administer Antitoxin as soon as possible to neutralize

unbound botulinum toxin
Clostridium tetani
 Habitat and transmission

Habitat Transmission
• The spores are found in soil, The most typical focus of the infection is a
human and animal faece globally. Puncture wound
• Disease is rare in developed
countries due to widespread – Spores introduced into small wounds via
immunization contaminated soil
• Disease seen most often in older Neonatal tetanus
individuals who have not received
their immunization boosters – spores enter through a contaminated
regularly, and whose immunity umbilicus or circumcision wound
has waned
 Pathogenesis
• Toxin spreads from the infected site by diffusing
Incubation period into the adjacent muscle tissues transported by
– varying from four days to several the lymphatic system into the blood stream
weeks. • It binds to ganglioside receptors on the nerve
ending, and a fragment of the bound toxin is
– shorter incubation period is
taken into the nerve cell
usually associated with more severe
• Blocks release of inhibitory mediators (e.g.,
disease and wounds closer to the
glycine and γ-aminobutyric acid [GABA]) at
brain
spinal synapses
• Causes severe prolonged muscle
Tetanus toxin (tetanospasmin) spasms(uncontrolled stimulation of the
produced by the vegetative cells. muscles)
 Diseases
Tetanus
• a generalized or localized hypertonia The disease can be
of the striated musculature frequently • mild, with good response to treatment
accompanied by clonic paroxysmal with sedatives and muscle-relaxing drugs
muscular spasms or contractions - very low mortality rate
– Localized tetanus involves muscle • severe, with moderate response to drugs
rigidity and spasms near the site of - a 20 to 40% fatality rate
the infected wound. • very severe, with poor response to
– Generalized tetanus, affect the treatment and a 50 to 90% fatality rate
entire body.
Usually the initial manifestation of External stimulus;
tetanus is generalized. noise or bright light precipitates a painful
spasm, and convulsions
 Diseases
Trismus
– spastic paralysis, muscle spasms
often first involve the site of
infection
– In the early stages of the disease
• rigid contraction/spasm of the
jaw muscles so that the mouth
cannot open (trismus
/lockjaw• )
 Diseases

Risus sardonicus/rictus grin

• an abnormal, sustained spasm
of the facial muscles that
appears to produce grinning
 Diseases

• Opisthotonos
– spasm of the strong extensor
muscles of the back causing
the head and lower limbs to
bend backward and the trunk
to arch forward.
 Diagnosis
• Clinically
– unique, recognizable, classic signs.
• Toxicity and neutralization tests
• Gram
.
 Laboratory diagnosis
– Gram positive
– occur singly or in pairs
– spores oval and terminal or
subterminal (drumstick or
tennis racket appearance)
– spores are common in soils
 Laboratory diagnosis
• Confirm bacteriologically by isolation from
the infected wound.
– They are motile
– Optimal growth occurs at 370C
– swarm
– Media containing 3 to 4%
agar are more conducive to formation of
discrete colonies.
– On blood agar, the colonies are flat,
translucent, and grey with a matted
surface, with a narrow zone of clear
(beta-type) hemolysis.
• Colonies have irregular and rhizoid margins
 Management
• Treatment:
– Prompt administration of antitoxin to neutralize any toxin not yet bound to
neurons is the first order of treatment.
– Treatment with human hyperimmune globulin (tetanus immune globulin)
– The organism is sensitive to penicillin
– debridement of necrotic tissue at the entry wound.
• Prevention:
– Active immunization with tetanus toxoid (formalin-inactivated toxin) prevents
tetanus
– It is usually administered to children as a triple vaccine with diphtheria toxoid
and pertussis antigens (DPT).
Clostridium difficile
 General characteristics
– Gram-positive rods in chains (up to six cells )
– spores are oval and subterminal
– motile
– liquefies gelatin but nonproteolytic
– negative for lecithinase and lipase
 Habitat and Transmission
Habitat Transmission
– a minor component of the Endogenous
normal flora of the large – antimicrobial drugs have been reported as
intestine in about 5% of predisposing factor(clindamycin,
individuals ampicillin, and the cephalosporins)
– responsible for about one forth – Proliferates when antimicrobial treatment
of antibiotic-associated suppresses more predominant species in the
diarrheas (AAD) in gut
hospitalized patients
– responsible for almost all Exogenous
cases of pseudomembranous – among hospitalized patients and
colitis (PMC). occasionally on the hands of hospital
personnel.
 Pathogenesis
– Pathogenic strains produce two
• The pseudomembranous exudate
toxic polypeptides, designated A and
B. • composed of mucus, fibrin,
inflammatory cells and cell debris
– Toxin A is an enterotoxin that
causes excessive fluid secretion and overlying an ulcerated epithelium
stimulates an inflammatory
response, • The severity of disease varies widely
– Toxin B is a cytotoxin, disrupts from mild diarrhoea through varying
protein synthesis and causes degrees of inflammation of the large
disorganization of the epithelial intestine to a fulminant
cytoskeleton. pseudomembranous coilitis
Diseases
 Diagnosis
• Clinical • Gram
– pseudomembranes during – Gram-positive straight rods in
rectosigmoidoscopy chains (up to six cells)
• Presence of C. difficile toxins in a – spores oval and subterminal
stool sample.

• Tissue culture cytotoxicity assays

• Anaerobic culture
 Management

– Discontinuance of the predisposing drug

– Fluid replacement usually lead to resolution
– Oral administration of metronidazole or vancomycin
 Lab diagnosis of anaerobes
• Specimen collection
– characteristics that strongly indicate the presence of anaerobes: foul
odour, sulfur granules, accumulation of gas
– best obtained by tissue biopsy or by aspiration of pus using a needle
and syringe
– specimens should not be refrigerated and, the amount of time they
remain at room temperature should be minimized
– specimen should be injected into an oxygen-free transport tube or vial
 Processing
• Macroscopic examination of specimen
– foul-smelling, necrotic tissue or exudate black, sulfur granules
– vortex to ensure even distribution of the material

• Microscopic examination (Gram staining)

– smear should be air-dried and fixed with methanol
– use 0.1% basic fuchsin as the counterstain
– multiple distinct morphologic forms (polymicrobic infection)
– presence of leukocytes (inflammatory response)
 Processing
Inoculation
– non selective media 5% sheep blood, MacConkey, and chocolate
agar plates
– media not exposed to oxygen (Prereduced, anaerobically
sterilized (PRAS)plated media)
– thioglycollate or cooked meat broth and placed immediately into
an

fresh plated media should be stored in an anaerobic chamber,

anaerobic jar, anaerobic bags or pouches
 Processing
Incubation
• Anaerobic chambers contain
• a catalyst – palladium-coated alumina
pellets, removes residual oxygen
• a desiccant – Silica gel absorbs water
• anaerobic gas (5% hydrogen, 5% to
10% CO2, and 85% to 90% nitrogen)
• an oxidation-reduction indicator-
methylene blue or resazurin
 MLS 400 quiz 1
• Ahmed is a 43 year-old man who works in
the Tamale abattoir. His main job is removing
hide from the slaughtered cattle. Ahmed
reported to the Tamale Teaching Hospital
with a 2-cm lesion on his left arm. The lesion
began as a painless papule that enlarged
within a few days, ulcerated and formed a
black crust (eschar).
 Answer all questions in 20min on A4 sheets

1. State the bacteria likely to be the cause of Ahmed ‘s infection.

2. Briefly describe how you will obtain appropriate specimen for lab
diagnosis of the infection.
3. State the Gram staining characteristics of the likely causative agent.
4. State one selective medium and appropriate conditions you will
recommend for culturing the likely agent.
5. Briefly describe the colonial characteristics of the likely causative agent
on blood agar.
6. List the virulent factors associated with the causative agent and indicate
the role of the listed virulent factors in the pathogenesis of the infection.