CHAPTER - 3

GRAM POSITIVE RODS

 ACKNOWLEDGMENT
• ADDIS ABABA UNIVERSITY
• JIMMA UNIVERSITY
• HAWASSA UNIVERSITY
• HARAMAYA UNIVERSITY
• UNIVERSITY OF GONDAR
• AMERICAN SOCIETY OF CLINICAL PATHOLOGY
• CDC- Ethiopia
 Learning objective:

At the end of this chapter the students will be able to:

– List the medically-important species of Gram positive rods
– Describe general characteristics of Gram positive rods
– Recognize diseases caused by Gram positive rods
– Describe the virulent factor of pathogenic species of Gram
positive rods
– Discuss pathogenicity, clinical manifestations, laboratory
diagnosis, prevention & control of members of the Gram
positive rods
Gram Positive Rods
– It includes
1. Genus: Bacillus
2. Genus :-Clostridium
3. Genus:- Corynebacterium
4. Genus Listeria
1. Genus Bacillus:
General characteristics
– Large gram positive (young/fresh culture) bacilli with square
end.
– Aerobic or facultative anaerobe
– Spore former
– Include thermophilic,psychrophilic, acidophilic, alkalophilic,
halotolerant, halophilic
– Majority are harmless, saprophyticus
– Some opportunistic or obligate pathogens of animals are
present
– At least 48 species are known but only B. anthracis and B.
cereus cause disease in humans.
Natural habitat
– Saprophytes, widely distributed in natural environment
(mainly in soil and water)
– Contaminants of operating rooms and surgical dressings,
pharmaceutical products and dried foods
I. B. anthracis
B. anthracis produces a single antigenic type of capsule and
several exotoxins.
B. anthracis is responsible for the disease anthrax.
Virulence factors
Capsule, plasmid encoded
Anthrax toxin (A-B toxin)
consists three distinct components/Thermo stable
proteins
Edema factor (EF)
Lethal factor (LF)
Protective antigen (PA)
Pathogenesis
B. anthracis causes anthrax which is mainly a disease of
sheep, cattle, goats and other herbivores with humans
becoming infected only after coming into contact with
infected animals or their skins.

Human infections (zoonoses) can occur from handling

infected animals or coming into contact with skins containing
anthrax spores
e.g. when using skins as clothing, water-carrying containers,
or sleeping mats.

Other sources of infection include animal hair, bones and

the bedding of infected animals.

Less commonly, infection is caused by eating infected

meat.
 B. anthracis…Cont’d
Depending on the source and site of infection, B. anthracis can cause:
Cutaneous anthrax (commonest form):
– Bacilli enter damaged skin, producing a blister (‘malignant
pustule’) which usually ulcerates and eventually forms a dry black
scab surrounded by oedema.
– Fatal septicaemia, toxaemia, and meningoencephalitis may
develop, especially in non-immune persons. Ocular anthrax may
also occur.
Pulmonary anthrax:
– Caused by inhaling large numbers of B. anthracis spores
(‘woolsorters’ disease).
– Infections are usually fatal.
Enteric anthrax:
– A severe form of gastroenteritis with fever, abdominal pain and
bloody diarrhoea, due to ingesting infected meat.
– Septicaemia often develops.
– Meningoencephalitis: Usually as a complication of septicaemia
and occasionally as primary anthrax meningoencephalitis.
Clinical Findings
– Skin infection (malignant pustule)
– Mediastintis, sepsis, meningitis
– Hemorrhagic pulmonary edema
– Hemorrhagic pneumonia
 Laboratory Diagnosis
Specimen: fluid or pus from a local lesion, blood, and
sputum.
Microscopic
• Stained smears from the local lesion or of blood from
dead animals often show chains of large gram-positive
rods.
• Loeffler’s polychrome (McFadyean) methylene blue
stained smear
– Square ended blue-black bacilli surrounded by a
pink/purple capsule

• Anthrax can be identified in dried smears by

immunofluorescence staining techniques.
Culture
When grown on blood agar plates, the organisms produce
non-hemolytic gray to white mucoid colonies with a rough
texture and a ground-glass appearance.

Comma-shaped outgrowths (Medusa head appearance) may

project from the colony.

Gram stained smear from culture shows large gram-positive

rods.

 Grow on nutrient medium under aerobic/anaerobic

conditions
Biochemical test:
Ferment glucose, maltose, sucrose, trehalose, and
dextrine, with acid production but not gas.

Positive for Nitrate reduction test, Catalase test, starch

hydrolysis, VP and gelatine liquefaction.

In semisolid medium, anthrax bacilli are always non motile,

whereas related nonpathogenic organisms
(eg, B. cereus) exhibit motility by "swarming."

Demonstration of capsule requires growth on bicarbonate-

containing medium in 5–7% carbon dioxide.
Lysis by a specific anthrax -bacteriophage may be helpful
in identifying the organism.
Serological test
ELISA measure antibodies against edema and lethal
toxins,
Acute and convalescent sera obtained 4 weeks apart
should be tested.
A positive result is a fourfold change or a single titer of
greater than 1:32.
Prevention & control

• Disposal of animal carcasses by burning or by deep burial

in lime pits

• Decontamination of animal products

• Wearing of protective clothes & gloves for handling

infected materials.
Eg. Animal’s hair, hide bone

• Immunization of domestic animals with live attenuated

vaccines.

• Immunization of individuals with high occupational risk

E.g. Leather factory
 II. Bacillus cereus

• B. cereus is predominantly responsible for food poisoning in

humans
• B cereus produces toxins that cause disease that is more an
intoxication than a food-borne infection.

Pathogenesis
• B. cereus produces enterotoxins that causes food poisoning
• It causes two distinct forms of food poisoning
– the emetic type, associated with fried rice
– the diarrheal type, associated with meat dishes and sauces.

• B cereus is an important cause of eye infections, severe

keratitis, endophthalmitis, and pano phthalmitis.
• Typically, the organisms are introduced into the eye by foreign
bodies associated with trauma.

• B cereus has also been associated with localized infections and

with systemic infections, including endocarditis, meningitis,
osteomyelitis, and pneumonia; the presence of a medical device
or intravenous drug use predisposes to these infections.

• Occasionally B. cereus causes opportunistic infections in

immunocompromised persons,
– e.g. pneumonia, bacteraemia, wound infections.

Clinical manifestation
Food poisoning caused by B. cereus is manifested by nausea,
vomiting, abdominal cramps, and occasionally diarrhea and is
self-limiting, with recovery occurring within 24 hours.
 Lab diagnosis

Suspected food (eg, rice, meat, vegitable) and vomitus of

patients are cultured on ordinary media.
Smear from colonies show large gram positive sporing bacilli
B. cereus, unlike B. anthracis, is motile, non-capsulate,
and produces haemolytic colonies on blood agar.
The organism is non-lactose fermenting, producing pale
colonies on MacConkey agar.
On egg-yolk agar, B. cereus gives a strong lecithinase
reaction. It rapidly liquefies gelatin stabs.
 Mannitol egg-yolk phenol-red polymyxin agar (MYPA) is
recommended as a selective medium for the isolation of B.
cereus from faeces, vomit, or food.
After overnight incubation at 35–37 ºC, large 3–7 mm flat,
dry grey-white colonies surrounded by an area of white
precipitate are produced.

Treatment
– B. cereus produces beta-lactamase and is resistant to
penicillin and cephalosporins.
– Antimicrobials with activity against B. cereus include
gentamicin, erythromycin, vancomycin and clindamycin.

Prevention and control

– Rice should not be kept warm for a long period of time
 2. Genus Clostridium
General characteristics
Large, gram positive (-ve or variable in old cultures)
Majority are motile, but C. perfrengens is non-motile
Anaerobic, rod shaped, spore forming (central, sub-terminal
or terminal spores),
Ferment organic compounds
Acid, alcohols, gas production during fermentation of
sugars
Foul smelling products from fermentation of aminoacids
and fats
Produce extra-cellular enzymes
Degrade biological molecules
Role in invasion and pathogenesis
play an important role in nature in biodegradation and the
carbon cycle
Cause tetanus, botulism, pseudo-membranous colitis, food
poisoning and gas gangrene
Most are saprophytes
Found in soils, aquatic sediments, skin, intestinal tract, and
feces of animals
Include four medically important Spp:
C. botulinium, C. tetani, C. perfrigens and C. difficile
Classification
– Based on shape and position of spore
– Based on biochemical properties
• Predominantly saccharolytic Clostridia (eg.C.
perfringens)
• Predominantly proteolytic Clostridia (eg. C. butulinum
A,B,F)
• Slightly proteolytic Clostridia (eg. C. tetani)
• Sacchrolytic Clostridia (eg. C. butulinum C,D,E)
• Neither proteolytic nor saccharolytic Clostridia (eg, C.
cochlearua)
• NB. Saccharolytic: capable of breaking the glycosidic
bond in saccharide.
 A. Clostridium tetani
Characteristics
Terminal spores within a swollen sporangium
Drumstick/ tennis racquet appearance
Potent toxin-neurotoxin
Organism multiply locally and symptoms appear remote
Most strains are motile (peritrichous flagella)
Horse and humans are susceptible,
cause tetanus
Virulence determinants
Tetanus toxin/tetanospasmin
Plasmid encoded
Tropism for inhibitory motor neurons
Block the release of inhibitory neurotransmitters
Heat labile, O2 labile
Pathogenesis

Tetanus results from trauma or a puncture wound leading to

tissue contamination.
Tetanus is a non-invasive disease occurring because of the
release of exotoxins.

C. tetani produces a spasmogenic toxin that fixes to

gangliosides thereby blocking the release of the
neurotransmitter glycine.

Glycine normally prevents contraction of antagonistic

muscles; therefore, muscle spasms and convulsions
(lockjaw) may occur.

Cardiac failure can lead to death in approximately 55-65% of

affected persons.
Clinical manifestation

– Neonatal and adult tetanus

– incubation period may range from 4–5 days to as many
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.
– Any external stimulus may precipitate a tetanic
generalized muscle spasm.
– 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.
 Lab diagnosis

Specimen
are usually wound swabs, or excised bits of tissue from
necrotic depth of wound
Microscopy
Gram stain : drum stick appearance
Culture
C. tetani is a strict anaerobe with a temperature range of 14–
43 ºC (37 ºC optimum).
Blood agar:
When isolated (only very occasionally), C. tetani
produces a fine film of feathery growth.
Use a hand lens to examine the plate.
On fresh blood agar, C. tetani is haemolytic (alpha first
followed by beta haemolysis).
Antitoxin test
If growth occurs on blood agar, subculture on a blood
agar antitoxin plate (half the plate covered with
antitoxin).
Incubate the plate anaerobically.
The haemolysis produced by C. tetani is inhibited by
the antitoxin.
Robertson’s cooked meat medium (RCMM):
– C. tetani is slowly proteolytic.
– If clostridial growth occurs (check a Gram smear), divide the
culture and heat one half at 80 ºC for 30 minutes and cool.
– Subculture both the unheated and heated cultures on fresh
blood agar and incubate anaerobically.
– Culture in cooked meat broth produce blackening of meat
and foul smelling product.
Prevention & control
– Active immunization with toxids
– Proper care of wounds contaminated with soil
– Prophylactic use of antitoxin
– Administration of penicillin
 B. C. perfringens

oval, sub-terminal and non-bulging spores

Non-motile bacteria
Virulence factor:
Exotoxin, Enterotoxin, and hydrolytic substance
Based on surface antigens and major lethal toxins (,,,)
produced, five types of C. perfringens (A–E) are recognized.
Pathogenesis:
Human disease is caused by types A and C (other types
cause disease in
animals).
All types of C. perfringens produce alpha toxin.
Type B and C strains also produce beta toxin.
C. perfringens type A1: Causes gas gangrene
(myonecrosis), anaerobic cellulitis, puerperal infection and
septicaemia.

Note: Other species of Clostridium that occasionally

cause gas gangrene include C. novyi and C.
septicum.

C. perfringens type A2: Causes food-poisoning, usually

within 8–12 hours of ingesting contaminated meat.

C. perfringens type C: Causes a severe form of jejunitis

(necrotizing enterocolitis), known as pigbel.
The source of infection is usually insufficiently cooked
pig meat.
The condition is often fatal especially in young
children.
 Clinical features

Wound infection and gas gangrene/clostridial myonecrosis

the infection spreads in 1–3 days to produce
crepitation in the subcutaneous tissue and muscle,
foul-smelling discharge,
rapidly progressing necrosis,
fever, hemolysis, toxemia, shock, and death.
Lab diagnosis
Specimen: consist of material from wounds, pus, and
tissue.
Microscopic examination
The presence of large gram-positive rods in Gram-
stained smears suggests gas gangrene clostridia;
spores are not regularly present.
Culture
Material is inoculated into chopped meat-glucose
medium and thioglycolate medium and onto blood agar
plates
incubated anaerobically.
The growth from one of the media is transferred into
milk.
A clot torn by gas in 24 hours is suggestive of C
perfringens.
 Biochemical tests

• Clostridia are catalase and oxidase negative.

• The most useful biochemical reactions which help to identify C.
perfringens and other pathogenic Clostridium species can be
tested by culturing the organism anaerobically on lactose egg
yolk medium
• This medium tests for lecithinase C activity, lipase
hydrolysis, lactose fermentation and proteinase activity.

– Lecithinase C activity: Seen as an opacity in the

medium due to the breakdown of lecithin in the egg yolk.

– Lipase hydrolysis: Seen as a pearly (fatty) layer

covering colonies and sometimes extending into the
medium A restricted intense area of opacity is also
produced in the medium.

– Lactose fermentation: There is a reddening in the

medium. The colonies become red on exposure to air.

– Proteinase activity (proteolysis): Shown by an area of

clearing around the colonies due to the breakdown of
casein in the milk by the enzyme proteinase.
On lactose egg yolk medium, C. perfringens:
Produces lecithinase C (alpha toxin)
Ferments lactose
Does not hydrolyze lipid
Shows no proteinase activity

Nagler reaction
Used for final identification of C. perfringens
It is about toxin production and neutralization by specific
antitoxin. The technique is referred to as the Nagler reaction
In medium containing lecithin, the opacity that can be
produced by C. perfringens can be inhibited by applying
specific antitoxic serum to the medium which will inactivate
the lecithinase.
C perfringens rarely produces spores when cultured on agar
 Treatment:
Penicillin
Prompt and extensive wound debridement
Polyvalent antitoxin

Prevention & Control

Early and adequate cleansing of contaminated wounds and
surgical debridement, together with the administration of
antimicrobial drugs directed against clostridia (eg, penicillin),
C. C. butulinum

– Oval, sub-terminal endospores

– Motile (perithricous flgella)
– Spore, relatively heat resistant (survive improper canning
and growth encouraged in this anaerobic environment)
– Grow best in neutral or low acid
– In decaying vegetation, intestinal tract of birds, mammals,
sedments of lkes, ponds, soil.
– 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 characteristics is diminished at acid pH or
high salt concentration.
– C. botulinum can be found in soil, water, and sewage.
Sub terminal spores

Fig. Clostridium botulinum

 Virulence factors

Botulism toxin/ neurotoxin

it is heat labile
Seven toxin types (A-G)/serotypes
Human illness is caused by mainly Type A, B, & E, and
rarely by F
Type E linked with fish products
Type A & B associated with variety of foods
Type D cause botulism in mammals ----- important!!!
Types C and D are encoded by bacteriophage that infect the
bacteria.
Not all strains produce toxin
Affect mainly peripheral nervous system
Prefer stimulatory motor neurons
Weak/ flaccid paralysis
The toxin is formed in food when C. botulinum spores
contaminate food.
Under anaerobic conditions, e.g. in tinned meat or fish
(not acid fruits), the spores germinate and the bacilli
multiply,producing toxin.
 Pathogenesis

Food-borne Botulism
Botulism is not infection but an intoxication resulting from the
ingestion of food in which C. botulism has grown (spores
have germinated) & produced toxin
rare and usually fatal
Result from an ingestion of a lethal preformed neurotoxin,
about 18-36 hours following ingestion of preformed toxin,
These protein exotoxins are often released in an inactive
form, then proteolytic cleavage activates them.
Type A is the most potent exotoxin known
These toxins block the release of the neurotransmitter acetylcholine
resulting in double vision, slurred speech, decreased saliva, difficult
swallowing and general weakness.
The toxin causes paralysis in about 20% of those affected.
Death usually occurs from respiratory failure.
Infant butulism
Rarely C. botulinum causes infantile botulism in which
the bacteria colonize the gut of infants and produce
toxin which is absorbed.
Wound botulism
The bacteria growing in the necrotic tissue of the
wound
 Clinical findings
 Sign & symptoms begin: 18 - 36 hrs after ingestion of toxic
food
 Visual disturbances (blurred vision)
 Difficulty to swallow
 Speech difficulty
 Gastrointestinal symptoms are not prominent
 No fever, dizziness and dryness of the mouth
 Descending weakness of skeletal muscles
Death occurs from respiratory paralysis or cardiac arrest
The patient remains fully conscious until shortly before
death.

N.B. Patients who recover do not develop antitoxin in the blood

In Infant botulism
constipation, weak sucking ability, generalized weakness
occur in infants at 5-20wks of age
Lab diagnosis
Specimens:
Include suspected food and patient’s faeces and serum (to
demonstrate toxin).
In infant botulism, C botulinum and toxin can be
demonstrated in bowel contents but not in serum
C. botulinum may be grown from food remains and tested
for toxin production, but this is rarely done and is of
questionable significance.
Toxin may be demonstrated by passive hemagglutination or
radioimmunoassay.
The antigenic type of toxin is identified by neutralization with
specific antitoxin in mice.
Culture and biochemical reactions

C. botulinum is a strict anaerobe.

Grows best at 30–35 ºC.

Robertson’s cooked meat medium (RCMM):

Inoculate the emulsified specimen (in 0.1% peptone water) in
several containers of RCMM.

Heat half of them at 80 ºC for 30 minutes (spores remain).

Incubate the heattreated and untreated inoculated RCMM at
35 ºC for 3–5 days.

Types A, B and F blacken and digest cooked meat medium

(proteolytic reaction) and produce hydrogen sulphide gas
(types C, D, and E do not).
Blood agar subculture from RCMM (anaerobic culture):
C. botulinum produces large semi-transparent colonies
with a wavy outline.
Most strains are beta-haemolytic.

Lactose egg yolk milk agar:

C. botulinum hydrolyzes lipid (pearly opalescence).
Types A, B, and F show proteinase activity (area of
clearing around the colonies).
Lactose is not fermented
Prevention and control
 D. Clostridium difficile

Slender bacilli - with large, oval, subterminal spores

Members of the intestinal flora
Nosocomial pathogen
Virulence factor
Two toxin: Toxin A (enterotoxin), Toxin B (extremely
lethal/cytopathic toxin)
Pathogenesis
Pseudomembranous colitis
Although many antibiotics have been associated with
pseudomembranous colitis, the most common are
ampicillin and clindamycin.
Administration of antibiotics results in proliferation of drug-
resistant C difficile that produces two toxins.
Toxin A, a potent enterotoxin that also has some
cytotoxic activity, binds to the brush border membranes
of the gut at receptor sites.
Toxin B is a potent cytotoxin.
Both toxins are found in the stools of patients with
pseudomembranous colitis.
Not all strains of C difficile produce the toxins, and the
tox genes apparently are not carried on plasmids or
phage.
 Antibiotic-Associated Diarrhea
• The administration of antibiotics frequently leads to a mild
to moderate form of diarrhea, termed antibiotic-
associated diarrhea.
• This disease is generally less severe than the classic
form of pseudomembranous colitis.
• As many as 25% of cases of antibiotic-associated
diarrhea may be associated with C difficile.
Clinical feature
• mild to moderate form of diarrhea, termed antibiotic-
associated diarrhea
• Plaques and microabscesses may be localized to one
area of the bowel.
• The diarrhea may be watery or bloody, and the patient
frequently has associated abdominal cramps,
leukocytosis, and fever
 Lab diagnosis

– detection of one or both C difficile toxins in stool

– endoscopic observation of pseudomembranes or
microabscesses in patients who have diarrhea and have
been given antibiotics.
Treatment
– The disease is treated by discontinuing administration of the
offending antibiotic and orally giving either metronidazole or
vancomycin.
3. Genus Corynebacterium

– non-spore-forming gram-positive bacilli.

– Corynebacterium species tend to be clubbed or irregularly
shaped
– These bacteria have a high guanosine plus cytosine content
– Are members of the normal flora of skin and mucous
membranes of humans.
– Other corynebacteria are found in animals and plants.
–
– Comprises the toxin-producing pathogen C. diphtheriae, C.
ulcerans and several commensals (diphtheroides) which
normally colonize the skin, nasopharynx, oropharynx, GIT
and UGT.
– Corynebacterium diphtheriae is the most important member
of the group, as it can produce a powerful exotoxin that
causes diphtheria in humans.
 Corynebacterium diphtheriae
C. diphtheriae is non-capsulate, non-motile, and does not
form spores.

Characteristically, they possess irregular swellings at one

end that give them the "club-shaped“f appearance .

Irregularly distributed within the rod (often near the poles) are
granules staining deeply with aniline dyes (metachromatic
granules) that give the rod a beaded appearance.

Individual corynebacteria in stained smears tend to lie

parallel or at acute angles to one another.

True branching is rarely observed in cultures.

Arranged in angular fashion like Chinese lettering or
cuneiform arrangement
C. diphtheriae biovars
There are four biovars (biotypes):
gravis, intermedius, mitis, and belfanti.
Originally these names were used to describe the severity of
disease.
It is now known that toxigenic and non-toxigenic strains
occur in all C. diphtheriae biovars.
In the investigation of diphtheria, it is not necessary to
differentiate these biovars.
 Virulence factor:

– All toxigenic C diphtheriae are capable of elaborating the

same disease-producing exotoxin.
– Diphtheria is caused by toxin-producing strains of C.
diphtheriae.
Pathogenesis
C. diphtheriae causes:
– Nasal, nasopharyngeal and tonsillar diphtheria, especially in
young children.
• Often there is marked oedema of the neck.
• Infection is by inhaling respiratory droplets.
– Cutaneous (skin) diphtheria
• Usually develops when C. diphtheriae infects open
wounds.
• Infection of the skin rarely leads to the serious
complications associated with diphtheria of the throat.
 Lab. Diagnosis:
Specimens:
Include throat, and, or nasopharyngeal swabs to confirm a
diagnosis of throat diphtheria, and a skin swab if cutaneous
diphtheria is suspected
Microscopy:
C. diphtheriae is Gram positive but usually stains unevenly
and weakly.
It is markedly pleomorphic.
Long, thin, and curved forms can be seen
short rods and rods enlarged at one end (clubshaped).
They often appear in clusters, joined at angles like Chinese
letters
In Albert stained smears, particularly from Loeffler serum
or Dorset egg cultures, C. diphtheriae often appears beaded
due to the presence of dark staining granules in the rods
These granules, known as volutin or metachromatic
granules, are energy-storing inorganic polyphosphate
units.
In some strains the granules form at the ends of the
rods.

In toluidine blue stained smears, the organisms stain pale

blue and the granules dark red-purple.
 Culture
C. diphtheriae is an aerobe and facultative anaerobe.
Temperature range for growth is 20–40 ºC with an optimum
of 35–37 ºC.

Loeffler serum medium and Dorset egg medium:

C. diphtheriae grows rapidly on these media, producing
significant growth in 4–6 hours.
The characteristic morphological features of C. diphtheriae,
especially granule formation, are well developed.
Tellurite blood agar:
This medium is widely used as a primary medium for
isolating C. diphtheriae from throat and nasopharyngeal
swabs.
C. diphtheriae reduces tellurite and produces grey or grey-
black colonies after 24–48 h incubation.
Commensal diphtheroid colonies are grey, non-haemolytic
Tinsdale medium:
After 24–28 h incubation,C. diphtheriae colonies are
grey-black, raised, and surrounded by a dark brown
area.
The brown colour is due to the hydrogen sulphide
produced from the cystine interacting with the tellurite.
Occasionally commensal diphtheroids and other
respiratory tract commensals may grow on Tinsdale’s
medium but the colonies are not surrounded by a
brown halo like those of C. diphtheriae.
Biochemical tests

All C. Diphtheriae reduces nitrate to nitrites

Toxigenic strain of C. Diphtheriae is:
Catalase and.
Oxidase negative.
Urease negative.
Ferments glucose and maltose with acid production
A few strains of gravis and mitis biovars ferment sucrose.
C. diphtheriae gravis ferments starch with acid production.

virulence
Toxigenicity of C. diphtheriae can be tested by the Elek gel
precipitation test.
Elek gel precipitation test method
A rectangular strip of filter paper soaked in antitoxin is
placed on the surface of a serum agar plate containing
20% horse serum while the medium is still in fluid form.

When the medium solidifies, the testing strain is

streaked across the plate at the right angles to the filter
paper strip and then incubated as 370C for 24-48hrs.

Toxin produced by bacterial growth diffused in agar

medium and produces line of precipitation where it
meets antitoxin molecules (dispersed from the filter
paper) in optimum concentration.

The line of precipitation radiates from the intersection

of the strip and the bacterial growth.

 Toxin-producing C. diphtheriae is identified by the

presence of precipitin lines and arcs of identity
Fig. Elek plate toxigenicity test.
Treatment
The treatment of diphtheria rests largely on rapid
suppression of toxin-producing bacteria by
 antimicrobial drugs (Penicillin or Erythromycin)

 early administration of specific antitoxin against the

toxin formed by the organisms at their site of entry

and multiplication.
 Diphtheria antitoxin

Prevention and control

Active immunization(DPT)
Passive immunization
Genus Listeria
General characteristics
• An intracellular pathogen
• Is a small pleomorphic, coccoid,Gram-positive bacilli
• Six species of listeria are recognized

– which are differentiated by a small number of

biochemical tests

– It includes L. monocytogenes, L. innocua, L. ivanovi, L.

welshimeri, L. sceligeri, and L. grayi

• Almost all human listeria infection are caused by L.

monocytogenes
 L. monocytogenes
• is a Gram positive non-capsulate, small rod or
coccobacillus
• often stains unevenly and is easily decolorized.
• When seen in groups it can resemble diphtheroids.
Motility:
• At 35–37 ºC L. monocytogenes is nonmotile or weakly
motile
• at low temperature (18–22 ºC), it is motile with a
characteristic tumbling and rotating motility in broth
cultures.
• Is a natural pathogen of wide range of animals, birds,
fish, tick and crustacea. The organism appear to be
saprophytic in soil.
• Common sources of infection are contaminated meats,
chicken, soft cheeses and vegetables.
Virulence factor
• Internalin (membrane protein)
– Probably facilitate ingestion of the organism by
machrophage, endothelial cells, etc.
• Listeriolysin O (haemolytic protein)
– Responsible for the disruption of the phagolysosome
membrane
– It is a major virulence factor
• Phospholipases
• Help cell to cell spread of the organism by dissolving cell
membrane
Pathogenesis
• Development of infection depends on host susceptibility, gastric
acidity, inoculum size, and virulunce factors
• L. monocytogenes causes meningitis and septicaemia mainly in
neonates, pregnant women, the elderly and immunosuppressed
persons.
• Listeriosis in pregnancy may lead to abortion and stillbirth.
Clinical manifestation
• Neonatal infection
– Infection of foetus in early pregnancy results in a condition
called granulomatous infantiseptica leading to abortion
– Characterized bt the formation of dissiminated abscess and
granulomas in multiple organ
Lab. Diagnosis
• Specimens:
– Mainly cerebrospinal fluid and blood for culture.
• In rare positive smear preparation, extra-and interacellular
gram positive bacilli are seen
Culture
• L. monocytogenes is an aerobe and facultative anaerobe.
• It is unusual in that it is capable of growth at refrigeration
temperatures.
• The temperature range for growth is 3–45 ºC with an
optimum of 30 ºC.
Blood agar:
• L. monocytogenes produces small, grey, translucent drop-
like colonies surrounded by a small zone of indistinct beta-
haemolysis
• Incubation for up to 48 h may be required to produce visible
growth.
• Check the morphology of the colonies by examining a Gram
smear.
Clear tryptose agar (or Mueller Hinton agar):
• Colonies appear pale blue-green when viewed from the side
(45 ºC angle) with a beam of white light.
• The bacteria act as a diffraction grating, reflecting back the
blue part of the spectrum.
Biochemical test

• Catalase and -methl-D-mannoside positive

• Indole, oxidase, D-xylose and urease negative.
• Ferments glucose and maltose with acid production.

Note: The characteristic motility and cultural characteristics of

L. monocytogenes are usually sufficient to identify it without
the need to use many biochemical tests.
Treatment
• Penicillin or Ampicillin, ether alone or with Gentamicin are the
drugs of choice
• Erythromycin is useful in patients allergic to penicillin
Prevention and control
• Prevention and control are difficult
• Hygienic food habit is beneficial
 REFERENCE

1. Richard A. Harvey, Pamella C. Champ, Microbiology,

Lippincott’s illustrated reviews, 2nd ed.
2. Benson’s microbiological application, Laboratory

. 2001
manual in general microbiology, 8th ed
3. Sherris, Medical microbiology, an introduction to
infectious disease. 4th ed. 2004.
4. Monica Cheesbrough, District LaboratoryPractice in
Tropical Countries Part II. 2nd ed. 2006