GRAM POSITIVE RODS

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 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
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 Gram Positive Rods
A. Genus: Bacillus
B. Genus: Clostridium
C. Genus: Corynebacterium
D. Genus; Listeria

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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,
halophilic
• Majority are harmless, saprophytic
• 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. 4

Natural habitat: Saprophytes, widely distributed in natural

 I. B. anthracis
 B. anthracis is responsible for the disease anthrax.
 B. anthracis produces a single antigenic type of capsule and
several exotoxins.
Virulence factors
 Capsule, plasmid encoded
 Anthrax toxin (A-B toxin)
 consists three distinct components/Thermo stable
proteins
Edema factor (EF)
Lethal factor (LF)
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Protective antigen (PA)
 Pathogenesis of Anthrax toxin

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 Pathogenesis..
 B. anthracis causes anthrax which is mainly a disease of
sheep, cattle, goats and other herbivores.
 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.
 Other routs can be eating infected meat or inhalation of
spore.
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 B. anthracis…Cont’d
A. 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.
B. Pulmonary anthrax

• Caused by inhaling large numbers of B.

anthracis spores (‘woolsorters’ disease).
• Infections are usually fatal.
C.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
8
occasionally as primary anthrax meningoencephalitis.
 Laboratory Diagnosis
Specimen: fluid or pus from a local lesion, blood, and sputum.
Microscopic
• Stained smears of the specimen; 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

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 Culture

 Blood agar:
 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:
 large gram-positive rods

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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.
 Anthrax bacilli are non motile.
 Demonstration of capsule requires growth on bicarbonate-
containing medium in 5–7% carbon dioxide.
• Can be identifying using specific anthrax –bacteriophage, PCR,
rapid serological test.

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 Treatment and prevention

Treatment: penicillin, Doxycycline and ciprofloxacin.

Prevention:
 Ciprofloxacin or doxycycline used as prophylaxis
 vaccine(90% effective)
 Incinerating animals that die of anthrax, rather than
burying them, will prevent the soil from becoming
contaminated with spores.

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 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.
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 Pathogenesis…
• B cereus is an important cause of eye infections, severe
keratitis
• Typically, the organisms are introduced into the eye by
foreign bodies associated with trauma.
• B cereus has also been associated with localized infections
and systemic infections, (endocarditis, meningitis,
osteomyelitis, and pneumonia)
• the use of a medical device or intravenous drug use
predisposes to these infections
• Clinical manifestation of Food poisoning; nausea, vomiting,
abdominal cramps, and occasionally diarrhea and is self-
limiting, with recovery occurring
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 Lab diagnosis
 Suspected food (eg, rice, meat, vegitable), stool and
vomitus of patients are cultured on ordinary media.
 Smear from colonies show large gram positive sporing
bacilli
 B. cereus is motile, non-capsulate
 on blood agar;
agar beta haemolytic colonies
 on MacConkey agar:
agar non-lactose fermenting/pale colonies
 On egg-yolk agar, B. cereus gives a strong lecithinase
reaction. It rapidly liquefies gelatin stabs.

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• 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.
– gentamicin, erythromycin, vancomycin and clindamycin can
be.
Prevention-Keeping
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shifera.M food below 5 ºC/above1657
ºC
 2. Genus Clostridium
General characteristics
 Large, gram positive (-ve or variable in old cultures), rod
shaped
 Majority are motile, but C. perfrengens is non-motile
 Anaerobic, 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
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 Produce extra-cellular enzymes; Role in invasion and

 General characteristics…
 Most are saprophytes
 Found in soils, aquatic sediments, skin, intestinal tract, and
feces of animals
 Include four medically important Spp:
 C. tetani, C. botulinium, C. difficile, and C. perfrigens
 Cause tetanus, botulism, pseudo-membranous colitis, food
poisoning and gas gangrene

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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)

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 A. Clostridium tetani
General Characteristics
 Round,terminal spores within a
swollen sporangium, non capsulatd
 Drumstick/ tennis racquet appearance Virulence determinants

 Potent toxin-neurotoxin  Tetanus toxin/

 Organism multiply locally and tetanospasmin

symptoms appear remote  Plasmid

encoded
 Most strains are motile (peritrichous  Tropism for
inhibitory motor
flagella) neurons
 Horse and humans are susceptible  Block the
 cause tetanus release of inhibitory
neurotransmitters
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 Heat labile, O2
stable
 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 such as glycine, gamma aminobutayric acid
(GABA).
 Glycine normally prevents contraction of antagonistic
muscles; therefore, muscle spasms and convulsions (lockjaw)
may occur.
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• Lack of inhibitory signals to the motor neurons and constant
release of acetylcholine to the muscle fibers leads to
irreversible contraction of the muscles and spastic paralysis.
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 Clinical manifestation
– Neonatal and adult tetanus
– localized, cephalic, and generalized tetanus
– incubation period may range from 4–5 days to several 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.
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• The patient is fully conscious
and pain may be intense.
• Death usually results from
interference with the
mechanics of respiration and
Cardiac failure (55-65%).
• The mortality rate in
generalized tetanus is very high.

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Specimen Lab diagnosis
 are usually wound swabs, or excised bits of tissue from
necrotic depth of wound
Microscopy: gram positive rods/ spore; drum stick appearance
Culture
 C. tetani is a strict anaerobe with a temperature range of
14–43 ºC (37 ºC optimum).
*Blood agar:
 C. tetani produce characteristic swarming growth &
Haemolytic (alpha first followed by beta haemolysis).

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 Antitoxin test
a. In vitro hemolysis inhibition test (tetanolysin)
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.
B. In vivo mouse inoculation test (tetanospasmin).

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 Robertson’s cooked meat medium (RCMM)

• C. tetani being proteolytic turns the meat particles black and

produces a foul odor.
Prevention & control
– Active immunization with toxoids (DTP vaccine)
– Proper care of wounds contaminated with soil
– Prophylactic use of antitoxin
– Administration of penicillin

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 B. C. perfringens
 oval, sub-terminal bulging spores, Non-motile

Virulence factor:
Exotoxin, Enterotoxin, and hydrolytic substance

Based on surface antigens and major lethal toxins (,,,ι )

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.
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• C. perfringens type A1: Causes gas gangrene (myonecrosis),
anaerobic cellulitis, puerperal infection and septicaemia.
– Note: occasionally by 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 and A: Causes a severe form of
jejunitis (necrotizing enterocolitis), known as Darmbrand”
(meaning “fire bowels”) or pigbel.
 The source of infection is usually insufficiently cooked pig
meat.
 The condition is often fatal especially in young children.
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 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.
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 Lab diagnosis
 Specimen: consist of material from wounds, pus, tissue,
blood
• Microscopic examination

 The presence of large gram-positive rods in Gram-

stained
 spores are not regularly present.

Culture
 chopped meat-glucose medium and thioglycolate medium
and blood agar plates =incubated anaerobically.
 The growth from one of the media is transferred into
milk.
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 A clot torn by gas in 24 hours is suggestive of C

• Clostridium species can be tested by culturing the organism
anaerobically on lactose egg yolk medium
• 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
• 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.
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On lactose egg yolk medium, C. perfringens:
 Produces lecithinase C (alpha toxin)
 Ferments lactose
 Does not hydrolyze lipid
 Shows no proteinase activity
Nagler reaction
C. Perfringens on lactose egg yolk medium
 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.
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 Treatment, Prevention and Control

• Surgery is the mainstay of prophylaxis and treatment of

gas gangrene(Prophylactic use of the antibiotic).
• Use of antibiotics and wound hygiene are the key factors
that help in prevention of C. perfringens infections.
• penicillin may be given for prophylaxis.
• No vaccine is available against these diseases

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 C. C. botulinum

• Oval, sub-terminal endospores

• Motile (perithricous flgella)

• Spore, relatively heat resistant, with standing 100 °C for several

hours.
– (survive improper canning and growth encouraged in this anaerobic
environment)

• Heat resistance characteristics is diminished at acid pH or high

salt concentration
• Grow best in neutral or low acid

• Can found in decaying vegetation, intestinal tract of birds,

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mammals, sedments of lakes, ponds, soil.
 Virulence factors
 Botulism toxin/ neurotoxin (heat labile)
 Seven toxin types (A-G)/serotypes
 Human illness is caused by mainly Type A, B, & E, and rarely
by F
 Type A & B associated with variety of foods (Type A is the
most potent exotoxin known)
 Type C, D, and E cause botulism in other mammals
 Not all strains produce toxin
 Affect mainly peripheral nervous system
 Prefer stimulatory motor neurons
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 Weak/ flaccid paralysis
 Pathogenesis of Botulinum toxin

Botulinum toxin acts by binding to synaptic vesicles of cholinergic nerves, thereby

preventing the release of acetylcholine (Ach) at the peripheral nerve endings, including
neuromuscular junctions. This results in a lack of stimulus to the muscle fibers,
irreversible relaxation of the muscles, and flaccid paralysis.
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Food-borne Botulism (rare and usually fatal)
 Botulism is not infection but an intoxication resulting from the
ingestion of food in which C. botulism has grown (spores have
germinated) & produced toxin
 Result about 18-36 hours following ingestion of preformed toxin,

Infant botulism
 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
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 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
 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
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 Lab diagnosis
 Specimens: 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/RIA

 The antigenic type of toxin is identified by neutralization with
specific antitoxin in mice.

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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.
 Types A, B and F blacken and digest cooked meat medium
(proteolytic reaction) and produce hydrogen sulphide gas

 (but not C, D, and E).

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 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

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 Prevention and control
 Spores of C. botulinum are widely distributed in soil
 They often contaminate vegetables fruits and other foods
 When such foods are canned / preserved, they either:
must be sufficiently heated to destroy spores or boiled for 20min
before consumption
 Strict regulation of commercial canning
 Careful observation of the cans. Eg. Swelling of the cans .

Treatment: Trivalent (A, B, E,) anti - toxins are available (IV)

 Antibiotics therapy: Metronidazole with penicillin are used to
prevent multiplication of C. botulinum in the gastrointestinal
tract and in the wound thus halting production and release of
toxins.
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 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)

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 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.
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46
 Not all strains of C difficile produce the toxins.
 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.

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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

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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.

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3. Genus Corynebacterium
– non-spore-forming gram-positive bacilli

– Corynebacterium species tend to be clubbed or

irregularly shaped
– Are members of the normal flora of skin and
mucous membranes of humans.
– Other corynebacteria are found in animals and
plants.

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 – 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.

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 Corynebacterium diphtheriae
 C. diphtheriae is non-capsulate, non-motile, and non spores
former.
 Characteristically, they possess irregular swellings at one end
that give them the "club-shaped“ 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.
 Arranged in angular fashion like Chinese lettering or cuneiform
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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.

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 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.

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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.

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 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 enlarged at one end (clubshaped).

 They often appear in clusters,

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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
 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.
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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

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,
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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.
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diphtheriae.
 Biochemical tests
 All C. Diphtheriae reduces nitrate to nitrites
 Toxigenic strain of C. Diphtheriae is:
 Catalase positive
 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;
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Toxigenicity of C. diphtheriae
shifera.M
can be tested by the Elek
60
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
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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

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 Fig. Elek plate toxigenicity test.

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 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

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 4. Genus Listeria
General characteristics
• An intracellular pathogen (facultative)
• Is a small pleomorphic, coccoid, Gram-positive bacilli
• More than twenty(20) species of listeria are recognized.
(differentiated biochemical tests)
• Almost all human listeria infection are caused by L.
monocytogenes

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 L. monocytogenes

• Gram positive non-capsulate, small rod or coccobacillus,

stains unevenly
• When seen in groups it can resemble diphtheroids.
• 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.
• At 35–37 ºC L. monocytogenes is non motile or weakly motile
• at low temperature (18–22 ºC), it is motile tumbling and
rotating motility in broth cultures.
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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

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cell membrane shifera.M 67
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.

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Clinical manifestation
• Neonatal infection

– Infection of fetus in early pregnancy results in a

condition called granulomatous infantiseptica
leading to abortion
– Characterized by the formation of dissiminated
abscess and granulomas in multiple organ

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 Lab. Diagnosis
• Specimens: cerebrospinal fluid and blood
• 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.

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****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.
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• The bacteria act as a diffraction grating, reflecting back the

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.

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