Gram Positive Rods

Objectives

 After completion of this session students will be able to:

1. Describe the general characteristics of gram positive rods
2. Name common pathogens among gram positive rods
3. Explain the pathogenesis, clinical features, microbiological
diagnosis & Rx of infections associated with gram positive rods

Contents
 Introduction
 Spore forming Gm+ rods: Bacillus, Clostridium
 Non spore forming Gm+ rod: Corynebacterium
Introduction
• There are four medically important genera of gram-positive
rods:
▫ Bacillus, Clostridium, Corynebacterium, and Listeria.
▫ They are not closely related, nor do they cause similar
clinical conditions.
 Intro…
1. Bacillus and Clostridium are ubiquitous, and they
form spores and can survive in the environment/soil for
many years.

2. The genus Corynebacterium includes C. diphtheriae,

the cause of the toxin-mediated disease diphtheria, as
well as several usually harmless human commensals.
▫ C. diphtheriae, that attacks the membranes of the throat
and releases a toxin that damages the heart and the
nervous system.

3. L. monocytogenes causes meningitis and sepsis in

newborns, pregnant women, and immunosuppressed
adults. It also causes outbreaks of febrile gastroenteritis.
1. THE GENUS BACILLUS
• Large, aerobic, G+ve, bacilli, occur in chain.
• Ubiquitous in nature – soil, water and airborne dust.
• Most spp of are harmless saprophytes (anthracoids).
• Important pathogens; Bacillus anthracis & B. cereus

Diarrheal/emetic type
 Bacillus anthracis
• Disease: Anthrax, primarily a disease of animals (sheep, cattle,
horses) causing septicemia & death.

▫ Bacteriology: Capsule composed of poly-D-

glutamate(aa): antiphagocytic.

• Habitat and Transmission: Habitat is soil.

▫ Transmission is by contact with infected animals or
inhalation of spores from animal hair and wool.

▫ Anthrax spores have the potential for use in biological

warfare (terrorism) because of their ability to survive and
spread easily in air and can be inhaled.
 B. anthracis

• Pathogenesis: Capsule + plasmid-coded exotoxins

consists of three proteins:

1. Edema factor (EF): an adenylate cyclase; w/c is

responsible for the severe edema.
2. Lethal factor (LF): kills cells by inhibiting a signal
transduction protein involved in cell division
3. Protective antigen (PA): mediates the entry of the
other two components into the cell.
▫ Used in the production of protective anthrax vaccines.
Infections are classified according to the portal of entry as;
•Dermal/cutanous anthrax,
•Primary inhalational anthrax, and
• Intestinal anthrax.
─Sepsis can develop from the primary infection focus.
 B. anthracis

There are 3 clinical types:

1. Cutaneous anthrax: (95% of the cases)
▫ It occurs in butchers, farmers & veterinarians.
▫ Organisms enter through small abrasions, multiply locally
producing a malignant pustule.

2. Pulmonary anthrax (wool sorter's disease):

▫ Rare disease, occurs by inhalation of spores.
▫ It occurs in persons who handle wool or animal hairs.

3. Intestinal anthrax:
▫ Very rare; organisms ingested in infected meat with invasion
and ulceration of the GI mucosa.

 From all these sites invasion of the blood stream may

occur giving rise to septicemic anthrax.
• Laboratory Diagnosis

▫ Specimens to be examined are fluid or pus

from a local lesion, blood, and sputum,
 Gram-stained smear,
 Aerobic culture on blood agar.
 B. anthracis is non-motile, in contrast
to other Bacillus species.
 Serology: rise in antibody titer is
diagnostic.

• Treatment: Penicillin G (no significant

resistance).
▫ Tetracycline, erythromycin be used in
pts with allergy to penicillin
 B. anthracis

Prevention:

1. Protective clothing & gloves for handling potentially

infected materials.
2. Disposal of animal carcases by burning or by deep burial
in lime pits.
3. Autoclaving of animal products.
4. Active immunization of domestic animals with live -
attenuated vaccines.
5. High risk persons immunized with a non-living vaccine
(Vaccine consisting of protective antigen (PA) )

▫ French biologist Louis Pasteur demonstrated that the bacteria

caused the disease and in 1881 developed an effective vaccine
against anthrax in animals.
 2. Genus Clostridium

▫ Are obligate anaerobic of greatest clinical importance.

▫ Gram-positive, blunt-ended rods.
▫ They form endospores.

• They are associated with soft tissue and skin

infections (cellulitis and fascitis), and antibiotic-
associated colitis and diarrhea.

• These organisms also synthesize some of the most

potent exotoxins known;
▫ For example, the toxins of specific clostridial species
cause botulism, tetanus, gas gangrene, and
pseudomembranous colitis.
 Clostridia…

• Clinically significant species of Clostridium include;

1. C. perfringens: causes histotoxic (tissue destructive)

infections (myonecrosis=gas gangrene) and food poisoning
2. C. tetani: causes tetanus (lockjaw)
3. C. botulinum: causes botulism
4. C. difficile: causes pseudomembranous colitis associated
with antibiotic use.

• Clostridia cannot use free oxygen as the terminal electron

acceptor in energy production.
Important Features of Clostridium Species
 Clostridium perfringens

• C. perfringens is a large, rod-shaped, nonmotile, gram-

positive, encapsulated bacillus.

• It is ubiquitous in nature, with its vegetative form as part of

the normal flora of the vagina and GIT.
• Its spores are found in soil.
• When introduced into tissue, C. perfringens can cause
anaerobic cellulitis, and myonecrosis (gas gangrene).

• Some strains of C. perfringens also cause a common form of

food poisoning.
 C. perfringens…

Pathogenesis

• C. perfringens secretes a variety of toxins;

▫ Exotoxins (twelve: alpha, beta…),
▫ Enterotoxins, and
▫ Degradative/hydrolytic enzymes (proteases, DNases,
hyaluronidase, and collagenases, which liquefy tissue and
promote the spread of infection)

• The disease processes by C. perfringens result from a

combination of infection with toxins and degradative
enzymes.
Toxins and degradative enzymes
produced by C. perfringens.
ET = enterotoxin
 C. perfringens…
Clinical significance

1. Myonecrosis (gas gangrene)

• Clostridial spores are introduced into tissue, eg. by

contamination of open wounds with infected soil, or by
endogenous transfer from the intestinal tract.

• Alpha toxin and other exotoxins are secreted and

extensive cell killing ensues.
▫ Production of enzymes that break down ground substance
facilitates the spread of infection.

• Fermentation of tissue carbohydrates yields gas, and

accumulation of gas bubbles in the subcutaneous spaces
produces a crinkling sensation on palpation (crepitation);
hence, the name gas gangrene.
 C. perfringens…

• The exudates are copious and foul

smelling.

• As the disease progresses, increased

capillary permeability leads to the
exotoxins being carried by the
circulation from damaged tissue to
other organs, resulting in systemic
effects such as shock, renal failure,
and intravascular hemolysis.

• Untreated clostridial myonecrosis is

uniformly fatal within days of the
initiation of gangrene
Gas gangrene of arm
 Other clinical findings of C. perfringens

1. Anaerobic cellulitis: This is a clostridial infection of connective tissue in

which the spread of bacterial growth along fascial planes (fasciitis) does not
involve invasion of muscle tissue.

2. Food poisoning: Typically, the onset of nausea, abdominal cramps, and

diarrhea occurs with in hours after eating contaminated food with clostridial
enterotoxin . Fever is absent and vomiting rare.

3. Enteritis necroticans: Outbreaks of a necrotizing bowel disease with high

mortality (>50%) caused by C. perfringens have been sporadically reported.

4. Clostridial endometritis: This condition is a grave complication of

incomplete abortion, or the use of inadequately sterilized instruments.
Gangrenous infection of uterine tissue is followed by toxemia and
bacteremia.
 Laboratory diagnosis

1. Gram stain, of specimens from diseased tissue usually

show vegetative clostridial forms (large, gram-positive
rods), accompanied by other bacteria and cellular debris.

2. Culture anaerobically on blood agar: C. perfringens

grows rapidly, producing colonies with a unique double
zone of hemolysis.

3. In food poisoning, the organism can be sought in

suspected food and patient's feces.
C. perfringens
A. Colonies on blood agar showing Gram stain
double zone of hemolysis. Arrow points to a large gram-
B. Photomicrograph of Gram stain positive rods
 Treatment and prevention

1. Removal of foreign material and devitalized tissue, and

exposure of the wound to O2. Hyperbaric oxygen
chambers increase the tissue O2 tension in the affected part
and probably inhibit the pathologic process.

2. If debridement is unable to control the progression of the

gangrene, amputation, when anatomically possible, is
still mandatory in gangrene.

3. Supplementary to this is the administration of antibiotics

in high dose. C. perfringens is sensitive to penicillin.
Clostridium tetani

• Found in the intestine of man, animals & in manured soil.

• It causes tetanus in man & animals.
Morphology:
• Gram-positive, long, chain bacilli with
round terminal spores giving the
characteristic ‘drum-stick’ appearance.
• They are motile.
Cultural characters:
• Strict anaerobes, grow on nutrient agar on which colonies
are surrounded by a clear zone of haemolysis due to its
tetanolysin toxin.
• The organism grows on Robertson cooked medium.
Antigenic structure:
• The neurotoxins produced by all
toxigenic strains of C. tetani are
serologically identical.
• 10 types of antigenic variation among
flagella identified.

Virulence:
• Human disease is caused by
tetanospasmin (neurotoxic
exotoxin).
• Toxins are produced by vegetative
cells of C. tetani.
• Toxin production appears to be under
a control of plasmid gene.
 C. tetani…
Transmission
• Spores are widespread in soil.
• The portal of entry is usually a wound
site, e.g., where a nail penetrates the
foot.
▫ The introduction of C. tetani spores into
even small wounds via contaminated soil
is probably a common occurrence.

• Growth of C. tetani is completely local,

but it produces a powerful neurotoxin
that is transported to the CNS, where it
causes spastic paralysis (strong
muscle contractions) .
Pathogenesis

• Tetanus toxin, called tetanospasmin, is an extremely

potent toxin (single antigenic polypeptide/A & B fragments/)

• It is transported from an infected locus by retrograde

neuronal flow or blood.
• The heavy fragment (B) mediates binding to neurons and cell
penetration of the light fragment (A).

• The A fragment blocks neurotransmitter release at inhibitory

synapses, and thus causes severe, prolonged muscle
spasms.
• Tetanospasmin estimated human lethal dose = 2.5 ng/kg.
Mechanism of tetanus toxin
 Clinical significance

• Incubation period: 4 days - several weeks.

• A shorter period is usually associated with more
severe disease and wounds closer to the brain.

• Tetanus presents as a spastic paralysis, in

which muscle spasms often first involve the site
of infection.

• Three clinical forms: local (uncommon),

cephalic (rare), generalized (most common).
▫ Generalized tetanus: descending pattern of
trismus (lockjaw), stiffness of the neck,
difficulty swallowing, rigidity of abdominal
muscles spasms continue for 3-4 weeks.
▫ Complete recovery may take months.
 C. tetani…

• In the early stages of the disease, the

jaw muscles are affected, so that the
mouth cannot open (trismus, or
lockjaw).
Open lower-leg fracture following a traffic accident; the
portal of entry of C. tetani
• Gradually, other voluntary muscles
become, and any external stimulus
(for example, noise or bright light)
precipitates a painful spasm, and
sometimes convulsions.

• Death, which occurs in 50-60%of

cases, is usually the result of paralysis
of chest muscles leading to
respiratory failure.
Tetanus involving back muscles

Opisthotonos, a pronounced arching of the back due

to spasm of the strong extensor muscles of the back.
 C. tetani…

• Injury: e.g. nail prick, surgical wound, gun

shot wound, infected umblical stump leading
to tetanus neonatorum.
▫ Neonatal tetanus, one of the cause of neonatal
mortality in developing countries is caused by
contamination of the umbilical stump.
 Laboratory diagnosis
• Diagnosis is mainly rests on the
clinical picture & history of
contaminated wounds.

Lab
▫ Gram staining : Wound exudate are
examined microscopically for the
presence of gram-positive bacilli with
drum-stick appearance.

▫ Culture : The exudate is cultured on

blood agar & incubated anaerobically,
on Robertson cooked meat medium.

▫ The organism can be identified by its

pathogenicity to laboratory animals.
 Treatment and prevention

1. Treatment
• Prompt administration of antitoxin
(TAT) to neutralize any toxin not yet
bound to neurons is the first order of Rx.

• Penicillin is given in big doses to inhibit

the growth of C. tetani & stops further
toxin production. It may also control
associated pyogenic infection.

• Debridement of necrotic tissue at the entry

wound.
2. Prevention

• Tetanus is a totally preventable disease.

• Active immunization with tetanus toxoid
(TT) (formalin-inactivated toxin) as DPT
prevents tetanus.

• Booster doses given to military personnel

before / during war, for pregnant women to
guard against labor infection & to provide
maternal immunity for the new born.

Passive immunization:
• Antitoxin given to wounded persons, without
previous history of vaccination.
• Antitoxin prophylaxis should be
accompanied by active immunization with TT
Clostridium botulinum
 C. botulinum

Morphology
• Gram-positive large straight rods, motile, non capsulate, spores
are oval central or sub-terminal with the ability to produce the
neurotoxin botulinum.
• Can contaminate food or open wounds,
or can be ingested as spores.
Typing:
• 8 serotype (A-H) known, each with a serologically distinct, but
pharmacologically similar toxin.
• Human botulism is usually due to types A, B & E.

• Disease: Botulism (a serious form of food poisoning).

• Habitat and Transmission: Habitat is the soil and freshwater
sediments. Organism and botulinum toxin transmitted in
improperly preserved food.
Pathogenesis
• Although C botulinum types A and B have been implicated
in cases of wound infection and botulism, most often
the illness is not an infection.

• Rather, it is an intoxication resulting from the ingestion of

food (canned meat or fish) in which C. botulinum has grown
and produced neurotoxin (the most potent poison
known).

• During growth of C. botulinum & during autolysis of the

bacteria, toxin is liberated into the environment.
 Pathogenesis…

• The toxin is absorbed from the gut

and binds to receptors of
presynaptic membranes of motor
neurons of the peripheral nervous
system and cranial nerves.

• The toxin acts by blocking

release of acetylcholine at
synapses and neuromuscular
junctions; which prevents muscle
stimulation that in turn resulting in
lack of muscle contraction and
eventually flaccid paralysis.

• Death results from respiratory or

cardiac failure.
 C. botu…
• Laboratory Diagnosis: Presence of toxin in patient's
serum or stool or in food. Detection of toxin involves either
antitoxin in serologic tests or production of the disease in
mice.

• Treatment: polivalent antitoxin to types A, B, and E made

in horses. Respiratory support may be required.

• Prevention
▫ Failure to sterilize food during preservation allows spores to
survive. The toxin is heat-labile; therefore, foods eaten without
proper cooking are usually implicated.
▫ Observing proper food preservation techniques,
cooking all home-canned food, and discarding bulging
cans.
Summary: Important Features of Pathogenesis by
Clostridium Species
3. Corynebacterium diphtheriae

• Disease: Diphtheria.
• Characteristics:
▫ Club-shaped Gm+ rods arranged in V or
L shape (resemble Chinese letters).
Arrow points to a "club-shaped" gram-
▫ Aerobic, non-spore-forming, non-motile. positive rod

▫ Black colonies on tellurite plate

• Transmission
▫ Humans are the only natural host
(human throat).
▫ Aerosol dissemination spreads the
organism from person to person.
 Pathogenesis

• Organism secretes an exotoxin that inhibits protein synthesis

(irreversibly blocks translation ).
• Toxin has two components: a B or binding portion and an A or
enzymatically active portion.

• Most prominent sign of diphtheria is thick, gray, adherent

pseudomembrane over the tonsils and throat caused by
death of mucosal epithelial cells.
• There are three prominent complications;
▫ Extension of the membrane in to the larynx and trachea, causing
airway obstruction.
▫ Myocarditis accompanied by arrhythmias and circulatory collapse.
▫ Swelling of lymph nodes is common.
 Diphtheria with marked
swelling of the lymph
nodes.

Pseudomembrane
 C. diphtheriae…

• Laboratory Diagnosis
▫ Gram-stained smear and culture.
▫ Black colonies on tellurite plate.
▫ Toxin production test by disease produced in laboratory
animals.

• Treatment: Antitoxin made in horses should be given

immediately to neutralize unbound toxin in the blood.
▫ Penicillin G or erythromycin kill the organism but neither
is a substitute for antitoxin.

• Prevention: Toxoid vaccine, usually given to children in

combination with tetanus toxoid and pertussis vaccine
(DPT).
 So far we have discussed

• Gram positive • Gram negative • Gram positive

cocci cocci rods
– Bacillus
▫ Staphylococci ▫ N. gonorrohea • B. anthracis
 S. aureus ▫ N. mengitidis
• B. cerus
– Clostridium
▫ Streptococci
• C. perferengis
 S. pyogens
 S. pneumonia • C. tetani
• C. botilinum
─ C. diphtheriae