MICROBIOLOGY LECTURE 7 – Gram Positive Spore and Non-

Sporeforming Bacilli
Notes from Lecture
USTMED ’07 Sec C – AsM

Gram Positive Sporeforming Bacilli

- Clostridium
- Bacillus

CLOSTRIDIUM
- Obligate anaerobes
- Gram positive bacteria
- Form endospores
- Responsible for many cases of food spoilage, even in 3. Pronounced arching of the back due to spasm of the
canned foods strong extensor muscles of the back (opistholonus)
- Clostridia often can ferment amino acids to produce
ATP by oxidizing one amino acid and using another as Other findings:
an electron acceptor in a process called the Stickland 4. abdomen boardlike
reaction 5. extremities stiff and extended (spastic paralysis)
- This reaction generates ammonia, hydrogen sulfide,
fatty acids, and amines during the anaerobic
6. spasm provoked by touch, bright light, noise, or
movement of the patient
decomposition of proteins
7. afebrile, except in the presence of complications
- These products are responsible for many unpleasant
odors arising during putrefaction
Laboratory diagnosis:
- Clostridium species
- no microbiological or serologic diagnosis
* C. tetani
- organism rarely isolated from the wound site
* C. botulinum
* C. perfringens
* C. difficile

A. Clostridium tetani
- causes tetanus (lockjaw)

Sources:
soil, dust, animal or human feces, plaster, unsterile suture,
rusty scissors, nails or pins
Produces a terminal spore ie. at the end of the rod giving the
organism the characteristic appearance of a “tennis racket”
Portal of entry:
* deep punctured wound
Treatment:
* dental caries
- Tetanus immune globulin to neutralize unbound toxin
* chronic otitis media
- Metronidazole/Pen-G-Na
* umbilical stumps
- Muscle relaxant (benzodiazepines)
* septic abortion
Prevention:
Factors favoring germination of spores:
1. Active immunization – use of tetanus toxoid
1. necrotic tissue
(formaldehyde treated toxin) as part of childhood
2. poor blood supply in the wound immunization and booster doses every 10 years
2. Passive immunization
Pathogenesis: Tetanus toxin o for grossly contaminated wounds
Tetanospasmin (exotoxin) produced o immunoglobulin (associated with allergic
by vegetative cells at wound site reactions– serum sickness reactions)
o human immunoglobulin

Carried intra-axonally (retrograde)

to the CNS
B. Clostridium botulinum
- causes botulism
Binding to ganglioside receptors
Sources:
1. Canned or vacuum packed foods without adequate
sterilization
Blocking release of mediators (e.g.
glycine) at spinal synapses • alkaline vegetables such as green beans,
peppers, and mushroom
2. Smoked fish
3. they are proteases that cleave the proteins involved 3. Honey (raw)
in mediator release
4. has only one antigenic type Botulinum toxin
5. along with botulinum toxin, it is among the most
toxic substance 1. produced by spores who survived due to
6. responsible for the clinical manifestation inadequate sterilization
2. preformed toxin from germinated spores in an
Clinical Findings: anaerobic environment
3. relatively heat-labile (inactivated by boiling for
several minutes

Pathogenesis: Botulinum toxin

• absorbed from the gut and carried via blood to
peripheral nerve synapses, where it blocks the
release of acetylcholine
• it is a protease that cleaves the proteins involved in
acetylcholine release
Due to rigid contraction of the jaw muscle • along with tetanus toxin, it is among the most toxic
1. Risus sardonicus (left panel) substances known
• there are 8 immunologic types of toxins A-G, Types
2. Lockjaw (right panel) A,B,E are the most common in human illness
 • Minute amounts of the toxin are effective in the
treatment of certain spasmodic muscle disorders such
as toticollis, “writer’s cramp” and blepharospasm
• Also used to remove wrinkles in the face

Clinical Forms/Manifestations
1. Wound botulism – when spore contaminate a
wound, germinate and produce toxin at the site
- associated with drug abuse especially skin- 2) Enterotoxin (heat labile protein)
popping with black tar heroin o acts on the lower portion of the small
2. Infant botulism – in which the organism grow in intestine
the gout and produce toxin
- associated with honey ingestion in infants o enterotoxin producing strain are heat
- a cause of floppy baby syndrome (poor resistant (spores remain viable for ova an
muscle tone) hour at 100°C)
- constipation
- feeding problem
- lethargy
- sudden infant death syndrome

Classic botilinum
• symptoms occur 12-36 hours after ingesting toxin-
containing foods
• no fever or sign of sepsis
• progressive paralysis of striated muscle ensues
• difficulties in focusing vision, in swallowing and other
cranial nerve function (ptosis)

Laboratory Diagnosis
- can be cultured and identified by standard anaerobic method
3) Degradative enzymes
Treatment
Trivalent
• antitoxin (Types A,B, E) from horse to neutralize
unbound botulinum toxin (serum sickness in 15% of
recipients)
• ventillatory support

Prevention
• proper sterilization of all canned and vacuum-packed
foods

C. Clostridium perfringens

Causes:
• Gas gangrene (myonecrosis)
• Food poisoning
• Anaerobic cellulites
• Enteritis necroticans
• Clostridial endometritis Clinical significance:
• Other causes of gas gangrene 1) Myonecrosis (gas gangrene)
o C. histolyticum o introduced into tissue by contaminated
o C. septicum
with dirt or endogenous transfer from the
o C. novyi intestinal tract
o severe open wound (compound features,
Transmission: crushing injuries are prime predisposing
• spores are located in the soil conditions)
• vegetative cells are members of the normal flora of o fermentation of tissue carbohydrates fields
the colon and vagina. gas and accumulations of gas bubbles
• gas gangrene is associated with war wounds, produce crinkling sensation on palpation
automobile and motorcycle accident (crepitation)
• septic abortion o exudates are copious and foul smelling
o increased capillary permeability leads to
Pathogenesis hematogenous spread of exotoxin resulting
• organisms grow in traumatized tissue (especially in shock, renal failure, and intravascular
muscle) hemolysis
• produce variety of toxins 2) Anaerobic cellulites
o affects connective tissue
Toxins elaborated by C. perfringens o does not involve muscle invasion
1) Exotoxin – 12 exotoxins designated by Greek letters. o surgical intervention is generally
α toxin
unsuccessful because of rapidity of spread
o the most important, a lecithinase
of the infection
(phospholipase C3) 3) Food poisoning
o symptoms occur 8-18 hours after ingestion
of contaminated foods (meat, chicken, fish
and their by-products)
o requires a large inoculum of 108 organism
or quarts
o nausea, abdominal cramps, and diarrhea
o afebrile patient
o self-limited (recovery within 1-2 days)
 4) Enteritis necroticans
o necrotizing bowel disease with high
mortality
o Other names: pigbel in New Guinea,
darmbrand in Germany
5) Clostridial endometritis
o gangrenous infection of uterine tissue
followed by bacteremia and toxemia
o grave complication of incomplete abortion
or use of inadequately sterilized
instruments.
Laboratory Diagnosis:
• gram stain of diseased tissue show vegetative
clostridial forms (large and gram-positive rods)
• rare inflammatory cells in exudates because of
extensive necrosis and intact host cells
• when cultured anaerobically on blood agar, rapidly
growing colonies with double zone of hemolysis
Clostridium perfringens
D. Clostridium difficile
- causes pseudomembranous colitis
Pathogenesis:
• a minor component of the normal flora of the large
intestine
• antibiotic suppresses the normal flora and leads to
proliferation of C. difficile
• Pathogenic strains produces 2 toxins
o Toxin A - an enterotoxin (causes excessive
fluid secretion)
o Toxin B – a cytotoxin (disrupt protein
synthesis and causes disorganization of
cytoskeleton
Clinical significance
• All antibiotics have been reported as predisposing
factor to clostridial antibiotic associated diarrhea and
pseudomembranous colitis
• most commonly implicated antibiotics are ampicillin,
clindamycin, and cephalosporins
Laboratory Diagnosis:
• anaerobic stool cultures
• demonstration of exotoxin, A and B from stool
extracts by ELISA
Treatment:
• withdrawal of the offending antibiotic
• oral metronidazole or vancomycin
• metronidazole is preferred because vancomycin may
result to vancomycin resistant enterococci
BACILLUS:
Two medically important species
A. Bacillus anthracis
- large gram positive rods with square ends, frequently
found in chains
- its antiphagocytic capsule is Composed of D-
glutamate (polysaccharides in other bacteria)
- causes anthrax
Three main forms of human disease:
1. cutaneous or malignant pustule (painless ulcer with a
black eschar; crust, scab following skin trauma)
2. pulmonary (inhalation)
• pulmonary or inhalational anthrax (Woolsorter’s
Disease)
• after being inhaled, organism moves rapidly to the
mediastinal lymph nodes where it causes hemorrhagic
mediastinitis
• because it leaves the lung so rapidly, it is not
transmitted by the respiratory route to others
• patients has flu-like symptoms progressing to bloody
pleural effusion, septic shock and death
3. gastrointestinal (from ingested contaminated meat)
• symptoms include vomiting, abdominal pain and
blood diarrhea
Laboratory Diagnosis
• large gram-positive rods in chains
• spores not seen in smears of exudates
• ELISA test for the antibodies to the toxins
Treatment
• Pen-G-Na
• Doxycycline
• Ciprofloxacin
Prevention
• use of prophylactic drugs (ciprofloxacin or
doxycycline during outbreak)
• cell-free vaccine for high-risk people
• incinerating animals that die of anthrax rather than
burying them to prevent soil contamination
B. Bacillus cereus
• causes food poisoning
• reheat fried rice as culprit
• portal of entry is the gastrointestinal tract
Pathogenesis
• Two toxins produced by B. cereus
1. Cholera toxin-like – stimulates adenylate cyclase
causing increased cyclic AMP within the enterocyte
2. Staphylococcal enterotoxin-like: it is a superantigen
Clinical Findings:
• Two syndromes:
1. One has a short incubation period (4 hours) and
consist primarily of nausea and vomiting
 2. The other has a long incubation period (18 hours) and 4. Cutaneous
features of watery, non-bloody diarrhea resembling 5. Bull neck
clostridial gastroenteritis

Laboratory Diagnosis
• not usually done

Treatment
• symptomatic

Prevention
• Rice should not be kept warm for long periods

Gram Positive Non-sporeforming Bacilli Laboratory Diagnosis

1. Corynebacterium • it can be associated from selective medium like
2. Listeria Tinsdale’s agar
3. Erysipelothrix • characteristic bands and polychromatic granules
on methylene blue staining
• chinese character or picket fence appearance

CORYNEBACTERIUM
Treatment
• small, slender, pleomorphic gram-positive rods • neutralization of toxin using horse serum
• non-motile antitoxin
• unencapsulted • eradication of organism by antibiotics
• occur in characteristic clumps that look like chinese (Penicillin, erythromycin)
characters or picket fence patterns
• human pathogen (grow aerobically on standard Prevention
laboratory media such as blood agar • active immunization using a toxoid

A. Corynebacterium diphtheriae
• causes diphtheria B. Diphtheroids
B. Diphtheroids • common commensal
• unable to produce exotoxin • few cause disease in immuno-compromised
• common commensals of the nose, throat, patients
nasopharynx, skin, urinary tract and • can be recovered from infections such as
conjunctivitis endocarditis of prosthetic valves, lung abscess
and urinary tract infection
• presence of multiple lung resistance
• susceptible to vancomycin
A. Corynebacterium diphtheria

Epidemiology:
• found in the throat and nasopharynx of carriers
and those with diphtheria
• primarily a localized infection (throat)
• spread by respiratory droplets for carriers or
convalescent patients
LISTERIA
• non- sporeforming gram positive rods
• occurs as diplobacilli or in short chains
• avid intracellular parasites
• catalase positive
• tumbling motility in liquid medium

Pathogenesis: Epidemiology
• Diphtheria is caused by the local and systemic • widespread among animals in nature
effects of a single exotoxin that inhibits • can occur as sporadic cases or in small
eukaryotic protein synthesis epidemics (food borne)
• only those strains that are lysogenic for a B o * ground meat, poultry, dairy products
phage can produce toxin and are therefore • humans are asymptomatic intestinal carriers
virulent • infection most common in pregnant women,
fetus or newborns, and in immuno-compromised
Clinical significance host
1. Tonsillar diphtheria
Pathogenesis
• facultative, intracellular parasite
• organism attaches to and enters mammalian
cells by phagocytosis and once internalized
escapes from the phagocytic vacuole by
elaborating a membrane-damaging toxin called
listeriolysin O

Clinical significances
• septicemia More frequent
• meningitis
2. Nasal diphtheria
• granulomatous skin lesion
• lymphadenitis
• endocarditis

Laboratory Identification
• organism isolated on blood, CSF and other
clinical specimens on blood agar
• positive motility
3. Laryngeal • production of catalase
Treatment
• ampicillin
• cotrimoxazole

Prevention
• Proper food preparation and handling
• Organism sensitive to penicillin, erythromycin
and tetracycline

ERYSIPELOTHRIX

• gram positive filamentous rods causes

erysipeloid
• disease commonly seen on people who handle
animal products (butchers,
veterinarians,fishermen)
• organism sensitive to penicillin, erythromycin
and tetracycline

-fin-

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