Aerobic Spore-Forming Bacilli

Bacillus
Bacillus anthracis

Bacillus cereus

Bacillus subtilis

stearothermophilus

> Food-borne illness

> Usually motile & not susceptible to penicillin

> Anthrax = primarily disease of herbivorous animals (sheep & cattle, horses,
hogs & goats)
> A gram (+), aerobic, spore forming bacillus
> First isolated by Robert Koch in 1877

> Can cause 2 clinical syndromes:

> present in the air, dust,

a. short incubation period of 4 hrs

brackish water

= severe nausea & vomiting & is frequently mistaken for

staphylococcal food poisoning
= after the ingestion of fried rice

contaminant
> seen in bacteremias & eye

> spores are used to

infections in heroin addicts

evaluate the efficacy of

> Humans accidentally encounter the disease in an agricultural setting, usually

= abdominal cramping & diarrhea

> usually respond to B-lactam

autoclaving & other

development of skin infection that become generalized

Morphology:

= confused w/ clostridial food poisoning

antibiotics

sterilization procedures

> In smears from blood & tissues of infected animal = organism usually singly
or in pairs
> Ends appear square, corners so sharp, when in chain, leaving an oval opening
between the organisms
> Encapsulated during growth in infected animal, but capsules cant be
demonstrated in vitro unless the organisms are cultured on a bicarbonate
containing medium in the presence of 6% CO2
> Spores are formed in culture, in soil, & in the tissues & exudates of dead
animals but not in the blood or tissues of living animals
Cultural Characteristics:
> Inoculated on 5% blood agar plates, blood free of antibiotics= demonstration
of characteristic colonial morphology
> pH 7.0 to 7.4 under aerobic conditions = maximal growth
> 37C = optimal temp for maximal growth
> After 24 hrs incubation: large, raised, opaque, grayish white, plumose colonies,
2-3 mm in diameter, with irregular, fringelike edge
> No hemolysis is produced

b. longer incubation period of 17 hrs

> common laboratory

Laboratory identification:
> Virulent strains are the only organisms that produce rough colonies when
grown in the absence of increased CO2 & mucoid colonies when grown on
sodium bicarbonate medium in an atmosphere of 5% CO2
> Differential susceptibility of B. anthracis and B. cereus to penicillin is the basis
for the string of pearls reaction that separates virulent and avirulent B. anthracis
from other aerobic spore formers
> After 3-6 hr incubation on surface of solid medium containing 0.5ug/ml
penicillin: cells become large & spherical & occur in chains, when viewed on
agar surface resemble string of pearls
> Confirmatory Test: inoculation of a suspension of organisms from an agar
plate to the mouse = death after 2-5 days; organisms recovered from heart blood
Resistance:
> Due to spores, extremely resistant to adverse chemical & physical environment
> Temp of 120C for 15 mins = inactivate the spores
> Spores remain viable for years in contaminated pastures & remain a source of
infection for long periods of time
Antigenic Structure:
> 3 antigens:
a. capsular polypeptide = with high molecular wt
consisting exclusively of D-glutamic acid
b. polysaccharide somatic antigen = component of the
cell wall & contains equimolar amounts of N-acetylglucosamine & D-galactose
c. complex protein toxin

Determinants of Pathogenicity:
Depends on 2 impt virulence factors:
a. poly (D-glutamic acid) capsule
b. exotoxin
> Capsules interfere with phagocytosis and impt in early stages of infection
> Antibodies against the capsular antigen are produced, but not protective
against the disease
> Lethal effects of exotoxin consists of 3 distinct & serologically active proteins:
a. protective antigen (PA)
b. edema factor (EF)
c. lethal factor (LF)
> Proteins act synergistically to produce systemic effects of anthrax
> No toxic activity if functions individually
> Toxic effects appear when:
> PA + EF = produces localized edema in the skin of
test animals
> PA + LF = death in test animals
Clinical Infection:
> Soil = ultimate reservoir of anthrax infection
> Terminal stages of the disease=bacilli are shed in large no. from all orifices of
the infected animal
> In animals, anthrax is severe & usually takes the form of septicemia
> In humans:
a.)

agricultural cases = result from direct contact with animals dying of

anthrax
b.) industrial cases = result from contact with contaminated animal products
Pathogenesis:
> Humans become infected in one of three ways:
1. Cutaneous route = organisms gain access thru

small abrasions or cuts &

multiply locally with a rather dramatic inflammatory response

2. Inhalation = organisms are inhaled, multiply in the

lung, & are swept to the

draining hilar lymph nodes, where marked hemorrhagic necrosis may occur
3. Ingestion = rarely, organisms are ingested in infected meat, with resultant
invasion & ulceration of the GI mucosa

Clinical Manifestations:
A. Cutaneous anthrax = 95% of human cases
= begins 2-5 days after infection as small, papules that
develops w/in a few days into small vesicles filled w/
dark
bluishblack fluid
= rupture of the vesicle reveals a black eschar at the
base
w/ a very prominent inflammatory ring of reaction around
the eschar (malignant pustule)
= lesions found on the hands, forearms, or head
B. Pulmonary infection = known as wool-sorters disease
= in patients who handle raw wool, hides or horsehair
= acquire the disease by inhalation of spore
= symptoms are of respiratory infection w/ fever,
malaise, myalgia & unproductive cough
= after several days will develop into a severe infection
with marked respiratory distress & cyanosis--- death
occurs after 24 hrs
C. GI infection = nausea, vomiting & diarrhea
= occasionally hematemesis
= usually followed with shock and death
> Anthrax infection in humans provides permanent immunity; second attacks are
rare
Laboratory Diagnosis:
> Specimens for culture should be obtained from malignant pustule, sputum or
blood
> Gram stain & fluorescent-antibody stain= useful in making presumptive
diagnosis
Treatment:
Penicillin = curative for cutaneous anthrax
Pulmonary anthrax = diagnosis is usually made post- mortem; same w/ GI
anthrax
= if diagnosis is made in sufficient time, give large doses of
Penicillin IV
= in patients allergic to penicillin, erythromycin or
tetracycline

Treatment:
> Clindamycin = DOC
> Also susceptible to: Aminoglycosides, Vancomycin, Tetracycline

can be given
Prevention:
> Active immunization = to herbivorous animals preventing anthrax
> For the protection of humans in high risk work situations, a non-living vaccine
consisting of aluminum hydroxide-adsorbed supernatant material from

& Erythromycin

fermentor cultures of a toxigenic but encapsulated strain of B. anthracis is used

= requires multiple doses over long periods of time

Clostridium
> Gram (+) spore-forming bacilli
> Most species are obligate anaerobes
> Pathogenic species produce soluble toxins that are extremely potent
> Present in soil & GIT of humans & animals
> The pathogenic clostridia can be divided into 4 major groups, according to the types of disease they
produce:
Enterotoxigenic
Histotoxic clostridia
clostridia
Clostridium tetani
Clostridium botulinum
> Clostridium botulinum, is
> The histotoxic clostridia
> Clostridium tetani, the
the etiologic agent of
characteristically cause a
causative agent of
botulism, w/c results from the
variety of tissue infection,
> The enterotoxigenic
tetanus, causes disease
ingestion of a powerful
usually subsequent to
clostridia produce food
thru a potent exotoxin
exotoxin previously formed by
wounds or other types of
poisoning & more severe
that is produced during
the organism in contaminated
traumatic injury
forms of GI disease
limited growth w/in tissue food

Histotoxic Clostridia (Clostridium

Clostridium difficile & Antibiotic

perfringens)

associated Colitis

Clostridium tetani

Clostridium botulinum
> Produces the most potent exotoxin
> Neurotoxin = cause of botulism, a severe
neuroparalytic disease characterized by
sudden onset & swiftness of course,
terminating in profound paralysis & pulmonary
arrest
> 8 serologically distinct botulinum toxins: A,
B, C1 , C2, D, E, F & G

> Causes clostridial myonecrosis

> Causes pseudomembranous enterocolitis

> Foodborne botulism = most common form of

> Obligate anaerobe

botulism an intoxication caused by the

> Selective agar containing cefoxitin, cycloserine,

ingestion of preformed botulinum toxin in

= severe form of muscle infection

fructose, & egg yolk= aids in its isolation from feces

contaminated food

= AKA gas gangrene or clostridial myositis

> Isolated from both human & animal feces

> Latin botulus (sausage)= fatal food

> Produces 2 major toxins:

> Causative agent of tetanus

poisoning syndome associated w/ eating of

> 3 important histotoxic clostridia:

1. Toxin B or cytotoxin = produces cytopathic effect in

> Follows a puncture wound & is

sausage

1. C. perfringens

vitro on tissue culture cells

characterized by severe muscle

> Organisms can also contaminate traumatic

2. C. novyi

2. Toxin A = potent enterotoxin w/c causes severe

spasms esp. the jaw (trismus or

wounds (wound botulism) & also the GIT of

3. C. Septicum

damage to the intestinal mucosa & excess fluid response

lockjaw)

infants (infant botulism)

Clostridium perfringens
> Cultured from 60-90% of cases of clostridial myonecrosis
> 5 types: A to E = separated according to their production of 4
major lethal toxins
> Type A = primarily responsible for disease in humans:
clostridial myonecrosis), less severe wound infections
&
a common form of food poisoning
= found in almost all GIT of animals

Morphology:
> Young cultures = gram(+);

Morphology:

Morphology:

older cultures are gram(-)

> Straight to slightly curved Gram(+) rod

> Short, plump, strongly Gram (+) rod

> Spores are terminally located =

w/rounded end

> Length varies according to the stage of growth, nutritional &

drumstick appearance

> Motile w/ peritrichous flagella

ionic composition of the medium

> Spore does not stain with

> Produces heat-resistant spores that are oval

> Rapidly growing organisms= coccoid or cubical

Gram stain = appears colorless

& subterminal & tend to distend the bacillus

> Older cultures = more elongated

round structure

> Spores are produced more consistently

> Non-motile; does not produce spores in ordinary media

> Has numerous peritrichous

when the organism is grown on alkaline

> Capsules demonstrated by direct exam of smears from

flagella that convey active motility

glucose gelatin media at 20-25C

wounds

to the organism
Cultural Characteristics:

> Spores usually not produced at higher temp.

> Obligate anaerobe, moderately

fastidious
> Optimal temp is 37C, optimal pH
is 7.4
Cultural Characteristics:

> Swarming of the organisms

> Cultures on BAP after 24hrs incubation: circular & smooth, 2-4

occurs on BAP

mm in diameter

> Edge of the colony appears

> As colonies increase in size with age, the periphery loses

translucent, finely granular sheet

symmetry & projects towards periphery of the medium

w/ a delicate filamentous

suggesting appearance of a colony of motile bacteria showing

advancing edge

Cultural Characteristics:

swarming

> Faint Beta hemolysis is

> Strict anaerobe

> Produces characteristic pattern of hemolysis on BAP

observed

> On BAP, all strains except type G are B-

> Egg-yolk media = precipitation (opalescence)

> Organism does not ferment any

hemolytic

> Milk media = stormy fermentation"

carbohydrates

+D10
Resistance:

Resistance:

> Degree of resistance to various physical &

> Spore conveys resistance to

chemical factors depends on a specific strain

various disinfectants & heat

& serologic type of organism

> Not destroyed by boiling for 20

> Type A is more resistant than types B, C,

mins.

and D.

> Autoclaving at 120C for 15 mins

> Type E is the least heat resistant

= preferred method for sterilizing

> The spores may survive several hours at

contaminated materials

100C & up to 10 mins. at 120C.

Antigenic Structure:

Antigenic Structure:

> Flagella (H), Somatic (O), and

1. Exotoxin = Types A, B, C (alpha), C(beta),

Antigenic Structures:

spore antigens

D, E, F & G

> Has 12 different toxins, all are protein in nature & antigenic

> Strains of the organism have

> Other soluble substances or minor antigens are enzymes with

been differentiated into ten types

defined substrates: e.g. = collagenase (K-antigen),

on the basis of their flagellar

deoxyribonuclease (v-antigen), hyaluronidase (u-antigen)

Determinants of Pathogenicity:

antigens

& produce 3 different toxins

Determinants of Pathogenicity:

> Alpha toxin = toxin w/ primary importance in

Determinants of Pathogenicity:

> *classic exotoxin is not released during the

> Tetanus toxin

life of the organism but appears in the

clostridial

myonecrosis
> Toxin is a lecithinase C (or phospholipase C) w/c splits lecithin

= neurotoxin (tetanospasmin)

to phosphorylcholine & diglyceride; activated by Calcium &

magnesium ions & also hydrolyzes sphingomyelin
> Disruption or leakage of cell membranes can explain the lysis
of erythrocytes, destruction of tissue, and edema observed this
disease

= except for strains of types C

& D, a single toxin is produced by each type
= strains C & D are complex

medium only after death & autolysis of the

organism*

= intracellular toxin released

by cellular autolysis
= heat labile protein that is
inactivated by heating for 20
mins at 60C
> Causes muscle spasms of the
masseter muscles w/ trismus

> Types of Botulism:

1. Food-borne botulism is a lethal food
poisoning that
results from ingestion of the neurotoxin in
incompletely processed food contaminated
with the
organisms

flexion of the upper extremities &

2. Infant botulism is related to the ingestion

extention of the lower extremities

by infants of C. botulinum spores, the

with arching of the back

multiplication of

(opisthotonus)

organisms w/in the GIT, & subsequent

absorption
of toxin
3. Wound botulism, which is the least
common, is a neuroparalytic illness
associated with wounds that
show little clinical evidence of active infection
4. Unclassified botulism occurs in persons
over the age of 1 yr who have symptoms of
clinical botulism w/ no identifiable vehicle of
transmission
> At present, food-borne botulism occur in
outbreaks after the consumption of homepreserved
> After ingestion of preformed botulinum
toxin--absorbed from the stomach & Small bowel

but toxin
reaching the colon is slowly absorbed--lymphatic
system----bloodstream---functional
disturbance of the peripheral nervous
system---inhibition of the release of
acetylcholine----toxin acts on the myoneural
junction to produce complete paralysis of the
cholinergic nerve fibers at the point of release
of the acetylcholine
Clinical Infection:
> Trauma associated with deep & lacerated or crush wounds of
muscle & w/ vascular damage of major vessels & capillary beds
is one of the important factor predisposing to clostridial
myonecrosis
> The basis for the requirement of trauma w/ ischemic or
necrotic areas is the anaerobic nature of the organism, w/c
require a reduced oxygen tension & oxidation-reduction
potential for growth
Pathogenesis:
> In areas of reduced O2 tension the pyruvate of muscle is
incompletely oxidized & lactic acid accumulates, causing a drop
in pH, activates endogenous proteolytic enzymes resulting in

Pathogenesis:

tissue necrosis
> Multiplication of the organisms is accompanied by the

> Most significant feature: setting

production of soluble toxins---toxins diffuse from initial site of

of the wound where

growth & attack healthy muscle & surrounding tissues

reduction potential is properly

> Tissues are then destroyed---spread of infection to new

poised to permit multiplication of

necrotic areaedema fluid produced & enzymes on tissue

organism & toxigenesis

components & gas accumulated from metabolism of the

> Tetanus neonatorum

oxidation-

organism increase the pressure w/in the muscle bundles

= most severe form of tetanus

> Circulation is impaired----further decreasing oxidation-

= usually results from cutting

reduction potential & pH---provide new areas for growth of the

the umbilical cord w/ unsterile

organism

instruments or from improper

> Tissues are then destroyed---spread of infection to new

care of umbilical stump

necrotic areaedema fluid produced & enzymes on tissue

> Aerobic bacteria has important

components & gas accumulated from metabolism of the

contributing factor: removes O2 &

organism increase the pressure w/in the muscle bundles

promotes growth of C. tetani

> Circulation is impaired----further decreasing oxidation-

> After germination of the spores,

reduction potential & pH---provide new areas for growth of the

toxin is elaborated & gains

organism

entrance to the CNS

Clinical Manifestations:
> 3 categories of increasing levels of severity of wound
infection:
1. Simple wound contamination
2. Anaerobic cellulitis
3. Clostridial myonecrosis
_______________
1. Simple Wound Contamination:
> One or more histotoxic clostridia may be present w/o
pathologic process
> Clostridia present may be non-toxigenic
2. Anaerobic Cellulitis:
> More serious form of wound infection

Clinical Manifestations:

> Clostridia infect tissue that is already severely compromised

> Incubation period = 4-10 days

Clinical Manifestations:

> Organism spread subcutaneously but do not invade healthy,

> Earliest manifestation: muscle

> Incubation period & clinical manifestations

intact muscle

stiffness, followed by trismus or

are similar to all types of botulinum toxin

> Patients not in extremely toxic condition, overall prognosis is

lockjaw

> The shorter the incub. period, the poorer the

more better than clostridial myonecrosis

> As the disease progress,

prognosis

3. Clostridial Myonecrosis:

tetanospasm cause clenching of

> Symptoms begin 12 to 36 hrs after ingestion

> Anaerobic infection of muscle in which organisms are invasive

the jaw (risus sardonicus), arching

of contaminated food or as late as 8 days

& the infection is associated w/ profound toxemia, extensive

of the back (opisthotonus), flexion

after.

local edema, variable amount of gas, massive tissue damage,

of the arms & extension of the

> Type E botulism appears to have a shorter

and death in untreated cases

lower extremities

incubation period than Types A & B.

> After injury----incubation period of 12-24 hrs---symptoms occur

> Respiratory complications:

> Severe nausea & vomiting are frequently

+A14Initial symptom: pain in affected area w/c increases in

aspiration pneumonia &

observed with type E rather than types A & B

severity as the infection spreads

atelectasis are common

> Weakness, dizziness, lassitude are often

> There is local edema & blood stained exudate

Occasionally the spasms are

early complaints

> Pulse rate rises disproportionately more than the temp.

intensified to produce bone

> Constipation is common rather than

> Skin color changes & finally becomes black

fractures

diarrhea

> Death occurs rapidly in untreated cases

> Poor prognosis is associated

Cranial nerve palsies are usually the

Uterine Infection:

w/:

presenting symptoms: diplopia (double

> Usually involving the gravid uterus

1. short incubation period,

vision), dysphagia, dysphonia

> Source of the organism may be exogenous or endogenous

2. rapid development from muscle

> The pupils are dilated & the tongue is very

> Septicemia & intravascular hemolysis may occur and lead to

spasm to tetanospasms,

dry & furry

secondary renal failure

3. injury close to the head, ext

> In type E intoxication, abdominal distention

> The disease progresses rapidly & has a high mortality rate

remes of age &

is very common leading to mistaken diagnosis

Clostridial Septicemia:

4. frequency & severity of

of acute abdomen

> Invasion of the bloodstream may occur in association with

Clinical Manifestations:

convulsions

> Weakness of the muscle groups (particularly

malignancy +A14Usually no history of external trauma

> Antibiotic associated diarrhea

> Patients who recover usually

of the neck, proximal extremities & respiratory

> Septicemia may follow biliary tract or GI surgery

> Pseudomembranous colitis = severe disease of the

return to a completely normal

musculature) is often observed leading to

> C. septicum or C. perfringens is usually the etiologic agent

GIT characterized by exudative plaques with underlying

state after variable period of

sudden resp. paralysis, airway obstruction &

> Death may occur in less than 24 hrs if untreated

necrosis of the mucosal surface of the intestine

stiffness

death

Immunity:
> Adequate immunization of
pregnant patients
= important for passive immunity
for the newborn
Laboratory Identification:
> Clinical material should be
transported containing CO2.
> Inoculated immediately on both
solid media & anaerobic culture
media such as chopped meat &
incubated under anaerobic
condition
> Rapid motility of the organism is
also helpful in the isolation
> Final proof of the isolation of a
toxin production when injected into
mice & its neutralization in mice
previously inoculated with antitoxin
Laboratory Diagnosis:
> Early diagnosis must be made on clinical grounds

Laboratory diagnosis:

Laboratory Diagnosis:

> Direct smear & gram stain of material deep w/in the wound =

Laboratory Diagnosis:

> made clinically because isolation

> As soon as botulism is suspected

provide information for differential diagnosis

> Colonoscopy & sigmoidoscopy w/ biopsy = invasive

of the organism can occur in the

clinically,specimens should be submitted

> Cultures & smears (from tissue, aspirates, or deep swabs)

procedures

absence of disease & also

immediately to the lab.

should be taken from affected muscle

> Culture of stool specimen for C. difficile

because it is possible to have the

> Diagnosis rests on identification of the toxin

Cultures from 2 or 3 sites is advantageous if the area is

> Elisa tests for toxin A & B

disease but be unable to isolate

or isolation of the organism from the

extensive

> Latex agglutination test = rapid screening test

the organism

specimen.

Treatment:
1. Simple wound
= removal of necrotic tissue & by cleansing
= admin. of antibiotics rarely required
2. Anaerobic cellulitis
= opening the involved area, removing all necrotic

Treatment:

tissue, cleansing thoroughly & giving

> Designed to prevent the further

antibiotics

3. Clostridial myonecrosis

elaboration & absorption of toxin

= intensive & immediate therapy is needed

> Administer antitoxin= human

= mortality rate of 15-30% & highly dependent on anatomic

tetanus immune globulin

location of the infection (Surgical removal of all infected &

> Debridement of the wound &

necrotic tissue is the most important modality)

removal of any foreign bodies

4. Clostrial myonecrosis is common in the ff areas:

> Large doses of Penicillin should

buttocks, thigh & shoulder

be given

> High dose penicillin= DOC

> Tetracycline or Metronidazole

> Clindamycin or Metronidazole = in patients allergic to Penicillin

can also be used

> 2 infection with E. coli= treated w/ gentamycin or tobramycin

> Barbiturates & diazepam = mild

> Clostridial myonecrosis involving the trunk treated with

tetanospasm

hyperbaric oxygen will have 50% survival rate

> Curare-like agent = paralyze

> Hyperbaric O2= patient is given 7 intermittent exposures to

patients muscles so that

100% O2 in a chamber pressurized to 3

atmospheres of absolute pressure
= has direct inhibitory effect on the organism &

respiratory function may be

Treatment:

maintained by positive pressure

> Discontinuing the antimicrobial agent

apparatus

Treatment:

toxin production but alpha antitoxin is not

> Maintaining fluid & electrolyte balance

> Tracheostomy= to minimize

> Immediate administration of antitoxin

inactivated

> voiding drugs that slow intestinal motility

respiratory complications

> Support of respiratory, cardiovascular &

> Administering anti-C.defficile drug = Vancomycin (DOC)

> Careful control of the

renal systems

tissues surrounding the infection----preventing

> Metronidazole & Bacitracin also effective

environment to reduce auditory &

> Guanidine hydrochloride = enhances

spread of infection

> Approx. 20-39% relapse after treatment

visual stimuli

acetylcholine

= increase the oxidation-reduction potential of the \

release

Prevention:
> Early & adequate wound debridement
> Adequate cleansing, removal of necrotic tissue
> Administration of prophylactic antibiotic (Penicillin)
= reduces the risk of anaerobic infection
> Food Poisoning:

Prevention:

> 8-24 hrs after ingestion of contaminated food = acute

Prevention:

> Homemaker should be alerted to use

abdominal pain & diarrhea

> Active or passive immunization

sterilized containers & pressure cookers in the

> Symptoms lasts for 12 to 18 hrs & recovery is usually

> Active immunity: tetanus toxoid

canning of all foods as to kill any C. botulinum

complete

(DPT)

spores

> Usually results from the ingestion of meat dishes, such as

> Passive Immunity :

> Before eating home-canned foods, it should

roasts, poultry, fish, & stews that are heavily infected w/ C.

Administration of antitoxin: human

be boiled for 1 min. or heated at 80C for 5

perfringens

tetanus immune globulin

mins. to destroy any toxin