CLINICAL BACTERIOLOGY 2nd Semester

MLS 223 | MODULE 5 S.Y. 2022-2023

UNIT 2: SPORE-FORMING GRAM-POSITIVE BACILLI Clostridium perfringens

CLOSTRIDIUM SPECIES
GENERAL CHARACTERISTICS
• Gram-positive bacilli
o Gram-variable Þ prolonged incubation
• Spore-forming
o Larger than diameter of organism which they
are formed Þ centrally, subterminally,
terminally.
o Swollen
• Most are motile with peritrichous flagella
o Exc. C. perfringens, C. innocuum, C.
ramosum
• Anaerobic (based on aerotolerance test)
Obligate Strict obligate NOT capable of growth on
anaerobe anaerobes agar surfaces exposed to
O2 levels above 0.5%.
C. heamolyticum, C. novyi
type B, C. tetani, C.
botulinum, Clostridioides
(formerly Clostridium)
difficile
Moderate grow when exposed to
obligate oxygen levels ranging from
anaerobes about 2% to 8% (average,
3%)
C. perfringens
Aerotolerant anaerobes limited growth on agar in
room air or 5% -10% CO2
incubator; grow best under
anaerobic conditions.
C. histolyticum, C. tertium
and C. carnis
• Catalase (-)
• Normal flora of GIT tract Þ cause endogenous and
exogenous infections
Classification of Clostridium species Based on
Pathogenesis
Gas gangrene/ Histotoxic • C. perfringes (80%)
• C. novyi
• C. septicum
• C. histolyticum
• C. sordellii
• C. sporogenes
• C. bifermentans
Tetani • C. tetani
Botulinum • C. botulinum
• C. baratii
• C. butyricum
• C. argentinense
Miscellaneous • C. clostridioforme grp
• C. ramosum
• C. innocuum
• C. tertium
Difficile • Clostridioides difficile
(old name: Clostridium
difficile)
Old name: Clostridium welchii
Common name: Welch’s bacillus
GENERAL CHARACTERISTICS
• G+ large bacilli with blunt ends
• Spores: central or eccentric (pushed to one side)
within a swollen sporangium Þ “BOX CAR”
appearance
• NM, Encapsulated
• Anaerobic Þ moderate obligate anaerobes
o Grows in the absence of O and presence of
O (2-8%, ave. of 3%)
HABITAT AND TRANSMISSION
• Spores found in soil, GIT of animals and humans
• Transmission:
o DIRECT INOCULATION of spores Þ breaks
in the SKIN
o INGESTION
PATHOGENESIS AND CLINICAL MANIFESTATIONS
1. Gas gangrene
2. Food poisoning
3. Necrotizing enterocolitis
4. Endometritis
GAS GANGRENE - Acquired by wound
(anaerobic contamination with the spores
myonecrosis) of C. perfringens
o Spores from soil, feces of
man/ animals
- Trauma/ traumatic breaks on
the skin Þ sharp obj produces
a deep-penetrating wound
o E.g. nail, rose thorn,
gunshots, surgical
procedures (septic
abortion)
- Trauma to host tissue Þ
damage muscles and impairs
blood supply Þ
hypoxia/anoxia
- Lack of oxygenation Þ spore
germination Þ growth of
anaerobic clostridia &
elaboration of exotoxins
o Gangrene – necrosis of
tissues due to lack of blood
supply
o Gas gangrene – causes by
C. perfringens
- Anaerobic environment
stimulate spores to germinate
Þ becomes vegetative cell/
bacilli Þ bacilli produce toxins
and enzymes in the tissues Þ
MYONECROSIS
o Myonecrosis – tissue death
in muscles
CHARACTERIZED BY :
Extensive necrosis of muscle and
connective tissue

COSCA 1
SIGNS AND SYMPTOMS:
Generalized fever, pain, edema,
and blood-tinged exudate in
the infected tissue
- Tissue becomes:
o discolored, purple, then
black, general tissue
destruction and bubbles
on the skin Þ gas
formation
o filled with bubbles of gas Þ
CO2 and H2 accumulate
VIRULENCE FACTORS
v EXOTOXINS
a) Alpha toxin (lecithinase,
phospholipase C)
§ Necrotizing,
hemolytic exotoxin
§ Hydrolyze lecithin and
sphingomyelin Þ cell
membrane of host
cells (incl.
erythrocytes,
leukocytes, muscle
cells) Þ necrosis
b) Theta (θ) toxin
§ Vascular damage
§ Oxygen-labile
cytolysin Þ rapid
tissue destruction –
§ pore-forming activity
§ alter capillary
permeability Þ toxic
to heart muscle
v ENZYMES —
COLLAGENASE, PROTEASE,
LIPASE causing more local
tissue necrosis and systemic
toxemia
1. Collagenase
§ Breaks down collagen
in connective tissues
Þ spread
2. Protease
§ Act on proteins/
proteinaceous
materials
3. Lipase
§ Hydrolyzes lipids (i.e.
skin)
v CARBOHYDRATE
FERMENTATION
Ability of C. perfringens to
ferment carbohydrates
§ Carbohydrates in
tissues of host are
utilized
FERMENTATION: anaerobic
breakdown of carbohydrates
Products:
Ø Acids
Ø Gas (large amounts)
§ Bubbles Þ swelling
Þ compression of
tissues Þ disrupt
blood flow Þ
favorable for the
growth of C.
perfringens
PREVENTION AND CONTROL
1. WOUND CLEANSING –
reducing the number pf
organisms/spores at the site
of infection
2. PROPHYLACTIC
ANTIBIOTIC THERAPY –
immediately followed after
wound cleansing; to kill or
eliminate the causative
agent/bacterial cells
3. SURGICAL PROCEDURES –
if there are manifestations of
gas gangrene (necrosis on the
tissue);
a. Debridement (removal) of
diseased tissue eliminates
the conditions that
promote spread of
gangrenous infections
- Supplemented by large doses
of broad-spectrum
antimicrobial agents in the
intestines/body cavity Þ
limited tissue can be removed
b. Amputation – surgical
removal of a part of the
body Þ extensive
myonecrosis
4. HYPERBARIC OXYGEN
THERAPY
- Breathing pure oxygen
using a hyperbaric
chamber (inclusion or
room)
- 3x the normal air
pressure Þ the higher air
pressure allows the lungs
absorb more oxygen Þ
inhibits growth and
reproduction of
organisms Þ promote
healing of wounds
- Growth of organisms are
inhibited by the presence
of oxygen
PERFRINGENS - Acquired by contamination of
FOOD POISONING food products with the spores
o From the soil
o Feces of man and
animals
COSCA 2
 - Frequently involved are: - Properly wash the
o Animal flesh (meat, meat/vegetables during
poultry, fish) preparation
o Vegetables (beans) not - Reducing the number of
cooked thoroughly to spores before cooking
destroy spores 2. ADEQUATE
o HIGH-PROTEIN FOODS REFRIGERATION
– provide nutritional - After cooking, do not
requirement for amino subject it to temperature
acids abuse/ keep the food at
- After cooking Þ spores survive warm temp. for a long time
Þ foods are maintained bet. 15 - Divide into smaller
to 16°C/ warm temp. (favors portions, cool, and
germination of spores to refrigerate
vegetative cells) RECAP from M5 Unit 1 Part A:
- Foods are cooled Þ spore B. cereus – cause of food
germinate and the germinated poisoning. Two types:
cells multiply (especially if • Emetic form – clinically
unrefrigerated) resembles Staphylococcal
MECHANISM: food poisoning
Ingestion of vegetative cells in • Diarrheal form – clinically
contaminated food Þ reach the GIT (manifestations and
Þ cells undergo sporulation incubation period)
(vegetative cells transform back resembles C. perfringens
into spores) Þ release enterotoxin food poisoning
(during sporulation) NECROTIZING - Food-borne infection
SIGNS AND SYMPTOMS: ENTEROCOLITIS (or o Acquired from
Occur in 8 to 16 hours up to 24 necrotizing bowel contaminated foods
hours after ingestion. disease/ NBD) o Transmissible by
o Crampy abdominal pail inanimate objects
o Foamy, foul-smelling - Necrosis of the intestinal
diarrhea mucosa of either/both the
o Nausea large and small intestine
o Fever & vomiting CHARACTERIZED BY :
(uncommon) Acute inflammation with focal to
- Recovery is rapid and death is widespread necrosis of the
extremely rare. intestinal mucosa.
VIRULENCE FACTOR SIGNS AND SYMPTOMS:
v C. Perfringens ENTEROTOXIN o Abdominal cramps
(CPE) o Abdominal distention
§ Responsible for the o Vomiting
symptoms of food poisoning o Bloody diarrhea
with C. perfringens o Shock (fluid & electrolyte
§ Released during sporulation problems)
of organisms Þ intestine o Death
§ Inserts into enterocyte - Other names depending on the
membranes to form pores region:
leading to: § Germany: “fire in the
• alterations in intracellular bowel”
calcium and membrane § New Guinea: “pig bel”
permeability. PREVENTION AND CONTROL
• leads to loss of cellular 1. GOOD COOKING HYGIENE
fluid and 2. ADEQUATE
macromolecules. REFRIGERATION
§ Penetrate and embed
themselves into the cell Clostridium tetani
membrane of the host Þ Common names:
creating pores and • Drumstick bacillus
channels/holes Þ leakage of • Lollipop bacillus
contents Þ cell death. • Tennis racquet bacillus
PREVENTION AND CONTROL • Tack head bacillus
1. GOOD COOKING HYGIENE GENERAL CHARACTERISTICS
• Gram+
COSCA 3
 • Spore-forming
• Spores: round and terminal on one end within a
swollen sporangium
• Motile, Nonencapsulated
• Strict obligate anaerobes Þ CANNOT grow in air;
grow with <0.5% O conc.
HABITAT AND TRANSMISSION
• SPORES found in the soil, GIT of man and animals
• Transmission:
o WOUND CONTAMINATION with spores from
soil or feces
PATHOGENESIS AND CLINICAL MANIFESTATIONS
TETANUS - Acquired by spore
Gk. tetanos = to stretch contamination of wound
o Necrotic tissue and poor
blood supply in the
wound Þ spore
germination & growth of
anaerobic clostridia Þ
Any type of injury
elaboration of
that breaks the
exotoxins.
skin can lead to
o cuts from a rusty can,
tetanus
tattoos, animal bites,
infections if,
splinters, surgical
spores of C.
wounds, injection drug
tetani enter the
use, body piercing, or
body through
lacerations.
broken skin, and
- Incubation period: 4 to 10
the person is not
days (shorter incubation
vaccinated.
period Þ more SERIOUS
condition)
CHARACTERIZED BY :
Spastic paralysis – severe
muscular spasms.
- The muscles are paralyzed in
a state of contraction; do
not relax
- Muscular spasm – muscles
contract uncontrollably
SIGNS AND SYMPTOMS:
1. Trismus (lock jaw) – early
effect of the disease due to
the rigid contraction of the
Trismus masseter muscle of the
face.
§ 1st manifestation on any
site of infection
§ Affects the muscle of the
jaw
Risus sardonicus
2. Risus sardonicus (sarcastic
smile) – sustained trismus;
patient looks eerily as though
he is smiling
3. Opisthotonus – involvement
Opisthotonus
of spinal musculature
§ Continuous back
spasms Þ extreme
arching of the back,
flexion of the arms,
extension of the legs
§ Contractions are
intermittent & extremely
painful Þ break the
vertebrae
- Respiratory failure ensues,
and without treatment,
results in high mortality rate
(up to 70%).
Tetanus neonatorum 4. Tetanus neonatorum
(neonatal tetanus) – fecal
contamination of the
umbilical cord/ use of
contaminated instruments
(e.g., umbilical stumps)
§ spastic paralysis of the
paravertebral muscles
§ locks the back into a
rigid, arched position
§ abnormal flexion of the
arms and the legs
VIRULENCE FACTORS
v TETANOSPASMIN
- Responsible for the
manifestations of tetanus
(small amt. required to
initiate symptoms)
- Primary virulence factor
- Released by vegetative
cells at the site of
infection
o Spread via blood
steam
o Bacilli remain at the
site of infection
- NEUROTOXIN
- Heat-stable
- Plasmid-mediated
- Binds to peripheral nerves
(presynaptic membranes
of motor neurons) Þ
reach the CNS
- Binds to gangliosides Þ
Mechanism of blocks release of
tetanospasmin inhibitory
neurotransmitters (glycine
and γ- aminobutyric acid
or GABA) Þ muscles
released from inhibition &
begin to contract
uncontrollably Þ spastic
paralysis
- Binding of the toxin is
irreversible Þ recovery
requires generation of
new axon terminals.
v TETANOLYSIN
- Hemolysin Þ responsible
for lysis of RBCs and
other cell types
- In the lab: C. tetani
produces a β-hemolysis
on BAM
COSCA 4
 - Causes local tissue • Strict obligate anaerobes Þ CANNOT grow in air;
necrosis at sites of grow with <0.5% O conc.
vegetative growth HABITAT AND TRANSMISSION
o lowers oxygen • Spores found in the SOIL Þ contaminate
tension in tissue VEGETATION
PREVENTION AND CONTROL • Transmission:
1. ROUTINE ACTIVE o INGESTION of preformed toxin or spores in
IMMUNIZATION food
- Tetanus toxoid o WOUND CONTAMINATION with spores
- Usually combined with PATHOGENESIS AND CLINICAL MANIFESTATIONS
o DPT vax for BOTULISM
primary - CHARACTERIZED BY: Flaccid paralysis
immunization in o The muscles are maintained/paralyzed in a
childhood state of relaxation; muscles do not contract
o DT for adults 1. Classical botulism/ Food-borne botulism
- completely prevent 2. Infant botulism
tetanus 3. Wound botulism
- 5 doses = 2, 4, 6, & 18 CLASSICAL - Acquire by ingestion of
months BOTULISM or preformed toxin/ contaminated
- 1 dose = bet. 4-6 y/o FOOD-BORNE food
- Booster = every 10 yrs BOTULISM - FOOD INTOXICATION
- Naturally acquired (L. botulis = sausage: - Associated with improperly
tetanus infections do not the disease was canned goods and vacuum-
provide immunity to earlier associated packed food; home-canned,
subsequent infections. with spoiled sausage) low acid foods
2. ANTITOXIN THERAPY o Vegetables
- Patient with symptoms o Fruits
of tetanus should o Condiments
immediately receive o Fish
antitoxin therapy - Inadequate sterilization Þ
- Prepared from horse fails to kill spores during
blood canning process
- Administration of human - Canning Þ creates little to no
tetanus immune globulin air environment; favors
(TIGH) anaerobic germination of
- Neutralizes circulating spores into vegetative cells Þ
toxin; ineffective against produce toxins
toxin already fixed in the SIGNS AND SYMPTOMS:
CNS Lack of muscle contraction
3. ANTIBIOTIC TREATMENT o Droopy eyes
- Penicillin strongly o Dysphagia (difficulty in
inhibits the growth of C. swallowing)
tetani and stops further Dysphonia (difficulty in
toxin production. speech)
- To kill and eliminate o Diplopia (double/blurry
bacterial cells vision)
4. WOUND CLEANSING o Weak muscles
- done immediately after o Dizziness, no sensory or
vaccination mental lapse
- to reduce the number of Later symptoms:
organisms o Descending muscular
contaminating the paralysis
wound site o Cardiac arrhythmias &
blood pressure instability
Clostridium botulinum o Death results from
Common name: Canned good bacillus respiratory paralysis
GENERAL CHARACTERISTICS (unless there is
• Large, Gram-positive mechanical ventilation)
• Spore-forming INFANT BOTULISM - Occurs in infants bet. 3 weeks
• Spores: oval and subterminal in location within a to 8 months
swollen sporangium, “SNOW SHOE” appearance - Acquired by ingestion of
• Motile, Nonencapsulated spores C. botulinum in
contaminated honey

COSCA 5
 o Soil, household dust, 2. Administration of large doses of horse C. botulinum
etc. antitoxin Þ neutralize free toxin
- Spores will germinate in GIT Þ 3. Avoid giving honey to infants less than 12 months of age
vegetative cell Þ toxin
- MICROBIAL ANTAGONISM: Clostridioides difficile
Adults do not acquire the Formerly Clostridium difficile
disease—have a normal flora in HABITAT AND TRANSMISSION
the GIT • Normal GIT flora in humans (2-10%); higher rates
SIGNS AND SYMPTOMS: among hospitalized persons & infants
o Lack of muscle • Disease acquired ENDOGENOUSLY
contraction PATHOGENESIS AND CLINICAL MANIFESTATIONS
o Constipation ANTIBIOTIC- - Drug-resistant C. difficile
o Hypotonia ASSOCIATED super infect the intestine
o Poor muscle tone DIARRHEA (AAD) & Þ normal flora disrupted
o Lethargy PSEUDOMEMBRANOUS by antibiotics
o Feeding problems COLITIS (PMS, or - Predominant symptom:
- Sudden infant death Antibiotic associated DIARRHEA
syndrome (SIDS) or “crib colitis) o 5-10 days into
death” antimicrobial
WOUND BOTULISM - Acquires by spore therapy/after therapy
contamination of wounds a o Mild & watery or
puncture, germinate and bloody
produce botulinum toxins. o Accompanied by
- Symptoms similar to food- abdominal cramping
borne botulism o Leukocytosis
- Increased in injecting drug o Fever
users - Colon Þ inflamed &
VIRULENCE FACTOR sloughs off loose,
v BOTULINUM TOXIN (BOTULIN) membrane-like patches
§ Neurotoxin – binds to the peripheral nerves (pseudomembranes) Þ
§ Heat-labile, phage-mediated consists fibrin and
• Strains that have been lysogenized can inflammatory cells
produce the toxin - Not arrested Þ cecal
§ Most potent exotoxins (with tetanospasmin) (cecum) perforation &
§ 7 serotypes (A to G) death
• Human botulism: A, B, D , F (least common VIRULENCE FACTOR
• Bird and animals: C and E v POLYPEPTIDE TOXINS: Toxin A (enterotoxin) and
• Produced by C. argentinense: G Toxin B (cytotoxin/ cytopathic toxin)
§ Blocks the release of excitatory § Cause necrosis of intestinal epithelium
neurotransmitter acetylcholine • Act in cytoplasm of enterocytes Þ disruption
at the peripheral nerve endings, of intercellular tight junctions Þ altered
including neuromuscular membrane permeability & fluid secretion
junctions, resulting in lack of § Within hours of contact w/ enterocytes cell,
muscle contraction ---flaccid rounding & neutrophilic infiltration appear.
paralysis. PREVENTION AND CONTROL
1. Infected persons shed large numbers of spores in their
Cosmetic injection of botulinum toxin (botox). stools Þ increased precautions Þ prevent spread to
Botulinum toxin in controlled doses can be used to temporarily other patients on antibiotic therapy.
minimize wrinkles. 2. Vaccination of C. difficile toxoid & restoration of normal
flora by mixed culture lactobacilli & yeasts.
PREVENTION AND CONTROL 3. Mild, uncomplicated cases Þ withdrawal of antibiotics &
1. Proper methods of preserving and handling canned replacement therapy (lost fluids & electrolytes)
foods.
- Adequate pressure cooking or autoclaving in the
canning process kills spores, & heating food at 100°C
for 10 minutes before eating destroys the toxin.
- Food from damaged cans or those that present
evidence of positive inside pressure should not even
be tasted because of the extreme toxicity of C.
botulinum toxin.
- Bulging cans or bottles that look or smell spoiled
should be discarded.

COSCA 6