MICROBIOLOGY

Sporeformers
Dr. Quiles
SPOREFORMERS o With similar hemolytic and necrotizing
Theta Toxin effects as alpha toxin but is not a lecithinase
Anaerobic Spore-forming Bacteria Aerobic Spore-forming Bacteria
Clostridium perfringens- most common Bacillus anthracis o Acts in intestines
Clostridium tetani Bacillus cereus o Causes intestinal stasis, loss of intestinal
Clostridium botulinum Bacillus subtilis mucosa
Beta Toxin
Clostridium difficile o Responsible for necrotic lesions in
necrotizing enterocolitis
 These are bacteria which form spore
o Protoxin
 These bacteria are very resilient to the environment o Activated by trypsin
 Some of them are part of the normal flora Epsilon Toxin
o Increased vascular permeability in GI wall
 Some of them come from soil and produce disease conditions
o Produced by Type E Clostridium perfringens
CLOSTRIDIUM SPECIES CHARACTERISTICS o Has necrotic activity and increased vascular
Iota Toxin
 Large; motile (most with peritrichous flagella); some species permeability
aerotolerant
 Natural habitat: soil or intestinal tract of animals and humans; water o Produced primarily by Type A strains
o Heat labile toxin
contaminated by feces
o Trypsin enhances toxic activity
 They possess spores. However in Clostridium perfringens , they
o Causes alteration of membrane permeability
cannot grow spores in culture media o Cytotoxic and Enterotoxic
 Location of spores: central, subterminal, or terminal Enterotoxin o Released during spore formation
 Most of them are obligate anaerobes/strictly anaerobic. Some of o Acts as superantigen that stimulates T
them are aerotolerant like Clostridium tertium and Clostridium lymphocytes activity
histolyticum o Antibodies against enterotoxin are non-
 Gram (+) bacilli except Clostridium ramosum, gram (-) protective
 Part of the normal flora of GIT → Saprophytes
 Ferments sugars; many can digest proteins → basis for dividing
Clostridia into Saccharolytic and Proteolytic group CLINICAL SYNDROMES
 Pathology: Botulism, Tetanus, Gas gangrene, Pseudomembranous  Bacteremia – Transient bacteremia or can be rapidly fatal
colitis  Soft tissue infections – Cellulitis, and Fasciitis
 Survive adverse environmental condition  Uterine infection – following abortion or prolonged labor
 They grow rapidly in an enriched anaerobic media  Myconecrosis (Gas gangrene)
 Produce a lot of toxins o Introduced into the skin by trauma or surgery → 1 week
→ intense pain → 2 days → extensive muscle necrosis,
Clostridium perfringens hemolysis and bleeding shock, renal failure and death
 Formerly known as C. weichii o Gas in the tissue due to the metabolic activity of the
rapidly dividing bacteria
 Non-motile
 Enteric Infections
 Large, rectangular, gram (+) bacillus
MOT: Ingestion of meat products
 Rarely produce spores
Short incubation period (8-24 hrs)
 Capable of rapid spreading growth on laboratory media
Abdominal cramps, watery diarrhea w/o
 Capable of beta hemolysis on blood containing media (double zone) Food Poisoning
fever and Nausea and vomiting
 Most common and most invasive among Clostridial species Clinical course: 24 -48 hrs
 Multiply rapidly inside the host
 Produce invasive infection Necrotizing eneterocolitis or pig-bel
 Subdivided into 5 types (A to E) based on toxins produced Due to beta toxin producing Type C
o Type A – cause most human disease Abdominal pain, bloody diarrhea,
o Types B – E – do not survive in soil → colonize intestinal intestinal perforation → Shock and
Enteritis
tract mainly of animals Peritonitis
Necroticans
Sweet potatoes → contains heat resistant
Major Lethal Toxins:
trypsin inhibitor → protect beta toxin
o A lecithinase (phospholipase C) ; produced by
from trypsin activation
all types but greatest amount produced by Type
A
o Lyses erythrocytes, platelets, leukocytes &
endothelial cells LABORATORY DIAGNOSIS
Alpha Toxin o Produced by all 5 types of Clostridium perfringes  Microscopy of clinical specimen → gram (+) bacilli in the absence of
o Responsible for increased vascular permeability leukocytes
with massive hemolysis and bleeding tissue
 Culture – rapid growing
destruction, hepatic toxicity and myocardial
dysfunction (hypotension and bradycardia) →  Recovery of bacilli on food → 10 bacteria/gram of food
Gangrenous tissue and sepsis  From feces → >10 bacteria/gram of feces

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 MICROBIOLOGY
Sporeformers
Dr. Quiles
TREATMENT AND PREVENTION Clostridium botulinum
 Soft tissue infections → Surgical debridement and high dose  Large, fastidious, sporeforming anaerobic bacilli
Penicillin  Four groups: Types I and II → common cause of human infection
 Hyperbaric oxygen  Seven botulinum toxins: A, B, C alpha, D, E, F, G
 For food poisoning → Self-limiting → No need for antibiotics  Toxins A, B, E, F → human diseases
 Type E – fish products
Clostridium Tetani  Types A, B or F → infant botulism
 Large, motile sporeforming bacilli, gram (+)  Other species that produce botulinum toxins
 Produces round terminal spores → “drumstick appearance” o Clostridium butyricum → Type E toxin
 Strict anaerobes without capsule o Clostridium baratii → Type F toxin
 Proteolytic but cannot ferment carbohydrates o Clostridium argentinense Type G toxin – do not cause
 Commonly found in the environment particularly in the soil human disease
 Rarely cause human infection
TOXINS
 Tetanospasmin-neurotoxin BOTULINUM TOXIN
o Binds with gangliosides (synaptobrevin) → blocks release  Neurotoxin subunit
of inhibitory transmitters (GABA, glycine) at the inhibitory o Very specific for cholinergic nerves
synapses → unregulated excitatory synaptic activity in the o Blocks the release of Ach at the peripheral synapse →
motor neuron → spastic paralysis flaccid paralysis
 Tetanolysin  Non-toxic subunit
o O2 labile hemolysin (similar to activity of C. perfringens o Protect the neurotoxin from inactivation of gastric acid
hemolysin)
o Serologically-related to streptolysin O Receptor: sialic acid receptors and glycoproteins
Target SNARE proteins (synaptobrevin, SNAP 25, syntaxin) in the neurons →
CLINICAL FORMS OF TETANUS inhibit release of Ach → no muscle contraction → flaccid paralysis
 Incubation period: 4-5 days to as many as weeks Types A & E toxin cleave SNAP 25
Type B toxin – cleave synaptobrevin
 Generalized tetanus
o Most common form
BOTULISM
o Involvement of the masseter → trismus or lock jaw
o Sustained trismus → Sardonic smile (Risus sardonica)  Classical (food-borne) botulism
o Persistent back spasm → opisthotonus o Commonly associated with home canned foods and
occasionally preserved fish
 Localized tetanus
o Food may even a small taste can cause full-blown clinical
o Confined to the muscle at the side of primary infection
disease
o Variant → Cephalic tetanus → Poor prognosis
o Incubation period: 18-36 hours after ingestion
 Neonatal tetanus (Tetanus neonatorum)
o Manifestation:
o Initial site of umbilical stump → generalized
 Weakness and dizziness → blurred vision with
fixed dilated pupils, dry mouth, constipation,
LABORATORY DIAGNOSIS
abdominal pains → flaccid paralysis (bilateral,
 History and physical examination
descending paralysis) → respiratory paralysis
 Microscopy – unsuccessful recovery of bacteria
→ Death
 Culture – slow growing
 No fever
 Infant botulism
TREATMENT
o Infants, 1 y/o (1-6 months)
 Wound debridement o Non-specific symptoms like failure to thrive or
 Penicillin or metronidazole – Drug of Choice constipation → flaccid paralysis → respiratory arrest
o Metronidazole preferred over penicillin because Pen o Associated with unpasteurized honey
inhibits GABA → CNS excitability o Poor feeing, weakness, signs of paralysis (“floppy baby”)
 Passive immunization with human tetanus Ig – Anti toxin o Implicated with SIDS (Sudden Infant Death Syndrome)
 Active immunization with tetanus toxoid → Infection does not  Wound botulism
confer immunity o Symptoms identical to food-borne botulism but longer
incubation period (1-4 days) and GI symptoms less
PREVENTION prominent
 DPT vaccine (Diphtheria, Pertussis and Tetanus toxoid) given 3 doses  Inhalation botulism → concentrated botulinum toxin → aerosolized
(2,4,6 months) → bioterrorism
 Boosters every 10 years
LABORATORY DIAGNOSIS: Culture

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 MICROBIOLOGY
Sporeformers
Dr. Quiles
TREATMENT Bacillus anthracis
 Gastric lavage – to eliminate the organism from the GIT  Large, gram (+) bacilli in long chain → bamboo fishing rod
 Penicillin – drug of choice appearance
 Trivalent Botulinum Antitoxin – to neutralize circulating toxin  Produce spores, encapsulated
 Adequate ventilatory support  Microscopic appearance: Serpentine chains of bacilli → medusa
head or curled hair look appearance
Clostridium difficile  Could be major agent of bioterrorism
 Responsible for antibiotic-associated pseudomembranous
enterocolitis ANTIGENIC STRUCTURE
 Colonizes the small intestine of a small percentage of healthy  Capsular Ag-Polypeptide capsule
individual o Consists of D-glutamic acid
 Antibiotic alters the normal enteric flora → overgrowth of the o Antiphagocytic
organism → produce toxin → disease  Somatic Ag-Polysaccharide cell wall
 New strain: o Plays no role in virulence
o Causes disease in US and Europe → Highly virulent →  Exotoxin component
more severe disease, higher mortality, increased risk of o Factor I – Edema factor
relapse and more complications o Factor II – Protective Ag
o New strain produce toxin → binary toxin → facilitate o Factor III – Lethal factor
adherence to surface of epithelial cells  Virulent strains carry genes for 3 toxin components on a large plasmid
o Resistant to fluoroquinolones → pXO1
 Individual toxins → protective antigen (PA), edema factor (EF), lethal
TOXINS factor (LF) → non-toxic individually
 Enterotoxin (Toxin A)  When combined individual toxins from important toxins
o Chemotactic for neutrophils with infiltration of PMNs into o PA → binds with 2 host cell receptors → formation of
the ileum → release of cytokines complex → enhances endocytosis
o Have a cytopathic effect → increase permeability of o LF → Zinc-dependent proteases → cleave mitogen-activated
intestinal wall → diarrhea with hemorrhagic necrosis protein kinase → cell death
 Cytotoxin o EF → Calmodulin-dependent adenylate cyclase → increased
o Causes depolarization of actin with destruction cellular intracellular cAMP levels → edema
o PA + EF → Edema toxin
cytoskeleton
o PA + LF → Lethal toxin
Antibiotics that can cause Pseudomembrane Enterocolitis:
 Ampicillin
 Virulence is due to:
 Clindamycin
o Capsule
 Lincomycin
o Edema toxin – fluid accumulation
 Fluoroquinolones o Lethal toxin – promotes lysis of macrophages and release
of TNF
LABORATORY DIAGNOSIS  PA – most immunogenic
 Detection of the cytotoxin or enterotoxin or toxin genes in the feces  Both EF and LF – inhibits host’s innate immune system
by nucleic acid amplification technique
 Isolation by stool culture
ANTHRAX
 Primarily a disease of herbivores (sheep, cattles, horse)
TREATMENT
 Infections on human are rare
 Discontinue the drug if it has negative side effect (e.g. diarrhea)
 Forms:
 In severe cases → Vancomycin or Metronidazole
o Cutaneous
 MOT: Inoculation of spores into breaks in the
Clostridium septicum skin or mucous membrane
 Cause non-traumatic myonecrosis  Painless papule at site of inoculation 12-36 hrs
 Often exist in patients with diabetes, leukemia, and Colon CA after entry → vesicle → pustule → necrotic
 Breach in intestinal mucosa and compromised host defenses → ulcer with blackened scab (eschar)
spread to tissues → proliferate → gas formation → death  Painful lymphadenopathy and massive edema
 Exhibit swarming on culture media may develop
 20% mortality
Clostridium sordelii o Gastrointestinal
 Implicated in fatal toxic shock syndrome associated with natural  Invasion of the upper intestinal tract → ulcers
childbirth or medically-induced abortions in the mouth and esophagus → regional
lymphadenopathy, edema, sepsis
Clostridium tertium  Invasion of cecum → nausea, vomiting →
 Associated with traumatic wound infections progress rapidly to systemic disease
 Grow aerobically unlike other Clostridia spp.  Almost 100% mortality

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 MICROBIOLOGY
Sporeformers
Dr. Quiles
Anthrax continued…..  At risk are:
o Pulmonary (Woolsorter’s disease) o People who eat contaminated food
 MOT: inhalation of spore into lungs o With penetrating injuries
 Early stage: non-specific symptoms → fever, o Those who received IV injections
non-productive cough, myalgia, malaise o Immunocompromised patients
 Second stage: rapid worsening of course  Resistant to penicillin and cephalosporin
Fever, edema, enlargement of mediastinal
LN → Sepsis, meningitis, or hemorrhagic TREATMENT
pulmonary edema  For gastroenteritis → fluid replacement
Pneumonia rarely develops  Other infections → Immediate treatment with Vancomycin,
Shocks and death within 3 days of initial
Clindamycin, Ciprofloxacin or Gentamicin
symptoms
 For vancomycin and clindamycin, maybe with or without
aminoglycosides
LABORATORY DIAGNOSIS
 Gram stain → large, gram (+) rods
Bacillus subtilis
 Culture → gray to white colonies with ground glass appearance;
 Exhibit motility by swarming
comma shaped outgrowth → “Medusa head”
 Common laboratory contaminant
 Demonstration of spore (culture at low CO2 atmosphere and
 Cause infection only in immunocompromised individual
malachite green or india ink)
 M’Fadyean methylene blue stain
 Direct fluorescent antibody
REFERENCES

PREVENTION  Microbiology Manual (2018)

CONTROL
 Disposal of animal carcasses by burning or by deep burial of lime pits
 Decontamination of animal products by autoclaving
 Protective clothing and gloves for handlers
 Active immunization with live attenuated vaccine

TREATMENT
 Penicillin + Gentamicin
 Ciprofloxacin or doxycycline combined with any of the ff:
o Rifampicin
o Vancomycin
o Clindamycin
o Clarithromycin
o Imipenem

Bacillus cereus
 Low virulence and is an opportunistic pathogen
 Causes:
o Food poisoning
 Mediated by one of 2 toxins:
 Heat labile enterotoxin- increased
cAMP → diarrheal form; meat
dishes and sauces
 Heat stable enterotoxin-
superantigen → emetic form; rice
dishes
o Eye infections
 Traumatic eye infections (keratitis,
endopthalmitis, panopthalmitis)
 Due to 3 toxins:
 Necrotic toxin – heat labile
 Cereolysin – hemolysin
 Phospholipase C – lecithinase
o Sepsis

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