Anaerobic, Spore-forming,

1
Anaerobic, Spore-forming, Gram-Positive Rods
?????? ? ? ?
2
Clostridium - anaerobic, gram-positive rods
capable of forming endospores (1) presence of
endospores, (2) strict anaerobic metabolism,
(3) inability to reduce sulfate to sulfite, (4)
gram-positive cell wall structure. - Spores are
rarely Clostridium perfringens, Clostridium
ramosum, - Aerotolerant Clostridium tertium,
Clostridium histolyticum - Some clostridia
gram-negative C. ramosum, Clostridium
clostridioforme
BOX 39-1. Important Clostridia
Organism Historical Derivation
Clostridium closter, a spindle
C. botulinum botulus, sausage (the first major outbreak was associated with insufficiently smoked sausage)
C. difficile difficile, difficult (difficult to isolate and grow refers to the extreme oxygen sensitivity of this organism)
C. perfringens perfringens, breaking through (associated with highly invasive tissue necrosis)
C. septicum septicum, putrefactive (associated with sepsis and a high mortality)
C. tertium tertium, third (historically, the third most commonly isolated anaerobe from war wounds
C. tetani tetani, related to tension (disease caused by this organism characterized by muscle spasms)
3

• The remarkable capacity of clostridia to cause
  diseases
• ability to survive adverse environmental
  conditions through spore formation
• (2) rapid growth in a nutritionally enriched,
  oxygen-deprived environment,
• (3) production of numerous histolytic toxins,
  enterotoxins, and neurotoxins.

• Ubiquitious soil, water, sewage
• Tetanus
• Botulism
• Gas gangrene
• Diarrhea and colitis

4
Table 39-1. Pathogenic Clostridia and Their
Associated Human Diseases
Species Human Disease Frequency
C. difficile Antibiotic-associated diarrhea, pseudomembranous colitis Common
C. perfringens Soft-tissue infections (e.g., cellulitis, suppurative myositis, myonecrosis, gas gangrene), food poisoning, enteritis necroticans, septicemia Common
C. septicum Gas gangrene, septicemia Uncommon
C. botulinum Botulism Uncommon
C. tetani Tetanus Uncommon
C. tertium Opportunistic infections Uncommon
C. baratii Botulism Rare
C. butyricum Botulism Rare
C. clostridioforme Opportunistic infections Rare
C. histolyticum Gas gangrene Rare
C. innocuum Opportunistic infections Rare
C. novyi Gas gangrene Rare
C. sordellii Gas gangrene Rare
C. sporogenes Opportunistic infections Rare
5
Clostridium Perfringens
PHYSIOLOGY AND STRUCTURE

• large (0.6 to 2.4 1.3 to 19.0 µm),
  rectangular, gram-positive rod
• spores rarely observed either in vivo or after
  in vitro cultivation.
• nonmotile clostridia
• rapidly spreading growth on laboratory media
• organism grows rapidly in tissues and in
  culture, is hemolytic, and is metabolically
  active,

• Growth of Clostridium perfringens on sheep blood
  agar.
• flat, spreading colonies and the hemolytic
  activity of the organism.
• zone of complete hemolysis (caused by the
  ?-toxin) - wider zone of
• partial hemolysis (caused by the a-toxin)

6
PATHOGENESIS AND IMMUNITY
Table Distribution of Lethal Toxins in
Clostridium perfringens Types A to E
Type Lethal Toxins Lethal Toxins Lethal Toxins Lethal Toxins
  Alpha Beta Epsilon Iota
A - - -
B -
C - -
D - -
E - -
- The production of one or more major lethal
toxins by C. perfringens (alpha a, beta ß,
epsilon e, and iota ? toxins) - subdivide
isolates into five types (A through E).
7
Table. Virulence Factors Associated with
Clostridium perfringens
Virulence Factors Biologic Activity
a-Toxin Lethal toxin phospholipase C (lecithinase) increases vascular permeability hemolysin produces necrotizing activity, as seen in myonecrosis
ß-Toxin Lethal toxin necrotizing activity
e-Toxin Lethal toxin permease
?-Toxin Lethal binary toxin necrotizing activity adenosine     diphosphate (ADP) ribosylating
d-Toxin Hemolysin
?-Toxin Heat- and oxygen-labile hemolysin cytolytic
?-Toxin Collagenase gelatinase necrotizing activity
?-Toxin Protease
µ-Toxin Hyaluronidase    
?-Toxin Deoxyribonuclease hemolysin necrotizing activity
Enterotoxin Alters membrane permeability in ileum (cytotoxic, enterotoxic) superantigen
Neuraminidase Alters cell surface ganglioside receptors promotes capillary thrombosis
8
a-Toxin
Lethal toxin phospholipase C (lecithinase)
increases vascular permeability hemolysin
produces necrotizing activity, as seen in
myonecrosis

• Lecithinase (phospholipase C)
• lyses erythrocytes, platelets, leukocytes,
  and endothelial cells.
• massive hemolysis
• increased vascular permeability and bleeding
  (augmented by destruction of platelets)
• tissue destruction (as found in myonecrosis)
• hepatic toxicity, and myocardial dysfunction
  (bradycardia, hypotension)

• Growth of Clostridum perfringens on egg-yolk
  agar.
• The a-toxin (lecithinase) hydrolyzes
  phospholipids in serum and egg yolk, producing an
  opaque precipitate (right).
• This precipitate is not observed when the
  organism is grown in the presence of antibodies
  against the toxin (left).
• Nagler's reaction is characteristic of C.
  perfringens.

9
ß-Toxin
Lethal toxin necrotizing activity

• responsible for intestinal stasis, loss of
  mucosa with formation of the necrotic lesions,
  and progression to necrotizing enteritis
  (enteritis necroticans ??? ?????, pig-bel).
• e-Toxin, Lethal toxin permease, a protoxin, is
  activated by trypsin and increases the vascular
  permeability of the gastrointestinal wall.
• ?-Toxin, the fourth major lethal toxin, Lethal
  binary toxin necrotizing activity adenosine
  diphosphate (ADP) ribosylating, is produced by
  type E C. perfringens. This toxin has necrotic
  activity and increases vascular permeability.

Enterotoxin - Type A strains. Alters
membrane permeability in ileum (cytotoxic,
enterotoxic) superantigen produced during
the phase transition from vegetative cells to
spores released with the formed spores
when the cells undergo the terminal stages of
spore formation (sporulation). released
enterotoxin binds to receptors on the brush
border membrane of the small intestine
epithelium in the ileum (primarily) and jejunum
but not duodenum. altered membrane
permeability and loss of fluids and ions.
acts as a superantigen simulating T lymphocyte
activity.
10
EPIDEMIOLOGY

• inhabits the intestinal tract of humans and
  animals
• widely distributed in nature, particularly in
  soil and water contaminated with feces
• Spores are formed under adverse environmental
  conditions and can survive for prolonged periods.
  
• - Type A C. perfringens responsible for most
  human infections, including soft-tissue
  infections, food poisoning, and primary
  septicemia.
• - Type C C. perfringens responsible for one
  other important infection in humans-enteritis
  necroticans (?????)

11
CLINICAL DISEASES
Soft-Tissue Infections (?????)

• cellulitis (???? ???)
• gas formation in the soft tissue

- Clostridia can be introduced into tissue during
surgery or by a traumatic injury. - Five days
after the injury, the skin became discolored and
bullae and necrosis developed.
(2) suppurative myositis (?????)
accumulation of pus in the muscle planes,
but muscle necrosis and systemic symptoms are
absent. (3) myonecrosis (???) or gas gangrene
(????). Clostridial myonecrosis
life-threatening disease, histotoxic clostridia
- intense pain, - develops within a week
after clostridia are introduced into tissue by
trauma or surgery - rapidly by extensive
muscle necrosis, shock, renal failure, an death,
often within 2 days of initial onset. - Gas
found in the tissue is caused by the metabolic
activity of the rapidly dividing bacteria (hence
the name gas gangrene)
12
Food Poisoning

• a short incubation period (8 to 24 hours),
• a clinical presentation that includes abdominal
  cramps and watery diarrhea but no fever, nausea,
  or vomiting
• a clinical course lasting 24 to 48 hours.
• ingestion of meat products (e.g., beef, chicken,
  turkey) contaminated with large numbers (108 to
  109 organisms) of enterotoxin-containing type A
  C. perfringens.
• Holding contaminated foods at temperatures below
  60C (46C is optimal) allows spores that
  survived the cooking process to germinate and
  multiply to high numbers.
• The refrigeration of food after preparation
  prevents this bacterial growth. Alternatively,
  reheating of the food can destroy the heat-labile
  enterotoxin.

13
Necrotizing Enteritis (enteritis necroticans or
pig-bel)

• rare, acute necrotizing process in the jejunum
  characterized by acute abdominal pain, vomiting,
  bloody diarrhea, ulceration of the small
  intestine, and perforation of the intestinal
  wall, leading to peritonitis(???) and shock.
• mortality in patients 50.
• ß-Toxin produced by C. perfringens Type C
• Necrotizing enteritis is most common in Papua
  New Guinea
• eating undercooked, contaminated pork with
  sweet potatoes
• heat-resistant trypsin inhibitor ?
  protects the ß-toxin from inactivation by
  trypsin.

Septicemia
- When C. perfringens is isolated in the blood
from patients with significant infections (e.g.,
myonecrosis, necrotizing enteritis), the organism
is typically associated with massive hemolysis.
14
LABORATORY DIAGNOSIS

• Microscopic detection of gram-positive rods in
  clinical specimens,
• in the
  absence of leukocytes, characteristic morphology.
  
• culture anaerobes.
• growth on agar media or in blood culture broths
• food poisoning more than 105 organisms/g food
  or more than 106 bacteria/g feces
• Immunoassays detection of the enterotoxin in
  fecal specimens

TREATMENT, PREVENTION, AND CONTROL
- C. perfringens soft tissue infections
(suppurative myositis and myonecrosis)
treated aggressively with surgical débridement
and high-dose penicillin therapy Hyperbaric
oxygen treatment - results are inconclusive

• Antibiotic therapy for clostridial food
  poisoning is unnecessary
• self-limiting disease (i.e., the diarrhea
  washes the bacteria out of the intestines and the
  normal intestinal flora reestablishes itself).

• Prevention and control of C. perfringens
  infections are difficult
• organisms are ubiquitous.
• wound care and the judicious use of prophylactic
  antibiotics

15
Clostridium tetani
PHYSIOLOGY AND STRUCTURE

• large (0.5 to 1.7 2.1 to 18.1 µm), motile,
• spore-forming rod (round, terminal spores
  drumstick)
• extremely sensitive to oxygen toxicity, strict
  anaerobe
• when growth is detected on agar media,
• it typically appears as a film over the
  surface
• of the agar rather than discrete colonies
• proteolytic but unable to ferment carbohydrates.
  
• Tetani related to tension (disease caused by
  this organism characterized by muscle spasms)

PATHOGENESIS AND IMMUNITY
Two toxins 1) tetanolysin oxygen-labile
hemolysin, clinical significance unknown,
inhibited by oxygen and serum
cholesterol 2) tetanospasmin plasmid-encoded,
heat-labile neurotoxin
nonconjugative.
16

• Tetanospasmin (A-B toxin) 150,000Da
• - Endogenous protease cleavage
• Disulfide bond and noncovalent forces hold the
  two chains together
• B-chain (heavy chain) carbohydrate-binding
  domain, carboxyl-terminal portion, binds to
  specific sialic acid receptors (e.g.,
  olysialogangliosides) and adjacent glycoproteins
  on the surface of motor neurons.
• endosomal vesicles and transported in the neuron
  axon to motor neuron soma located in the spinal
  cord ? endosome ? acidified ? conformational
  change of the heavy chain ? passage of the toxin
  light chain into the cytosol
• A-chain (light chain) zinc endopeptidase
• ? cleaves core proteins involved in the
  trafficking and release of neurotransmitters
• ? inactivates inhibitory neurotransmitters
  glycine and gamma-aminobutryic acid (GABA)
• ? spastic (rigid) paralysis (??? ??) toxin
  binding is irreversible, so recovery depends on
  whether new axonal terminals form

17
EPIDEMIOLOGY

• ubiquitous, fertile soil, transiently colonizes
  the gastrointestinal tracts of many animals
• relatively rare high incidence of
  vaccine-induced immunity
• many deaths in developing countries where
  vaccination is unavailable or medical practices
  are lax, more than 1 million cases occur
  worldwide, with a mortality rate ranging from 30
  to 50

CLINICAL DISEASES

• Generalized tetanus (??? ???) most common form
• - Involvement of the masseter muscles (???,
  trismus or lockjaw, ????, ?? ??????? ??), risus
  sardonicus (??)
• early signs drooling, sweating, irritability,
  and persistent back spasms (??, ??)
  (opisthotonos)
• - autonomic nervous system is involved in
  patients with more severe disease cardiac
  arrhythmias, fluctuations in blood pressure,
  profound sweating, and dehydration.

18
localized tetanus (??? ???) - musculature at the
site of primary infection. cephalic tetanus
(? ???)- primary site of infection is the head,
very poor.
Neonatal tetanus (tetanus neonatorum) initial
infection of the umbilical stump
?
generalized mortality in infants exceeds 90,
and developmental defects in survivors almost
exclusively a disease in developing countries.
LABORATORY DIAGNOSIS

• - Diagnosis of tetanus basis of the clinical
  presentation
• Microscopic detection - frequently unsuccessful
• Culture results are positive in only
  approximately 30 of patients with tetanus
• disease can be caused by relatively few
  organisms
• slow-growing bacteria are killed rapidly
  when exposed to air
• Neither tetanus toxin nor antibodies to the toxin
  are detectable in the patient
• toxin is rapidly bound to motor neurons
  and internalized

19
TREATMENT, PREVENTION, AND CONTROL
Treatment of tetanus débridement of the
primary wound (which may appear innocuous)
use of metronidazole- eliminate the vegetative
bacteria passive immunization with human
tetanus immunoglobulin (TIG-tetanus antitoxin)
- binding free tetanospasmin molecules
vaccination with tetanus toxoid (TD)
penicillin should not be used Vaccination a
series of three doses of tetanus toxoid (DPT)
? booster doses every 10 years, is
highly effective in preventing tetanus.
20
Clostridium botulinum
PHYSIOLOGY AND STRUCTURE

• heterogeneous group of large (0.6 to 1.4 3.0
  to 20.2 µm),
• fastidious (???? ????), spore-forming, anaerobic
  rods.
• Seven antigenically distinct botulinum toxins (A
  to G)
• human disease is associated with types A,
  B, E, and F.
• - Toxin production is associated with specific
  groups

Table Clostridium botulinum Classification and
Toxin Production
Group Neurotoxin Type Phenotyptic Properties
I A, B, F Proteolytic, saccharolytic
II B, E, F Nonproteolytic, saccharolytic
III C, D Weakly proteolytic, saccharolytic
IV G Weakly proteolytic, asaccharolytic
21
PATHOGENESIS AND IMMUNITY

• Like tetanus toxin
• A-B toxin , 150,000-Da
• heavy or B chain
• - large, nontoxic subunit passage through
  the digestive tract
• - carboxyl-terminal portion binds specific
  sialic acid receptors and glycoproteins of motor
  neurons
• ? stimulates endocytosis of the toxin
  molecule
• ? Acidification of the endosome
• ? N-terminal, heavy chain mediated release
  of the light chain
• light or A chain - zinc-endopeptidase
  activity
• ? inactivates the proteins that regulate
  release of acetylcholine,
• ? blocking neurotransmission at
  peripheral cholinergic synapses.
• ? flaccid (???) paralysis.

22
EPIDEMIOLOGY
- soil and water samples

• classic or foodborne botulism
• consumption of home-canned foods
  (types A and B toxins)
• consumption of preserved fish (type E
  toxin).
• (2) infant botulism
• consumption of foods (particularly
  honey) contaminated with botulinum spores.
• (3) wound botulism - unknown, but the disease is
  very rare.
• (4) inhalation botulism. major concern in this
  era of bioterrorism
• Botulinum - concentrated for purposes
  of aerosolization as a biologic weapon.

23
CLINICAL DISEASES
Foodborne Botulism

• 1 to 2 days after consuming the contaminated
  food.
• initial signs blurred vision with fixed,
  dilated pupils(???), dry mouth (indicative of the
  anticholinergic effects of the toxin),
  constipation(??), and abdominal pain. Fever is
  absent.
• flaccid paralysis Bilateral descending
  weakness of the peripheral muscles
• respiratory paralysis - death.

Infant Botulism

• neurotoxin produced in vivo by C. botulinum
  colonizing
• 
  the gastrointestinal tracts of infants.
• the symptoms are initially nonspecific (e.g.,
  constipation, weak cry, or "failure to thrive").
  - flaccid paralysis and respiratory arrest can
  develop
• mortality very low (1 to 2)
• Sudden infant death syndrome

Wound Botulism
- toxin production by C. botulinum in
contaminated wounds
24
LABORATORY DIAGNOSIS

• culture C. botulinum from the feces of patients
  with foodborne disease and from the implicated
  food if it is available.
• toxin is detected in the serum of more than 90
  of infants with botulism.

• Isolation of C. botulinum from specimens (feces,
  food) heating the specimen for 10 minutes at
  80C to kill all non-clostridial cells ? Culture
  of the heated specimen on nutritionally enriched
  anaerobic media ? the heat-resistant C. botulinum
  spores to germinate
• lipase production ? iridescent film on
  egg-yolk agar, digest milk proteins, hydrolyze
  gelatin, ferment glucose
• - Demonstration of toxin production mouse
  bioassay

1. C. botulinum is isolated from feces
2. toxin activity is detected in feces or serum.
3. stool cultures in virtually all patients,
4. wound exudate or serum.

25
TREATMENT, PREVENTION, AND CONTROL
Treatment

1. adequate ventilatory support Ventilatory
   support is extremely important in reducing
   mortality
2. elimination of the organism from the
   gastrointestinal tract, through the judicious use
   of gastric lavage and metronidazole or penicillin
   therapy
3. the use of trivalent botulinum antitoxin versus
   toxins A, B, and E to bind toxin circulating in
   the bloodstream..

Prevention
(1) destroying the spores in food (virtually
impossible for practical reasons), (2)
preventing spore germination (by maintaining the
food in an acid pH or storage at 4C or colder),
(3) destroying the preformed toxin (all
botulinum toxins are inactivated by heating at
60C to 100C for 10 minutes). (4) Infant
botulism has been associated with the consumption
of honey contaminated with C. botulinum spores,
so children younger than 1 year should not eat
honey.
26
Clostridium difficile
Physiology and Structure

• Gram-positive, spore-forming rod, Strict anaerobe

Virulence

• Two toxins
• enterotoxin (toxin A)
• - chemotactic for neutrophils,
• - stimulating the infiltration of
  polymorphonuclear neutrophils into the ileum
• with release of cytokines.
• - cytopathic effect disruption of the
  tight cell-cell junction,
• increased permeability of
  the intestinal wall, and subsequent diarrhea.
• 2) cytotoxin (toxin B).
• - actin to depolymerize, with the
  resultant destruction of the cellular
  cytoskeleton both in vivo and in vitro.

27
Table Virulence Factors Associated with
Clostridium difficile
Virulence Factor Biologic Activity
Enterotoxin (toxin A) Produces chemotaxis induces cytokine production with hypersecretion of fluid produces hemorrhagic necrosis
Cytotoxin (toxin B) Induces depolymerization of actin with loss of cellular cytoskeleton
Adhesin factor Mediates binding to human colonic cells
Hyaluronidase     Produces hydrolytic activity
Spore formation Permits organism's survival for months in hospital environment
EPIDEMIOLOGY

• Ubiquitous
• normal intestinal flora in a small number of
  healthy people, hospitalized patients
• spores can be detected in hospital rooms of
  infected patients
• disease develops in people taking antibiotics
• normal enteric flora ? overgrowth of
  resistant organisms
• or
  susceptible to the exogenous acquisition of C.
  difficile
• -proliferate in the colon and produce their
  toxins ? diseases

28
CLINICAL DISEASES
Toxin-producing C. difficile ? responsible for
antibiotic-associated gastrointestinal diseases
after antibiotic treatment, clindamycin,
penicillins, cephalosporins - relatively
benign, - self-limited diarrhea to severe,
- life-threatening pseudomembranous colitis
(??? ???).
Antibiotic-associated colitis caused by
Clostridium difficile. histologic section of
colon shows an intense inflammatory response,
with the characteristic "plaque" (black arrow)
overlying the intact intestinal mucosa (white
arrow)
Antibiotic-associated colitis lumen of the
colon. white plaques of fibrin, mucus, and
inflammatory cells overlying the normal red
intestinal mucosa.
29
Diagnosis - enterotoxin or cytotoxin in a stool
specimen from a patient immunoassays.
in vivo cytotoxicity assay using tissue culture
cells
and specific neutralizing antibodies for the
cytotoxin

• Treatment
• Discontinuation of the implicated antibiotic
  (e.g., ampicillin, clindamycin
• Severe diarrhea or colitis specific therapy
  with metronidazole, vancomycin
• Relapses may occur the spores are resistant
• ? second course of treatment with the same
  antibiotic is frequently successful.
• commonly exists in hospitals, particularly in
  areas adjacent to infected patients (e.g., beds,
  bathrooms).
• spores of C. difficile are difficult to
  eliminate