Week 5 Flashcards
TAXONOMY Clostridia
Histotoxic clostridia
* characteristically cause variety of tissue infections
* usually subsequent to wounds or other types of traumatic injury
* most commonly involved: C. perfringens, C. novyi, C. septicum, C.
histolyticum, C. bifermentans
Enterotoxigenic clostridia
* produce food poisoning and more severe forms of gastrointestinal disease e.g., C. perfringens, C. difficile
Clostridium tetani
* causative agent of tetanus
* causes disease through exotoxin produced during limited growth within tissue
Clostridium botulinum
* causative agent of botulism
* results from ingestion of exotoxin previously formed by organism in contaminated food
GENERAL CHARACTERISTICS Clostridia
- Gram-positive, may stain irregularly or appear Gram-negative in older
cultures - Straight or curved rods (short coccoid to long filaments) with rounded,
tapered, or blunt ends; may appear singly, in pairs, or in chains - All species can form endospores
− round or oval
− terminally, subterminally or centrally located
− usually “bulging” (wider than bacterial body)
− useful in identification - C. tetani - round terminal spores giving characteristic “drumstick” appearance
- C. perfringens - forms spores with difficulty
- All motile by peritrichous flagella, few species non-motile e.g. C.
perfringens , C. ramosum, C. innocuum - Most species are non-encapsulated but C. perfringens has a capsule* Most species are obligate anaerobes but a few species are
aerotolerant e.g. C. histolyticum - Clostridia lack cytochrome system and thus react negatively in the Cytochrome Oxidase Test
- Energy for ATP generation obtained from substrate-level
phosphorylation - All species are Catalase negative
- Many species are saccharolytic
−ferment sugars
− produce reddening of meat particles in Robertson meat medium with rancid smell - Many species are proteolytic
− produce enzymes that digest proteins
−cause blackening and digestion of meat particles in Robertson meat medium with foul smell - Pathogenic species produce soluble toxins, some of which are
extremely potent
NATURAL HABITAT Clostridia
- distributed widely in nature
- found in soil, freshwater, marine sediments throughout the world* some species psychrophilic or thermophilic, but most are mesophilic
- several Clostridium spp. reside in lower intestinal tracts of humans and other animals as part of the normal microflora
VIRULENCE FACTORS - CLOSTRIDIUM
PERFRINGENS
- 5 types: A, B, C, D, E on basis of 12 exotoxins
- type A most common in human infections, other types important pathogens of domestic animals
- α-toxin
− enzyme, phospholipase C or lecithinase C
− produced by all 5 types, activated by Ca2+ and Mg2+ ions
− causes cell lysis due to lecithinase action on lecithin in mammalian cell membrane
− has primary importance in pathogenesis of clostridial myonecrosis
− has lethal, dermonecrotic, haemolytic activities - Other toxins:
−collagenase
− proteinase
− hyaluronidase
− deoxyribonuclease −several have haemolytic activity −some have necrotising or lethal effects on laboratory animals
VIRULENCE FACTORS
CLOSTRIDIOIDES DIFFICILE
- Produces 2 exotoxins which are important in the development of
Antibiotic-Associated Diarhoeae (AAD) - toxin A
− Strong chemoattractant for human granulocytes
− Causes transient rise in their intracellular calcium concentration
− Activated granulocytes release inflammatory mediators within lamina propria
− These damage epithelial cells of gut mucosa and cause excess fluid response - toxin B
− Produces cytopathic effect in vitro on tissue culture cells
− Effect can be inhibited by specific antisera to purified toxin B
VIRULENCE FACTORS - CLOSTRIDIUM TETANI
- has flagella (H), somatic (O), spore antigens
- 10 types on basis of flagellar antigens
- tetanus toxin - tetanospasmin
−extremely toxic neurotoxin, responsible for all symptoms in tetanus
−intracellular toxin released by cellular autolysis
− heat-labile protein inactivated by heating at 60oC for 20 minutes
−single polypeptide chain composed of 3 domains: A, B, C
− on release from bacterium, cleaved by clostridial proteases to 2 subunits: light chain (A); heavy chain (BC) which are linked by a single disulphide bond
−light A chain is associated with the toxic activity
−fragment B of heavy chain forms channels in lipid membranes
−fragment C of heavy chain contains a ganglioside-binding site
−tetanospasmin attaches to peripheral motor nerve endings
−travels along nerves to central nervous system (CNS)
− binds to gangliosides in the CNS
− blocks inhibitory impulses to motor neurones
− patients develop prolonged muscle spasms of both flexor and extensor muscles
VIRULENCE FACTORS - CLOSTRIDIUM
BOTULINUM
- Botulinum toxin
− neurotoxin
− heat-labile protein
− not released during life, appears only after death and autolysis of organism
−single polypeptide chain
−cleaved by bacterial proteases into di-chain molecule composed of: - heavy chain (responsible for binding)
- light chain
−chains held together by non-covalent bonds and a disulphide bond
− production governed by specific bacteriophages −acts primarily by binding to synaptic vesicles of cholinergic nerves
− prevents release of acetylcholine at peripheral nerve endings
(including neuromuscular junctions)
− patients develop flaccid, descending paralysis
CLINICAL SIGNIFICANCE - CLOSTRIDIUM PERFRINGENS
Wound and soft tissue infections
* Infection of exogenous or endogenous origin
* Exogenous infection from soil entering tissue
* Endogenous infection from:
−faecal flora present on skin or on particles of clothing carried
into wound
−clostridia escaping from bowel when its integrity disrupted by disease, injury, surgery
Wound infections - 3 categories
* Simple wound contamination
− histotoxic clostridia present without obvious pathologic process
* Anaerobic (crepitant) cellulitis
−clostridia infect tissue already compromised as result of ischaemia or direct trauma
− growth extensive, gas normally prominent feature
− patients not in extremely toxic condition, prognosis considerably better than for clostridial myonecrosis
* Clostridial myonecrosis (gas gangrene)
− organisms invasive, infection associated with profound toxaemia
−extensive local oedema, presence of gas, massive tissue damage
−skin colour changes (may become black)
− death in untreated cases
CLINICAL SIGNIFICANCE OF
CLOSTRIDIOIDES DIFFICILE
- One of most commonly detected enteric pathogens
- Important cause of nosocomial infections in hospitals and nursing homes
- Antibiotic-associated diarrhoea (AAD)
− benign, self-limited diarrhoea
−subsides following discontinuation of antibiotic usage - Antibiotic-associated colitis (AAC)
−intestinal symptoms more severe
− diarrhoea may persist - pseudomembranous colitis (PMC)
−severe, potentially lethal
−characterised by exudative plaques with underlying necrosis of mucosal surface of intestine
− may also be caused by Clostridium perfringens
−antimicrobial agents of all classes and anticancer
chemotherapeutic agents implicated in development
− most commonly reported agents: penicillins, cephalosporins
CLINICAL SIGNIFICANCE - CLOSTRIDIUM TETANI
- Causative agent of tetanus
- Toxigenic disease, associated with puncture wounds that do not appear as serious infections
- Now largely disease of non-immunised humans and animals
- Incubation period from 4 to 10 days (directly related to distance of
primary wound infection from CNS) - May occur in localised form, developing in muscles adjacent to site of inoculation (including cephalic form)
- Earliest manifestation muscle stiffness, followed by spasm of masseter muscles (lock jaw - trismus)
- As disease progresses tetanospasms produce:
− characteristic grimace (risus sardonicus)
− contraction of back muscles causing backward arching (opisthotonos)
− flexion of arms
− extension of lower extremities - condition lasts several weeks, death may ensue during one of spasms
CLINICAL SIGNIFICANCE - CLOSTRIDIUM BOTULINUM
- causative agent of botulism
- neuroparalytic disease classified into 4 categories
- foodborne botulism
−intoxication caused by ingestion of preformed botulinum toxin in contaminated food - wound botulism
−rarer form
−results from elaboration of botulinum toxin in vivo after growth of organism in infected wound - infant botulism
− botulinum toxin elaborated in vivo in intestinal tract of infant
colonised with organism after ingestion of spores - unclassified botulism
−involves individuals older than 12 months with symptoms of
clinical botulism with no identifiable vehicle of transmission - length of incubation period related to dose of toxin
- symptoms begin 12 to 36 hours after ingestion of contaminated food or 8 days after
- death results from: −respiratory failure caused by paralysis of tongue or muscles of pharynx that occlude upper airway
−from paralysis of diaphragm and intercostal muscles
−secondary pneumonia caused by non-botulinum organisms
COLLECTION, TRANSPORT, AND STORAGE OF SPECIMENS Clostridia
- Material for anaerobic culture best obtained using needle and syringe
- Immediately following collection, specimens should be placed into adequate anaerobic transporter
COLLECTION, TRANSPORT, AND STORAGE OF SPECIMENS Clostridia
- Material for anaerobic culture best obtained using needle and syringe
- Immediately following collection, specimens should be placed into adequate anaerobic transporter
- Rubber-stoppered collection tube or vial, gassed out with oxygenfree CO2 or nitrogen recommended for transport
- Specimen for C. difficile culture and toxin assay:
−single, freshly passed stool (10mL to 20mL)
−should be placed in leakproof container
− processed within 2 hours
−if delay anticipated, specimen must be stored at 5oC for up to 2 days - specimens for C. botulinum culture and toxin assay:
−15mL to 20mL of serum (not whole blood)
−swab samples of wound
−25g to 50g of stool
−suspect food(s)
−should be transported as previously described - Only 1 in 3 specimens taken from infected site yield growth of C. tetani, therefore multiple specimens are needed
CULTURE MEDIA Clostridia
Enrichment media
* Enriched Thioglycollate Medium
* Chopped meat glucose or chopped meat medium e.g. Robertson meat medium
* Supplemented Brain-Heart Infusion broth
Selective media
* Anaerobe Phenylethyl Alcohol (PEA) blood agar medium
* Anaerobe Colistin-Nalidixic Acid (CNA) agar medium
Non-selective media
* Anaerobe Blood Agar medium (AnBAP)
Spore selection technique
* heat or alcohol treatment - useful for isolating spore-forming
bacteria while inhibiting non-spore-forming bacteria
* spores of clostridia (or of Bacillus spp.) resist heat or alcohol
treatment, whereas vegetative cells killed
* after treatment, spores germinate in appropriate broth medium,
produce growth under appropriate conditions
INCUBATION CONDITIONS Clostridia
- inoculated plates should be:
−immediately placed into an anaerobic environment
−incubated at 35oC to 37oC
−for 48 hours
−re-incubated for another 2 to 4 days to allow slow-growing
organisms to form colonies
