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ANAEROBIC GRAM POSITIVE BACILLI

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Myles Rentoria

on 4 May 2015

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Transcript of ANAEROBIC GRAM POSITIVE BACILLI

* Obligate anaerobic
*Sporeforming
*Gram (+) Bacilli, Catalase (-)
*Motile with Pertrichous flagella except:
- C. perfringens
- C. ranosum
- C. innocum
*have Swollen sporongia except:
- C. perfringens
- C. bifermentans
*non-encapsulated except:
- C. perfringens
Clostridia
*toxins usually gain access to the body through ingestion or via open wounds except: C. difficle

*they produce collagenase (spreading factor), hyaluronidase, lecithinase (cell destruction) or phospholipase (spreading factor)

* histotoxic clostridia:
- C. perfringers - C. novyi -C. septicum
-C. histolyticum -C. sporogenes
-C. bifermetans
Clostridia
*has a single Hemolytic reaction except: C. perfringens
*CHO fermenters except
- C. tetani
- C. histolyticum
*encountered in Exogenous anaerobic infections or intoxications
Clostridium perfrigens
+Gas gangerene Bacillus
+formely known as Clostridium welchii
+spores are seldom seen, but is oval, central to subterminal
+boxcar-shaped, gray to white colonies in BAP
+characterized by double zone of hemolysis (alpha & beta zones)
+has characteristic "STORMY FERMENTATION OF MILK" (using lithmus milk medium)- production of acid and gas
Clinical Infections:
1. Gas Gangerene/ Myonecrosis ("eatingsore")
- a life threatening destruction of muscle and other tissues; necrotizing infection of skeletal muscles
- organisms contaminate wounds thru trauma, frostbite or surgery
-accompanied by pain,swelling, serous discharge, discoloraation and tissue necrosis
+Hyperbaric oxygen theraphy- high concentration of O2 at inc. pressure( treatment procedure)
Clostridium perfrigens
+produced deoxyribonuclease- lowers viscosity of exudates; for morbolity of bacteria
* Lecithinase (+) - detected using egg yolk agar (EYA)
* Nagler test (+) - Lecithovitallin rxn (lecithinase C) on EYA
* Reverse CAMP test (+)- arrow head zone of hemolysis
*V.F.: a-toxin and enterotoxin
Clostridium tetani
*the endospores found in hospital environments, in soil and dust, and feces of many farm animals
*on BAP, colonies are with matte surface, narrow zone of B-hemolysis
*V.F. : tetanospamin (nuerotoxin)

Clostridium tetani
* tack head Bacillus
*soil and environmental inhabitants
*motile; produce heavily swarming anaerobically; sporeforming bacilli
*gelatinase indole (+); lecithinase and lipase (-)
*terminal spore and swollen sporongia- Drumstick/lollipop/tennis racket" appearance
Clostridia
Clostridium
perfringens
Clostridium
tetani
ANAEROBIC GRAM POSITIVE BACILLI
INDICATORS OF ANAEROBIC BACTERIA:
+ FOUL ODOR

+PRESENCE OF SULFUR GRANULES

+BRICK-RED FLOURESCENCE

+ABSENCE OF SUPEROXIDE DISMUTASE (SOD)
* Actinomyces spp., Propionibacterium spp., Eubacterium nodatum
*Prevotella or Porphyromonas
Clostridia

2. food poisoning\Enteritis ("Pig-bel")
+ ingestion of enterotoxin contaminationed food
+diarrhea (foul-smelling stool) and crampy abdominal feeling

+Tetanospasmin: is an endopeptidase that selectively cleaves the synaptic vesicle membrane protein synaptobrevin: it causes tensions or cramping and twisting in skeletal muscles surrounding the wound and tightness of jaw muscles
Clinical Infection/Disease
+tetanus- " Trismus"/ lock jaw (risus sardonicus smile)
+the organism (spore) estabslishes a wound infection and elaborates the potent toxin that mediates generalized muscle spasms
+tetanus neonatorum- contaminated instruments used for newborns
+sympotomatology: muscular rigidity: jaws, neck and lumbar region. difficulty in swallowing rigidity of the abdomen, chest back and limbs
Clostridium botulinum
+ Canned good bacilli
+it is found in soil and aquatic sediment
+it is charaterized by the presence of subterminal spore; B-hemolytic on BAP
+is a proteolytic bacteria and has ability to oxidized one amino acid with the second amino acid as the electron acceptor (stickland reaction)
Clostridium botulinum
+there are 7 types of botulism toxin
(A-G)
+ V.F. botulism toxin nuerotoxin( most potent toxin known to man)
+botulism factor: selectively cleaves the synaptic vesicle membrane protein, synaptobrevin, thus preventing exocytosis and release of the nuerotransmitter acetylcholine
Clinical Infection:
*botulism- result from ingestion of performed toxin in nonaacidic vegetables, meat based food or mushroom foodstuffs (botulism toxin A- most common cause
+ absorption of the toxin leads to nearly complete paralysis of respiratory and other essential muscle groups
+clinical diagnosis of botulism is confirmed by demonstration of botulism toxin in serum,feces, vomitus, or gastric contents, stool and tissue biopsy (wound botulism
+infant botulism- is an actual infection by ingesting the organism from honey or via breast feeding.
Clostridium difficile
*C.A. of pseudomembranous colitis and nosocomial diarrhea
*microscopy reveals chains up to cells aligned end to end
*endospores may be oval and subterminal
by: Rentoria, Flores & Antonio
Clostridium difficile
*"horse stable" odor and nonhemolytic- BAP
*ferment fructose forming acid- (+) yellow ground glass colonies- CCFA
*infection control dilemma among hospitalized patients
*it is acquired in the hospitals by individuals receiving antibiotics- antibiotics assoc. colitis
*V.F.: Toxin A ( weak enterotoxin) & toxin B (potent cytotoxin)
SPECIMEN COLLECTION & TRANSPORT
+Specimens: blood, CSF abscess
+material for anaerobic culture is the best obtained by tissue or by aspiration using a needle & syringe
+food & fecal specimens suspected of C.perfringens food poisoning should be transported at 4 degree celcius
+air is removed from the sealed jar using mercury

+all specimens should be held at room temperature pending processing in the laboratory, because refrigeration can oxygenate the specimen
+feces for C. difficile culture and toxin assay should be liquid or unformed; solid formed, or rectal swabs are adequate to detect carriers but do not detect enterocolitis
+crucial factor in the final success of anaerobic cultures is the transport of the specimen (lethal effect of oxygen)
Laboratory Diagnosis
1. Gram stain
2. Culture – Anaerobic blood agar, thioglycollate, Egg Yolk Agar (EYA), Cycloserine Cefoxitin Fructose Agar (CCFA), Peptone – Yeast Glucose broth (PYG), Brucella/ Blood Agar (BRU/BA), PRAS
- Primary plates should be freshly prepared or used within 2 weeks of preparation.
- Plates stored for longer periods accumulate peroxides and become dehydrated (growth inhibition)
- Swarming and double zone of hemolysis on BRU/BA is observable.

•- EYA is used to detect lecithinase activity – (+) insoluble opaque whitish precipitate within the agar.
- PYG detects volative fatty acid.
- Cycloserine and cefoxitin inhibit gram (-) coliforms.
•- Lipase (+) organisms produce a colony that is covered with an iridescent, multicolored sheen “appearance of gasoline on water or mother –of pearl”
•- Reazurin or methylene blue – indicators for anaerobic jars
3. Direct Nagler Test
•- Using EYA plate + C. perfringens type A antitoxin
•-
Result:
(+) inhibition of lecithinase reaction produced by C. perfringens

4. Mouse Neutralization Test
• - Is a definitive identification test for C. botulinum
• - It detects the presence of neurotoxins in serum or feces.
5. Reverse CAMP test
• - To confirm C. perfringens
•- (+) result: arrowhead at the intersection of the 2 streaks.

PROCEDURE FOR IDENTIFICATION OF ANAEROBIC BACTERIA
-the anaerobic gram (-) bacteria are the major normal flora in the colon, out numbering aerobes by 1000:1
-Inoculation of organisms should start with CAP followed by BAP.
-PRAS (prereduced and anaerobically sterilized) – provides a semisolid agar and anaerobic environment
-Bile disk is utilized for some strains of B. fragilis
-SPS disk is utilized for rapid identification of P. anaerobius.
-Propionobacterium acnes is associated with endophthalmitis after cataract surgery.
-B. ureolyticus requires formate and fumarate for growth in broth culture.
-Kanamycin, colistin and vancomysin – preliminary antibiotics for testing
-Laked kanamycin-vancomycin agar – selective medium for anaerobes.
Bacteriodes fragilis •
- Gram (-) anaerobic bacteria; nonmotile; pleomorphic with vacuoles; encapsulated
- The most commonly encountered bacteria in anaerobic condition
- Catalase (+); growth in 20% bile; beta lactamase producers; saccharolytic •
- Significant cause of intraabdominal anscesses.
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