ANAEROBIC GRAM POSITIVE BACILLI

by: Rentoria, Flores & Antonio

ANAEROBIC GRAM POSITIVE BACILLI

INDICATORS OF ANAEROBIC BACTERIA:

* Actinomyces spp., Propionibacterium spp., Eubacterium nodatum

+ FOUL ODOR

+PRESENCE OF SULFUR GRANULES

+BRICK-RED FLOURESCENCE

+ABSENCE OF SUPEROXIDE DISMUTASE (SOD)

*Prevotella or Porphyromonas

Clostridia

Clostridia

* 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

•- 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

*has a single Hemolytic reaction except: C. perfringens

*CHO fermenters except

- C. tetani

- C. histolyticum

*encountered in Exogenous anaerobic infections or intoxications

Clostridia

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.

*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

SPECIMEN COLLECTION & TRANSPORT

-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.

+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

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.

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.

+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)

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 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)

Clinical Infection:

*C.A. of pseudomembranous colitis and nosocomial diarrhea

*microscopy reveals chains up to cells aligned end to end

*endospores may be oval and subterminal

*botulism- result from ingestion of performed toxin in nonaacidic vegetables, meat based food or mushroom foodstuffs (botulism toxin A- most common cause

Clostridium

tetani

+ 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 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

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)

* 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

+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

perfringens

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

2. food poisoning\Enteritis ("Pig-bel")

+ ingestion of enterotoxin contaminationed food

+diarrhea (foul-smelling stool) and crampy abdominal feeling

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

+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