Anaerobes = Bacteria that do not require oxygen for growth & reproduction Oxygen is detrimental to them

Obligate ( strict) anaerobe = tolerates no more than 0.5% oxygen

Aerotolerant (moderate) anaerobe = tolerates small amounts (2-5%) oxygen

( Most anaerobes are moderately obligate, and tolerate 2-8% oxygen),= important

Facultative anaerobes = Bacteria that can grow in the presence or absence of oxygen

• FA^ - grow well on non-selective anaerobic media (i.e., ANA-BAP/CDC-ANA/ ANABRU)

• FA ^ Ex - Escherichia coli ,Streptococci, Staphylococcus aureus

Clues that a patient has an anaerobic infection

• Foul-smelling

• Gas bubbles

• Black Blood

• human/animal bite or abdominal surgery

• Sulfur granules

Acceptable Specimens

• Sterile body sites

• blood, bone marrow, deep wounds, CSF, other body fluids/ deep tissue

• Aspirates specimens= Suprapubic (urine), Culdocentesis ( uterus), Transtracheal ( lung)

Unacceptable Specimens

• Contaminated body sites

• Ex- Specimens for routine culture superficial wounds/sores/ulcers swabs mouth/throat/sputum specimens stool/rectal swab (unless for C. difficile) vaginal/cervical/urethral swabs clean catch/catheterized urine

Anaerobic collection/transport

• Best specimen: needle aspirate

• Swabs only if transported in anaerobic transport media/system

• Specimens should be transported ASAP to

  the lab

• Specimens should be plated ASAP, or held

  under anaerobic condition

Anaerobic Incubation System ( terms)

Anaerobic chambers: best; anaerobes never exposed to air; expensive

Anaerobic jars: Gas pak or evacuation/gas replacement---less expensive; anaerobes exposed to air

Anaerobic bags: least expensive; accommodates one or two plates, but anaerobes exposed to air

Gas Pak System

• Components = Plastic jar, lid with rubber seal, gas generating envelope, palladium catalysts

• Anaerobiasis indicator :methylene blue strip ;undergoes a redox reaction

  blue: oxygen- white: no oxygen

If Gas Pak is working

• lid feel warm

• moisture on the side of the jar

• Methylene blue indicator loses its blue color and turns white

• ( Resazurin indicator may be used; it turns from white to pink in absence of oxygen)

Gas Pak not working

• Lid crack

• Rubber seal is broken

• Catalyst not working

• Gas generating envelop defective

Important Notes

• The palladium catalysts, if not a part of the gas generating system, must be dried after each use in an 160-170F oven for at least 2 hours to remove moisture

• Pellets must be periodically replaced when contaminated with byproducts of anaerobiasis

• GAS PAK REACTION look at powerpoint 22

Anaerombic Media

• PRAS media: Pre-Reduced, Anaerobically Sterilized media = Contains hemin and vitamin K, necessary for the growth of most anaerobes

• Primary isolation media(PIM) = CDC-ANA/ ANA- BAP/ BRU ANA, BBE, KVLB, PEA/CNA, Enriched thio/ chopped meat

  Optional selective PIM Media

• CCFA = (Cycloserine Cefoxitin Fructose Agar): used only when Clostridiodes difficile culture requested

• EYA = (Egg Yolk Agar): used if Clostridium suspected (based on gram stain or diagnosis)

  
  

ANA- BAP ( CDC- ANA/ BRU- ANA)

• Components = SBA + hemin/ Vit. K

• Purpose = nonselective/ most anaerobes grow well on

• Draw back = Facultative anaerobes also grow well on it

BBE= Bacteroides Bile Esculin

• Components = 20% bile and Esculin

• Purpose = Select and differentiate the B. fragilis group

KVLB = Kanamycin Vancomycin, Laked Blood

• Components = laked SBA and antibiotics

• Purpose = enhance pigment of Prevotella / Recovers B. frag group

PEA = Phenylethyl-alcohol

• Components = SBA + PEA

• Purpose = Inhibit gram negative facultatives, select for gram positive anaerobes, inhibits swarming proteus

CNA = Colistin Naladixic Acid

• Components = SBA + antibiotics

• Purpose = same as PEA

• Many labs use PEA for anaerobes and CNA for aerobic culture

Liquid Media = Enriched Thio

• Components = broth / hemin and vitamin k

• Purpose = holding broth

• Look over slide 32

EYA = Egg Yolk Agar

• Components = egg yolks, provide fatty acids

• Purpose = detect lipase/ lecithinase production

• Lipase positive =iridescent sheen 0r pearly layer

• Lecithinase positive opaque precipitate under the colony

• Used to ID Clostridium species

  CCFA = Cycloserine Cefoxitin Fructose Agar

• Components= Antibiotics, furctose, indicator

• Purpose= select for Clostridiodes difficlile

• horse stable odor yellow colonies (fructose-positive

Commonly Used Rapid Biochemicals

• Catalase test: 3% or 15% (best) H202

• Catalase postive= bubbles

• Catalase negative = no bubbles

• Spot Indole test: reagent: para-dimethyl cinnamaldehyde

• Indole positive = blue-green color

• Indole negative = pink color

Differentiation Disks

• Used to determine if the anaerobe is

  sensitive or resistant to antibiotics, such as

  Kanamycin (k), Vancomycin (Va), Colistin

  (Cl)

• Sensitive = zone of inhibition ( no growth)

• Resistant = no zone of inhibition (growth)

The presumptive ID of Anaerobes

• Some anaerobes are rarely cultured, but presumptively identified by clinical symptoms of the patient.

• clostridium tetani = small gram positive bacilli with round, terminal spores, resemble a tennis racket or drum stick

• Clostridium botulinum- gram positive bacilli with oval, subterminal spores

• Clostridium perfringens (when the cause of food poisoning)

  Gram Stain

• Clostridium tetani = small gram positive bacilli with round, terminal spores, resemble a tennis racket or drum stick

• Clostridium botulinum = gram positive bacilli with oval, subterminal spores

• Propionibacterium = pleomorphic gram positive bacilli ; beaded, clubbed, palisades, diphtheroidal

• Fusobacterium nucleatum = pale staining gnb with tapered ends

• Fusobacterium necrophorum = pale staining gram negative bacilli with rounded ends, may be pleomorphic with coccoid bodies and chains

• Peptostreptococcus = gram positive cocci, single, pairs &chains

• Veillonella = Tiny,gram negative cocci , pairs and packets

Polymicrobic anaerobicinfections = several anaerobes present in the same infected body site/specimen
Endogenous= Human normal flora ( inside the body)

• Ex = Bacteroides, Clostridium perfringens, Clostridiodes difficile, Peptostreptococcus

• body site = mouth, GI tract, genital, skin

Endogenous infection

• normal flora: mouth ,genital and GI tract

• Veillonella

• Peptostreptococcus

• Prevotella & Porphyromonas

• Fusobacterium

• Bacteroides species

• “Lumpy jaw”: Actinomyces israelii

Endogenous Contaminant

• Propionibacterium species ( Cutibacterium)

• normal flora: skin and mouth

• most common anaerobic contaminant isolated from blood cultures

• caused by improperly cleaning skin prior to collection of blood cultures

• however, may cause endocarditis

• thus, must evaluate its recovery from blood

Exogenous = live outside the human body ( soil- other animals, water- plants)

• Ex - Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Clostridiodes difficile

Exogenous infection

• cause by clostridium species

• Not usually recovered in culture, but diagnosed by patient symptoms /DAD

  • Clostridium tetani tetanus

  • Clostridium botulinum botulism

  • Clostridium perfringens food poisoning

  • Clostridiodes difficile : colitis/diarrhea-AAD

Clostridium botulinum

• Wound: trauma to skin

• Food: especially spores in home canned vegies

• Infant: especially spores in honey

• Bacteria grow and produce the most potent neurotoxin known that inhibits the release of acetylcholins, causing a flaccid paralysis

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