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
6