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5-Anaerobes (2)

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



B

5-Anaerobes (2)

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