LAB MICRO 4

anaerboes & parasites

Define different types of isolates based on atmospheric requirements:

• Obligate Aerobe: only aerobic growth; oxygen required

• Facultative Anaerobe: both aerobic & anaerobic growth; greater growth in presence of oxygen

• Obligate Anaerobe: only anaerobic growth; cannot grow in the presence of oxygen (toxic)

• Aerotolerant Anaerobe: anaerobic growth; but continues in presence of oxygen

• Microaerophilic: aerobic growth; oxygen required in low concentration

• Capnophilic: aerobic growth; CO2 required in high concentration

What clinical clues are associated with anaerobes?

• Infectious process next to mucosal surfaces with normal anaerobic flora in GI, female genital tract, & oral cavity

• Foul odor or gas

• Tissue necrosis & abscess formation

• Failure to respond to antibiotics that are not active against anaerobic bacteria

Specimen collection requirements for anaerobes

• Avoid contamination by anaerobic normal flora of mucosal surfaces

• Sterile body fluids only (blood, pleural & peritoneal fluids, aspirates)

• Anaerobic transport media – agar gel with buffered mineral salts & oxygen free

• Room temp 15-20 minutes or longer if in anaerobic vial

• DO NOT INCUBATE, may cause overgrowth

• DO NOT REFRIGERATE, cold increases oxygen diffusion

Anaerobic growth methods

• anaerboic chamber

• anaerobic jars & sealed boxes

• anoxomat

• oxyplate

• anaerobic indicators

Describe Anaerobic Chamber

• materials introduced through small doors in airlock while operator works through arm ports in airtight sleeves

  • 85% N2, 10% H2, 5% CO2 as compressed gases

Describe Anaerobic Jars & Sealed Boxes:

• Water mixed with chemical packet containing sodium bicarbonate & sodium borohydride, hydrogen & CO2 generated

• Hydrogen & atmospheric oxygen in jar react with palladium catalyst & combine to form water, removing oxygen

• Atmosphere contains: <5% oxygen, need 80-90% N2, 5% hydrogen, 5-10% CO2

• Anaerobic indicator placed in jar to monitor atmospheric conditions

  • Methylene Blue = blue → colorless

  • Resazurin = pink → colorless

Describe Anoxomat

•  jar evacuation & replacement system uses compressed gas to create anaerobic atmosphere

  • 80% N2, 10% hydrogen, 10% CO2

  • Palladium pouch = removes any O2, forming water

Describe OxyPlate

• each petri dish becomes anaerobic chamber

  • Culture medium contains enzyme (oxyrase) which combines oxygen with hydrogen to remove oxygen as water is formed

Describe anaerobic indicators

• Methylene Blue = blue → colorless

• Resazurin = pink → colorless

• Clostridium perfringens: place inoculated plate in anaerobic environment, growth = anaerobic conditions maintained

Why do we use anaerobic culture media?

• Contains hemin, vitamin K to enhance anaerobic growth – selective most important because anaerobic infections are polymicrobial (infection caused by 2 or more species)

Anaerobic Blood Culture (CDC):

• Anaerobic Blood Culture (CDC): enriched (5% sheep blood, hemin, K1), differential based on hemolysis

  • general purpose medium for cultivation of all anaerobic bacteria

Bacteroides Bile Esculin Agar (BBE)

• Bacteroides Bile Esculin Agar (BBE): enriched (bile salts 20%, esculin, ferric ammonium citrate), selective, & differential (esculin hydrolysis = black colonies & iron)

  • Isolation & presumptive ID of bacteroides fragilis based on bile tolerance & esculin hydrolysis

Kanamycin-Vancomycin Laked Sheep Blood Agar (KVL)

• Kanamycin-Vancomycin Laked Sheep Blood Agar (KVL): enriched (laked sheep blood, kanamycin, vancomycin, hemin, K1), selective, differential (pigment)

  • Isolation of anaerobic bacteria bacteroides & prevotella 

  • Prevents growth of facultative gram neg & gram pos bacteria

  • Lysed blood enhances pigment formation

Phenylethyl Alcohol Agar (PEA)

• Phenylethyl Alcohol Agar (PEA): enriched (antibiotics), selective (inhibits gneg rods)

  • Isolation of most anaerobic organisms (gpos & gneg) from mixed cultures

  • Inhibits facultative anaerobic gram neg bacteria like enterobacterales

Colistin Nalidixic Acid Agar (CNA)

• Colistin Nalidixic Acid Agar (CNA): inhibits gram neg organisms & grows most gpos anaerobic & facultative anaerobic bacteria

Egg Yolk Agar

• Egg Yolk Agar: detect proteolytic enzymes (lipase & lecithinase) produced by clostridium

  • Lecithinase = opaque zone around colony

  • Lipase = blue green sheen on agar surface around colony

Broths with Reducing Agents

• Broths with Reducing Agents: thioglycollate & cooked meat 

  • Oxygen tension decreases toward bottom of tube allowing growth of anaerobic isolates without incubation in anaerobic conditions

Describe aerotolerance testing

• Bacteria must first be demonstrated to be an anaerobe

• Inoculate onto anaerobic BAP & increased CO2 CAP 

• Growth only on anaerobic BAP = obligate anaerobe

• Growth on aerobic CAP, but better on anaerobic BAP = aerotolerant anaerobes

Anaerobic GNB

• Bacteroides sp – GNCB

• Prevoltella sp – GNCB

• Porphyromonas sp – GNCB

• Fusobacterium sp – GNR with tapered ends

• Definitive ID with MALDI-TOF or MOLECULAR 

Bacteroides fragilis

• GNCB

• BBE = brown

  • 20% bile tolerant

  • Bile esculin + (can hydrolyze esculin)

• KVL = growth, vancomycin & kanamycin & colistin RESISTANT

• Indole -

Prevotella

• GNBC

• BBE = NO GROWTH, inhibited by bile

• KVL = GROWTH, vanco/kana/colistin RESISTANT

• Indole -

• Black colonies

Porphyromonas

• GNBC

• BBE = NOW GROWTH

• KVL = NO GROWTH, vancomycin SENSITIVE

• Indole +

• Black colonies

Fusobacterium sp

• GN fusiform, bread crumb colonies

• F. nucleatum: common, cause brain abscess

• F. necrophorum: serious, lemierre’s

• BBE: NO GROWTH

  • Inhibited by 20% bile

• KVL: NO GROWTH

  • Vancomycin RESISTANT

  • Kanamycin, colistin SENSITIVE

• Most common are indole +

Anaerobic GNC

• Veillonella: most common infections in mouth & urogenital

  • Nitrate reduction +

  • Glucose fermentation -

• Acidaminococcus: most common infections in GI 

  • Nitrate reduction -

  • Glucose fermentation -

• Megasphaera: most common infections in GI 

  • Nitrate reduction -

  • Glucose fermentation +

Anaerobic GPB

• C. perfringes

• C. tetani

• C. botulinum

• C. difficile

• C. septica

C. perfringes

• Clostridium perfringens: Gas gangrene (myonecrosis), post-abortion sepsis, food poisoning, histotoxin & enterotoxin

  • Double zone beta hemolysis

  • Boxcar shaped cells

  • Setup on EYA 

    • Lecithiase +

    • Lipase -

  • Subterminal spores

  • Reverse CAMP +

  • Nagler test (anti-lecithinase) +

  • Tributyrin test -

C. tetani

• Clostridium tetani: tetanus, neurotoxin

  • Terminal drumstick spores

  • Lecithinase -

  • Lipase -

C. botulinum

• Clostridium botulinum: botulism, neurotoxin

  • Subterminal oval spores

  • Motile with peritrichous flagella

  • Lecithinase +

  • Lipase +

C. difficile

• Clostridioides difficile: antibiotic associated diarrhea, hard to culture

  • Yellow ground glass colonies on CCFA with horse stable odor

  • Commercial antigen kits for rapid detection of toxin (enterotoxin & cytotoxin)

C. septicum

• Clostridium septica: NF of GI tract suggests colon cancer in blood culture

  • Virulence = histotoxins 

  • Swarming medusa head colonies

  • Lecithinase & lipase -

Cutibacterium acne

• Cutibacterium acne: 

  • GP pleomorphic rods – anaerobic diphtheroid

  • Cat +

  • Indole +

  • NF of skin, most commonly isolated anaerobic GPB

  • Opportunistic pathogen – recovered from shoulder joints

Chart for Clostridium

describe the AST used for anaerobic ID

• Kirby-bauer disc setup for ID

• Disc antimicrobial concentrations utilized for anaerobic ID ONLY, not interchangeable for routine organisms

Definitive ID of anaerobic organisms

• Biochemical test batteries

• Metabolic end product analysis using gas liquid chromatography

• Cell wall fatty acid profiling by gas liquid chromatography

• MALDI-TOF

• Molecular testing

Why might we collect parasite stool samples?

• Based on patient signs & symptoms, clinical history, & travel to endemic areas

• Cysts found in formed stool

  Trophs found in watery stool

Where do we find parasitic organisms?

• GI tract is most common site

• CSF – acanthamoeba sp

• Eye – acanthomoeba spp, loa loa

• Skin – onchocerca volvulus

• Blood – plasmodium sp, babesia sp.

• Fresh fecal samples in clean sterile wide mouth containers 

• Stool collection vials – clean vial with no preservatives or some preservatives

• Liquid samples examined within 20 minutes of passage if not in preservative, formed within 24 hours

What type of analysis do we do for O & P (ova & parasite) examination?

• Fresh for quick wet mount – saline & 1% iodine

• Preservatives – increase recovery of many that degrade quickly

  • Formalin

  • PVA (polyvinyl alcohol)

  • SAF (sodium acetate-acetic acid formalin)

  • Schaudinn’s Fluid (PVA & mercuric acid)

• Macroscopic Analysis – visual determination of eggs, worms, proglottids, color

• Microscopic – 

  • wet mount (motile trophozoites, cysts, helminth eggs)

  • Trichrome stain – ID of protozoan trophozoites or cysts

  • Modified Acid Fast stain – oocytes of cryptosporidium

What symptoms do we see in parasitic infections?

• Diarrheal disease (bloody, watery, purulent, bowel obstruction, belching, steatorrhea)

• Eosinophilia

• Skin rash, itching

• Hepatosplenomegaly, lymphadenopathy

• Jaundice

• UTI symptoms

• Inflammation of lungs, cough 

• Fever, chills, night sweats, malaise, muscle pain

What kind of treatments may interfere with parasite testing?

• Mineral oil 

• Bismuth

• Antibiotics (tetracycline) – wait 2 weeks before sample collection

• Barium – wait 5-10 days

• Antimalarial agents

What are the standard recommendations for parasite stool samples?

• 3 specimens (2 before laxative, 1 after)

• Amebiasis – 6, detects 90% of infections (cost is issue)

• Post treatment collection to confirm clearance (3-4 weeks protozoa, 5-6 wks taenia)

• Determine specimen collection # based on lab testing performed (ELISA, microscopy, fresh vs concentrated vs permanent smear, pooling)

• Samples submitted within 10 day period, NEVER same day OR 1 per day 3 consecutive days

Pros & Cons of fresh stool

• Pros: 

  • no chemical preservatives & low cost

  • Motile trophoxoites

  • Perform all testing – ELISA, stains, direct examination, concentrations, permanent stains, acid fast stains

• Cons:

  • Time to receipt & processing can result in organism degradation

Pros & Cons of preserved stool

• Pros:

  • Easier diagnosis as preservations of organisms morphology

  • Delivery time not critical & home collection encouraged

  • Perform most testing – concentrations, permanent stains, ELISA, acid fast

• Cons:

  • Cost of vials & disposal of mercuric chloride preservatives

  • If not mixed, organisms may not be preserved