Anaerobes

Anaerobic Jars

- rigid polycarbonate (plastic) containers with a sealable lid - culture plates, tubes, id strips and antimicrobial susceptibility tests can be placed in these jars - limit exposure of plates to air to 15 minutes maximum during setup and examination - reactivate catalyst after use

Cooked meat (chopped meat) broth

- solid meat particles initiate growth of bacteria; reducing substances lower oxidation-reduction potential (Eh) - nonselective for cultivation of anaerobic organisms; with addition of glucose, can be used for gas-liquid chromatography

Principal Anaerobe Defenses against oxygen toxicity

- superoxide dismutase - catalase

Clostridium tetani

- tetanus (lockjaw)- tetanus neurotoxin - swarming colonies and rods with round, terminal spores and a "drumstick" or "tennis racket"

Ground State (oxygen toxicity)

- triple oxygen 3O2: S=1 electronic ground state of molecylar oxygen (O2) - most stable and common allotrope of oxygen - molecules of triplet oxygen contain two unpaired electrons, making triplet oxygen and unusual example of a stable and commonly encountered diaradical

Bacteroides bile esculin agar (BBE)

- triplicase soy agar base with ferric ammonium citrate and hemin - bile salts and gentamicin act as inhibitors - selective and differential for Bacteroides fragilis group - also grows Bilophila, F. mortiferum/varium

Prevotella sp. (nonpigmented)

- ubiquitous oral cavity - dental biofilms - esophagus and stomach - bile (-) - catalase (-) - indole (-) - KVC: RRV

Caveats for Testing for PFE

- use fresh, actively metabolizing organism - pure culture essential - do not expose to oxygen for more than a few minutes - some enzymes are inactivated by oxygen

Gram Negative rods anaerobes

- wide variety of infections - part of normal flora GI tract, female genital tract and oropharynx

Gram positive, nonspore-forming bacilli

Actinomyces Bifidobacterium Eubacterium Eggerthella Collinsella Lactobacillus Mobiluncus Atophhium Propionibacterium

Disadvantages of Pre-formed Enzyme Kits

+ "one size fits all" + 80 to 120 very different organisms in data base - all tests not appropriate for every species - more tests in the kit= greater the opportunity to generate profile number - most problems with GPB and clostridia

Bacteroides fragilis

- KVC: RRR - bile resistant: grow in broth with 20% bile - catalase: (+); indole (-) - alpha-flucopidase (+) - hydrolyzes esculin

Porhyromonas

- KVC: RSR - indole (+) - pigmented colony (fluoresces brick red) - sensitivity to SPS

Bacteroides ureolyticus group

- KVC: SRS - nitrate (+) - urease (+) - requires formate/ fumarate - pits agar - asaccharolytic, reduces nitrate and requires formate and fumurate - small, transparent to translucent, corrode aga

Prevotella sp. (pigmented)

- Provetella intermidia - KVC: RRS - indole (+) - pigmented colony (fluoresces brick red) - lipase (+)

Performed Enzyme testing

- Rapid ANA II: qualitative micro method employing conventional and chromogenic substrates for identification of medically important anaerobic bacteria - Rapid ID 32A: ideal solution for simplified and standardized identification method (miniaturized version of current existing identification techniques) - CRYSTAL: miniaturized identification system that requires only one step for inoculation (oil addition) - API ZYM: detection of enzymes has been applied to 81 bacteria belonging to several species - Vitek: fully automated system that performs bacterial identification and antibiotic susceptibility testing (identifies over 75 anaerobic bacteria in only 4 hours incubation, rapid computer- assisted results, identifies anaerobic bacteria under aerobic conditions) - Microscan: Provide first-time accuracy with direct MIC for trusted detection of emerging and critical antimicrobial resistance - Individual Tests (Rosco, Key Scientific)

Anaerobic blood agar (CDC)

- prepared with Columbia, Schaedler, CDC, Brucella, or brain-heart infusion base supplemented with 5% sheep blood, 0.5% yeast extract, hemming, L-cystine and vitamin K - nonselective medium for isolation of anaerobes and facultative anaerobes

Identification Methods

- VPI Biochemicals - PRAS Biochemicals: measure pH to determine acid production; usually with indicator (phenol red or bromthymol blue) - GLC (Gas-liquid chromatography): separate and identifty anarerobic metabolic end products (i.e. volatile fatty acids) and nonvolatile organic acids of carbohydrate fermentation and amino acid degradation - API 20A: profile identification - Preformed enzymes (PFEs) - spot tests: > catalase: 15% H2O2 > indole: paradimethyl-aminocinnamaldehyde reagent > urease disk > nitrate: inoculate, incubate and add reagents > oxidase: all anaerobes are oxidase negative; only bacteria that use oxygen have oxidase (enzyme performs any redox reaction where oxygen is an electron acceptor) - MALDI-TOF

Microaerophiles

- a microorganism that requires oxygen to survive, but requires environments containing lower levels of oxygen than are present in the atmosphere (~20% concentration) - many are also capnophiles, require an elevated concentration of CO2 - Campylopbacter, Helicobacter and Borrelia

Obligate aerobes

- aerobic organisms that require oxygen to grow - need oxygen because they cannot ferment or respire anaerobically - gather at the top of the tube (O2 concentration is highest) - cellular respiration: use oxygen to oxidize substances (sugars and fats) in order to obtain energy - during respiration: use O2 as the terminal acceptor - advantage of yielding more energy than obligate anaerobes, but face high levels of oxidase stress - Nocardia, Mycobacteria, Pseudomonas

Aerotolerant anaerobes

- anaerobic bacteria do not require oxygen - grows better in the absence of oxygen but can survive in atmospheres containing molecular oxygen - concentration of oxygen that an aero tolerant anaerobe can tolerate varies from one species to another - thrive best in anaerobic environment - grows very poorly in ambient air (~ 21% O2); grow well under anaerobic conditions - Lactobacillus, Clostridium

Specimen Transport (anaerobic)

- aspirates are preferable to swabs - capped syringes may be used as a transport system - if using swabs, PRAS Cary-Blair deeps are superior to dry swab system - best obtained by tissue biopsy or by aspiration using needle and syringe - rubber-stoppered collection vial containing agar indicator system - vial is gassed out with oxygen-free carbon dioxide (CO2) or nitrogen - all held at room temperature= refrigeration can oxygenate specimen

Clostridium difficile

- associated with antibiotic-associated diarrhea and pseudomembranous entercolitis - produces a toxin, is able to proliferate resulting in diarrhea or colitis - toxin test for toxin A, enterotoxin, toxin B and cytotoxin - "horse-stable" odor - yellow ground glass colonies on CCFA agar - KVC: SSR - (+) proline - chartreuse fluorescence - subterminal spore - glutamate dehydrogenase (GDH)

Pre Reduced Anaerobically Sterilized (PRAS) media

- boiled free of molecular oxygen - reducing agent then added - autoclaved anaerobically - dispensed anaerobically - stored anaerobically

Clostridium botulinum

- botulism - botulin neurotoxin- most potent toxin (toxin BoNT) - diagnosisof neurotocin in serum, feces, gastric contents, vomitus or suspect food poisoining or environmental ( bioterrorism) - mouse bioassay

Laked kanamycin-vancomycin blood agar (LKV)

- brucella agar with kanamycin, vancomycin, vitamin K and 5% laked blood - selective for isolation of Provetella and Bacteroides spp. - grows some Fusobacteria - inhibits gram-positive organism and Enterics

Hydrogen peroxide

- causes damage to the microorganism or to media on which they are to grow

Anaerobic Bags or Plastic pouches

- commercially available and can hold one to three plates - plates, gas- generating system and indicator are sealed in a disposable, gas-impermeable plastic bag - convenient for small labs and for drop-in samples - limit exposure of plates to air to 15 minutes max. during setup and examination - commercially available envelope with hydrogen and CO2 generator that is activated either by adding water (GasPak) or by moisture on the agar plates - production of heat (detected by touching top of the jar) and moisture on the walls= catalyst and generator envelope - methylene blue or resazurin - "evacuation-replacement": draw vacuum of mercury; 2 times (80-90% N2, 5-10% H2 and 5-10% CO2)

Egg yolk agar

- egg yolk base - nonselective for determination of lecithinase and lipase production by clostridia and fusobacteria

Cycloserine cefoxitin fructose agar (CCFA)

- egg yolk base with fructose, cycloserine (500 mg/L) and cefoxitin (16 mg/L); neutral red indicator - selective for Clostridium difficile

Lactobacillus spp.

- facultative or obligate anaerobes - normal flora of the mouth, intestinal tract and vagina - long chains of GPB - catalase (-)

Clostridium perfringens

- gas gangyre (myenecrosis) - demonstrates a double-zone hemolysis - Nagler test (+) - lecitihinase (+) - reverse cAMP (+) - spores present - KVC: SSR - indole (-) - catalase (-)

Clostridium species

- gram positive bacilli produces endospores that survive adverse environmental conditions and germinate when conditions are favorable for bacterial growth - endospore-forming, obligatory anaerobic (or aerotolerant), catalase (-), gram positive bacteria - KVC: SSR

Natural defenses against oxygen toxicity

- hemoglobin - superoxide dismutase - catalase - peroxidase - vitamin C - vitamin E - uric acid

Superoxide Anion

- highly reactive free radicals capable of causing severe damage to enzyme system, proteins, lipids and bacterial cell walls

Hydroperoxy Radical

- hydroxyl radicals are short-lived but can cause severe damage to enzymes, proteins and lipids

Advantages of Pre-formed Enzymes

- identification of asaccharolytic organisms - growth in the system not required - rapid (15 min-4 hr) - less expensive than conventional tests

Oxygen Kills!

- kills time: procedure that fail due to excessive exposure to oxygen result in delayed reports - kills confidence - kills credibility - kills budgets: committed time and money to work-up a culture, however, you did not obtain the results you needed (material and time are wasted) - kills media: cause of media detonation; manufacturing and storage, media accumulate toxic products of oxidation that will inhibit growth of many anaerobic pathogens - kills people: culture results are compromised by processing the specimen in air and on oxidized media, the loser is the patient

Principal causes of media detoriation

- light - oxygen - dehydration or hydration

Considerations for Culture Media made in Air

- make the medium fresh and use within 7 days - do not add reducing agents to a medium made or stored in air - do not store any cultural medium in the presence of light

Facultative anaerobes

- makes ATP by aerobic respiration if O2 is present but is also capable of switching to fermentation - concentrations of oxygen and fermentable material in the environment influence the organism's use of aerobic respiration vs. fermentation to derive energy - can grow aerobically and anaerobically - Staphylococcus, Enterobacteriaceae

Positive pressure

- manometer shows if you have a positive pressure

Gas

- mixture of: 5% H2 5% CO2 90% N2 - do NOT exceed 5% H2 for safety reasons - you can test for the presence of hydrogen by flowing the gas over good catalyst (catalyst will heat up if hydrogen is present)

Identification using Rapid Methods

- more meaningful to provide clinical with rapid, presumptive identification - presence or absence of anaerobes - presence or absence of B. fragilis groups especially important for choice of therapy - limited for identification

Eggerthella lenta

- nitrate (+) - urease (+) - red fluorescence - catalase (-)

Clostridium septicum

- nonhemolytic - swarming colonies - "medusa head" appearance - lecithinase (-) - esculin (+)

Fusobacteria

- normal flora in the respiratory and gastrointestinal tracts - typically isolated from oral cavity (dental biofilms) - chartreuse fluorescence - KVC: SRS - F. nucleatum: long, slender, GNR with tapered ends; indole (+) and lipase (-); bread crumb like and greening of agar - F. necrophorum: pleomorphic GNR; indole (+) and lipase (+) - Leptotrichia- very large fusiforms with one pointed and one blunted ends; colonies are large gray convulated (oral cabity or urogenital tract)

Bifidobacterium spp.

- normal intestinal flora - rods are pleomorphic and may have two forks - KVC: SVR - may be difficult to identify

Anaerobic phenylethyl alcohol agar (PEA)

- nutrient agar base, 5% blood, phenyl ethyl alcohol - selective for inhibition of enteric gram negative rods and swarming by some clostridia - grows about all anaerobes - inhibits Enterics

Anaerobic Chambers

- optimal anaerobic incubation system - allows all laboratory manipulation to occur in anaerobic environment - chambers are equipped with gloves (glove boxes)- use airtight rubber gloves to perform their work inside the chamber - key components: anaerobic gas mixture, palladium catalyst, indicator (methylene blue or resazurin), desiccant, incubator and entry lock - no exposure to oxygen - work flow can be organized in a mass production fashion - reactivate the catalyst (every 6 months) - do not use desiccants in an anaerobic chamber - use activated charcoal to absorb H2S - do not bring plates out of chamber for insepction - gas mixture: 5% CO2, 10% hydrogen, 85% nitrogen and palladium catalyst

Obligate anaerobes

- organisms that can grow only in the complete absence of molecular oxygen - require anaerobic conditions for growth - oxygen and its derivatives (e.g. hydrogen peroxide) are toxic to these organisms - Bacteroides, Clostridium

Anaerobic bacteria

- organisms that grow in the absence of O2 - vary in their ability to tolerate O2

Damage to Anaerobes by Oxygen

- oxidation of lipids in membranes - inactivation of enzymes - direct genetic damage

Oxygen damage to cultural media

- oxidation of organic constituents - formation of hydrogen peroxide - damage is accelerated by reducing agents e.g. Cysteine - damage is not reversed by "reducing" the media

Water

- oxygen intermediates eventually turn into water

Catalyst

- palladium - inactivated by moisture, H2S and bacterial metabolic products - fresh (i.e. new) or reactivated catalyst should be used each time - reactivated by heating in an oven at 160C for 2 hours - pellets should then be stored at room temperature in a dry container desiccator

Thioglycollate broth

- pancreatic digestion of casein, soy broth, and glucose to enrich growth of most bacteria - thioglycollate and agar reduce Eh - may be supplemented with hemming and vitamin K1 - nonselective for cultivation of anaerobes, facultative anaerobes and aerobes

Peptone- yeast extract- glucose broth (PYG)

- peptone base, yeast extract, glucose, cysteine (reducing agent), resazurin (oxygen tension indicator), salts - nonselective for cultivation of anaerobic bacteria for gas-liquid chromatography

Actinomyces israelii

- periodontal disease - sulfur granules may be present - gram positive rods have pleomorphic, beaded and branching appearance - older colonies have a "molar-tooth" morphology

Critical Issues

- pure culture - not from a shared plate - less than 72 hours old - proper density of organism - media or origin - minimize exposure for air

Bacteroides fragilis group

-saccharolytic, bile-resistant, non pigmented - B. fragilis (indole -, catalase +) - B. thetaiotaomicron (indole +, catalase +) - B. vulgatus (indole-, catalase +, esculin-) - B. ovalus

Algorithm for isolation and identification of anaerobic bacteria

1. direct gram stain 2. streak for isolation (BBE, LKV, PEA, BAP, CHOC, THIO, EYA) 3. examine individual colonies for distinctive morphologies 4. Gram stain and subculture to BAP, CHOC (Aerotolerance test: slow growing aerobes such as Capnocytophaga, Eikenella, Actinobacillus) > Gram Negative: kanamycin, vancomycin, or colistin in 1st quadrant, nitrate disk in 2nd quadrant > Gram positive cocci: nitrate and SPS > Gram positive rod: nitrate

Singlet state (oxygen toxicity)

= single oxygen: dioxygen (O2) and dioxidene, a gaseous inorganic chemical with the formula O=O which is a quantum state where all electrons spin paired - kinetically unstable at ambient temperature however the rate of decay is low

Propionibacterium acnes

Cutiebacterium acnes - KVC: SSR - proline (+) - contaminant like coagulase- negative staphylococci - "anaerobic diptheroids" - pleomorphic, gram positive rod - catalase (+) - indole (+)

Keeping the chamber anaerobic

The Triangle - hydrogen - positive pressure - active catalyst