Lecture 13 – Anaerobic Methods PDF

BIOL 2010 Lecture 13 – Anaerobic methods BAILEY AND SCOT T’S – CHAPTER 40 DIAGNOSTIC MICROBIOLOGY – CHAPTER 22 In this lecture Explain the characteristics and pathogenesis of commonly encountered clinically significant microorganisms, including the most frequently isolated species by body system...

BIOL 2010 Lecture 13 – Anaerobic methods BAILEY AND SCOT T’S – CHAPTER 40 DIAGNOSTIC MICROBIOLOGY – CHAPTER 22 In this lecture Explain the characteristics and pathogenesis of commonly encountered clinically significant microorganisms, including the most frequently isolated species by body system Anaerobes Gram Positive and Negative Cocci Gram Positive Bacilli Gram Negative Bacilli Discuss bacterial virulence, as related to these organisms Anaerobes Defined Anaerobes Able to replicate in the absence of oxygen Ambient air Contains approximately 21% oxygen and 0.04% carbon dioxide Obligate (strict) anaerobes Killed in the presence of oxygen Almost immediately Oxygen free radicals lead to cell destruction Bactericidal Require low oxidation-reduction (redox) potential Anaerobes Defined (Cont.) Capnophilic Increased carbon dioxide (CO2) (5%–10%), reduced oxygen (O2) to 15% Conditions still sufficient to allow aerobic organisms to replicate Microaerophilic Oxygen reduced to 5% or less Anaerobes Defined (Cont.) Facultative anaerobes Preferentially use oxygen as an electron acceptor if available but can grow well without oxygen Aerotolerant (moderate anaerobes) Oxygen steals electrons used in metabolism. Bacteriostatic Classification of Bacteria on the Basis of Their Relationship to O2 and CO2 Why Some Organisms Are Anaerobes Molecular oxygen has a direct toxic effect to some anaerobes. Lack enzymes to break down toxic products O2−: superoxide anion H2O2: hydrogen peroxide OH−: hydroxyl radical O2 + e− O2− (superoxide anion) O2 + e− + 2H+ H2O2 (hydrogen peroxide) Why Some Organisms Are Anaerobes (Cont.) Strict aerobic and facultative anaerobic bacteria can protect themselves. Possess enzymes to break down toxic products Superoxide dismutase Catalase 4O2− + 4H+  2H2O2 + O2 via superoxide dismutase 2H2O2  2H2O + O2 via catalase Where Anaerobes Are Found Exogenous Endogenous Organisms exist outside of the Organisms exist inside the bodies bodies of animals. of animals and become sources of Infections develop when the agent infection. is ingested or has gained entrance Example: Bacteroides, through trauma. Porphyromonas, Prevotella, Example: Clostridium, Sarcina, Fusobacterium, Clostridium, Fusobacterium, and others Propionibacterium, and anaerobic cocci Important Concepts Need proper education about which sites are appropriate for anaerobes Includes collection of actual site of infection, not just the surface Prevent misleading and nonuseful clinical results Requires rapid processing Why? Toxicity of oxygen Drying of specimen Specimens for Anaerobic Bacteriology ACCEPTABLE SPECIMENS UNACCEPTABLE SPECIMENS CSF Sputum Abscesses Swabs of surface organs (ex. Aspirations Throat, gingival, skin, perirectal) Urine (Ex. Midstream) Surgical specimens Large bowel contents (ex. stool, Body Fluids colostomy bags) Biopsy Vaginal swabs Specimens for Anaerobic Bacteriology Ideally needle and syringe Less contamination and can expel any air However – this poses a needlestick injury hazard Best to inject specimen into pre-reduced anaerobically sterilized (PRAS) media Once in laboratory, vortex to mix sample and plate to appropriate isolation media. Use a few drops on PRAS media and streak for isolated colonies Vital since most anaerobic infections are polymicrobial. Need isolated colonies to verify relevant organisms PRAS Transport Medium Specimens for Anaerobic Bacteriology Specimen should be collected to avoid exposure to oxygen Swabs Not recommended and should only be used if under prereduced conditions Tissue Place small piece into PRAS media or reduced transport medium. Blood Aseptic specimen collection and subsequent inoculation of both anaerobic and aerobic blood culture bottles Processing of Clinical Samples Plates inoculated for anaerobic culture must be incubated in anaerobic conditions at 35-37C It is important not to keep inoculated plates out at room temperature more than 1 hour. Maintaining environment keeps cultures viable. Options for incubation systems: Anaerobic chambers Anaerobic bags or pouches Anaerobic jars Processing of Clinical Samples Anaerobic incubation systems utilize a combination of: Catalyst – palladium pellets which removes residual oxygen from chamber Must be “reactivated” after use, as they get inactivated by water and toxic end products of anerobes Dessicant – absorbs water which is formed Anaerobic gas – mixture of H2, CO2 and Nitrogen Quality control of Anaerobic systems – important to ensure anaerobic conditions met Physical reactions: Condensation (does not apply to all systems) Chemical indicators: Methylene blue or resazurin strips (must turn white within several hours) Biological indicators: Obligate aerobe and anaerobes Processing of Clinical Samples Resazurin: Pink Methylene blue: Blue Both must turn white to indicate anaerobic conditions Anaerobic chamber Large, Oxygen-free chamber Advantages: Less susceptible to oxygen Can work with cultures at any time Can accommodate microscopes and other equipment Cost-effective over time Chamber already in anaerobic condition Disadvantages: “Cuffs” may not fit all May be large – difficult to maneuver at first Anaerobic jar Anaerobic Jar system – many varieties available Envelope gas generator used Water-generating system: Catalyst required, water needs to be added to the envelope. Often re-usable catalysts Can look for water to assess if system is working Waterless gas-generating system: NO water or catalyst required Anaerobic jar Ex. Anoxomat system Gas evacuation system Anaerobic environment within 3 minutes! Anaerobic jar Advantages: MANY varieties to suit lab needs Low cost for smaller labs Disadvantages: Some envelope systems can take 30-45 minutes to obtain anerobic environment Plates must be removed from system for evaluation (this exposes bacteria to oxygen) Some bacteria are VERY sensitive and will die immediately upon exposure to oxygen Anaerobic pouch Many varieties available Accommodates a few plates inside the bag The bag contains an oxygen removal system and a seal Advantages Can be used to transport specimens Could be examined for growth inside the bag (i.e. no exposure to oxygen) Disadvantages Only accommodates a few plates Not feasible to use as a stand-alone anaerobic system Identification of Anaerobes The method and level of identification usually depends on Size and capabilities of the laboratory Clinical Site of infection (Ex. Sterile site like blood or a wound infection) Type of infection (i.e. is it polymicrobial?) Most laboratories will perform presumptive Identification Quick, less costly May be sufficient for treatment Definitive ID Can be complex and expensive Presumptive identification of Anaerobes Explore cultural characteristics Presentation on media Look out for clues (Ex. Smell) Aerotolerance test Gram Stain Quick biochemical tests Anaerobic disks Catalase Spot Indole Presumptive ID – Direct Microscopic examination Direct microscopic exam (or direct smear) includes preparing a gram- stained smear of clinical specimen Reasons for performing direct microscopic examinations Helps rule in and rule out pathogens Establishes a polymicrobial or monomicrobial infection Provided presumptive identification Aids in selection of culture media Determines the quality of specimen collected Can serve as a valuable quality control tool Presumptive ID – Direct Microscopic examination When Gram stain technique is used use methanol as fixative instead of heat. It is important to recognize morphology that may indicate presumptive identification Gram-positive bacilli—Clostridium Thin gram-negative bacilli—Fusobacteria Tiny gram-negative cocci that are gram-variable Veillonella Gram-negative coccobacilli Bacteroides, Porphyromonas, Prevotella Presumptive ID –Microscopic examination Copyright © 2019 by Elsevier Inc. All rights reserved. Presumptive ID - Culture Choice of anaerobic media is extremely important. Anaerobes have special nutritional requirements. Vitamin K Hemin Yeast Extract CDC blood agar provides the best recovery. Specimens submitted for anaerobes typically also have a request for aerobic culture. CHOC, BAP, MAC and in some laboratories CNA (selective media for gram- positive bacteria) Media for Anaerobes Anaerobic Blood Agar Bacteroides Bile Esculin Agar Brucella Blood Agar Kanamycin-Vancomycin-Laked Blood Agar Phenylethyl Alcohol Agar Colistin Nalidixic acid Agar Anaerobic Broth Media for Anaerobes Anaerobic Blood Agar An enriched medium containing horse or sheep red blood cells for enrichment and detection of hemolysis, hemin, vitamin K (required by some Porphyromonas spp.), and yeast extract Growth Supports growth of almost all obligate and facultative anaerobes Best for anaerobic gram-positive cocci Media for Anaerobes Brucella Blood Agar An enriched medium containing sheep red blood cells for enrichment and detection of hemolysis, casein peptones, dextrose, hemin, vitamin K and yeast extract Growth Supports growth of almost all obligate and facultative anaerobes Best for anaerobic gram-negative bacteria Media for Anaerobes Kanamycin-Vancomycin Laked Blood Agar (KVLB) A selective medium containing Kanamycin - inhibits most facultative gram-negative bacilli Vancomycin - which inhibits most gram-positive organisms Laked blood - accelerates production of brown-black pigmented colonies by certain Prevotella spp.); Growth Primary use: Detects Prevotela and other pigment producers well Supports growth of Bacteroides Also grows anything that is resistant to the antibiotics (ex. Yeasts, other gram negative bacillis) Media for Anaerobes PEA and SNA Selective, differential media PEA contains phenylethyl alcohol as selective ingredient CAN contains colistin, nalidixic acid as selective ingredient Both contain Blood as differential ingredient Growth Suppresses growth of gram negative contaminants (Enterobacteriaceae) Grows almost all obligate anaerobes (gram-positive and gram-negative) Grows gram-positive, facultative anaerobes Media for Anaerobes Bacteroides Bile Esculin Agar (BBE) Selective, differential media 20% bile inhibits most anaerobes Gentamicin inhibits most aerobes Esculin is differential ingredient – hydrolysis demonstrates black color Growth Primarily Bacteroides spp. (which appear black) SOME strains of Fusobacterium, Klebsiella pneumoniae, Enterococcus, yeast will break through Media for Anaerobes Anaerobic Broth Examples: Cooked Meat, Thioglycollate Growth Supports growth of almost all types of bacteria – not a wise idea to use as the sole media or broth Why? Recall: Thioglycolate growth pattern corresponds atmospheric requirements (Image) Media for Anaerobes – example Identify clues from culture Indications of the Presence of Anaerobes in Cultures Foul odor when opening an anaerobic jar or bag Characteristic colonial morphology on anaerobic agar plates but not on aerobic culture plates Unique morphology on Gram stain of clinical specimens or isolates Good growth on Bacteroides bile esculin (BBE) agar Double zone of hemolysis on blood agar Brick-red fluorescence on kanamycin vancomycin laked blood (KVLB) agar Identify clues from culture Describe colony morphology and growth on selective media Sometimes, colonies are tiny and require a microscopic aid (Ex. Image) Describe the Gram stain reaction and cell morphology Set up an aerotolerance test Inoculate pure culture/subculture plate and add appropriate disks Aerotolerance Testing Determines whether a microorganism isolated under anaerobic conditions is a strict anaerobe or a facultative anaerobe Inoculated with a short streak onto a chocolate agar plate for incubation in a CO2 incubator and an anaerobic blood agar incubated anaerobically Fluorescence Ultraviolet (UV) light Porphyromonas and Prevotella spp. Brick-red fluorescence (picture A) Fusobacterium nucleatum and C. difficile Chartreuse fluorescence (picture B) Veillonella spp. Red fluorescence Dependent on culture medium Quick Biochemical Tests Catalase test 15% hydrogen peroxide added to organism on slide Bubbling is positive. Separates Clostridium (negative) from Bacillus (positive) Spot indole test Dilated cardiomyopathy with ataxia (DCMA) reagent Filter paper saturated with para-dimethylaminocinnamaldehyde Turns a blue or green color (positive) Pink or orange color (negative) Rapid urease test Rapid motility test Special Potency Antimicrobial Disks Also called KVC disc test or anaerobic disc test Determines/confirms gram stain reactions for ID purposes NOT to be used as susceptibility test Vancomycin disk (5ug) Susceptibility indicates gram-positive bacillus or Porphyromonas Kanamycin disk (1000ug) Susceptibility indicates Bacteroides other than fragilis group Also Bilophila, Fusobacterium, Veillonella Colistin disk (10ug) Susceptibility indicates same as kanamycin disk plus Prevotella. Special Potency Antimicrobial Disks Procedure: Streak out organism on a suitable plate Place discs on HEAVILY inoculated area of the plate (usually first quadrant of the plate Separate discs sufficiently (see image) Incubate and read in 24-48 hours. Interpretation: a zone of inhibition ≥10 mm is considered susceptible  Special Potency Antimicrobial Disks Vancomycin Kanamycin Colistin Most likely (presumptive) ID S V R Pink-staining Gram Positive Bacilli S R R Porphorymonas spp. R R R Bacteroides fragilis group Could also be: Parabacteroides or Prevotella spp. R V V Prevotella sp. R R S Prevotella sp. R S S Other anaerobes ex. Fusobacterium etc. S: Susceptible, V: Variable, R: Resistant Note that other information must be considered to gain a comprehensive ID of the organism (Ex. Cultural characteristics, clinical site, gram stain) Other Special Potency Disks Nitrate disk Ability to reduce nitrate Sodium polyanethol sulfonate (SPS) disk Peptostreptococcus anaerobius is susceptible. Peptoniphilus asaccharolyticus is resistant. Bile disk Growth in 20% bile Bile-tolerant, gram-negative bacillus is most likely a member of the B. fragilis group. Special Potency Antimicrobia l Disks Lecithinase and Lipase Reactions Egg yolk agar (EYA) – can determine lecithinase, lipase and Proteolytic Enzymes Lecithinase Lecithinase cleaves lecithin found in egg yolk Releases insoluble fat Forms opaque zone around colony (Top image) Used to ID C. perfringens Lipase Hydrolyzes triglycerides and diglycerides to fatty acids and glycerol Positive organisms are iridescent, multicolored sheen (bottom image) F. necrophorum Proteolytic Reactions Organisms that produce proteolytic enzymes (proteases) exhibit Completely clear zone Often narrow around their colonies Best observed by Holding plate up to strong light source Reminiscent of the complete clearing seen with β-hemolytic organisms on SBA plates Summary of Presumptive ID Presumptive identifications are based on Gram stain reaction and morphology, Colony appearance on different media Results of simple, inexpensive, and rapid test procedures Review results together to formulate presumptive ID Presumptive ID generally sufficient for treatment of anaerobes Summary of Presumptive ID Definitive ID Techniques Biochemical-based multitest systems Preformed enzyme-based systems Cellular fatty acid analysis by high-resolution gas-liquid chromatography (GLC) Matrix-assisted laser desorption ionization Time-of-flight mass spectrometry 16S ribosomal RNA gene sequencing Knowledge Check 1 Which of these is TRUE about anaerobic chambers? a. Due to its size, it is easy to use for all people b. Plates must be removed from the chamber for observation c. Is useful to transport anaerobic cultures d. Catalysts need to get re-activated. Knowledge Check 2 Which discs would be appropriate to set up for an anaerobic gram positive cocci? a. Kanamycin, Vancomycin, Colistin b. No discs c. Kanamycin, Vancomycin and Nitrate d. SPS and Nitrate disc Knowledge Check 3 Which of these is NOT part of presumptive ID of anaerobes? a. Spot Indole b. Catalase c. Biochemical multi-test systems d. Direct Smear observation Knowledge Check 4 Which of the following describes an aerotolerance test? a. Inoculate a CHOC plate for CO2 incubator and anaerobic incubator b. Inoculate a Brucella plate for CO2 incubator and anaerobic incubator c. Inoculate a CHOC plate for CO2 incubator and Brucella plate for anaerobic incubator d. Inoculate a Brucella plate for CO2 incubator and CHOC plate for anaerobic incubator Knowledge Check 5 Which of the following would likely NOT support the growth of Bacteroides (Anerobic gram negative rod)? a. MacConkey Agar b. BBE agar c. Brucella Agar d. KVLB Agar