Anaerobes of Clinical Importance Part One MLAB 2434 – Microbiology Keri Brophy-Martinez

Concepts in Anaerobic Bacteriology Air = about 21% O2 and 0.03% CO2 CO2 Incubator = about 15% O2 and 5%-10% CO2 Microaerophilic System = 5% CO2 Anaerobic System – 0% O2

Concepts in Anaerobic Bacteriology Obligate (strict) anaerobes Grow ONLY in the absence of molecular oxygen Killed by exposure to air Aerotolerate (moderate) anaerobes Can tolerate exposure to air for several hours but perform metabolic activities

Concepts in Anaerobic Bacteriology Facultative anaerobes Do not require O2, but use it if available.

Why Anaerobes? Oxygen is toxic because it combines with enzymes, proteins, nucleic acids, vitamins and lipids that are vital to cell reproduction Anaerobes do not have enzymes for protection against the toxic effects of molecular oxygen, so oxygen can have a bacteriostatic or even bactericidal effect on them

Why Anaerobes? Substances produced when oxygen becomes reduced are even more toxic, producing such things as hydrogen peroxide and superoxide anion Anaerobes require environments with low oxidation-reduction potential (redox), so they must live in areas where the redox potential is low

Where Anaerobes are Found Anaerobes are thought to be the earliest forms of life All life on earth was anaerobic for hundreds of millions of years Today they are found in soil, fresh and salt water, and in normal flora of humans and animals

Where Anaerobes are Found Anaerobes that live outside the body are called “exogenous anaerobes” (Example: Clostridium species) Anaerobes that live inside the body are called “endogenous anaerobes” Advantages: barrier to colonization by pathogenic organisms, source of fatty acids, vitamins and cofactors, help mature neonate immune system Disadvantages: opportunists for immunocompromised Most anaerobic infections are from endogenous sources

Anaerobic Anatomical Sites for Endogenous Anaerobes Mucosal surfaces such as linings of oral cavity, GI tract, and GU tract Respiratory Tract – 90% of bacteria in the mouth are anaerobes If mucosal surfaces are disturbed, infections can occur in the oral cavity and in aspiration pneumonia Sometimes cause “bad breath”

Anaerobic Anatomical Sites for Endogenous Anaerobes Skin – frequently these normal skin anaerobes contaminate blood cultures GU Tract – anaerobes rarely cause infection in the urinary tract, but cervical and vaginal areas have 50% anaerobes GI Tract – Approximately 2/3’s of all bacteria are in the stool; only cultured anaerobically if Clostridium difficle is suspected

Factors that Predispose Patients for Anaerobic Infections Trauma to mucosal membranes or skin Interruption of blood flow Tissue necrosis Decrease in redox potential in tissues Prior antibiotic therapy when organism was resistant Immunosuppresion

Virulence Factors Polysaccharide capsule Adherence factors Clostridial toxins/exoenzymes Hyaluronidase Lipases Proteases/Proteinases/ Phospholipases/ Permeases Necrotizing toxins

Indications of Anaerobic Infections Usually purulent (pus-producing) Close proximity to a mucosal surface Infection persists despite antibiotic therapy Presence of foul odor Presence of large quantities of gas (bubbling or cracking sound when tissue is pushed) Presence of black color or brick-red fluorescence Distinct morphologic characteristics in gram-stained preparation

Collection, Transport and Processing Specimens for Anaerobic Culture Any specimen collected on a swab is usually not acceptable because of the possibility of having normal anaerobic organisms Must be transported with minimum exposure to oxygen

Specimens for Anaerobic Culture Aspirates Should be collected with needle and syringe Excess air expressed from syringe Specimen injected into oxygen-free transport tube or vial

Specimens for Anaerobic Culture Tissue must be placed in an oxygen-free transport bag or vial Usually ground for best results Blood aerobic AND anaerobic bottles are collected for most blood culture requests

Unacceptable Specimens for Culture Swabs collected from throat, nasopharyngeal, gingival, rectal, vaginal, cervical, urethral, decubitus ulcers, feet and other exposed wounds Sputum Voided or catheterized urine

Processing Clinical Samples for Anaerobic Culture Must be placed in an anaerobic chamber or holding device while awaiting processing Procedures Macroscopic exam of specimen Foul odor Presence of “sulfur granules” Black pigmentation Gram stain Distinct morphology Increased WBCs

Processing Clinical Samples for Anaerobic Culture Inoculation of anaerobic media Require enriched media for growth Utilize pre-reduced media Eliminates dissolved O2 in media Reducing agents lower redox potential Inoculate nonselective, selective and liquid enrichment media Anaerobic incubation

Typical Anaerobic Media Anaerobic blood agar (BRU/BA) Supports growth of all obligate and facultative anaerobes Bacteroides bile esculin agar (BBE) Supports growth of bile-tolerant anaerobes, such as Bacteroides, Prevotella, Porphyromonas, Fusobacterium species Kanamycin-vancomycin-laked blood agar (KVLB) Supports growth of Bacteroides and Prevotella spp.; certain facultative gnr will also grow Phenylethyl alcohol agar (PEA) Supports growth of all obligate and gram positive facultative anaerobes, inhibits enteric gnr Cycloserine-cefoxitin-fructose agar (CCFA) Selective for C. difficile Anaerobic broth, such as thioglycollate (THIO) or chopped meat Supports growth of all types of bacteria; obligate aerobes near the top, obligate anaerobes at the bottom and facultative anaerobes throughout

Anaerobic Media Bacteroides fragilis on KVLB (left) and BBE agar (right)

Anaerobic Incubation Anaerobic chambers Anaerobic jars Sealed box which provides an oxygen-free environment for inoculation and incubation of culture Anaerobic jars Gas-Pak envelopes generate CO2 and H2, which combines with O2 H2 is explosive; palladium catalyst MUST be used Anaerobic bags or pouches All systems must have an oxygen indicator system in place Methylene blue strips Resazurin

Anaerobic Incubation Anaerobic chambers

Anaerobic Incubation Anaerobic GasPak System Anaerobic bags/ containers

Interpretation of Cultures Primary cultures are examined after 48 hours of incubation If no growth, reincubate for up to 5 days before discarding

Indications of Anaerobes in Cultures Foul odor when opening anaerobic jar or bag Colonies on anaerobically incubated media but not on aerobic media Good growth on BBE Colonies on KVLB that are pigmented or fluorescent Double zone of hemolysis on blood agar

Anaerobic Culture Workup If observe growth on media or liquid media Check aerotolerance Subculture a colony to BAP, incubate in ambient air and subculture a colony to Ana BAP, incubate anaerobically After 24 hours, determine if organism is obligate anaerobe or facultative anaerobe

Interpretation of Cultures If the aerotolerance test confirms an anaerobe, evaluate colony morphology Consider: Color/pigment, surface, density, consistency, form, elevation, margins, fluorescence, pitting of agar, double zone of beta hemolysis, odor, swarming, molar tooth/breadcrumb, ground glass/fried egg Number of different types of colonies Quantitation Type of media supporting growth

Interpretation of Cultures Gram stain suspicious colonies Note gram reaction, shape, presence of spores, filamentous etc

Location of Spores Terminal Subterminal

Presumptive Identification of Anaerobes Aerotolerance Fluorescence Special-potency antimicrobial disks Catalase test Spot indole test Motility test Lecithinase and lipase reactions Presumpto plates

Definitive Identification of Anaerobes PRAS (Pre-reduced Anaerobic System) and non-PRAS biochemical test media Biochemical-based and preexisting enzyme-based minisystems Gas-liquid chromatographic (GLC) analysis of metabolic end products Fatty acid analysis Alcohols Molecular testing

Antimicrobial Susceptibility Not routinely performed Drugs of choice Chloramphenicol, metronidazole, cephalosporins Perform beta-lactamase testing Gram negative rods

Treatment Protocols Surgical therapy Hyperbaric oxygen Antitoxins Draining abscesses, removing dead tissue, eliminating obstructions Hyperbaric oxygen Oxygen is forced into necrotic tissues, killing anaerobes Antitoxins Used in cases of tetanus and botulism to neutralize the neurotoxins produced by C. tetani and C. botulinum

References http://www.labsupplyoutlaws.com/products/Lab-Equipment/Microbiology-Apparatus/Environmental-Systems-for-Microbiology/BD-BBL-Bio-Bag-Environmental-Chambers.htm Kiser, K. M., Payne, W. C., & Taff, T. A. (2011). Clinical Laboratory Microbiology: A Practical Approach . Upper Saddle River, NJ: Pearson Education. Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.