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Introduction to anaerobes. Introduction Believe it or not oxygen is a highly toxic substance. Some forms are more toxic than others. Use of oxygen, or.

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Presentation on theme: "Introduction to anaerobes. Introduction Believe it or not oxygen is a highly toxic substance. Some forms are more toxic than others. Use of oxygen, or."— Presentation transcript:

1 Introduction to anaerobes

2 Introduction Believe it or not oxygen is a highly toxic substance. Some forms are more toxic than others. Use of oxygen, or in some cases merely surviving in its presence requires mechanisms to deal with oxygen toxicity. Charged, electron withdrawing oxygen free radicals are referred to as reactive oxygen species (ROS). ROS are potent oxidants that can at least break bonds and cause damaging mutations (sometimes carcinogenic). All organisms capable of aerobic respiration depend on 2 protective mechanisms: 1) oxygen reducing cytochrome oxidase proteins (vs cytochrome oxidases involved in anaerobic respiration) that terminate their electron transport systems, and 2) two enzymes that work together to detoxify a particular ROS by-product of aerobic respiration: superoxide (O -2 )

3 Introduction 2 H O -2  Superoxide dismutase (SOD)  H 2 O 2 2 H 2 O 2  Catalase  2 H 2 O + O 2 Together these enzymes convert the ROS superoxide into harmless water and molecular oxygen. Obligate anaerobes produce neither of these enzymes. The term obligate anaerobe does NOT necessarily imply that oxygen is bacteriacidal, although this is the case for some unusual “strict obligate anaerobes” such as the “Methanogens”. Medically important obligate anaerobes can survive the presence of oxygen but cannot grow in it (ie. oxygen is bacteriastatic to them). Actually, they can grow in [O 2 ] less than 0.5%.

4 Introduction Strict obligate anaerobes could not function as pathogens due to limiting concentrations of oxygen in every body tissue. Consider the difficulty of manipulating samples and culturing strict obligate anaerobes! Obligate anaerobes are also sensitive to positive “redox potential” values (redox is short for oxidation-reduction). Positive redox values (+Eh) indicate a tendency to oxidize (withdraw electrons) as does oxygen (aerobic env.). Negative redox values (-Eh) tend to reduce (donate electrons) which is indicative of anaerobic environments, and favorable to obligate anaerobes. They prefer Eh < -50mV. Reducing conditions are maintained in media using “reducing agents” such as thioglycolate or the amino acid cysteine. Reducing agents are synonymous with the idea of “anti-oxidants.”

5 Introduction Obligate anaerobes (I will just call them anaerobes from now on) fall into 2 metabolic groups. The fermenters do not conduct respiration at all and require no terminal electron acceptor. They are slow growing as fermentation is a relatively inefficient means of acquiring energy. Among the anaerobes, fermenters are relatively oxygen tollerant (generalization). Medically important anaerobes are fermentors. Other anaerobes conduct anaerobic respiration. Here a non-oxygen terminal electron acceptor is used. They inhabit sub-surface environments (soil, benthose, other) and generate reduced compounds such as sulfide, ammonia, methane, and reduced metals such as ferrous iron. They are cool but not medically important so who cares, right?

6 Introduction Anaerobes can be found in or on virtually any body location, but are most prevalent in the oral cavity, GI tract, genitourinary tract, all mucous membranes and even skin. These guys utilize your body chemistry and other microbes to shelter them from oxygen or “scrub” oxygen from their surroundings. Mucous membranes possess natural reducing agents such as vitamins, poysaccharides, lipids and proteins. Anaerobes can submerge in the mucous as a O 2 shelter Anaerobes often exist in a microbial community which includes aerobic organisms that consume or scrub oxygen from the surroundings. These “biofilms” are stratified with anaerobes being in a lower layer of the film.

7 Introduction Anaerobes relationship with the human host is varied. Some anaerobic species have important human mutualistic function, including normal colon flora that aid in digestion (Bacteroides), and acidification of the female genitorurinary tract (Lactobacillus). Others are now considered commensal, but all of the above at least control growth of opportunistic pathogens. Types of human pathology associated with anaerobic bacteria include intra-abdominal infections, pulmonary infections, pelvic infections, brain abscesses, skin and soft tissue infections, oral infections, and bacteremia and endocarditis. With the exception of the Clostridia, the mechanism by which the anaerobes cause human pathology is not well understood

8 Methods An anaerobic environment can be provided in several ways. These include pre-reduced anaerobically sterilized media (PRAS), Gas Pak Jar, anaerobic pouch, anaerobic “glove box”, and “roll tubes” PRAS media are commercially available in agar plates, agar slants, or broths. They are manufactured in an oxygen free environment and packaged individually in air- tight sealed pouches or bags. Regular commercial media can be “pre-reduced” by placing it an anaerobic jar or glove box for a few hours prior to inoculation with clinical specimens The “Roll tubes” technique was developed at Virginia Tech’s Anaerobe Lab, and is excellent for maintaining a completely anaerobic environment. However, the technique is very tedious and rarely used in the clinical laboratory

9 Methods – Gas Pak The anaerobic jar method (e.g. Gas Pak® Jar) was introduced into the clinical laboratory over 30 years ago and was the most commonly used method until introduction of the anaerobic pouch Inoculated anaerobic culture media are placed in the “jar” An envelope (i.e. “Pak”) containing sodium borohydrite and sodium carbonate is placed in the jar to generate hydrogen and CO 2 Water is added to the envelope which is also placed in the jar. A methylene blue test strip is added, and the jar is sealed.

10 continued Hydrogen generated when water combines with the borohydrite chemically binds to oxygen trapped in the sealed jar to form water = an anaerobic environment A small wire basket attached under the lid contains aluminum pellets coated with palladium. This serves to rapidly catalyze the reaction. If the lid of the jar is not warm to the touch within 40 minutes after it is sealed, or if condensation does not form within the jar within 25 minutes, the jar should be opened and entire process be repeated Likewise, if the methylene blue indicator strip in the jar or pouch does not turn white within one hour, the process should be repeated

11 continued The sodium bicarbonate in the envelope releases carbon dioxide. This is unrelated to the production of the anaerobic environment, however, growth of most anaerobes in culture is enhanced by the presence of carbon dioxide Hydrogen sulfide and other gases produced by bacterial growth can “poison” the palladium catalysts when used over and over. To prevent this, the palladium catalyst should be periodically heated in a dry oven at o C to remove the contaminating gasses and thus regenerate the palladium’s catalytic abilities. This is referred to as “re- charging” the palladium.

12 Gas Pak® Jar H2OH2O H 2 CO 2 Sodium borohydrite Sodium bicarbonate Palladium catalyst in wire basket Foil Envelope “Gas Pak” Culture Plates Clamp Lid

13 Anaerobic Pouch Anaerobic pouches work similarly to gas pak jars and are currently most often used in the clinical laboratory They are sealable bags made of oxygen impermeable see- through plastic material They hold one to two agar plates or a few tubes The major advantage of anaerobic pouch is that plates and tubes can be examined without opening to pouch thus avoiding exposing them to oxygen if there is no growth They are also more convenient. See Figure 14-7 on page 729.

14 Anaerobic Chambers and Holding Jars Anaerobic glove boxes or chambers and holding jars are self-contained anaerobic chambers. Air is removed with a vacuum pump and replaced by oxygen free gas (usually 85% nitrogen, 10% hydrogen and 5% carbon dioxide) from compressed gas cylinders The anaerobic glove box has a sufficiently large volume to allow culture and media manipulation. Specimens and media can be passed in and out of an automatic entry lock Technicians place their hands in the gloves protruding into the chamber The main purpose of the anaerobic holding jar is to pre- reduce media and hold it under these conditions until there are enough specimens to fill a gas generating jar

15 Glove box

16 Culture of Anaerobes Specimens must be placed in a transport system that excludes oxygen (reducing agent, etc.), and be processed with a minimum exposure to oxygen Media must be less that 1 week old or be pre-reduced, in either case to reduce ROS concentration Tom: Pre-reduced media can be placed in a sealed cellophane bag in a refrigerator (???) for a few days or it can be used immediately. Anaerobe recovery media should include non-selective, selective and enrichment types. Vitamin K and hemin are commonly added to anaerobe culture media as these are absolute growth requirements for some clinically significant anaerobes For a non-selective medium, CDC suggest a modified blood agar (AnBAP) with the above as a general purpose medium. It will recover anaerobes but other pathogens as well, which is important. BHI is also good, among others

17 Culture of Anaerobes Thioglycollate enriched with vitamin K and hemin or chopped meat glucose are usually included as a “back-up” broth in case the anaerobic technique fails due to technical problems. Thioglycolate (S) is a good reducing agent and anaerobes such as Clostridia like meat. For selective media, anaerobic phenyl ethyl alcohol (PEA) agar can grow most anaerobic bacteria, as well as aerobic or facultatively anaerobic bacteria. The PEA inhibits the swarming of Proteus and growth of most other potentially contaminating enteric gram negative rods Kanamycin/Vancomycin is a good selective medium when Gram negative anaerobes such as Bacteroides, Prevotella and Fusobacterium are suspected. Kanamycin inhibits the enteric rods (but these anaerobic genera are resistant), and vancomycin inhibits Gram positive cells.

18 Aerotolerance Tests Anaerobic growth is no guarantee that the organism is an obligate anaerobe – could be facultative or aerotollerant. Colonies growing on agar plates incubated anaerobically must be subcultured to two different media to see if it is an obligate anaerobe One of the sub-cultured plates is incubated in air and the other is incubated in an obligately anaerobic environment

19 Example Aerotolerance Tests A A B B C C D D Four different “morphotypes” grew on the original an- aerobic plate. Each was subcultured to two non-selective enriched blood agar plates. The plate on the left was incu- bated anaerobically and the plate on the right was incubated in air. Organisms A and C are obligate anaerobes. Organ- ism B is a facultative anaerobe. Organism D is either an very strict obligate anaerobe or requires longer incubation (further testing is required)

20 Identification Identifying anaerobic bacteria to species is technically challenging in most cases. Fortunately species identification is often not necessary. A mixture of anaerobes in a clinical specimen is often reported as such with no further testing. With a few exceptions presumptive ID via morphology and a few simple tests is sufficient information to allow appropriate antimicrobial therapy Most anaerobes have a predictable antimicrobial sensitivity pattern; antimicrobial susceptibility testing of anaerobes is therefore rarely required Standard drugs such as penicillin, clindamycin or metronidazole are almost always effective against obligate anaerobes

21 Species Identification There are many commercially available identification “kits” for identifying obligate anaerobic bacteria. Some require standard inoculation and extended anaerobic incubation prior to reading results. Others utilizing heavy inocula give quick results from pre-existing enzymes. These therefore do not require anaerobic incubation. Reference labs ID anaerobes chromotographically on the basis of fatty acid methyl ester (FAME) profiles Other macromolecule probes or homology can be used as well

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