Anaerobes Lecture PDF

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Summary

- Endogenous, bacteria from the body cause infection. Usually opportunistic normal flora. - Exogenous, bacteria from the environment cause infection. Usually from trauma to a protective barrier allowing environmental bacteria to infect the body.Characteristics of Anaerobes- Facultative Anaerobes, do...

Description

MLSC-3131U, Clinical Microbiology II -

Endogenous, bacteria from the body cause infection. Usually opportunistic normal flora. Exogenous, bacteria from the environment cause infection. Usually from trauma to a protective barrier allowing environmental bacteria to infect the body.

Characteristics of Anaerobes -

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Facultative Anaerobes, do not require oxygen but will use it if present. Obligate Anaerobes, are intolerant to oxygen. Oxygen is a free radical that is highly reactive and anaerobes lack the protective enzyme required to protect the organism from oxidation and death. o Moderate, can tolerate 2-8% oxygen. o Strict, cannot tolerate any oxygen. Certain anaerobes are more aerotolerant because they may only have one of the two protective enzymes (catalase and superoxide dismutase). Thioglycolate broth is an enriched broth that is used to determine the aerotolerance of bacteria. Colonies that grow at the top of the tube are obligate anaerobes. Those that grow at the bottom are obligate anaerobes. Those that grow throughout the tube are facultative anaerobes.

Processing Anaerobic Specimens -

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The media is prereduced by placing the uninoculated media in an anaerobic jar in preparation for anaerobic specimen (wound swab) incubation. Typically subculturing does not require prereducing. Quality control is done for the media by testing if the new media can support anaerobic organisms. Quality control for anaerobic jars is done every time it is used. Done with methylene blue or resazurin pink. Methylene blue will be blue when oxygenated and clear when not oxygenated. Aerotolerant anaerobes are not used for anaerobic jar QC. These organisms can tolerate a little bit of oxygen and will not work for determining the atmospheric quality of the jar. Bacteria like Clostridium novyi are strict anaerobes and can be used along with Pseudomonas aeruginosa as a negative control.

Media for Anaerobes -

This media requires a reducing agent to maintain anaerobic conditions. Sodium thioglycolate is an example of this. Resazurin pink is used as an indicator for thioglycolate broth. It turns pink when the medium is not reduced (aerobic) and colourless when it is reduced (anaerobic). Media also typically includes enrichment ingredients.

MLSC-3131U, Clinical Microbiology II Primary Plating Media for Anaerobes -

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Enriched and Not Selective, Brucella agar (5% blood) which supports the growth of all anaerobes. Selective Media, selects only for specific bacteria using selective ingredients. o Bacteroides Bile Esculin, inhibits most other anaerobes from those that hydrolyse bile (selective ingredient). o Laked Kanamycin-Vancomycin, inhibits the growth of most aerobic and facultative anaerobic bacteria GNBs and GP organisms. Isolates Prevotella and Bacteroides. o Phenylethyl Alcohol Agar, supports the growth of GP and GN organisms. Inhibits facultative GNBs and reduces swarming of some Clostridium and Proteus. Enrichment Aerobic Broth, serve to support a swab with very few organisms. o Thioglycolate Broth, also known as cooked meat broth. Not selective and have potential for the overgrowth of facultative anaerobes, masking the strict anaerobes.

AnaeroGen System -

Serves to rapidly absorb oxygen from the jar and generate CO2. The reaction starts as soon as the pack is exposed to air. The pack must be placed in the jar and the jar must be sealed within one minute. The redox indicator strip must be placed in the jar prior to closing. Heat and condensation should occur if the jar is working.

Anaerobic Incubation -

Anaerobes should not be exposed to air before 24 hours of incubation. Anaerobes are the most sensitive during their log phase. Re-incubation should be done ASAP after examination. Most anaerobes will grow within 5 days and require at least 5 days for incubation. Enrichment broths should be gram stained and cultured if turbid.

Anaerobic Collection and Transportation -

Oxygen susceptible organisms prove to be problematic. Specialized collection approaches are needed. Anerobic swabs like amies swabs should not be used for anaerobic recovery because they are very expensive. Specimens must be transported to the lab within 24 hours and old samples are rejected. Syringes are the best for aspirates and fluids. Tissues samples are most preferably collected from biopsies. Irretrievable old specimens can still be processed if needed but the age of the sample is commented.

MLSC-3131U, Clinical Microbiology II -

Store samples at room temperature if they cannot be plated immediately. Otherwise, plate them immediately.

Unacceptable Specimens for Anaerobic Cultures -

Throat or nasopharyngeal swabs. Superficial skin material collected with swabs (aerobic skin flora). Sputum (mouth flora contamination) Aerobic swabs (aerobic flora bacteria). Urine. Vaginal and cervical swabs. Surfaces or edges of a wound. Stool.

Direct Detection of Anaerobes -

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The gram stain is very important because it can show bacterial spores which most anaerobes possess. Also visually shows us the type and relative quantity of organisms present including host cells (Epithelial cells and WBCs). Acts as quality assurance by guiding further workup. Foul Odours, very typical upon opening anaerobic jars containing anaerobic bacteria. Growth, growth on anerobic plates and no growth on CO2 incubated plates is typical of obligate anaerobes. Also, growth on BBE and LKV (selective media) are indicative of anaerobes. Hemolysis, anaerobes can show double zones of hemolysis on BAP (Clostridium perfringes).

Gram Staining Anaerobes -

Bacteroides fragilis, GNB in short rods that stain faintly. Fusobacterium spp, GNB that are very long and pointy (fusiform). Peptostreptococcus spp and Anaerobic Streptococcus, GPC in clusters and chains. Veillonella spp, tiny GNCs. Clostridium perfringes, GPB in rod box car shapes. Actinomyces iIsraeli, GPB that shows branching and beading.

Colonial Morphology of Anaerobes -

Bacteroides, translucent, grey and possess a unique odour. Fusobacterium spp, tiny, translucent and grey. Shows greening around the colonies after exposure to oxygen. Clostridium perfringes, large, transparent, irregular, flat and can show double zones of hemolysis. Actinomyces isrealii, has a molar tooth appearance.

MLSC-3131U, Clinical Microbiology II Clostridia -

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Spore forming GPBs. Spore location depends on the species, as some will be in the middle of the rod (central), at the end of the rod (terminal) or almost at the end (subterminal). Spores helps the bacteria survive in adverse and extreme conditions. Spores enter the body through wounds or ingestion. Clostridium tetani, causes tetanus and is very oxygen sensitive. It is difficult to grow, is motile and has terminal spores. Virulence factors include tetanospasmin (neurotoxin) and tetanolysin (hemolysin). Fully developed termina spore gives Clostridium tetani a drumstick appearance on a gram stain. A thin film is produced on blood agar since it swarms. Clostridium perfringens, full time pathogen that causes soft tissue infections like gas gangrene. Large rectangular GPB in a rod shape. Spores are rarely seen and shows rapid growth. This is somewhat aerotolerant and can grow at higher temperatures (43-47 degrees). Virulence factors include Alpha toxin (hemolysis). Gram stain shows GPB with a boxcar shape. Colonial morphology shows two zones of beta hemolysis. Can be identified using a reverse CAMP test. Will also show opaque growth on egg yolk agar. Clostridium botulinum, causes botulism from the bacteria’s powerful neurotoxin.

Non-Spore Forming Anaerobic GPB -

Actinomyces, Actinomyces shows growth in a molar tooth shape and causes sulfur granules in a wound. Propionibacterium acnes, normal flora clubbed shaped GPB, filaments resembling Corynebacterium. Catalase positive and partially aerotolerant.

Anaerobic GNB -

Include B. fragilis group, Porphyromonas, Prevotella and fusobacterium. Bacteroides, bile tolerant and positive on BBE and shows growth in the presence of Bile disks. Fusobacterium, long pointy rod. Bile disk sensitive/inhibited.

Anaerobic GPC -

Colonize the oral cavity, GI tract, GU tract and skin. Identified using anticoagulant SPS disks. Peptostreptococcus is inhibited while anaerobic streptococcus are resistant.

MLSC-3131U, Clinical Microbiology II Anaerobic GNC -

Non-motile, non-fermenting and reduces nitrate. Normal flora of the oral cavity but can cause endocarditis if it travels to the heart through the blood via oral wounds. Nitrate positive anaerobic GNC can be reported as Veillonella spp....