Anaerobic bacteria and their isolation

Noureen Saeed Senior technologist Clinical microbiology

Introduction
► There

is no universally accepted definition of anaerobic bacteria these require a reduced oxygen tension for growth and fail to grow on surface media when incubated in air or 5-10% CO2. ► Obligate anaerobes ► Aerotolerant anaerobe ► Facultative anaerobes. ► Capnophilic bacteria ► Microaerophilic bacteria

Anaerobes as normal flora
► Most

muco cutaneous surfaces of humans harbor a rich flora of anaerobic bacteria which varies at different anatomic sites in terms of concentrations and microbial species. ► Upper air ways ► saliva (approximately 108 /ml ) ► Gingival crevices (almost 1012 /ml) ► The gastrointestinal tract

Cont……..
► Small

intestine ► Terminal ileum and colon (peptostreptococcus species,bacteroides species, fusobacterium species clostridia and a variety of non spore forming gram positive rods (bifidobacterium, eubacterium etc)

Contd………
► Female

genital tract (105 -1011 /ml) this conc. Shows various shifts during various stages of the menstrual cycle that may be hormonally influenced. most of the organisms are anaerobes the dominant anaerobes are lactobacillus,peptostreptococcus and bacteroides species. (normal, healthy individuals) great variations are seen in case of pregnancy, gynecologic surgery and antibiotic therapy. ► Urethra in both sexes contains skin flora as well as bacteroides and fusobacterium

Why anaerobes are killed by oxygen?

Why anaerobes are killed by oxygen
Aerobic organisms uses oxygen as terminal electron acceptor and generates toxic oxygen reduction products including H2O2, hydroxy radicals, singlet oxygen and superoxide anions, such reactive metabolites destroy the lipids components of cells and damages the DNA, aerobes and facultative anaerobes are protected from these reactive by products by producing certain enzymes peroxidases and super oxide dismutases

Anaerobic infections
► Anaerobes

are causative agents of a variety of head and neck infections e.g peritonitis, sinusitis and brain abscesses ► Spillage of fecal contents into the peritoneum following appendicitis,penetrating trauma or cancer can lead to intra abdominal infections involving anaerobes ► Bacteremia due to anaerobes usually occur after obstetrics or gynecological infections these bacteria may also cause infections of bones, joints and grafts.

Pathophysiology
►Anaerobic

infections are usually endogenous that is they originate from the hosts own flora when there is a breech in the muco cutaneous barrier resulting in the displacement of normal flora. ►Only important exception is histotoxic clostridial syndrome such as botulism, some cases of gas gangrene in which the organisms is acquired from the environmental sources.

Clues to anaerobic infection
► Infection

adjacent to surfaces that harbors anaerobes as normal flora. ► Infections characterized by abscess formation or tissue necrosis. ► Gas formation ► Grams stain of exudates showing poly microbial flora or orgs with morphologic features of anaerobes. ► Foul odor ► Presence of sulfur granules

Microbiologic methods to establish Anaerobic infections

Specimen collection/transport
Liquid or tissue specimen is preferred swabs should be avoided. ► Successful recovery of anaerobes requires rapid delivery to the laboratory in a transport medium that maintains a moist, anaerobic atmosphere. A number of systems are available for transport of specimens for anaerobic culture these systems include enclosed tubes or vials with an anaerobic atmosphere and isotonic agar base, glass or plastic tubes with catalytic systems for generating anaerobic atmosphere (vacutainer anaerobic specimen collector), pre reduced anaerobically sterilized medium (remel) etc.

Anaerobic media

Anaerobic media

► ► ► ► ► ► ►

All general purpose non selective media having peptones, yeast extract, vitamin K, hemin, 5% sheep blood agar and reducing agent can be used for culturing anaerobes. Variety of broth and solid media are commercially available for the isolation of anaerobic bacteria Anaerobic blood agar Kanamycin vancomycin laked blood agar Phenylethyl alcohal agar Columbia colistin nalidixic blood agar medium Thioglycolate broth Chopped meat broth

Anaerobic incubation systems
►A

variety of systems are available for this purpose ► anaerobic jars ► disposable bags ► chambers and ► roll tube methods etc.

Incubation conditions:
No primary plate streaked for isolation of anaerobes should be exposed to air for longer than 15-20 minutes bc anaerobes may be killed by prolonged exposure to oxygen and may never be detected especially fusobacterium species. ► Incubation temperature is ordinarily 35-370C. although some anaerobes such as clostridia will grow more rapidly at higher temperatures 42-47 0C . ► Longer incubation periods are required for fastidious bacteria such as actinomycetes species or bilophila sp or bacteroides melaninogenicus group of organisms that require incubation beyond 72 hours.

Identification of anaerobes:
► The

grams reaction, morphology of vegetative cells the presence or absence of spores are all key features in the identification of anaerobic bacteria. ► The use of special potency antibiotic disks on the plates can help in preliminary identification of anaerobes usually colistin(CT), VA and Kanamycin disks are used, usually gram positive anaerobes are susceptible to VA and Kanamycin and resistant to CT. Kanamycin is useful for separating fusobacterium from bacteroides species. Fusobacteria are mostly sensitive

► Level

I identification: ► Examination of culture plates ► Colony morphology ► Hemolysis ► Pigment production ► Fluorescence ► Swarming/ spreading etc

► ► ► ► ► ► ► ►

Level II identification: further grouping or definite identification can be achieved by use of several tests including Bile test Nitrate disk test Formate fumerate (F/F) growth stimulation test Motility Ethanol spore test SPS susceptibility test Lipase reaction Naglers test

► Level

III identification systems: ► several systems are available including macro tube and micro tube methods, commercially available system can be used such as API anaerobes or rapid id ANA, minitek system, ATB 32A system, ANI card and anaerobe panel, microscan or vitek etc.

► Alternatively: ►a

novel method that is based on analysis of whole cell long-chain fatty acids profiles. In this method the organism is inoculated in peptone yeast broth and incubated these cells are harvested by centrifugation and than added with a mixture of methanol and NaOH than chromatography is performed and resulting profile is compared with other profiles in the system data base and a computer print out lists the identification of test isolate (microbial ID inc, Newark)

THANK YOU