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)
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