Micro 443 Mark S. Chandler, Ph.D.

Identification of Anaerobes
Obligate anaerobic bacteria are those that grow in the absence of free oxygen but fail to grow in
the presence of oxygen

Growth Response at 48 hours
Atmosphere Aerobes
a
Facultative anaerobes
b
Anaerobes
c,d
Air + + __
Anaerobic __ + +
a
Neisseria, Pseudomonas (obligate respirers)
b
Staphylococcus, Corynebacterium, enteric GNRs, etc., (Streptococcus, aerotolerant anaerobe/obligate fermenter)
c
Bacteroides, Fusobacterium, Clostridium, Prevotella, Porphyromonas, Peptostreptococcus, Viellonella, and many others
d
For good recovery of anaerobes, specimens are inoculated onto blood agar supplemented with vitamin K and hemin
(reducing agents such as thioglycolate and L-cysteine may be added to anaerobic transport media and certain culture media
to help maintain reduced conditions)


Anaerobes vary considerably in their tolerance to atmospheric oxygen. Some of the proteins
present in bacteria react with O
2
to form oxygen molecules that are toxic to cells. Toxic
forms of oxygen include singlet oxygen (O
2
*), superoxide anion (O
2

), hydrogen peroxide
(H
2
O
2
), and hydroxyl radical (OH). Enzymes that destroy these toxic oxygen products
include catalase, peroxidase, and superoxide dismutase. Low levels or complete absence of
superoxide dismutase is likely a major reason why oxygen is toxic to anaerobes.


Therefore rapid achievement and maintenance of low oxygen tension is essential for cultivation
of anaerobes use anaerobe jars and glove boxes in clinical labs.









Micro 443 Mark S. Chandler, Ph.D.

Common Medically Important Anaerobic Bacteria

GNR
Bacteroides fragilis group these are the most common anaerobes recovered from clinical
specimens. They are more resistant to penicillin and
cephalosporins than other anaerobes (resistance in other groups
is increasing). The bile-resistance of the B. fragilis group can be
used as a simple overnight test to screen for them. (See separate
description of presumptive identification).
Prevotella group includes the black pigmented Pr. melaninogenica
Porphyromonas
Fusobacterium spp.
Others -- 20 genera
GPR with spores
Clostridium perfringens does not produce spores in clinical specimens or on clinical media
Clostridium difficile
Clostridium tetani
Clostridium botulinum, plus more than eight other species
GPR no spores
Actinomyces spp.
Propionibacterium spp.
Several other genera
GPC
Peptostreptococcus spp.
GNC
Veillonella spp.

Anaerobic infections can involve virtually any organ when conditions are suitable.
Trauma, poor circulation, and tissue necrosis provide favorable conditions for anaerobes to
multiply.
Exogenous sources

Endogenous sources
Micro 443 Mark S. Chandler, Ph.D.


Important to isolate and identify the organism or group of organism as most anaerobic infections
are associated with high morbidity and mortality (presumptive ID OK). Treatment involves
antibiotics; prompt surgical intervention including debridement of necrotic tissue and/or
amputation may be important-
Laboratory isolation:
Initial Work-up for Anaerobe Subculture
Subculture each colony type
Incubate 48h
Air anaerobe jar




Conclusion #2 and # 4 are anaerobes


Micro 443 Mark S. Chandler, Ph.D.

Identification (ID) of Anaerobic GNRs
Physician must request species ID otherwise presumptive ID is done as species ID is costly
and takes several days to a week or more and treatment is usually necessary immediately.
Preliminary examination of isolates should include; Gram stain, colony morphology,
pigment, fluorescence (long wave UV), hemolysis, antibiotic susceptibility, and
biochemical screening.
Most of the clinically important GNRs can be placed into broad groups on the basis of these
relatively few tests.
For Presumptive ID
Antibiotic screening: Kanamycin, Vancomycin, Colistin (in lab we will use Penicillin)
Biochemical screening: Bile (growth in 20 % bile), spot indole, catalase, red
fluorescence, and black pigment.
Bacteroides fragilis group is resistant to all the above antibiotics and to bile. Catalase +
Prevotella are sensitive to bile, resistant to vancomycin and kanamycin, variable to
colistin and may produce black colonies.
Porphyromonas are susceptible to vancomycin and bile and resistant to colistin and may
fluoresce red.
Thus using this small battery of tests many anaerobes can be presumptively identified.
For species ID
Biochemical tests for definitive species identification.
Overnight biochemical tests that require growth; API strip
Also strips that test for a panel of preformed enzymes in ~4 hr; AnIdent
Old method;Gas liquid chromotography (GLC) of fatty acids from glucose
metabolism
Presumtive ID of Clostridium perfringens
Gram stain, hemolysis, lecithinase on Naglers egg yolk agar

Presumtive ID of Clostridium difficile

C. difficileis naturally resistant to many antibiotics
Selective and differential media available for isolation of C. difficile
CCFA medium has peptones and the antibiotics cycloserine and cefoxitin with
fructose and a pH indicator. C. dif grows giving yellow colonies with
yellow fluorescence
Direct detection of toxins in stool is often used in place of culture for diagnosis