Important Concepts in Anaerobic Bacteriology

A. Anaerobes Defined
B. Why Some Organisms Are Anaerobes
C. Where are Anaerobes Found
 OUTLINE OF CONTENTS

A. Specimen Quality (Selection)

B. Specimen Transport
C. Processing Clinical Samples
A. Specimen Quality (Selection)

1. Suitable Specimens for Anaerobic Culture

2. Unsuitable Specimens for Anaerobic Culture
3. Fecal Specimens for Clostridial Illness
 OUTLINE OF CONTENTS

A. Specimen Quality (Selection)

B. Specimen Transport
C. Processing Clinical Samples
B. Specimen Transport

1. Anaerobic transport system

i. Rubber-stoppered collection vial
ii. Oxygen-free Transport tubes
iii. Anaerobic pouch
2. Contents of Transport Media
i. Reducing agent (thioglycolic acid, Na thioglycolate, cystine)
ii. Redox indicator (resazurin, methylene blue)
 OUTLINE OF CONTENTS

A. Specimen Quality
B. Specimen Transport
C. Processing Clinical Samples
C. Processing Clinical Samples

1. Macroscopic Examination of the Specimen

2. Microscopic Examination of the Specimen
3. Inoculation to Plated or Tubed Media
4. Anaerobic Incubation
 Perceptual Elements

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ANAEROBES OF CLINICAL
IMPORTANCE
 OUTLINE

Anaerobes and their Diseases

1. Virulence Factors
2. Associated Disease
Procedures for Identifying Anaerobes
3. Cellular Morphology
4. Colony Morphology on Blood Agar
5. Other Characteristics and Presumptive Tests
6. Summary
 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Clostridium botulinum 2. Porphyromonas spp.
3. Prevotella spp.
3. Clostridium tetani
4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum D. Cocci
1. Peptococcus niger
B. Gram Positive Bacilli 2. Peptostreptoccus spp.
1. Actinomyces spp. 3. Veillonella spp.
2. Propionibacterium spp.
3. Bifidobacterium spp.
 Location of Spore

1. Terminal Spores
• Appear in the end of
the organism
2. Central Spores
• Appear in the middle
3. Subterminal Spores
• Appear between the
end and the middle of
the organisms
 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Clostridium botulinum 2. Porphyromonas spp.
3. Prevotella spp.
3. Clostridium tetani
4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
D. Cocci
1. Peptococcus niger
B. Gram Positive Bacilli
2. Peptostreptoccus spp.
1. Actinomyces spp.
3. Veillonella spp.
2. Propionibacterium spp.
3. Bifidobacterium spp.
 Clostridium perfringens
Gram Positive Spore-forming Bacilli

1
Clostridium perfringens

1. Virulence factor
a. α-toxin – type C food poisoning (enteritis necrotans)
b. Enterotoxin
2. Associated diseases
a. Bacteremia, Gas gangrene (myonecrosis) and food poisoning

Gas Gangrene
Clostridium perfringens

3. Cellular Morphology
a. Gram-variable straight rods with blunt ends
b. Spores are oval, subterminally located and swell cell
c. Boxcar, large square rods
Clostridium perfringens

4. Colonial Appearance
a. Large Irregulars Shape, Double zone of hemolysis
• alpha toxin – outer zone, partially hemolyzed
• theta toxin – inner zone is completely hemolyzed

Anaerobic blood agar plate

Clostridium perfringens

5. Other Characteristics and Presumptive Test

b. Lecithinase positive in Egg-yolk Agar (EYA)
c. Nagler Reaction
d. Reverse CAMP test
Clostridium perfringens

5. Other Characteristics and Presumptive Test

b. Lecithinase positive in Egg-yolk Agar (EYA)
C. perfringens
Lecithinase positive (opaque zone around the
colonies) and Lipase negative (no sheen)

B. fragilis
Lecithinase and Lipase negative

F. necrophorum
Lipase positive (iridescent,
multicolored sheen)
Clostridium perfringens

5. Other Characteristics and Presumptive Test

b. Lecithinase positive in Egg-yolk Agar (EYA)

Lecithinase (+) Lecithinase (-)

Clostridium perfringens

5. Other Characteristics and Presumptive Test

b. Lecithinase positive in Egg-yolk Agar (EYA)
c. Nagler Reaction
d. Reverse CAMP test
Clostridium perfringens

5. Other Characteristics and Presumptive Test

c. Nagler Reaction
• One-half of the surface is streaked Type A antitoxin

Zone of precipitation
Clostridium perfringens

5. Other Characteristics and Presumptive Test

b. Lecithinase positive in Egg-yolk Agar (EYA)
c. Nagler Reaction
d. Reverse CAMP test
Clostridium perfringens

5. Other Characteristics and Presumptive Test

d. Reverse CAMP test Streptococcus agalactiae
Clostridium perfringens

Bow-tie zone of Arrow-shaped zone

hemolysis hemolysis
Clostridium perfringens

5. Other Characteristics and Presumptive Test

b. Lecithinase positive in Egg-yolk Agar (EYA)
c. Nagler Reaction
d. Reverse CAMP test
Clostridium perfringens

6. Summary
i. Gas Gangrene and Food poisoning
ii. Boxcar, Large square rods
iii. Double Zone of Hemolysis
iv. Lecithinase - Positive
v. Nagler Reaction
vi. Reverse CAMP - Positive
 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Clostridium tetani 2. Porphyromonas spp.
3. Prevotella spp.
3. Clostridium botulinum
4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
D. Cocci
1. Peptococcus niger
B. Gram Positive Bacilli
2. Peptostreptoccus spp.
1. Actinomyces spp.
3. Veillonella spp.
2. Propionibacterium spp.
3. Bifidobacterium spp.
 Clostridium tetani
Gram Positive Spore-forming Bacilli

2
Clostridium tetani

1. Virulence factor
• Tetanospasmin
• a neurotoxin that causes a spastic type of paralysis with
continuous muscle spasms

Backward arching of the back muscles

Clostridium tetani

2. Associated disease
• Tetanus – paralysis with continuous muscular spasms
• Trismus (lockjaw)
• Risus sarcodinicus (distorted grin)
• Breathing difficulty

Backward arching of the back muscles

Clostridium tetani

3. Cellular Morphology
a. Gram-variable straight rods with blunt ends
b. Swollen terminal spores
c. Drumstick appearance
Clostridium tetani

4. Colony Morphology
a. Smoothly swarming but slow growing
b. Narrow zone of beta hemolysis

Anaerobic Blood Agar

Clostridium tetani

6. Summary
i. Tetanus
ii. Swollen terminal spores “drum stick appearance
iii. Swarming and with narrow zone of β-hemolysis
 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Clostridium tetani 2. Porphyromonas spp.
3. Prevotella spp.
3. Clostridium botulinum
4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
D. Cocci
1. Peptococcus niger
B. Gram Positive Bacilli
2. Peptostreptoccus spp.
1. Actinomyces spp.
3. Veillonella spp.
2. Propionibacterium spp.
3. Bifidobacterium spp.
 Clostridium botulinum
Gram Positive Spore-forming Bacilli

3
Clostridium botulinum

1. Virulence factor
• Botulism toxin (neurotoxins)
• Result in a flaccid type of paralysis
• Strabismus “wandering eye” ,“frown lines”
2. Associated disease
a. Foodborne, Infant and wound botulism
Infant Botulism

Wound Botulism Foodborne Botulism

Clostridium botulinum

2. Associated disease
a. Foodborne Botulism – ingestion of preformed toxin
b. Infant botulism – ingestion of spores
c. Wound Botulism – contamination of wound with spores

Foodborne Botulism

Infant Botulism Wound Botulism

Clostridium botulinum

3. Cellular morphology
• Gram positive straight rods with blunt ends
• Swollen terminal spores
• Tennis racket appearance
Clostridium botulinum

4. Colony morphology
• Usually β-hemolytic
5. Other information
• Confirmation
i. Demonstration of neurotoxin in serum, feces, vomitus or
gastric contents
ii. Recovery of the organism in stool
Clostridium botulinum

6. Summary
i. Foodborne, Infant and wound botulism
ii. Swollen terminal spores “tennis racket appearance”
iii. β-hemolytic
iv. Confirmation by demonstration of neurotoxin or organism
 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Clostridium tetani 2. Porphyromonas spp.
3. Prevotella spp.
3. Clostridium botulinum
4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
D. Cocci
B. Gram Positive Bacilli
1. Peptococcus niger
1. Actinomyces spp.
2. Peptostreptoccus spp.
2. Propionibacterium spp.
3. Veillonella spp.
3. Bifidobacterium spp.
 Clostridium difficile
Gram Positive Spore-forming Bacilli

4
Clostridium difficile

1. Virulence factor
a. Toxin A (enterotoxin) – toxic to cells of the intestinal mucosa
b. Toxin B (cytotoxin) – necrosis of colonic mucosa
2. Associated Disease
• Antibiotic-associated diarrhea and pseudomembrane colitis

Pseudomembrane colitis
Clostridium difficile

3. Cellular morphology
• Thin rods, rare spores
4. Colony Morphology
a. Horse stable, barnyard odor; chartreuse fluorescence
b. Yellow “ground glass” in CCFA

Gram Stain CCFA

Clostridium difficile

5. Other information
a. Confirmed by demonstration of:
i. Toxin B (cyototoxin) in feces by tissue culture
ii. Organism in feces (liquid or unformed)
Clostridium difficile

6. Summary
i. Antibiotic-associated diarrhea and pseudomembrane colitis
a. Horse stable, barnyard odor; chartreuse fluorescence
b. Yellow “ground glass” in CCFA
c. Demostration of Toxin B (cyototoxin) in feces by tissue
culture and organism in feces (liquid or unformed)
 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Porphyromonas spp.
2. Clostridium tetani
3. Prevotella spp.
3. Clostridium botulinum 4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
D. Cocci
1. Peptococcus niger
B. Gram Positive Bacilli
2. Peptostreptoccus spp.
1. Actinomyces spp.
3. Veillonella spp.
2. Propionibacterium spp.
3. Bifidobacterium spp.
 Clostridium septicum
Gram Positive Spore-forming Bacilli

5
Clostridium septicum

1. Associated Disease
a. Associated with malignancies (colorectal cancer)
b. Neutropenic enterocolitis and myonecrosis

Myonecrosis in congenital neutropenia

http://www.gfmer.ch/genetic_diseases_v2/gendis_detail_list.php?cat3=1015
Clostridium septicum

2. Cellular morphology
• Thin rods, subterminal spores
3. Colony morphology
• Resembles “Medusa head”, β-hemolytic, smoothly swarming

Anaerobic Blood Agar

http://en.wikipedia.org/w/index.php?title=File:Clostridium_septicum.tif&page=1
 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Porphyromonas spp.
2. Clostridium botulinum
3. Prevotella spp.
3. Clostridium tetani 4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
B. Gram Positive Bacilli
D. Cocci
1. Actinomyces spp.
1. Peptococcus niger
2. Propionibacterium spp.
2. Peptostreptoccus spp.
3. Bifidobacterium spp.
3. Veillonella spp.
4. Lactobacillus spp.
 Identification of Clostridium Species

Double
Swar- Zone of Chartreuse Lecithi- Spore
Lipase Position
ming β- Fluorescence nase
hemolysis
C. perfringens - + - + - -ST
C. botulinum - - - - + ST
C. tetani + - - - - T
C. difficile - - + - - ST
C. septicum + - - - - ST
 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Porphyromonas spp.
2. Clostridium botulinum 3. Prevotella spp.
3. Clostridium tetani 4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
D. Cocci
1. Peptococcus niger
B. Gram Positive Bacilli 2. Peptostreptoccus spp.
1. Actinomyces spp. 3. Veillonella spp.
2. Propionibacterium spp.
3. Bifidobacterium spp.
Anaerobic
Gram Positive Bacilli

B
1. Actinomyces israelli

1. Associated disease
• Actinomycosis
• Sinus tracts and fistulae, which erupt to the surface and
drain pus. May contain “sulfur granules”
1. Actinomyces israelli

2. Cellular Morphology
• Gram positive, branching filamentous rods
3. Colony Morphology
• White, opaque and resemble “molar tooth”

Gram Stain Colony Morphology

 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Porphyromonas spp.
2. Clostridium botulinum
3. Prevotella spp.
3. Clostridium tetani 4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
D. Cocci
1. Peptococcus niger
B. Gram Positive Bacilli 2. Peptostreptoccus spp.
1. Actinomyces spp. 3. Veillonella spp.
2. Propionibacterium spp.
3. Bifidobacterium spp.
2. Propionibacterium acnes

1. Associated disease
• Associated with inflammatory process in acne
• Actinomycoses, bacteremia, subacute bacterial endocarditis
• Common contaminants of blood culture bottles

acne
2. Propionibacterium acnes

2. Cellular Morphology
• Pleomorphic rods with diptheroid appearance
• “Anaerobic diphtheroids”
3. Colony Morphology
• Small and white to large and yellowish tan

Gram Stain Colonial appearance

 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Porphyromonas spp.
2. Clostridium botulinum
3. Prevotella spp.
3. Clostridium tetani 4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
D. Cocci
1. Peptococcus niger
B. Gram Positive Bacilli 2. Peptostreptoccus spp.
1. Actinomyces spp. 3. Veillonella spp.
2. Propionibacterium spp.
3. Bifidobacterium spp.
3. Bifidobacterium spp.

1. Spectrum of Disease
• In mixed infections of abdomen, pelvis and GUT
• Actinomycoses
2. Colonial Appearance
• Small, white, convex, shiny with irregular edge
3. Bifidobacterium spp.

3. Colony Morphology
• Diptheroid
• End of cells may be spatulated or bifurcated “dog bones”

Gram Stain Scanning EM

Identification of Gram Positive Species

48-hr Rough Branched Catalase/

colony colony Bacilli Indole Comments
<1mm
Molar
A. israelii + + + - Tooth
Colony
Rods w/
Bifidobacterium - - - - forked
ends

P. acnes - - - +
Anaerobic
Gram Negative Bacilli

C
 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Clostridium botulinum 2. Porphyromonas spp.
3. Prevotella spp.
3. Clostridium tetani
4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
D. Cocci
B. Gram Positive Bacilli
1. Peptococcus niger
1. Actinomyces spp.
2. Peptostreptoccus spp.
2. Propionibacterium spp.
3. Veillonella spp.
3. Bifidobacterium spp.
Anaerobic Gram Negative Bacilli

1. Virulence Factor
• Polysaccharide Capsules 
inhibit phagocytosis
• Adherence factors 
adherence to cell surfaces
2. Spectrum of Disease
• Found in mixed infections
• Brain Abscess
• Peritoneal infections
 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Clostridium botulinum 2. Porphyromonas spp.
3. Prevotella spp.
3. Clostridium tetani
4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
D. Cocci
1. Peptococcus niger
B. Gram Positive Bacilli
2. Peptostreptoccus spp.
1. Actinomyces spp.
3. Veillonella spp.
2. Propionibacterium spp.
3. Bifidobacterium spp.
1. Bacteroides fragilis

3. Cellular Morphology
• Coccobacili or pleomorphic
4. Colony Morphology
• Gray and non hemolytic in BAP
• Large (>1mm) gray black colonies on BBE Agar

Gram Stain Bacteroides BBE

1. Bacteroides fragilis

5. Other Characteristics
• Growth in 20% bile (resistant to bile) and
bile esculin hydrolysis positive
• Saccharolytic
• Resistant to kanamycin, vancomycin and colistin
 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Prevotella spp.
2. Clostridium botulinum
3. Porphyromonas spp.
3. Clostridium tetani 4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
B. Gram Positive Bacilli
D. Cocci
1. Actinomyces spp.
1. Peptococcus niger
2. Propionibacterium spp.
2. Peptostreptoccus spp.
3. Bifidobacterium spp.
3. Veillonella spp.
2. Prevotella spp.

3. Cellular morphology
• Tiny coccobacilli
4. Colony morphology
• Fluoresces brick red on blood agar
• Black pigment in KVLB agar.

Ana BAP KVLB

2. Prevotella spp.

5. Other Lab Characteristics

• Ferments glucose and other carbohydrates (saccharolytic)
• Inhibited by 20% bile
• Susceptible to Colisten
 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Prevotella spp.
2. Clostridium botulinum
3. Porphyromonas spp.
3. Clostridium tetani 4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
D. Cocci
B. Gram Positive Bacilli
1. Peptococcus niger
1. Actinomyces spp.
2. Peptostreptoccus spp.
2. Propionibacterium spp.
3. Veillonella spp.
3. Bifidobacterium spp.
3. Porphyromonas spp.

3. Cellular Morphology
• Tiny coccobacilli
4. Colony Morphology
• Fluoresces brick red on blood agar
• No growth on KVLB

Ana BAP
3. Porphyromonas spp.

5. Other Lab Characteristics

• Do not ferment carbohydrates (assaccharolytic)
• Inhibited by 20% bile
a. Do not grow on KVLB due to susceptibility to vancomycin
 Organisms in Focus
A. Gram Positive Spore C. Gram Negative
Forming Bacilli Bacilli
1. Clostridium perfringens 1. Bacteroides fragilis
2. Prevotella spp.
2. Clostridium botulinum
3. Porphyromonas spp.
3. Clostridium tetani 4. Fusobacterium spp.
4. Clostridium difficile
5. Clostridium septicum
D. Cocci
1. Peptococcus niger
B. Gram Positive Bacilli 2. Peptostreptoccus spp.
1. Actinomyces spp. 3. Veillonella spp.
2. Propionibacterium spp.
3. Bifidobacterium spp.
4. Fusobacterium nucleatum

3. Gram stain
• Spindle-shaped with pointed ends
4. Colony Morphology
• Colony types
i. Bread crumblike
ii. Ground glass Gram Stain
• Greening on exposure to air
• Fluoresces chartreuse

Brucella Agar
Identification of Anaerobic Gram Negative Bacilli

Esculin Hydrolysis

Iridescent sheen
Growth on KVLB
Bile Resistance

Fluorescence

fluorescence
Chartreuse
Brick Red
V K Co

B. fragilis R R R + + + - - -

Prevotella R R V - - + + - -

Porphyromonas S R R - - - + - -
 Organisms in Focus

A. Gram Positive Spore C. Gram Negative

Forming Bacilli Bacilli
a. Clostridium perfringens a. Bacteroides fragilis
b. Clostridium tetani b. Porphyromonas spp.
c. Prevotella spp.
c. Clostridium botulinum
d. Fusobacterium spp.
d. Clostridium difficile

D. Cocci
B. Gram Positive Bacilli a. Peptococcus niger
a. Actinomyces spp. b. Peptostreptoccus spp.
b. Propionibacterium spp. c. Veillonella spp.
c. Bifidobacterium spp.
Anaerobic
Cocci

C
Cocci
1. Associated disease
• Infections occur throughout the
body mostly associated with
polymicrobial infection.

Veillonella infection
Cocci
2. Cellular Morphology
• Gram negative diplococci
(or in short
chains)
• Veillonella parvula
• Gram positive cocci
Peptostreptococcus
• Peptococcus niger
and
Peptostreptococcus
(Finegoldia magna)

3. Colony Morphology
• Red fluorescence Veillonella
• Veillonella parvula
 Organisms in Focus

A. Gram Positive Spore C. Gram Negative

Forming Bacilli Bacilli
a. Clostridium perfringens a. Bacteroides fragilis
b. Clostridium tetani b. Porphyromonas spp.
c. Prevotella spp.
c. Clostridium botulinum
d. Fusobacterium spp.
d. Clostridium difficile

D. Cocci
B. Gram Positive Bacilli a. Peptococcus niger
a. Actinomyces spp. b. Peptostreptoccus spp.
b. Propionibacterium spp. c. Veillonella spp.
c. Bifidobacterium spp.
Laboratory Diagnosis
 Examination

Biochemical Testing
 API 20A System
Gas-liquid chromatography
 Metabolic ends products from glucose metabolism
Gas chromatography
 Whole-cell long chain fatty acid methyl ester (FAME) analysis
Antibiotic Identification Disc
 Aids the preliminary grouping of anaerobes
 Kanamycin, 1 mg, Colistin, 10 µg, vancomycin 5 µg
1) Laked KV Blood Agar

For isolation of gram negative anaerobes

(Bacteroides, Prevotella and some Fusobacterium)
 Kanamycin inhibits facultative gram (-) bacilli
 Vancomycin inhibits facultative and obligate gram (+) bacteria
2) Antibiotic disk test

To determine for characteristic susceptability pattern to:

 Colistin (10 µg), Vancomycin (5 µg) and Kanamycin (1 mg)

Resistant:
Zone of inhibition of 10 mm
or less
Susceptible:
Zone greater than 10 mm

Ana BAP
2) Antibiotic disk test

To determine for characteristic susceptability pattern to

 Colistin (10 µg), Vancomycin (5 µg) and kanamycin (1000 µg)
Organism K Co Va
Bacteroidis fragilis R R R
Prevotella R V R
Porphyromonas R R S
Fusobacterium S S R
Viellonella S S R
Peptosteptococcus V R S
Propionibacterium S R S
 Cultivation

• Aerotolerance Test
• The colony is subcultured:
i. Aerobic agar plate
ii. Anaerobic blood agar plate
Colony Aerobe Capnophilic Facultative Obligate
Type Aerobe Anaerobe Anaerobe
Non-CO2 + - + -
CO2 + + + -
Anaerobic - - + +
 Organisms in Focus

A. Gram Positive Spore C. Gram Negative

Forming Bacilli Bacilli
a. Clostridium perfringens a. Bacteroides fragilis
b. Porphyromonas spp.
b. Clostridium tetani
c. Prevotella spp.
c. Clostridium botulinum d. Fusobacterium spp.
d. Clostridium difficile
e. Clostridum septicum
D. Cocci
B. Gram Positive Bacilli
a. Peptococcus niger
a. Actinomyces spp.
b. Peptostreptoccus spp.
b. Propionibacterium spp.
c. Veillonella spp.
c. Bifidobacterium spp.
Thank You 