LESSON 18.

ANAEROBIC BACTERIA
Foul odour upon Indicators of Anaerobic Bacteria
opening an anaerobic
jar of bag

Presence of sulfur
granules

Brick-Red
Fluorescence

Absence of Superoxide
Dismutase (SOD)

Growth on Anaerobic
Culture Plate

Double Zone of
Hemolysis incubated
anaerobically

Clostridium difficile An indicator of Anaerobic Bacteria with Foul odour upon

Fusobacterium opening an anaerobic jar of bag
Porphyromonas

Actinomyces An indicator of Anaerobic Bacteria with the presence of sulfur

Cutibacterium granules
Eubacterium nodatum

Prevotella / An indicator of Anaerobic Bacteria with Brick-Red

Porphyromonas Fluorescence

ENDOGENOUS ANAEROBES COMMON IN HUMAN INFECTIONS

ANAEROBE INFECTION

Actinomyces spp. Actinomycosis

Clostridium difficile Antibiotic-associated diarrhoea;

Pseudomembranous Enterocolitis

Bacteroides fragilis Bacteremia

Fusobacterium
Clostridium
Peptostreptococcus

Bacteroides Brain Abscesses

Prevotella
Porphyromonas
Fusobacterium
Clostridium spp.

Peptostreptococcus Female Urogenitary Tract Infection

Bacteroides
Clostridium
Prevotella bivia
Actinomyces israelii

Bacteroides Intra-abdominal Infections

Fusobacterium Liver Abscess
Clostridium perfringens Peritonitis
Peptostreptococcus Perineal and Perirectal Infections

Clostridium perfringens Myonecrosis

Clostridium novyi
Clostridium septicum

Peptostreptococcus Oral, Sinus, Dental Infections

Porphyromonas
Fusobacterium

Porphyromonas Aspiration Pneumonia and

Fusobacterium nucleatum Pleuropulmonary Infections
Peptostreptococcus
Bacteroides fragilis
Actinomyces

Clostridium spp.
Clostridium spp. Obligate Aerobes;
Gram (+) Spore-forming rod;
Catalase (+)

● Collagenase Virulence of Clostridium spp.

● Hyaluronidase
● Lecithinase (Cell
Destruction)
● Phospholipase

Clostridium perfringens Histotoxic Clostridia (Myonecrosis)

Clostridium novyi
Clostridium histolyticum
Clostridium septicum
Clostridium bifermentans

Clostridium septicum Marker for malignancy in the GI Tract

Clostridium septicum "Smooth-swarming" on plated media

Clostridium sordellii Only Urease (+) Clostridia

Clostridium spp. Form endospores anaerobically

Clostridium perfringens All Clostridium spp. are swollen Sporangia, except

Clostridium bifermentans

Clostridium perfringens All Clostridium spp. are non-capsulated, except

Clostridium perfringens All Clostridium spp. have Single Hemolytic Reaction, except
Clostridium perfringens All Clostridium spp. have Peritrichous flagella, except
Clostridium ranosum
Clostridium innocum

Clostridium tetani All Clostridium spp. are CHO Fermenters, except

Clostridium histolyticum

Clostridium noyvi This Clostridium spp. is killed immediately in Oxygen

Presence

Clostridium perfringens
Gas Gangrene Bacillus Common name of Clostridium perfringens

Clostridium welchii Former name of Clostridium perfringens

Clostridium perfringens This Clostridium spp. is the most commonly isolated in

blood cultures

Alpha Toxin Virulence Factor of Clostridium perfringens

Enterotoxin A & C

Endospores central to Clostridium perfringens under microscope

subterminal

"Boxcar-shaped" bacilli Clostridium perfringens under microscope

BAP: Culture of Clostridium perfringens

Dome-shaped
Gray to white colonies
Double Zone of
Hemolysis (Alpha &
Beta Zones)

Litmus Milk: Culture of Clostridium perfringens

"Stormy Fermentation
of Milk"

Positive (+) for: Biochemical Tests of Clostridium perfringens

Lecithinase (Egg Yolk
Agar)
Nagler Test
(Lecithovitalin Reaction
on EYA)
Reverse CAMP Test

Clostridium perfringens Very fermentative, non motile

CLINICAL INFECTIONS of Clostridium perfringens

GAS GANGRENE / ● Life threatening destruction of
MYONECROSIS ("EATING SORE") muscles and other tissues
● Necrotizing Infection of Skeletal
 Muscles
● Causes: Alpha Toxin
● Entry via wounds, trauma, frostbite,
surgery
● Accompanied by bullae (fluid-filled
blisters), pain, swelling, serous
discharge, discolouration, tissue
necrosis
● TREATMENT: Hyperbaric Oxygen
Therapy

FOOD POISONING / ● Ingestion of Enterotoxin in

ENTERITIS NECROTANS ("PIG-BELL") contaminated food (improper
storage)
● Symptoms: Foul-smelling stool
(Diarrhoea), Crampy abdominal
feeling
● Types of Food Poisoning
○ TYPE A - mild, self-limiting;
with diarrhoea and cramping
pain
○ TYPE B - Serious, rarely
encountered; bloody
diarrhoea and cramping pain

Clostridium tetani
Tack Head Bacillus Common name of Clostridium tetani

Terminal Spore and Clostridium tetani under the microscope

Swollen Sporangia

"Drumstick / Lollipop / Clostridium tetani under the microscope

Tennis Racket"
appearance

BAP: Culture of Clostridium tetani

Heavy "smooth
swarming" anaerobic;
grow slowly

Clostridium tetani In Culture with BAP, Colonies with matte surface with
narrow zone of B-Hemolysis

Gelatinase & Indole (+) Biochemical Tests of Clostridium tetani

Lecithinase & Lipase (-)

Neurotoxin Virulence Factor of Clostridium tetani

TETANUS Trismus ("Lock Jaw")

Risus Sardonicus ("Distorted Grin")
Ophistonos ("Muscle Spasms")

Tetanus Neonatorum Contaminated instruments used for newborns

Clostridium botulinum
Canned Good Bacillus Common name of Clostridium botulinum

Subterminal Spore Clostridium botulinum have

B-hemolytic (BAP) Clostridium botulinum have

Botulism Toxin Clostridium botulinum virulence factor, and most potent toxin
known to man

Toxin Type A Botox Treatment

Used to treat Strabismus (Wandering Eyes)

Botulism Double or Blurred Vision

Impaired Speech
Difficult in Swallowing
Weakness
Paralysis

Foodborne Botulism Ingestion of Preformed Toxin

Caused by Botulism Toxin Type A

Infant Botulism Actual infection caused by breastfeeding

Clostridium difficile
Most common cause: Antibiotic-associated diarrhoea and
Pseudomembranous Enterocolitis

Glutamate Produced by Clostridium difficile that is mportant for the toxin

Dehydrogenase determination of Clostridium difficile

Enterotoxin A Virulence Factor of Clostridium difficile

Cytotoxin B

Fermenters Fructose Pink-coloured medium turns yellow

forming Formic Acid
(CCFA)

BAP Culture of Clostridium difficile that has "Horse stable" or

Barnyard" odour

CCFA Culture of Clostridium difficile with Yellow, Ground Glass

Colonies

COLLECTION, TRANSPORT, STORAGE OF ANAEROBIC BACTERIA

Room Temperature Storage Temperature

Transported at 4°C Food and Faeces (Clostridium difficile)

Growth of other Improper collection

Anaerobes
Actual site of infection, Specimen is collected from
NOT swab of mucosal
surface

Aspiration and Biopsy is When to use a swab?

NOT POSSIBLE

0.5ml sterile Where to place a swab?

Thioglycolate broth

Transport of specimen Crucial factor in final success of anaerobic cultures

● Swab Unacceptable Specimens for Anaerobic Bacteria

● Sputum
● Bronchial Wash
● Faeces,
Ileostomy,
Colostomy
Effluents
● Gastric & Small
Bowel Contents

LABORATORY DIAGNOSIS
● Thioglycollate Media used
● Cycloserine
Cefoxitin
Fructose Agar
(CCFA)

● Pre-reduced Transport Media

Anaerobically
Sterilised
(PRAS)
● Amies

● Peroxides Growth Inhibitors

● Dehydration

Mercury What to use to remove air from a sealed jar?

BRU/BAP Swarming & double zone of hemolysis

Brucella Blood Agar /
Blood Agar Plate

EYA Detects the activity of Lipase and Lecithin (+ insoluble opaque

Egg Yolk Agar whitish precipitate within the agar)

PYG Detects volatile fatty acids

Peptone Yeast Glucose

CCFA Selective and Differential Medium

Cycloserine Cefoxitin
Fructose Agar
Cycloserine, Cefoxitin Inhibits Gram Negative Coliforms

Neutra Red pH indicator of CCFA

(Yellow in presence of
Acid pH)

Clostridium septicum "Smooth swarming" growth on plated media

Clostridium tertium Grow minimally on the aerotolerance plate, mistaken for a

facultative anaerobe

CULTURE MEDIA
NON-SELECTIVE CULTURE MEDIUM SELECTIVE CULTURE MEDIUM

Cooked Meat Anaerobic Organisms; Bacteroides Selective and Differential for

Broth with glucose can be used Bile Esculin Bacteroides fragilis
for Gas-Liquid Agar (BBE)
Chromatography

Anaerobic Anaerobes and Laked Prevotella, Bacteroides spp.

Blood Agar Facultative Anaerobes Kanamycin-
Vancomycin
Blood Agar
(LKVB)

EYA Lecithinase and Lipase Anaerobic Selective for inhibition of

PEA G-rods and swarming by
some Clostridium spp.

Peptone-Yeast Gas-Liquid Cycloserine Selective for Clostridioides

Extract Chromatography Cefoxitin difficile
Glucose Broth Fructose
Agar (CCFA)

Thioglycolate Anaerobes, Facultative

Anaerobes, Aerobes

LABORATORY DIAGNOSIS
Catalase Test Differentiate Clostridium (-) from Bacillus (+)

15% H2O2 Reagent of Catalase Test

Bubble Formation Positive Result of Catalase Test

Egg Yolk Agar Medium for Lipase and Lecithinase Test

Positive for Lipase Colony: "Mother of Pearl" or Gasoline on water appearance

Clostridium botulinum Organisms Lipase (+)

Clostridium novyi Type A
Clostridium sporogenes

Positive for Lecithinase Opaque Zone around colonies

Clostridium Organisms Lecithinase (+)
perfringens
Clostridium bifermentans
Clostridium sordellii
Clostridium novyi Type A

OTHER LABORATORY TESTS

Direct Nagler Test (+) yellowish egg colour

Mouse Neutralisation Definitive identification test for Clostridium botulinum

Test

Reverse CAMP Test Confirmatory Test for Clostridium perfringens

(+) reverse arrowhead

ANAEROBIC NON-SPORE FORMING BACTERIA (Culture:Anaerobic Blood Agar Plate)

ORGANISM GS REACTION DISTINGUISHED BY

Actinomyces spp. G (+) Anaerobic Young Colonies:

Actinomyces, Straight or
Slightly Curved Filamentous
Rods, may be beaded ot
banded
"Spider-like" or Wooly
Appearance
Old Colonies: "Molar Tooth"
(Actinomyces israelii)

Bacteroides fragilis G (-), Pale-staining, Gray-white, circular, smooth,

Pleomorphic rods; "Safety non-hemolytic
pin" appearance

Bacteroides ureolyticus G (-), Pale-staining, thin Colonies corrode (pit) the

rods; some curved agar

Bifidobacterium spp. Gram (+) Diphtheroid; Small, white, shiny, convex

Coccoid or Pointed shape; colonies
Bifurcated (forked) ends;
resemble "Dogs Bones"

Clostridium septicum G (+) rod (Young cultures) Rhizoid Margins resembling

or "Medusa Head"
G (-) rod (Old cultures);
Subterminal spores

Cutibacterium spp. G (+) Diphtheroid-like;

(Former: Propionibacterium) Palisade arrangement

Eubacterium G (+) Pleomorphic rod; Chartreuse colour

Seagull-wing shaped fluorescence

Fusobacterium nucleatum G (-) spindle-shaped rods; Green-coloured medium

subsp. nucleatum resemble Capnocytophaga upon air exposure;
"Breadcrumbs-like" colonies
Lactobacillus spp. Gram V rods; short
coccobacilli; resemble
Streptococci

Leptotrichia spp. G (-) large fusiform rods "Raspberry-like" colonies

Peptococcus niger G (+) cocci; Single, Pairs,

Tetrads

Peptostreptococcus G (+) Large coccobacillus in

anaerobius chains

Porphyromonas spp. G (-) coccobacilli

Prevotella spp. G (-) rods Brick-Red Fluorescence

Veillonella parvula G (-) tiny diplococci Red Fluorescence

ACTINOBACTERIA
1. Actinomyces
2. Bifidobacterium
3. Cutibacterium
4. Eggerthella
5. Mobiluncus

Bacteroides fragilis Significant cause of intra-abdominal abscesses

Microscopy: Pleomorphic with vacuoles; Encapsulated

Actinomyces israelii Diagnosis: Sulfur Granules

Cutibacterium acnes Contamination from patient's skin from poor site

preparation during phlebotomy

Lactobacillus acidophilus Important in Female Genitalia - protect from urogenital

infections
Can grow at pH 3-4 (Tomato Juice Agar)

Lactobacillus rhamnosus Most often associated with septicemia and endocarditis

Prevotella melanogenica gingival crevices

Porphyromonas gingival and peri-apical tooth

Fusobacterium Lemierre's Disease

necrophorum

Fusobacterium nucleatum Thin rods with tapered ends ➡ needle-shaped

morphology

Veilonella

Gardnella vaginalis Bacterial Vaginosis

Gardnella vaginalis Wet smears ➡ clue cells
"Fishy odour" vaginal discharge
Most common cause of Bacterial Vaginosis

Finegoldia magna Most pathogenic anaerobe cocci

Peptostreptococcus Re-Classified into Anaerococcus, Finegoldia, and

Peptoniphilus

IDENTIFICATION OF ANAEROBIC NON-SPORE FORMING BACTERIA

● Outnumbering aerobes 1000:1
● Inoculation of organisms start with CAP followed by BAP
● Selective Medium (Anaerobes): Laked Kanamycin-Vancomycin Agar
● Susceptible (S) : > 10 mm Zone of Inhibition
● Resistant (R) : < 10 mm Zone of Inhibition

Kanamycin Vancomycin Colistin

Bacteroides fragilis R R R

Bacteroides S R S
ureolyticus

Fusobacterium S R S

Porphyromonas R S S

Veilonella S R S

TREATMENT DESCRIPTION

Surgical Drainage + ● No cytochrome systems for oxygen metabolism

Antimicrobial Therapy ● Lack SOD and Catalase

B-Lactamase Producers ● Bacteroides and Prevotella

● B-Lactam - B-Lactamase inhibitor combinations
(Ampicillin-Sulbactam)

Penicillin G ● DOC for non-beta lactamase producing organisms

Most active drugs for ● Clindamycin and metronidazole

treatment of anaerobic ● Clindamycin: preferred for infections above the waist
infections

Alternative Drugs (2nd ● Cephalosporins - Cefoxitin, cefotetan

Line) ● Piperacillin
● Carbapenem antibiotics - Ertapenem, Imipenem,
Meropenem, Doripenem
● Tigecycline - Skin, Soft tissues, Intra-abdominal
infections