Clostridium perfringens: Isolation and Identification

Author: Leah Molai

N.dip Biomedical Technology: Clinical pathology

Isolation and Identification of Clostridium perfringens

INTRODUCTION
Scientific classification:
Kingdom: Bacteria
Division: Firmicutes
Class: Clostridia
Order: Clostridiales
Family: Clostridiaceae
Genus: Clostridium
Species: perfringens
Binomial name: Clostridium perfringens

Clostridium perfringens (formerly known as C. welchii) is an anaerobic, large Gram positive spore-
forming bacillus. C. perfringens is ubiquitous in nature and can be found in soil, water and
gastrointestinal tract (GIT) of humans and other vertebrates and insects. Clostridium difficile, C tetani, C.
botulinum, C. septicum, C. sporogenes and C. sordellii are some of the Clostridium species known to
man.
Sources of infection can be either:
Exogenous: injury, contamination of wound by animal faeces OR
Endogenous: from own GIT after disruption of mucous membrane, often due to a mixed infection which
can enhances an anaerobic environment, thereby encouraging the organism to grow.

Infections caused by C. perfringens can lead to:

 Skin/Wound infections: superficial skin infection, anaerobic cellulitis, myonecrosis or gas gangrene
and puerperal infections. A pus swab is required to confirm the cause of infection.
 Septicaemia (can lead to Disseminated Intravascular Coagulation). A Blood culture specimen will be
required to confirm this.
 Self-limiting food poisoning (watery diarrhoea) where enterotoxin was produced in the meat.
Incubation period is 6 to 24 hours after ingestion. If large dose is ingested it can lead to Necrotizing
Enteritis. A Stool specimen is required to confirm the diagnosis.

Pathogenecity:
Many enzymes and 4 major toxins divide C. Perfringens into 5 types ‘A to E’
1. Toxins:
 Alpha toxin/Phospholipase C (Lecithinase):
Lethal toxins present with all Clostridium perfringens types especially with Type A which causes
Myonecrosis. This lyses red blood cells, leucocytes, platelets and endothelial cells increasing vascular
permeability, massive haemolysis, bleeding, tissue destruction, hepatic toxicity and myocardial
dysfunction.
 Beta toxin:
 Present with C type which causes the necrotic lesions in Necrotizing Enterocolitis, from ingestion of e.g.
contaminated pork.
 Epsilon:
A protoxin which is activated by Trypsin, increases the vascular permeability of the gastrointestinal
wall.
 Iota:
Necrotic activity and increase in vascular permeability
 Enterotoxin (Type 2):
Food poising- not lethal as the others are. Disrupts ion transport in the ileum and jejunum.
2. Enzymes: help the organisms survive and spread throughout the body.
 Haemolysins
 Proteases
 Collagenases
3. Spore forming- Survives in adverse circumstances.
4. Tissue destruction- Destructs tissue- gas from carbohydrate fermentation compresses blood vessels
making them even more anaerobic.
5. Capsule- Evades defence mechanisms.

ISOLATION AND IDENTIFICATION

Scenario: A wound swab is received in a Microbiology laboratory. PRAS (Pre-Reduced Anaerobic
Sterilized) selective medium was used for transport.

Gram stain:
 Gram positive bacilli, large boxcar shaped, single
 Oval/sub-terminal spores that do not bulge the cell and are not visible on gram stain
 C.perfringens tends to looks similar to Bacillus species which are aerobe but also Gram positive bacilli,
large boxcar shaped in bamboo chains with central, oval spores which are visible as unstained areas
which do not swell the cell.

Culture:
The swab is inoculated on Pus plates: Anaerobic blood agar, Blood agar, MacConkey, Chocolate agar and
Thioglycolate broth. The anaerobic blood is incubated anaerobically while the other plates are incubated
capnophilically at 37oC for 24 to 48 hours.
Other media that can be used include: PEA agar (Phenyl Ethyl Alcohol agar), CNA (Colistin Nalidixic Acid)
and Anaerobic Blood agar are selective for gram positive organisms by inhibiting aerobic and facultative
anerobic gram negatives.

There will be growth only on the anaerobically incubated blood and Thioglycolate broth, an enriched
medium used to enhance the growth of anaerobes while incubating aerobically. Growth will be at the
bottom of the tube where the conditions are anaerobic. There will be no growth on the other plates.

Colony morphology on Sheep Blood agar:

 Large, smooth, regular, convex, slightly opaque, flat radially striated transparent border
 Can be rough flat vine leaf colony
 Typical Double zone of haemolysis:
-Zone of complete haemolysis due to Beta haemolytic Theta toxin (one of the minor toxins)
-Wider zone of partial haemolysis caused by Alpha haemolytic Alpha toxin (one of the major toxins)
 Mucoid (due to capsule)

Tests for presumptive identification of Clostridium perfringens

a) Catalase test:
Differentiates between: Aerobic Bacillus species =Positive
Aero-tolerant Anaerobe C. perfringens = Negative

b) SIM media:
Sulphide production: Positive
Indole production: Negative
Motility: Non motile (other Clostridia which are motile)

Confirmatory tests:
a) Litmus milk:
 Proteolytic Peptonization of casein: Negative
 Digestion of casein: Negative
 Coagulation of casein to clot: Positive
 Lactose fermentation: Positive
 Litmus indicator reduced: Positive
 Gas from lactose fermentation: Positive

b) Toxin production:
Toxin detection tests play an important role in differentiating gram positive bacilli as many of them
produce toxins.
Lecithinase and Nagler reaction positive due to Alpha toxin production and Lipase= Negative as they are
unable to produce the enzyme lipase which hydrolyses lipids into fatty acids and glycerol.

c) Reverse CAMP (Christie Atkins Munch and Petersen):

Reverse CAMP Positive (reverse camp positive organisms do not possess camp factor, but their
haemolysis is enhanced by camp factor positive organisms like Streptococcus agalactiae)

d) Gelatine Hydrolysis:
Gelatinase Positive

e) Robertson’s Cooked Meat Media:

Saccharolytic: Positive (Fermentation of Carbohydrates with acid and gas production)
Proteolytic: Negative (Unable to decompose proteins)

f) Identification by Antimicrobial disc:

Metronidazole:
All Anaerobes except Propionibacterium are susceptible to Metronidazole.
Other antibiotics:
Clostridium perfringens has a typical, constant result with the following discs:
Kannamycin (1000 µm) Susceptible
Vancomycin (5 µm) Susceptible
Colistin (10 µm) Resistant
 Other Methods employed for ID of Anaerobes:
a) Quad Plates:
Indicate 4 biochemical reactions:
1. Milk(Casein/Proteolysis): Negative
2. Glucose fermentation: Positive
3. DNAse hydrolysis: Positive
4. Starch Hydrolysis: Positive

b) API 20A:
A profile of 20 Biochemical reactions selected specifically for Anaerobes. Numerical values are assigned
according to the reactions that take place with a catalase test performed separately and also assigned a
value. The digit profile number will identify the organism using the API (Analytical Profile Index).

REFERENCES:

www.merck.com/mmhe/sec09/ch122/ch122d.html last accessed 30/07/2010

www.textbookofbacteriology.net/clostridia.html last accessed 30/07/2010
www.wikipedia.org/wiki/Clostridium_perfringens last accessed 30/07/2010
Anita Markotter - Microbiology Lecture notes (2010)