Clostridium on Blood Agar: Introduction, Pathogenecity, Laboratory Diagnosis and Treatment

Clostridium on blood agar

Introduction of Clostridium

Clostridium species on blood agar as shown above picture. The name Clostridium is derived from the word ‘Kloster’ meaning a spindle. Most species of this genus, Clostridium are saprophytes found in soil, water, and decomposing plant and animal matter whereas Cl. tetani and Cl. perfringens are commensals of the intestinal tract of humans and animals.#

Clostridia are  Gram-positive rods, motile ( except Cl. perfringens and Cl. tetani type VI),  non-capsulated ( except Cl. perfringens and Cl. butyricum), obligate anaerobes capable of producing spores that protect them in a harmful environment. Individual cells are rod shaped. The spores are usually wider than the rods and are located terminally or sub terminally. Most clostridia are motile by peritrichous flagella while others have capsule-like Clostridium perfringens characteristics. Clostridium consists of around 100 species that include common free-living bacteria as well as important pathogens There are five main species responsible for disease in humans. Cl. perfringens: gas gangrene; food poisoning C. tetani: tetanus, C. botulinum: botulism C. difficile: pseudomembranous colitis, and  Cl. sordellii : can cause a fatal infection in exceptionally rare cases after medical abortions.

Clostridia are Gram-positive, spore-forming rods, usually having sizes of 3-8 X 0.4-1.2 µm. They are highly pleomorphic. The shape and position of spores vary in different species and are useful for the identification of Clostridia. The spores may be at central in Cl. bifermentans , sub-terminal in Cl. perfringens , Oval and terminal in Cl. tertium, and spherical and terminal giving drum stick appearance in Cl. tetani.

Clostridium perfringens (Clostridium welchii)

Cl. perfringens is a relatively large Gram-positive short fat bacilli with blunt ends. It is encapsulated and non-motile. Anaerobic. It grows quickly on laboratory media on blood agar ( beta-hemolytic ) and has the following virulence factors – toxins, alpha-toxin – causes RBC rupture, edema, and tissue destruction, enterotoxin,  collagenase, and hyaluronidase.

Clostridium tetani

The anaerobic bacterium of the genus species Clostridium and is gram positive, slender bacillus and it has spherical terminal spores giving a drum stick appearance It is non encapsulated and motile with peritrichous flagella. It produces a potent biological toxin, tetanospasmin, and is the causative agent of tetanus a disease characterized by painful muscle spasms that can lead to respiratory failure and, in up to 40% of cases, death.

Clostridium botulinum

The worm botulism means sausage as the poorly cooked sausage was formerly associated with this type of food poisoning. This organism causes a severe form of food poisoning. It is a gram-positive rod, non-encapsulated, motile with peritrichate flagella, and produces sub-terminal, oval, bulging spores.

Clostridium difficle

It was first isolated from the feces of newborn infants and so named due to unusual difficulties involved in the isolation of this pathogen. It is a long, slender, Gram-positive rod containing oval and terminal spores.

Pathogenicity of Clostridium

It depends on the involvement of species of the genus, Clostridium and they are as follow-

  1. Cl. perfringenes: Gas Gangrene, food poisoning, necrotizing enteritis, gangrenous appendicitis, urogenital infections, brain abscess, meningitis, panophthalmitis, and puerperal infection.
  2. Clostridium tetani: Tetanus
  3. Clostridium botulinum: Foodborne botulism, infant botulism and wound botulism
  4. Clsotridium difficle: Antibiotic-associated colitis

Laboratory Diagnosis of Clostridium

The diagnosis should always be made clinically and laboratory tests are done to confirm it. Laboratory diagnosis may be made by demonstrating bacilli by microscopy, culture, or by animal inoculation. Specimens generally collected are wound swabs, exudate, or tissue from the wound.

1. Microscopy

Gram stain is a very useful tool for screening the genus and helpful for presumptive identifications of Clostridium species.

Requirements for Gram stain

a) Compound light microscope

b) Reagents and glasswares

  • Bunsen flame
  • Wire loop
  • Clean grease-free slides
  • Marker pen
  • Crystal violet (Basic dye)
  • Gram’s iodine(mordant)
  • 95% ethanol (decolorizing agent)
  • 1% safranin or dilute carbol fuchsin or neutral red

c) Quality control strains

Positive Control (PC) : Staphylococcus aureus (ATCC 25923)

Negative Control (NC): Escherichia coli (ATCC 25922)

d) Specimen (  Note:-5% sheep blood agar having growth of Clostridium  species used and growth of this organism in thioglycollate broth or Robertson’s cooked meat medium can also be used.)

Preparation of bacterial smear: from liquid culture

  • Take a clean, and grease-free slide for making a smear.
  • Take one or two loopful of the bacterial cell suspension and place them on the slide with a bacteriological loop.
  • Then with a circular movement of the loop, spread the cell suspension into a thin area.
  • Allow the smear to air dry.
  • Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.

Preparation of bacterial smear: from the solid medium

  • Take a clean, and grease-free slide for making a smear.
  • Take a loopful of 0.85% saline i. e. physiological saline and place it on the center of the slide.
  • With a straight wire touch the surface of a well-isolated colony from the solid media and emulsify in the saline drop forming a thin film.
  • Allow the smear to air dry.
  • Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.

Procedure

  1. Cover the smear with crystal violet and allow it to stand for one minute.
  2. Rinse the smear gently under tap water.
  3. Cover the smear with Gram’s iodine and allow it to stand for one minute.
  4. Rinse smear again gently under tap water.
  5. Decolorize the smear with 95% alcohol.
  6. Rinse the smear again gently under tap water.
  7. Cover the smear again gently with safranin for one minute.
  8. Rinse the smear again gently under tap water and air dry it.
  9. Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.

Observation

Positive Control:   violet color, round in shape in single, pairs and cluster

Test: red color and rod in shape

Negative Control: red in color and rod in shape

Result and Interpretation of Gram Stain

Gram-positive: purple or violet color

Gram-negative: Pink or red in color

Cocci: round in shape

Bacilli: rod in shape

Positive Control(PC): Gram-positive cocci in single, pairs and cluster

Test: Gram-positive bacilli or rods and also with evidence of spores as shown above picture

Negative Control(NC): Gram-negative bacilli

2. Culture

Liquid media for cultivation of anaerobes are –

  • Thiglyclolate broth and
  • Robertson’ cooked meat medium

Solid medium for cultivation of anaerobes are-

  • Blood agar
  • Cycloserine-cefoxitin-fructose agar (CCFA) for isolation of Clostridium difficile from stool
  • Selective media such as kanamycin-vancomycin (VK) blood agar
  • Obligate anaerobes can not grow in the presence of oxygen i.e. air. These organisms die rapidly on exposure to air, therefore for maintaining anaerobiosis various methods have been devised for anaerobic culture.

Principle

Mechanism of anaerobiosis achieves by following methods-

Displacement of oxygen

Absorption of oxygen

Combustion of oxygen or their combinations

Deep nutrient agar tube is the simplest method. Anaerobes grow in the depth of the medium, and the number of colonies becomes fewer towards the surface. Strict anaerobes will not grow within a centimeter of the surface.

Using reducing agents like glucose, ascorbic acid, cysteine, thioglycollate in the medium. Cooked meat particle also acts as s good reducing agent due to having glutathione e.g. Robertson’s cooked meat medium.

Another simple method of anaerobiosis in the application of a candle jar. A burning candle can not continue due to the exhaustion of oxygen inside the jar.

In laboratories, combustion involves the combining of oxygen with hydrogen to form water in the presence of a catalyst like palladium or palladinized asbestos. Anaerobic jars are a constant feature of anaerobic culture. They include the McIntosh and Fildes anaerobic jar, which has ilets to admit hydrogen and carbon dioxide, a vacuum pump for evacuating oxygen, and a catalyst fitted into the lid.

Similarly, a simpler but more expensive technique is the Gaspak system. This utilizes a transparent polycarbonate jar with a lid bearing a screened catalyst chamber. The catalyst, consisting of pellets of sodium borohydride, cobalt chloride, citric acid, and sodium bicarbonate is contained in the sachet. Water is added to the sachet and it is immediately placed in the jar, which is then sealed tightly. The resulting reaction liberates hydrogen and carbon dioxide. An indicator also adds to demonstrate anaerobiosis.

Requirements

  • Anaerobic culture system
  • McIntosh and Fildes anaerobic jar
  • Gaspak system
  • Robertson’s cooked meat medium/ Thioglycollate  broth
  • Blood agar plates
  • Control strains
  • Clostridium sporogenes (anaerobes)- a  48 hours thioglycollate broth culture
  • Pseudomonas aeruginosa (obligate aerobes)- a  48 hours thioglycollate broth culture

Procedure

  1. Inoculate the specimen in Robertson’s cooked meat medium and incubate for 48 hours.
  2. Divides each blood agar plate into two equal parts.
  3. Inoculate a loopful of each control organism into half-blood agar.
  4. Similarly, also inoculate test specimen of 48 hours incubated Robertson’s cooked meat medium into a half-blood agar plate.
  5. Stack the plates into the anaerobic jar, introduce the catalyst and quickly seal the lid.
  6. Incubate the plates at 37°C for 48 hours.
  7. Incubate one plate of control strains aerobically.
  8. Remove the plates and examine for growth.

Quality control

Pseudomonas aeruginosa does not grow anaerobically.

Clostridium sporogenes do not grow aerobically.

Observations

When anaerobiosis is complete, obligate anaerobes like Clostridium sporogenes will grow, while obligate aerobes like Pseudomonas aeruginosa will not grow.

Result and Interpretation

  • P. aeruginosa will not grow on the blood agar plate incubated anaerobically, while Cl. sporogenes will grow on the blood agar plate. P. aeruginosa is a strict aerobe that can not grow in the absence of oxygen.
  • P. aeruginosa will show growth on the aerobically incubated while Cl. sporogenes will not grow because Cl. sporogenes is a strict anaerobe that can not grow if oxygen is present.

3. Special Biochemical Tests

Test for Cl. perfringens

  • Litmus milk test( stormy fermentation): Positive
  • Nagler Reaction ( lecithinase  test) : Positive
  • Reverse CAMP test: Positive

4. Other Tests

  1. Animal pathogenecity- for Cl. perfringens
  2. Toxigenecity test: for Cl. tetani
  3. Demonstration of toxins- for Clostridium botulinum and  Clsotridium difficle

Prophylaxis

The commonly available methods are-

  • surgical
  • Antibiotics and
  • Immunization

Treatment of Clostridium

It also depends on the involvement of the organism and the type of infection. Antibiotics useful for the treatment of gas gangrene are metronidazole, penicillin, sulphonamides, tetracycline, and amoxicillin. Tetanus treatment consists of controlling spasms, maintaining the airway by tracheotomy, and attention to feeding. The antitoxin may be used to neutralize the unbound toxins. Antimicrobial therapy with penicillin or metronidazole should be started and continued for a week or more. Similarly, for the treatment of Cl. difficle, metronidazole is the choice of drug.

Further Readings

  1. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  2. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  3. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
  4. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
  5. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  6.  Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
  7.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  8. Topley & Wilsons’  Principle of Bacteriology, Virology, and immunology. Editors: M.T. Parker & L.H. Collier, 8th ed 1990, Publisher Edward Arnold publication, London.
  9. Medical Microbiology-The Practice of Medical Microbiology Vol-2-12th Edn. –Robert Cruickshank
  10. District Laboratory Practice in  Tropical Countries  –  Part-2-   Monica Cheesebrough-   2nd Edn Update
  11. https://www.encyclopedia.com/medicine/encyclopedias-almanacs-transcripts-and-maps/anaerobic-bacteria-culture-0e
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