Cultural, Biochemical characteristics, Epidemiology, Pathogenesis, Diagnosis and Control

1. Cultural, Biochemical characteristics,
Epidemiology, Pathogenesis, Diagnosis
And Control
Presented By:
Sangram P. Ramane
PhD Scholar
Roll no. 1447
DIVISION OF VETERINARY BACTERIOLOGY AND MYCOLOGY, IVRI
Assignment
On
Anaerobic Bacteria (VBM 705)

2. CONTENTS
 Introduction
 History
 Morphology and Cultural Characteristics
 Biochemical Characteristics
 Epidemiology
 Toxins Produced
 Pathogenesis, Lesions and Symptoms
 Diagnosis
 Treatment and Control

3. INTRODUCTION
 Not considered as great pathogen of animal
but is a secondary invader.
 Ability of organism to produce extensive
liquefactive necrosis in tissue is its
outstanding characteristic.
 Collagenase produced have medicinal value
and in past Proteinases produced by these
used experimentally for the debridement of
burns.
Clostridium histolyticum
Scientific classification
Kingdom: Bacteria
Division: Firmicutes
Class: Clostridia
Family: Clostridiaceae
Genus: Clostridium
Species: Cl. histolyticum
Binomial name
Clostridium histolyticum
(Weinberg and Séguin 1916)
Bergey, Harrison, Breed,
Hammer, and Huntoon 1923

4. Placement in Biochemical Classification
Clostridia
Both proteolytic
and saccharolytic
Proteolytic
predominantly
Saccharolytic
predominantly
Slightly
proteolytic but
not saccharolytic
Saccharolytic but
not proteolytic
Neither
proteolytic nor
saccharolytic
Cl. bifermentans
Cl. botulinum A,B,F
Cl. histolyticum
Cl. sordelli
Cl. sporogenes
Cl. perfringens
Cl. septicum
Cl. chauvoei
Cl. novyi
Cl. difficile
Cl. fallax
Cl. botulinum C,D,E
Cl. tertium
Cl. tetanomorphum
Cl. sphenoides
Cl. cochlearumCl. tetani
Introduction cont…

5. Placement in Morphological Classification Depending on
Position of Spore
Clostridia
Central and Sub
terminal
Oval and Terminal Spherical and Terminal
Cl. bifermentans, Cl. botulinum
Cl. histolyticum
Cl. sordelli, Cl. sporogenes
Cl. perfringens, Cl. septicum
Cl. chauvoei, Cl. novyi, Cl. fallax
Cl. difficile
Cl. tertium
Cl. cochlearum
Cl. tetani
Cl. tetanomorphum
Cl. sphenoides
Introduction cont…

6. Placement in Classification Depending on Pathogenecity
Clostridia
Gas Gangrene
Group
Food Poisoning
Group
Tetanus Group Acute Colitis
Cl. tetani Cl. difficileCl. perfringens,
Cl. botulinum
Cl. Perfringens
Cl. Septicum
Cl. novyi
Cl. histolyticum
Cl. fallax
Cl. bifermentans
Cl. sporogenes
Doubtful
Pathogenic
Less PathogenicEstablished
Pathogens
Introduction cont…

7. HISTORY
 First time described in 1916 by Weinberg and Seguin from war wounds
and found to be the associated with cases of gas gangrene.
 After isolating the bacteria was originally classified as a member of
the genus Bacillus by Weinberg and Seguin.
 Heller in 1922 renamed the species Weinbergillus histolyticus.
 Bergey et al in 1923 reclassified as a Clostridium species (Clostridium
histolyticum).

8. MORPHOLOGY & CULTURAL CHARACTERISTICS
 Gram positive bacillus measuring 5-8 x 0.5-0.7 µm
 Tend to occur in pairs.
 Readily sporulates in media producing large oval, sub terminal spores.
 Richly flagellate (peritrichous flagella), vigorously motile in young cultures
and in effusions
 Aero-tolerant (not strict anaerobes) and most strains will grow on blood
agar plates incubated in an atmosphere of air with 5-10% added CO2.
 Smooth and rough colonies. Rough have flat edges with rhizoids.

9. BIOCHEMICAL CHARACTERISTICS
 On Egg Yolk Agar: Negative for Lipase and Lecithinase activity
 Gelatin Hydrolysis: Positive, Urease, Nitrate and Indole: Negative,
Hemolytic for sheep RBCs
 Fermentation Pattern: By Hoogerheide (1987)
Pathogenic strains - Non saccharolytic
Non Pathogenis strains - Saccharolytic (Resembling Cl. Sporogenes)
Principle fermentation product - Acetic Acid
 In Milk: Variable reaction. Digests native and denatured proteins to
amino-acids with production of ammonia, that clots milk and later
digests the clot (Blanc & Pozerski, 1920)

10. Cl. histolyticum
on Egg Yolk agar
Gram staining of
Cl. histolyticum

11. EPIDEMIOLOGY
 Occasionally associated with gas gangrene in human and horses.
 Found to be pathogenic for small laboratory animals (guinea-pigs, mice and
rabbits), less so for rats.
 Clostridial gas gangrene:
Cl. perfringens most common cause (80-90% of cases),
Cl. novyi (40%),
Cl. septicum (20%),
Cl. histolyticum (10%),
Cl. bifermentans (10%),
Cl. fallax (5%)
 Invading organism and not found to be toxic on feeding.

12.  Thrives in feces and soil.
 Had been isolated from the soil of reindeer grazing areas (Polyakov, 1981).
Also from bone meal and gelatin.
 Cl. histolyticum spores are dormant in normal healthy equine skeletal
muscle and specific events must occur to trigger specific cascade of
pathological events (Vengust et al, 2003).
 Frank and Scott have reported two cases of Cl. histolyticum infection in
horses.
 Outbreak of Cl. histolyticum infections in injecting drug users (IDU) in
England and Scotland (Brazier et al, 2004).
It is believed that, somewhere along the supply chain, heroin is being
contaminated with spores. These spores have been shown to survive the
heroin ‘cooking up’ process that commonly involves heating in citric acid
(pH 2.1) prior to injection.
Epidemiology cont…

13. TOXINS PRODUCED
SN Toxin Biological Effect
1 Alpha  Lethal and necrotizing
2 Beta  Clostridiopeptidase A, Collagenase A, Collagenase I, Microbial
collagenase
 Catalyzes the hydrolysis of collagen
 Target is glycyl-prolyl sequence.
 Can attack on gelatin.
3 Epsilon  Hemolytic
4 Gamma  A cysteine-activated proteinase
 Attacks altered collagen (e.g. hide-powder or azocoll) but not
native collagen.
 Also can act on gelatin.
5 Delta  Clostripain
 Proteinase

14. PATHOGENESIS
 Infection requires 2 conditions to coexist.
First, organisms must be inoculated into the tissues.
Second, oxygen tension must be low enough for the organisms to proliferate.
These organisms are not strict anaerobes; 30% oxygen tension in the tissues
allows for free growth of these bacteria, but 70% oxygen tension restricts their
growth.
 Inoculation of organisms into low oxygen tension tissues is followed by an
incubation period that usually ranges from 12-24 hours. However, this period can
be as brief as 1 hour or as long as several weeks.
 Multiplication and production of exotoxins result in myonecrosis. Exotoxins
appear to be tissue-destructive soluble antigens.
 Mucosal permeability and tissue injury was produced by collagenase from Cl.
histolyticum (Herbert et al, 1995)

15. LESIONS
 Infections are characterized by a very low level of host inflammatory
response to organism.
 In fact, it is more of a response to the exotoxins than a classic immune
response to invading organisms.
 Purulence is often absent.
 Process of myonecrosis can spread as fast as 2 cm/h. This results in
systemic toxicity and shock that can be fatal within 12 hours.
 Overwhelming shock with accompanying renal failure usually leads to
death.

16.  In lab animals I/M injection of
culture:
Extensive local destruction,
Splitting of the skin,
Hemorrhagic muscle pulp,
Denudation,
Sometimes auto-amputation
through a joint.
 Neither putrid changes nor gas
formation occurred in the liquefying
area, which frequently spread far
beyond the injected limb.
View of underside of skin of rabbit.
Dark areas are of hemorrhage
beneath the intradermal injection.
Lesions cont…

17. SYMPTOMS
 Clostridial Myonecrosis shows increasingly severe pain beginning at the
injury site 24 h after infection is the first reliable symptom.
 Skin may initially be pale, but it quickly changes to bronze and then to a
purplish red.
 Infected region becomes tense and tender, and bullae filled with
reddish-blue fluid appear.
 Signs of systemic toxicity, including tachycardia, fever, and diaphoresis,
develop rapidly, followed by shock and multiple organ failure.

18. DIAGNOSIS

19. TREATMENT AND CONTROL
 The role of hyperbaric oxygen treatment remains unclear.
 Altemeier and Fullen reported a significant reduction in mortality
among patients with gas gangrene using penicillin and tetracycline
plus aggressive surgery in the absence of hyperbaric oxygen.
 Treatment of experimental gas gangrene has demonstrated that
tetracycline, clindamycin, and chloramphenicol were more effective
than penicillin.
 No vaccines reported against Cl. histolyticum.

20. PHARMACEUTICAL APPLICATION
 Collagenase Cl. histolyticum dismantles collagen.
 It is used as a powder-and-solvent injection kit for
the treatment for Dupuytren's contracture.
 Reported to break down the excess collagen that
causes Peyronie's disease.
 BioSpecifics Technologies developed the
preparation, which is manufactured and marketed
by Auxilium Pharmaceuticals and Pfizer as Xiaflex in
the US and Xiapex in Europe.
 COLLAGENASE (Santyl®) breaks down collagen in
damaged tissue and helps healthy tissue to grow.
Application of collagenase to a wound may help it
heal faster. A generic collagenase ointment is
available.

21.  Identification of Clostridium Species, Issue no: 3 Issue date: 14.07.08 Issued by:
Standards Unit, Evaluations and Standards Laboratory
 Dennis L.S., Alan L.B., Henry F.C., Dale E., Patchen D., Ellie J.C.G, Sherwood L.G., Jan V.H.,
Edward L.K., Jose G.M. and James C.W. (2005). Practice Guidelines for the Diagnosis
and Management of Skin and Soft-Tissue Infections. Guidelines for Skin and Soft-
Tissue Infections, CID 2005:41, 1373-1406
 Jarosław J, Aldona K, Ewa J, Gayane M. (2005). Determination of the cytotoxic effect of
Clostridium histolyticum culture supernatant on HeLa cells in the presence of protease
inhibitors. FEMS Immunology and Medical Microbiology 45:137–142.
 Edward L.H., Ines M., and Stephen Z. (1959) Toxicity of Enzymes From Clostridium
histolyticum.
 Shoki N. and Masaaki I. (1966). Toxigenicity of Clostridium histolyticum. Journal of
Baceriology, 91:2, 477-483.
 Oakley C.L. and Harriet G.W. The Alpha, Beta and Gamma Antigens of Clostridium
histolyticum (Weinberg & Sbguin, 1916)
 Leopold W. and Walter K. Studies on The Proteinase of Clostridium Histolyticum
 Hoogerheide J.C. Variability in Morphological and Biochemical Properties of
Clostridium Histolyticum (Weinberg And Seguin)
REFERENCES

22. … …THANK YOU