Gas gangrene is a severe polymicrobial wound infection. It is caused by various clostridial anaerobic microflora in association with pathogenic facultatively anaerobic bacteria (staphylococci, streptococci, gram-negative rods, etc.)
The History of Discovery
W. Welch and G. Nuttall first isolated the major causative agent of Clostridium perfringens gas gangrene in 1892. About 15 years earlier, in 1877, L. Pasteur and J. Joubert discovered the first member of the Clostridium septicum gas gangrene group. R. Koch then confirmed its ability to cause edema caused by gas gangrene. In 1894, F. Novy identified another species of clostridium, which was later named Clostridium novyi. M. Weinberg and P. Seguin finally identified Clostridium hystolyticum in 1916.
Classification of Pathogenic Clostridia
Clostridia of gas gangrene belong to the family Clostridiaceae, genus Clostridium. Among the disease causative agents are the species Clostridium perfringens, C. novyi, C. septicum, C. hystolyticum as well as C. sordelli, C. fallax, C. ramosum and some others.
Structure and Properties of Clostridia
Morphology of Clostridia
Typically, perfringens are gram-positive dense non-motile rods with rounded ends. It has a central or subterminal localised oval spore and forms a capsule within the infected host. Novyi with subterminal spores are rod-shaped motile peritrichous bacteria. They do not form a capsule. Septicum are non-capsulated polymorphic rods that can form long filamentous forms. Central or subterminal spores and peritrichous flagella are carried by micro-organisms. In morphology, the hystolyticum is similar to the two previous representatives.
Cultivation of Clostridia
Among all other clostridia, it is found that perfringens are the most aerotolerant. It is grown on iron sulfite agar, Schaedler agar and glucose blood agar in anaerobic jars, Kitt-Tarozzi, and other anaerobic media, similar to other anaerobic bacteria. Within the first 6-8 h of cultivation, Perfringens is able to blacken iron sulfite agar. Medium C.perfringens produces homogeneous turbidity in gas production in Kitt-Tarozzi. Novyi, C.septicum and C.hystolyticum are bacteria which are strictly anaerobic. Blood agar C.novyi forms rough fringed colonies with hemolysis on glucose. In the meat-peptone broth, septicum is easily cultivated. On glucose blood agar, the bacteria create a film. The colonies of agar stab cultures look like balls of wool.
Biochemical properties of Clostridia
Compulsory anaerobes are all clostridia. Perfringens ferments large amounts of acid and gas end products into glucose, sucrose, lactose, starch, and many other sugars. They liquefy gelatin, serum blood coagulation and milk, resulting in a sponge-like clot. Nitrates are reduced to nitrites by these bacteria. Butyric and acetic acids and large amounts of CO2, H2, H2S and other gases are produced.
Novyi ferments glucose, maltose and glycerol by producing acid and gas. They also liquefy gelatin and with small flakes, coagulate milk. Septicum slowly liquefies gelatin and uses hydrogen sulphide and ammonia-looking proteins. Acid and gas formation are metabolised with several mono- or disaccharides Hystolyticum does not ferment sugars, but reveals significant activity in proteolytics.
Antigenic structure of Clostridia
Serological Differentiation with C. Perfringens is based on the variation of microbial toxins in antigenic terms. Six main serovars are known, namely A, B, C, D, E, and F. Type A is further divided into many subtypes. For humans, types A, C and D are pathogenic; animals are affected by B, C, D, and E. Novyi consists of four antigenic variants, A, B, C and D, where the predominant human pathogenic variant is type A. According to their exotoxins, septicum may be differentiated into 6 serovars. Depending on toxin structure differences, hystolyticum has 5 antigenic variants.
Virulence factors of Clostridia
All clostridia produce extreme variety of virulence factors that predominantly display potent enzymatic activity. α-Toxin of C. perfringens or phospholipase C displays high lecithinase activity that damages cell membranes, enhances vascular permeability and develops necrotizing activity. β-Toxin is a potent necrotizing substance; ε-toxin increases vascular permeability in the gastrointestinal tract. θ-Toxin or perfringolysin O demonstrates polyfunctional hemolytic, dermonecrotizing and lethal properties. Other minor toxins also possess enzymatic properties. For instance, κ-toxin acts as collagenase, μ-toxin – hyaluronidase, δ-toxin develops hemolytic activity.
In addition, C. perfringens expresses potent enterotoxin. Novyi produces at least 8 distinct toxins with hemolytic, lecithinase, protease and hyaluronidase activities. Septicum has 4 major toxins: α-toxin with lethal, hemolytic and necrotizing activity, β-toxin with DNase activity, γ-toxin is hyaluronidase and δ-toxin is hemolysin. Hystolyticum expresses 5 toxins, among them are α-toxin with lethal and necrotizing activity, β-toxin with collagenase activity, γ-toxin with protease activity, δ-toxin with elastase activity, and ε-toxin renders hemolytic activity.
Resistance of Clostridia
The C. perfringens spores are resistant to boiling for 8 to 90 minutes. In the concentrations commonly used for disinfection the vegetative forms are most susceptible to hydrogen peroxide and phenol.
C. novyi spores survive for a 20-25 year period without losing their virulence in the natural surroundings. They are killed by direct sunlight in 24 hours and destroyed by boiling in 10 to 25 minutes. Spores are resistant to formaldehyde exposure for 10 minutes for a 3% solution.
Pathogenesis and Clinical Findings in Gas Gangrene
Clostridia remains in the intestines of both animals and humans (as a source of infection) and is discharged from the feces outside. The soil constantly has spores of clostridia. Thus, contact with particles of dust and soil inevitably leads to skin and mucosal tissues contaminated by spores of the closure. Gas gangrene occurs, in severe tissue trauma, septic abortion, and other similar situations, if soft tissue (muscles, adipose, or connective tissues) are severely damaged, when infected with sparrows of C. perfringens and others clostridia. Clostridial infection is therefore mainly transmitted through contact pathways.
The causative agents of anaerobic infections require certain conditions for their germination and overgrowth. The basic one is the presence of dead or damaged tissues resulting in low oxidation-reduction potential (state of anaerobiosis). Characteristic type of injury (deep narrow wounds or contaminated crashed tissues) as well as patient state of health predisposes to the emergence of gas gangrene (for instance, diabetes mellitus strongly impairs tissue oxygenation).
Progressive propagation of pathogenic anaerobes leads to further degradation of body tissues thus aggravating anaerobic conditions. Active spread of infection ensures relatively short incubation period – from several hours up to 4-5 days.
Gas gangrene targets primarily muscles and adipose tissue as they harbor a lot of potential substrates for microbial toxic enzymes (e.g., glycogen or phospholipids). As the result, exotoxins of clostridia cause expanding tissue necrosis and melting. It is followed by accumulation of gases like СО2 and Н2 in soft tissues that is detected as gas gangrene. Growing edema blocks local circulation, thereby enhancing anaerobic conditions and toxin production. Edema is characteristic for the first phase of the infection, and gangrene of the soft tissues progresses in the second phase.
Microbial exotoxins generate both local and systemic devastating effects, being spread throughout the body. The products of tissue decay render additional toxicity against host tissues. As the result of massive edema and tissue necrosis with gas formation, the skin over the affected limbs becomes pale, then reddish and cyanotic with extensive hemorrhages. Deep destructive changes in subcutaneous adipose tissue, muscles, and fascias require urgent surgical treatment and systemic antimicrobial and antitoxic therapy.
The immunity arisen in the course of anaerobic clostridial infections is maintained predominantly by antitoxic antibodies. They neutralize the activity of multiple microbial toxins. However, the immune response is non-protective being of low grade. Without complex intensive treatment it is impossible to prevent the rapid disease progression. Besides gas gangrene development, C. perfringens may cause severe necrotizing enteritis followed by deep damage of small intestine. It ensues from the action of clostridial β-toxin with potent cytotoxic and necrotizing activity. In addition, C. perfringens are not so rare agents of food poisonings (or food toxinfections). These disorders are related with production of enterotoxins by clostridia.
Laboratory Diagnosis of Gas Gangrene
The specimens for examination comprise the pieces of necrotic tissues, tissue fluids and wound discharges, surgical stitch materials, dressings, etc. As preliminary test, microscopical examination of wound discharge for C. perfringens or other clostridia is made on the ground of their typical morphological characteristics. Also immunofluorescence microscopy can be applied for direct identification of clostridia in clinical samples.
Culture isolation is elaborated in anaerobic conditions (e.g., in anaerobic jars). Identification of microbial species takes into account their growth on iron sulfite agar, fermentation of carbohydrates, gelatin liquefaction and other biochemical tests, microbial serological properties. To confirm the diagnosis, experimental injection of mice with broth culture filtrates for exotoxin detection as well as antitoxin-toxin neutralization reactions are performed.
Rapid diagnostic test for detection of clostridial exototoxins in clinical samples are based on ELISA test or indirect hemagglutination assay with erythrocyte antitoxic diagnosticum. Genetic typing of clostridia species is performed by PCR.
Treatment and Prophylaxis of Gas Gangrene
Treatment of gas gangrene comprises the intensive surgical management of wounds and injuries with removal of affected tissues up to limb amputations, massive antibiotic chemotherapy against anaerobic infection (e.g., with β-lactams, aminoglycosides, and metronidazole), infusion and detoxification therapy, administration of polyvalent purified and concentrated antitoxin against C. perfringens and other clostridia. Hyperbaric oxygen therapy, blood transfusions and administration of inhibitors of proteolytic enzymes are the additional supportive measures for gas gangrene treatment. Prophylaxis of gas gangrene is non-specific. It primarily includes the protection of wounds and injuries from contamination and their adequate surgical treatment.