Use of in-vitro antimicrobial susceptibility methods as an identification marker for vaccinal strains of Clostridium perfringens in vaccine production process

The aim of study was to differentiate vaccinal strains of Clostridium perfringens from other common environmental species through the use of biochemical tests, antibiotic susceptibility and MIC determination. Morphological and biochemical characteristics of Clostridium perfringens types B-11 and D-27 were investigated. Morphologically, both strains were gram-positive but in old cultures showed gram-negative character. The spores were large and ovoidal were located subterminally and centrally. There were little differences in the shape and colony characteristics. Clostridium perfringens type B-11 was larger bacilli, straight with rounded ends. Clostridium perfringens type B-11 produced thin, circular, raised, opaque and hemolytic colonies on blood agar. While in broth medium, it produced peptolytic, uniform and marked turbidity with gas production. Whereas type D-27 showed thicker colonies on agar as compared to type B-11. Biochemically, Clostridium perfringens types B-11 and D-27 were quite similar except for TSI which was utilized by B-11 but not by D-27. Both types produced beta hemolysis on blood agar, lacithinase activity reaction on egg yolk agar, gelatin liquefaction, anaerobic growth, nitrate reduction were positive whereas negative for catalase and indole. Both types of Clostridium perfringens were non-motile. Furthermore, both B-11 and D-27 types of Clostridium perfringens were fermented all sugars except mannitole and salicin. Reinforced Clostridial Medium (RCM), Brain Heart Infusion (BHI) and Muller Hinton agars (MHA) were used to select suitable medium for the sensitivity. All media were observed equally good for the use with various representative antibiotics, except for colistin sulphate, that showed no activity against the organisms. Twenty-five antibiotics from different groups were used in this study. Of these, 4 from cephalosporins, 6 from quinolone, 4 from penicillin, 1 from tetracycline, 3 from aminoglycosides and 7 from diverse groups. Antibiogram susceptibility of Clostridium perfringens of vaccinal types B-11 and D-27 was carried out. A clear difference was ascertained between vaccinal types and contaminants against the antibiotics used in this study. Among the antibiotics, penicillin's group i.e. amoxicillin and ampicillin showed an interesting feature in zone of inhibition between both vaccinal types B-11 and D-27 (22mm and 24mm and 20mm respectively). The extensive antibiogram susceptibility of Clostridium species envisages to explore selected antibiotics to which the Clostridium perfringens types B-11 and D-27 were resistant and other contaminant such as Escherichia coli was sensitive. During this study, the colistin sulphate allowed the growth of Clostridium perfringens types B-11 and D-27 (i.e. resistant) but did not allow the growth of Escherichia coli. Minimum inhibitory concentration (MIC) determined the antibiotic chose from the study as ID marker (i.e. colistin sulphate) revealed that MIC of 6.4 mug/ml is sufficient into inhibit the growth of contaminant, but allowed the growth of Clostridium perfringens types B-11 and D-27. Therefore, colistin sulphate is an antibiotic of choice for confirming the purity of cultures of Clostridium perfringens types B-11 and D-27. It is concluded that vaccine manufacturing process suffers from surrounding contamination, which results in less efficient vaccine. It is further identified that the vaccinal strains of Clostridium perfringens types B-11 and D-27 were very similar to each other by the biochemical, antibiotic susceptibility and MIC. During investigation the contaminant Escherichia coli was isolated and found highly susceptible to colistin sulphate while Clostridium perfringens types B-11 and D-27 were resistant to colistin sulphate. However, colistin sulphate could be used for purity of vaccine. In the light of results the recommendations and suggestions are set to improve the understanding of identification procedure of the member organisms of Clostridia, especially Clostridium perfringens types B-11 and D-27 for use of applied microbiology such as laboratory identification and vaccine production.