Use of antimicrobial susceptibility pattern as an identification marker of vaccinal and wild type strains of Bacillus anthracis in vaccine manufacturing process

This study aimed at differentiation of vicinal and wild Bacillus anthracis and other commonly related environmental bacilli through the use of biochemical tests, antibiotic susceptibility and determination of MICs. Extraneous contaminants of vaccine were obtained from the growth of vaccine batches on BHIA. The extraneous contaminants identified in this study were Bacillus megaterium, Bacillus cereus, Bacillus mycoides and Bacillus subtilis. All of the four contaminants were gram positive and motile. The haemolysis on blood agar was variable and catalase was positive except for Bacillus anthracis Lecithinase reaction on egg yolk agar was also variable whereas crease activity was absent in all the organisms including Bacillus anthracis. Gelatin hydrolysis and anaerobic growth were positive in all contaminants and Bacillus anthracis vicinal strain. Sodium Chloride (NaCI) at concentration of 6% was tolerated by B. megaterium and B. subtilis. Free or endospores were observed in all including B. anthracis Acid was produced from glucose, salicin and xylose in oxidation/fermentation reactions, whereas utilization of mannitol, maltose and lactase varied. The organisms have opaque to creamy ground glass consistency colonies on agar surfaces compared to broth where homogenous growth and pellicle formation were the main features and varied according to organism. Under the microscope; short to long chains made up of gram-positive rods were observed. Selection experiments for determination of suitable media for antibiotic sensitivity testing (AST) showed Muller Hinton Agar (MHA) and Disc Sensitivity Test Agar (DSTA) with highest consistency and reproducibility of the results. Forty four (44) antibiotics front different groups were used in this study. Of these 9 were from Cephalosporins group, G from quinolone group, 5 from penicillin group, 3 from B-lactam inhibitors combination group, 2 from tetracycline group, 04 from aminoglycoside group and 15 from diverse group. Antibiograrn of B. anthracis included both wild as well vicinal strain, whereas no significant difference could be ascertained between tile wild and vicinal strains against arty, of tile antibiotic used in this study. The extensive antibiogram aril of a number of Bacillary species envisage to explore e selected antibiotics to which the B. Anthracis is resistant and other common contaminant bacilli are sensitive. During this study the co-trimoxazole (trimethoprim + sulphamethoxazole) at the concentration of 25 ug/disc allowed the growth of B anthracis (i.e. resistant) but did not allow the growth of other four extraneous contaminants bacilli. Minimum Inhibitory Concentrations (MICs) determined of the antibiotic chosen from the study as "ID Marker" (i.e. co-trimoxazole) revealed that MIC of 25 and mug/ml is sufficient to inhibit growth of commonly contaminating bacilli of bacteriology laboratory capable of spoiling vaccine during its production, the above determined MIC allow growth of Bacillus anthracis but do not allow growth of Bacillus megatenum, Bacillus cereus, Bacilli mycoides and Bacillus subtilis. Co-trimoxazole is also therefore be taken as an antibiotic of choice in confirming the purity of cultures of B anthracis.