Bioprospecting and Characterisation of Potential Bacteriocin Producing Bacillus Species Isolated from Rastrineobola Argentea (Omena) with Activity Against Bovine Mastitis Bacterial Pathogens

Abstract

The inappropriate use of antibiotics has resulted in an increase in drug-resistant pathogenic bacteria. The global problem calls for alternative novel antimicrobial agents with a wide range of clinical significance. Bacteriocins offer an alternative to antimicrobials and have been tried out in food and other industries with limited success. This study sought to explore antimicrobial agents from bacteria inhabiting the commonly consumed Rastrineobola argentea from open-air markets in Kisumu Kenya and evaluate their potential for production of crude bacteriocins with antimicrobial activity against bovine mastitis bacterial pathogens. Dried samples were randomly collected from fifteen different fishermen at open-air markets in Kisumu. Using Nutrient agar, 60 bacteria isolates were isolated and characterized using standard microbiological procedures and 54 gram-positive isolates identified as Bacillus spp. were further tested for their bacteriocin production capacity. Each isolate was tested for crude bacteriocin production. The extracts were optimized and their physiological and physicochemical parameters determined. On the other hand, the 54 bacteriocin-producing isolates were characterized using molecular techniques by 16S rRNA gene sequencing. All the isolates demonstrated varied utilization of sugars, and 28 isolates showed antimicrobial activity towards the common bacterial standard test microorganisms. Further characterization of the isolates using the analytical profile index system showed that 28 isolates had antimicrobial activity against E. coli and S. aureus test microorganisms. The analytical profile index system indicated that 20 isolates (71%) were identified as members of the Bacillus spp. The crude bacteriocin extracts withstood the effect of the proteolytic protekinase K enzyme, while trypsin and lipase enzymes caused 40% and 15% loss of inhibition, respectively. When tested at different pH levels, the bacteriocin extracts had no inhibition at pH 3-5, but had varied inhibition at pH 6-9 across the test organisms and withstood temperatures of 50 °C–60 °C, showing no loss of activity. The bacteriocin activity decreased by 20% as the temperature increased from 70 °C to 80 °C. Temperatures of 100 °C and 121 °C resulted in a 40% and > 50% reduction in bacteriocin activity, respectively. Metal ions, Cu2+, Fe2+ and Zn 2+ had varying effects on bacteriocin activity against test micro-organisms. The blast analysis of the partial sequences revealed that twenty (20) isolates belonged to Bacillus subtilis, with similarities ranging between 96.2% and 99.7%; Bacillus mycoides represented 18% (5 isolates) with similarities 99.9% to 100%; and Bacillus pumilus constituted 11% (3 isolates) with similarities ranging from 98.4% to 100%. The study has demonstrated R. argentea collected from open air markets in Kisumu harbors to a large extent B. subtilis, followed by B. mycoides, B. pumilus and B. cereus which have antimicrobial activity against standard laboratory test E. coli, S. aureus, P. aeruginosa and K. pneumoniae, microorganisms and their corresponding counterparts isolated from mastitis infected animals. Additionally, the Bacillus species were able to produce enzymes, ferment carbohydrates, and survive in different temperature and pH conditions besides tolerating presence of metallic ions. R. argentea harbours Bacillus species from which bacteriocins can be obtained having activity against bovine mastitis bacterial pathogens.