Isolation, characterization and antibiogram of Bacillus cereus from milk products

Abstract

This study, conducted within the Department of Veterinary Public Health and Epidemiology (VPHE) at Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), undertook a comprehensive investigation into the prevalence, identification, characterization, and antibiotic resistance patterns of Bacillus cereus in milk products from various regions of Haryana, India. Using a systematic sampling strategy, eight tehsils spanning two agroclimatic zones were selected for sample collection. A total of 200 samples were obtained from randomly selected shops within these tehsils. Each sample underwent pre-enrichment in Brain Heart Infusion (BHI) broth with a 1:10 dilution to facilitate the growth of any existing B. cereus contaminants. Following pre-enrichment, streaking on selective PEMBA agar plates was performed for the isolation of B. cereus colonies. The presence of presumptive B. cereus colonies was confirmed through a series of biochemical tests, including gram staining, nitrate reduction, oxidase, indole, methyl red reduction, Voges Praskaur, and catalase tests. Molecular analysis using the 16SrRNA gene confirmed the presence of 88 positive B. cereus isolates. Further characterization involved the differentiation of B. cereus from Bacillus thuringiensis via the cry2 gene. Additionally, the study assessed the presence of virulence-associated genes, identifying gyrB, cytk, hblA, and nheA genes in the isolated strains. Geographical variation in B. cereus prevalence was observed, with higher rates detected in certain tehsils such as Kalka (68%) and Panchkula (60%). Antibiotic sensitivity testing using Tryptone Soya Agar (TSA) broth for enrichment and Muller Hinton Agar (MHA) plates with 14 antibiotic discs revealed widespread resistance among the isolates. Notably, all isolates exhibited resistance to Ampicillin, Cefazolin, Methicillin, Polymyxin-B, and Penicillin, while displaying maximum sensitivity to Amikacin, Gentamycin, Levofloxacin, and Meropenem. Alarmingly, all isolates displayed multiple drug resistance (MDR), indicating resistance to three or more classes of antibiotics. These findings underscore the urgent need for stringent food safety measures and surveillance protocols in dairy production and distribution. Effective interventions are imperative to mitigate the risk of B. cereus contamination and combat antibiotic resistance in milk products. Collaboration with the VPHE department at LUVAS and continued research efforts are essential to address emerging challenges and uphold food safety standards effectively.