Selection of candidate probiotic bacteria from chickens with antagonism against campylobacter

Abstract

Campylobacter is one of the most common causes of bacterial foodborne disease in humans worldwide. The effect of probiotics on Campylobacter control in poultry is inconsistent or rarely observed. For this reason, research on probiotics for Campylobacter control in poultry is required. Therefore, the objective of this study was to identify lactobacilli, Bacillus or Enterococcus strains isolated from Campylobacter-negative chickens which have the ability to inhibit the growth of Campylobacter in vitro and demonstrate favorable probiotic characteristics. A total of 602 bacterial isolates from Campylobacter-negative broiler chickens were assessed for their probiotic properties including Campylobacter inhibiting activity test, hemolytic activity test, acid and bile tolerance test, antimicrobial susceptibility test, and chicken intestinal mucus adhesion test. Additionally, determination of antimicrobial resistance genes and their location on the genome by whole genome sequencing was performed on the candidate probiotic bacteria. We identified 2 Limosilactobacillus reuteri isolates (isolate i 24.1/2 and i 24.2/2) that showed good probiotic properties. These isolates demonstrated Campylobacter inhibiting activity with Campylobacter inhibition zone diameter of 16 mm (i 24.1/2) and 15 mm (i 24.2/2) in agar well diffusion assay and they were negative for hemolytic activity test. In addition, the two isolates exhibited excellent acid tolerance (91.12% and 99.58% survival rates for i 24.1/2 and i 24.2/2, respectively) and bile tolerance (99.47% and 102.95% survival rates for i 24.1/2 and i 24.2/2, respectively). Furthermore, these isolates also showed the ability to adhere to chicken intestinal mucus with 80.10% (i 24.1/2) and 70.35% (i 24.2/2) adhesion efficiency. Even though vancomycin and ampicillin resistance was found in both isolates, it presents a minimal risk for horizontal resistance genes transfer because resistance to vancomycin is considered intrinsic resistance in most lactobacilli and ampicillin resistance in L. reuteri is probably caused by point mutations in the genes encoding penicillin-binding proteins. However, both candidate probiotic isolates still harbored plasmids that carried lnuA resistance gene. Thus, these candidates were not suitable to be used as a probiotic at present. But, if the plasmid carrying the antimicrobial resistance gene is removed from the candidate probiotic bacteria, which had been accomplished in the past, these candidates could be suitable for being used as a probiotic. Furthermore, in order to develop a new probiotic product, these candidates should be further evaluated for their efficacy as probiotics by in vivo experiments in chickens.