Identification and some biological characteristics of Bacillus strains isolated from poultry large intestine

Introduction. Censuses of phylogenetic diversity of bacteria colonizing the intestinal tract of clinically healthy poultry conducted over the past decade indicate that up to 60% of genera present in the gut microbiome contain spore-forming bacteria, accounting for 30% of total gut microbiota. Benefits associated with using probiotics containing Bacillus spore-forming bacteria have been documented. Analysis of the prevalence of hemolytic and potential biofilm-forming activity, as well as antibiotic resistance in poultry gut spore-forming microbiota is essential for understanding the true role of aerobic spore-formers of the Bacillus genus in avian gut microbiome ecology.

Objective. Identification and investigation of biological characteristics (hemolytic activity, potential biofilm-forming capacity and antibiotic resistance) of Bacillus bacterial isolates obtained from the large intestine of poultry.

Materials and methods. Spore-forming bacteria were isolated from cecal content through sample heat treatment. Phenotypic identification was performed using API 50CHB biochemical test panels (bioMérieux, France). Hemolytic properties were assessed using Columbia agar (HiMedia Laboratories Pvt Ltd., India) supplemented with 5% sterile defibrinated sheep blood; catalase activity was assessed using 10% hydrogen peroxide according to General Pharmacopoeia Monograph GPM.1.7.2.0012.15; antibiotic sensitivity was assessed with disk diffusion test involving standard antibiotic-impregnated disks (5–30 μg/disk). Biofilm-producing, spore-forming bacteria were tested qualitatively using brain-heart infusion agar (BHI; HiMedia Laboratories Pvt Ltd., India) supplemented with Congo red and 5% sucrose.

Results. It was established that the cecal aerobic spore-forming microbiota in poultry comprised B. licheniformis, B. subtilis/amyloliquefaciens, B. mycoides, B. megaterium and B. cereus. All tested isolates were catalase-positive and lacked α-hemolytic activity. Some isolates demonstrated β-hemolytic activity. The overwhelming majority exhibited biofilm-forming phenotypes and showed susceptibility to tested antibiotics.

Conclusion. Vegetative forms of Bacillus spore-forming bacteria may potentially persist in or temporarily associate with the complex gut ecosystem. Hemolytically active intestinal isolates cannot be considered safe until the effects of this virulence factor on animals are clarified. These findings provide a basis for selecting candidate Bacillus strains for probiotic development.

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