Evaluation and Selection of Bacillus Species Based on Enzyme Production, Antimicrobial Activity, and Biofilm Synthesis as Direct-Fed Microbial Candidates for Poultry

Abstract

Social concern about misuse of antibiotics as growth promoters (AGP) and generation of multidrug-resistant bacteria have restricted the dietary inclusion of antibiotics in livestock feed in several countries. Direct-fed microbials (DFM) are one of the multiple alternatives commonly evaluated as substitutes of AGP. Sporeformer bacteria from the genus Bacillus have been extensively investigated because of their extraordinary properties to form highly resistant endospores, produce antimicrobial compounds, and synthesize different exogenous enzymes. The purpose of the present study was to evaluate and select Bacillus spp. from environmental and poultry sources as DFM candidates, considering their enzyme production profile, biofilm synthesis capacity, and pathogen-inhibition activity. Thirty-one Bacillus isolates were screened for in vitro relative enzyme activity of amylase, protease, lipase, and phytase using a selective media for each enzyme, with 3/31 strains selected as superior enzyme producers. These three isolates were identified as Bacillus subtilis (1/3), and Bacillus amyloliquefaciens (2/3), based on biochemical tests and 16S rRNA sequence analysis. For evaluation of biofilm synthesis, the generation of an adherent crystal violet-stained ring was determined in polypropylene tubes, resulting in 11/31 strains showing a strong biofilm formation. Moreover, all Bacillus strains were evaluated for growth inhibition activity against Salmonella enterica serovar Enteritidis (26/31), Escherichia coli (28/31), and Clostridioides difficile (29/31). Additionally, in previous in vitro and in vivo studies, these selected Bacillus strains have shown to be resistant to different biochemical conditions of the gastrointestinal tract of poultry. Results of the present study suggest that the selection and consumption of Bacillus-DFM, producing a variable set of enzymes and antimicrobial compounds, may contribute to enhanced performance through improving nutrient digestibility, reducing intestinal viscosity, maintaining a beneficial gut microbiota, and promoting healthy intestinal integrity in poultry.

Assessment of the potential of the multi‐enzyme producer Bacillus amyloliquefaciens US573 as alternative feed additive

“…In this regard, our results clearly demonstrated that the B. amyloliquefaciens US573 strain produces simultaneously multiple extracellular hydrolytic enzymes, as was the case for the commercial feed additive Rovabio® Max (ADISSEO). This is likely to ensure a synergistic enzyme‐mediated improvement of nutrient bioavailability and feed conversion rate as a consequence of enhanced digestibility of the antinutritional factors present within feed ingredients of poultry diets …”

Bacillusprobiotics: an alternative to antibiotics for livestock production

“…The CE mechanisms by which Bacillus protects aquatic animals against pathogen infection can be extended to immunity enhancement. Li et al (2007) found that L. vannamei fed B. licheniformis exhibited a significant increase in phenoloxidase and superoxide dismutase activities, and showed a decrease in the population of Vibrio sp. in the intestine as compared with the control shrimp.…”

“…Several Bacillus sp. are able to produce a variety of extracellular enzymes including amylase, protease, lipase, phytase, cellulase and xylanase (Latorre et al 2016). These enzymes have been used in animal nutrition to assist in the digestion of feed and improve nutrient absorption.…”

Selection ofBacillus subtilisUS191 as a mannanase‐producing probiotic candidate

“…The Bacillus species have the advantage of possessing the spore‐forming ability coupled with acid and bile tolerance properties along with the antagonistic activity against major gut pathogens and susceptibility to most of antibiotics . In addition, they produce various extracellular hydrolytic enzymes, which can help in improving digestion and exhibit the ability to form biofilm contributing to the protection of the gut epithelium from adherence of opportunistic pathogenic microorganisms and helping to withstand harsh environmental conditions and physical stresses .…”