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

Juan D Latorre,Jose L Vicente,Xochitl Hernandez-Velasco,Amanda D Wolfenden,Anita Menconi,Billy M Hargis,Ross E Wolfenden,Lisa R Bielke,Guillermo Tellez

doi:10.3389/fvets.2016.00095

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.

Highlights

The continuous tendency to reduce the use of antibiotic growth promoters (AGP) in poultry production, due to social concern about generation of antibiotic-resistant bacteria, has resulted in the crucial necessity to find economically viable alternatives that can maintain optimal health and performance parameters under commercial conditions [1, 2]
The purpose of the present study was to evaluate and select Bacillus isolates from environmental and poultry sources as candidate Direct-fed microbials (DFM) based upon enzyme production profiles, pathogen-inhibition capacity, and biofilm synthesis, extending our understanding of the mechanism of action of Bacillus-DFM and its applicability in the poultry industry
Bacillus spores were isolated by heat treatment of intestinal, fecal, and environmental samples, eliminating the presence of vegetative cells

Summary

Introduction

The continuous tendency to reduce the use of antibiotic growth promoters (AGP) in poultry production, due to social concern about generation of antibiotic-resistant bacteria, has resulted in the crucial necessity to find economically viable alternatives that can maintain optimal health and performance parameters under commercial conditions [1, 2]. Among the microorganisms used as direct-fed microbials (DFM), Bacillus spores have been increasingly included as feed additives in poultry diets, due to their remarkable resistance to harsh environmental conditions, and have a long shelf life [5, 6]. Different studies have shown that Bacillus spores can be present, germinate, and survive in the gastrointestinal tract (GIT) of different animal species, suggesting that these bacteria could be considered facultative anaerobes and part of the metabolically active host microbiota [7,8,9,10]. Rate of survival and persistence of some Bacillus strains in the GIT may be related to their capacity to synthesize biofilms, thereby, protecting themselves against the harsh environmental conditions present in the gut [11]. One of the principal sources of enzymes and antibiotics from bacterial origin used by biotechnology companies are produced by different Bacillus strains, making this multifunctional microorganism useful inside or outside a host [12, 13]

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Conclusion