Whole-Genome Sequence and Interaction Analysis in the Production of Six Enzymes From the Three Bacillus Strains Present in a Commercial Direct-Fed Microbial (Norum™) Using a Bliss Independence Test.

Daniel Hernandez-Patlan,Guillermo Tellez-Isaias,Bruno Solis-Cruz,Oscar Ortiz Holguin,Pallavi Singh,Billy M Hargis,Xochitl Hernandez-Velasco,Ramiro Delgado,Catie Ausland,Juan D Latorre,Ruben Merino-Guzman,Miguel Morales Rodríguez

doi:10.3389/fvets.2022.784387

Abstract

The three Bacillus strains present in Norum™ were initially selected by their excellent to good relative enzyme activity (REA) production score for amylase, protease, lipase, phytase, cellulase, β-glucanase, and xylanase. Further studies confirmed that the three isolates also showed an antibacterial activity, Gram-positive and Gram-negative poultry pathogens. Norum™ (Eco-Bio/Euxxis Bioscience LLC) is a Bacillus spore direct-fed microbial (DFM). The Bacillus isolates were screened and selected based on in vitro enzyme production profiles. Moreover, in chickens fed high non-starch polysaccharides, this DFM demonstrated to reduce digesta viscosity, bacterial translocation, increase performance, bone mineralization, and balance the intestinal microbiota. In the present study, we present the whole-genome sequence of each of the three isolates in Norum™, as well as the synergistic, additive, or antagonistic effects on the enzyme production behavior of the three Bacillus strains and their combinations when grown together vs. when grown individually. The whole-genome sequence identified isolate AM1002 as Bacillus subtilis (isolate 1), isolate AM0938 as Bacillus amyloliquefaciens (isolate 2), and isolate JD17 as Bacillus licheniformis (isolate 3). The three Bacillus isolates used in the present study produce different enzymes (xylanase, cellulase, phytase, lipase, protease, and β-glucanase). However, this production was modified when two or more Bacillus strains were combined, suggesting possible synergistic, antagonistic, or additive interactions. The Bliss analysis suggested (p < 0.05) that the combination of Bacillus strains 1–2 and 1–2–3 had intermediate effects and predicted that the combination of Bacillus strains 2–3 could have better effects than the combination of all the three Bacillus strains. In summary, the current study demonstrated the need of selecting Bacillus strains based on quantitative enzyme determination and data analysis to assess the impacts of combinations to avoid antagonistic interactions that could limit treatment efficacy. These results suggest that using Bacillus strains 2–3 together could lead to a new generation of DFMs with effects superior to those already examined in Bacillus strains 1–2–3 and, therefore, a potential alternative to growth-promoting antibiotics. More research utilizing poultry models is being considered to confirm and expand the existing findings.

Highlights

Recent regulations to avoid antibiotics in animal production have led to the evaluation of new viable alternatives in terms of efficacy, costs, acceptability, and practicability [1]
The three Bacillus strains present in NorumTM were initially selected by their excellent to good relative enzyme activity (REA) production score for amylase, protease, lipase, phytase, cellulase, β-glucanase, and xylanase [7, 8]
Isolates AM1002 and AM0938 previously identified as Bacillus subtilis and Bacillus amyloliquefaciens by 16S rDNA sequence, respectively, were confirmed to be of the same genus and species

Summary

Introduction

Recent regulations to avoid antibiotics in animal production have led to the evaluation of new viable alternatives in terms of efficacy, costs, acceptability, and practicability [1]. Despite the numerous alternatives that have been evaluated to replace antibiotics in the modern poultry industry, the group of probiotics has shown promising results [2]. While not all Bacillus spores are heat tolerant, some isolates are the most strenuous life forms on the planet and can survive in scorching environments [4,5,6]. As a result of these findings, products containing Bacillus spores are sold as probiotics. They have potential advantages over more common LAB products because they can be used as directfed microbials (DFM). We have shown that the Bacillus spores used as DFM improved intestinal integrity, bone mineralization, and reduced ammonia excretion in turkey poults fed with a rye-based diet [13]. The sporulated form of these microorganisms can germinate in the aggressive conditions of the gastrointestinal tract and produce biofilms to protect themselves and tolerate the acidic pH, high osmotic concentrations of sodium chloride, and bile salts [15]

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