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Bacillus subtilis probiotics: The research for uniqueness and consistent results.

Pierre-Andre Geraert, Juan-Carlos Cabrera, Stephanie Pedrosa, Estelle Devillard, Stefan Jakob.

Ban of Antibiotic Growth Promoters: Complexity of alternative solutions

Stepping out of antibiotic growth promotor usage in poultry feeds is a growing challenge in lots of countries around the world. Due to extensive use of antibiotics, some bacteria also pathogenic for humans have developed antibiotic resistances, forcing animal producers to find alternatives to the antibiotics currently used in animal feeding to guarantee production efficiency and safe foods for consumers. A large range of products has shown some potential to help poultry growers to replace antibiotics in their productions.

Among all the solutions available, probiotics are known to be the most efficient ones because of their properties to balance the gut microbiota and therefore to act for good gut health and for a better absorption of nutrients. However, all probiotics are not same! With their ability to resist pelleting and gastric conditions, as well as their tremendous properties of gut microbiota stabilizers, Bacillus subtilis based probiotics appeared as one of the best alternatives to the use of in-feed antibiotics in poultry production to guarantee sustainable performance.

How to choose between all Bacillus subtilis based probiotics

Even within Bacillus subtilis based product, the large variability of the efficacy obtained underlines the importance of a methodology to differentiate the various strain as well as to select a new effective one. In order to evaluate the potential and to differentiate several Bacillus subtilis strains, an extensive screening process have been developed and applied. Within this process, in-vitro experiments were done as well as a complete phylogenetic characterization of the involved microorganisms. 

A serie of measurements were performed on various Bacillus strains stored at the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen- German Collection of Microorganisms and Cell Cultures), Braunschweig, Germany. Hemolytic activity of the strains was tested as described by the European Food Safety Authority using Tryptic Soy Agar containing 5% defibrinated sheep blood (Statens Serum Institute, Denmark). The incubation was done at 30˚C for 48 hours, and when there was a hemolytic activity, it was observed by the formation of a lysis halo in the plates. This halo was not present when we tested B.subtilis DSM29784 strain. In order to evaluate the potential activity of the different Bacillus subtilis strains against some pathogenic microorganisms, in vitro radial diffusion assays were performed. A suspension containing 105 spores of Clostridium perfringens ATTC13124 was added to the medium and 10 µl of culture fluid of the Bacillus strains were added to the test plate. The plate were incubated under anaerobic conditions at 37 °C overnight. Some lysis were visible around some of the wells: it was the case for Bacillus subtilis strain DSM29784.

Antimicrobial susceptibility was analyzed by determination of the Minimal Inhibitory Concentration of eight clinical relevant antibiotics representing the most abundant and critical antibiotics used for humans. The analyses were made by following the standard procedure recommended by CLSI 2012. Lastly, the effect on gut barrier was also evaluated using Caco2 cell transwell cultures with measurement of trans-epithelial electrical resistance under standard or IL-1 stimulation conditions. Gastric stability (under pH2 and pepsin supplementation) and germination time in intestinal conditions were also determined. To all these criteria, B. subtilis DSM29784 showed good results.

To complete the characterization of the various Bacillus strains, a sequencing and phylogenetic analysis based on the gyrB gene was done. The sequences were aligned using the ClustalW and a phylogenetic tree was made based on distance matrix calculated with the Kimura distance formula using MegAlign. Figure 1 shows the homology between the different strains. To allow comparison, if we take human and mice they share 92% of their genes. Thus, comparing different Bacillus subtilis strains may show from 79% to 97% homology precluding their individual efficacy.

Figure 1. Phylogenetic tree based on gyrB gene sequences of selected Bacillus reference strains (including DSM29784 strain).

DSM29784, A B. subtilis strain that provides consistency on in vivo performance even under challenged conditions

Table 1. Effect of Bacillus subtilis strains on growth performance in broilers from 1 to 35 days of age

Developing a consistent probiotic solution with actions on both performance and gut health is not so easy. Indeed, the final in vivo evaluation is necessary for full characterization, and it is important to determine the potential of a probiotic according to the environmental conditions. Indeed, it is often clearly admitted that challenging the animals, through built-up litter, enhancing densities, imbalanced protein diets might exacerbate the potential of probiotics. However, it should be kept in mind that challenges through very high feed intake, genetically inherent in nowadays used poultry strains, could induce more intestinal pressure on birds than “health” challenges. Indeed, recent trials performed with a new B. subtilis strain showed that the improvement of growth in low performing birds (1.6 kg BW at 35 days) was not better than the improvement measured in the highly performing ones (2.2 kg BW at 35 days). Table 1 shows the consistency of performance obtained with the new B. subtilis strain, over a range of dietary conditions and performance levels.

Necrotic enteritis, caused by Clostridium perfringens, is a critical intestinal disorder causing a lot of economic losses estimated to reach up to 6 billion$ per year worldwide. Together with dysbacteriosis or dysbiosis causing dis-balance of the intestinal microbiota, it has...

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