Antibiotic sensitive Bacillus strains having antimicrobial effect against E. coli and Clostridium perfringens and having high sporulation capacity

#The present application is the U.S. National Stage of International Application No. PCT/EP2013/057590, filed on Apr. 11, 2013, which claims the benefit of European Patent Application No. 12164087.4, filed on Apr. 13, 2012.

#Bacillus spp are used for probiotic solutions in the animal feed industry and positive effects of Bacillus based probiotics on production and health in production animals are well known (Spiehs et al., 2008; Cutting, 2011). Their usage is related to the ability of Bacillus to replace or reduce the use of antibiotics, which are used as growth promoters in the animal feed industry.

#However, there is an unmet need for Bacillus strains which do not have antibiotic resistance against antibiotics which are commonly used for humans. The present invention provides isolated Bacillus strains which are characterized by sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol and which also have antimicrobial activity against major pathogens such as E. coli and Clostridium perfringens. The strains further have a sporulation percentage of at least 80 when measured on day 2 making it possible to efficiently produce safe and useful Bacillus spores for animal feed production.

#FIG. 1 shows schematically the antimicrobial activity of 261 Bacilli strains. It is surprising that many Bacillus amyloliquefaciens strains have antimicrobial effect.

#The phase-out of antibiotic growth promoters in the European Union in 2006 has resulted in an increased need for cost-effective feed additives with high efficacy and thus the need for new probiotics. Bacillus-based probiotic feed additives are known for their positive effects on health and production in pigs and poultry. These products are relevant for the feed industry because spores are heat stable and can survive the pelletizing process at temperatures up to 90-95° C.

#Probiotics for pigs need to be safe for animals, humans and the environment and should increase growth and feed utilization of the animal. The objective of the present invention was to screen in three steps a wide range of aerobic endosporeforming bacteria (AEB) from different sources for their probiotic effect in pigs. The AEB were isolated from fermented food (Kantong, and Gergoush primary starters), pig feces, soil and different culture collections. 261 AEB isolates were identified by sequencing of 16S rDNA genes, and investigated for relevant antibiotic resistance by determination of the minimal inhibitory concentration (MIC) of several relevant antibiotics.

#Further analyses included bile and acid tolerance, pathogen inhibition, growth in different media, sporulation as well as interactions with animal cell lines to assess the likelihood of positive effects on tight junctions in the intestinal system. Results show a high difference between both species and strains. The isolated species were primarily of the genus Bacillus including B. amyloliquefaciens, B. subtilis and B. safensis from food sources, B. subtilis, B. pumilus, B. amyloliquefaciens, B. licheniformis, B. megaterium from feces and B. licheniformis and B. simplex from soil.