Protection activity of a novel probiotic strain of Bacillus subtilis against Salmonella Enteritidis infection Mongkol Thirabunyanon a,⇑ , Narin Thongwittaya b a Biotechnology Section, Faculty of Science, Maejo University, Chiang Mai 50290, Thailand b Faculty of Animal Science and Technology, Maejo University, Chiang Mai 50290, Thailand article info Article history: Received 30 September 2010 Accepted 12 August 2011 Keywords: Bacillus subtilis Disease Epithelial cells Infection Probiotic Salmonella Enteritidis abstract The activity of 240 bacterial isolates screened from the gastrointestinal tracts of native chickens were evaluated for use as a potential probiotic in food animal production in order to protect against animal diseases and reduce pathogenic contamination of human food products. In observing the antagonistic activity of 117 bacilli isolates, 10 of these isolates exhibited higher growth inhibition of seven foodborne pathogens, including Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Vibrio cholerae. Beneficial probiotic criteria from these isolates – which included non-pathogenicity, acid and bile salt tolerance, hydrophobicity, and adhesion to intestinal epithelial cells – exhibited that one isolate of NC11 had the most potential as a probiotic. 16S rRNA gene sequencing showed that this NC11 isolate was Bacillus subtilis. This B. subtilis NC11 was sen- sitive to all antibiotics and was not cytotoxic to intestinal epithelial cells. Reduction of S. Enteritidis attachment to the surfaces of intestinal epithelial cells via action of a cultured medium from B. subtilis NC11 was observed by scanning electron microscopy. B. subtilis NC11 cells, as well as the bacterial cul- tured medium or the cultured medium adjusted to pH 7, significantly inhibited S. Enteritidis invasion (P < 0.01) of intestinal epithelial cells. This study indicates that B. subtilis NC11 has characteristics of a potential probiotic, and exhibits strong inhibition activity against S. Enteritidis infection to intestinal epithelial cells. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Salmonella is a major foodborne pathogen that is found in poul- try products and can cause severe illness in humans. Diseases and syndromes such as enteric fever, bacteremia, focal infection, and enterocolitis are caused by this type of pathogenic bacterium. Human salmonellosis has become an important international pub- lic health and economic issue (Duguid and North, 1991; Velge et al., 2005). Human health protection through the elimination of foodborne pathogens from food animals and their products has been an increasing concern for all sectors of the food production chain (La Ragione and Woodward, 2003; La Ragione et al., 2001). The widespread use of antibiotics as therapeutic agents and growth promoters in animal husbandry has led to a worldwide in- crease in the antibiotic resistance of bacteria, an imbalance of nor- mal microflora, and drug residues in food products. Consequently these compounds have been banned as animal feed additives by the European Union (Perreten, 2003). One alternative method that has been recommended due to its successful application is the use of probiotics (Reuter, 2001). Our previous study (Samanya and Yamauchi, 2002) suggested that due to its probiotic potential, Bacillus subtilis var. natto is one strain that could be promoted as a biological product intended for humans or animals. There are many criteria that must be investigated before estab- lishing that a new strain of bacteria is a probiotic. These criteria must include the non-pathogenicity of the microorganism, its abil- ity to inhibit the growth of harmful organisms, its tolerance for acid and bile salt conditions, and its ability to adhere to intestinal epithelial cells (Salminen et al., 1998). Competitive exclusion is one of the modes of action of a beneficial probiotic that is exhibited to protect against infection from any pathogen in the intestinal epi- thelial cells of animal host. However, it is unclear whether the effective action of competitive exclusion by using probiotic Bacillus spp. will result in solo or combined actions in the gastrointestinal tract of the host, including immunomodulation, competition for adhesion sites, and production of antimicrobial agents (Patterson and Burkholder, 2003). Lactic acid bacteria (LAB) have been found to inhibit infection by Salmonella spp. to the intestinal epithelial cells (Tsai et al., 2005; Golowczyc et al., 2007). There was no report, however, of any effective action of probiotic Bacillus spp. in inhib- iting the infection of Salmonella in the intestinal epithelial cells. The aims of this study were to investigate the activity of isolates of Bacillus spp. for possible use as potential probiotics, and 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.08.008 ⇑ Corresponding author. Tel.: +66 5387 3535; fax: +66 5387 8225. E-mail address: mthirabun@yahoo.co.th (M. Thirabunyanon). Research in Veterinary Science 93 (2012) 74–81 Contents lists available at SciVerse ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc