International Journal of Antimicrobial Agents 40 (2012) 127–134 Contents lists available at SciVerse ScienceDirect International Journal of Antimicrobial Agents jou rn al h om epa ge: h ttp://www.elsevier.com/locate/ijantimicag Purification and genetic characterisation of the novel bacteriocin LS2 produced by the human oral strain Lactobacillus salivarius BGHO1 Milos Busarcevic a,∗ , Michèle Dalgalarrondo b a Laboratory for Molecular Genetics of Industrial Microorganisms, Institute of Molecular Genetics and Genetic Engineering, Belgrade, Serbia b UR 1268 Biopolymers Interactions Assemblies, National Institute of Agronomic Research, Functions and Interactions of Dairy Proteins, B.P. 71627, 44316 Nantes Cedex 3, France a r t i c l e i n f o Article history: Received 4 January 2012 Accepted 12 April 2012 Keywords: Lactobacillus salivarius Antimicrobial peptide Probiotic Oral cavity Pathogenic microorganisms a b s t r a c t The aim of this study was to investigate the antimicrobial potential of Lactobacillus salivarius BGHO1, a human oral strain with probiotic characteristics and a broad inhibitory spectrum both against Gram- positive and Gram-negative pathogens. Here we present the bacteriocin LS2, an extremely pH- and heat-stable peptide with antilisterial activity. LS2 is a novel member of the class IId bacteriocins, unique among all currently characterised bacteriocins. It is somewhat similar to putative bacteriocins from several oral streptococci, including the cariogenic Streptococcus mutans. LS2 is a 41-amino-acid, highly hydrophobic cationic peptide of 4115.1 Da that is sensitive to proteolytic enzymes. LS2 was purified from cells of strain BGHO1 by solvent extraction and reverse-phase chromatography. Mass spectrometry was used to determine the molecular mass of the purified peptide. N-terminal amino acid sequencing enabled identification of the LS2 structural gene bacls2 by a reverse genetics approach. Downstream of the bacls2 gene, two bacteriocin-like genes were found, named blp1a and blp1b, and one putative bacteriocin immunity gene named bimlp. We also present the identification of the 242-kb megaplasmid pMPHO1 by pulsed-field gel electrophoresis, which harbours the genes bacls2, blp1a, blp1b and bimlp. Two peptides with antimicrobial activity, whose approximate sizes corresponded to those of blp1a and blp1b, were identified only after culturing strain BGHO1 in a chemically defined medium. This study demonstrated the capacity of Lactobacillus salivarius BGHO1 to produce multiple bacteriocins and further established this strain as a promising probiotic candidate. © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. 1. Introduction Modern medicine is faced with an alarming increase in multi- ple antibiotic resistance among virtually all pathogenic bacteria. An urgent need for novel antimicrobial agents has therefore been initi- ated by many research groups to develop alternatives to antibiotic treatment. One such alternative is bacteriocin-producing probi- otic strains of lactic acid bacteria (LAB). Probiotics, as defined by the World Health Organization (WHO), are ‘live microorgan- isms which, when administered in adequate amounts, confer a health benefit on the host’. LAB, a group of Generally Regarded As Safe (GRAS) microorganisms, have a long-documented history of being used as probiotics to control gastrointestinal diseases, where they can act by altering the gut microflora as well as by modulation of the host’s immune response and the production of antibacterial substances [1]. Bacteriocins are a diverse group ∗ Corresponding author. Present address: Functional Genomics Lab, Cluster in Biomedicine (CBM), Strada Statale 14–km 163,5, AREA Science Park, 34149 Trieste, Italy. Tel.: +39 348 468 0089; fax: +39 040 375 7710. E-mail addresses: milos.busarcevic@cbm.fvg.it, mbusarac@gmail.com (M. Busarcevic). of ribosomally synthesised antimicrobials produced by bacteria, which display either narrow- or broad-spectrum antimicrobial activity [1]. The oral cavity is a structurally complex niche inhabited by extremely diverse microbiota [2]. The incidence of oral diseases caused by bacteria is at a dramatic pandemic scale [3]. In addition to their direct involvement in the pathogenesis of caries and peri- odontitis, oral pathogens are associated with general death risk, gastric cancer and cardiovascular disease [4–6]. The study of bacterial replacement therapeutic strategies deploying bacteriocinogenic probiotics has increased over the last two decades. Several probiotic LAB strains of non-oral ori- gin were previously demonstrated to control the growth of oral microorganisms, including cariogenic streptococci [7]. However, it is reasonable to expect that probiotic bacteria of oral origin would be better adapted to colonise and persist on dental and mucosal sur- faces, where they could contribute to the host’s health. Until now, only seven oral cavity bacteriocins have been fully characterised [8–11] and only one oral strain, Streptococcus salivarius K12, has been successfully used as a probiotic in the oral cavity [8]. Owing to the increasing problem of antibiotic resistance, novel oral bac- teriocins and probiotics could be a promising therapy and deserve further research [12,13]. 0924-8579/$ – see front matter © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. http://dx.doi.org/10.1016/j.ijantimicag.2012.04.011