Int. J. Mol. Sci. 2013, 14, 4640-4654; doi:10.3390/ijms14034640 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article Incidence of Bacteriocins Produced by Food-Related Lactic Acid Bacteria Active towards Oral Pathogens Georgia Zoumpopoulou 1, Eudoxie Pepelassi 2, William Papaioannou 3, Marina Georgalaki 1, Petros A. Maragkoudakis 1,†, Petros A. Tarantilis 4, Moschos Polissiou 4, Effie Tsakalidou 1 and Konstantinos Papadimitriou 1,* 1 Laboratory of Dairy Research, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece; E-Mails: [email protected] (G.Z.); [email protected] (M.G.); [email protected] (P.A.M.); [email protected] (E.T.) 2 Department of Periodontology, School of Dentistry, National and Kapodistrian University of Athens, 2 Thivon Str., Athens 11527, Greece; E-Mail: [email protected] 3 Department of Preventive and Community Dentistry, School of Dentistry, National and Kapodistrian University of Athens, 2 Thivon Str., Athens 11527, Greece; E-Mail: [email protected] 4 Laboratory of Chemistry, Department of Science, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece; E-Mails: [email protected] (P.A.T.); [email protected] (M.P.) † Present address: European Commission, Joint Research Centre, via E. Fermi 2749, Ispra 21027, Varese, Italy; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +30-210-529-4661; Fax: +30-210-529-4672. Received: 8 October 2012; in revised form: 1 February 2013 / Accepted: 18 February 2013 / Published: 26 February 2013 Abstract: In the present study we investigated the incidence of bacteriocins produced by 236 lactic acid bacteria (LAB) food isolates against pathogenic or opportunistic pathogenic oral bacteria. This set of LAB contained several strains (≥17%) producing bacteriocins active against food-related bacteria. Interestingly only Streptococcus macedonicus ACA-DC 198 was able to inhibit the growth of Streptococcus oralis, Streptococcus sanguinis and Streptococcus gordonii, while Lactobacillus fermentum ACA-DC 179 and Lactobacillus plantarun ACA-DC 269 produced bacteriocins solely against Streptococcus oralis. Thus, the percentage of strains that were found to produce bacteriocins against oral bacteria was ~1.3%. The rarity of bacteriocins active against oral LAB pathogens produced by food-related Int. J. Mol. Sci. 2013, 14 4641 LAB was unexpected given their close phylogenetic relationship. Nevertheless, when tested in inhibition assays, the potency of the bacteriocin(s) of S. macedonicus ACA-DC 198 against the three oral streptococci was high. Fourier-transform infrared spectroscopy combined with principal component analysis revealed that exposure of the target cells to the antimicrobial compounds caused major alterations of key cellular constituents. Our findings indicate that bacteriocins produced by food-related LAB against oral LAB may be rare, but deserve further investigation since, when discovered, they can be effective antimicrobials. Keywords: lactic acid bacteria; food; oral; Streptococcus; bacteriocin; FT-IR 1. Introduction Probiotics, defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” [1], have become a major topic of lactic acid bacteria (LAB) research over the last two decades. Although a number of health-promoting effects arising from the use of probiotics have been demonstrated, so far the main field of research has been the gastrointestinal tract [2]. Probiotics have been poorly investigated from the oral health perspective. However, this is gradually changing [3]. Dental plaque is a biofilm that develops naturally on the teeth and is composed of about 700 bacteria species that take part in the complex ecosystem of the oral cavity [4]. Interactions among these bacterial species, from the early stages of colonization to the formation of mature supragingival and subgingival plaque, are the main etiological agents of oral infectious diseases, such as dental caries, gingivitis and periodontitis [5]. Dental caries is a multifactorial disease of bacterial origin that is characterized by acid demineralization of the tooth enamel as a result of the proliferation of streptococci from the mutans group [6]. Gingivitis and periodontitis are largely caused by specific Gram-negative bacterial infections. Such infections lead to the destruction of the connective tissue and the disruption of the underlying alveovar bone and periodontal ligament supporting the teeth [7]. Moreover, in recent years it has been suggested that oral bacteria are associated with many systemic diseases, such as pneumonia and cardiovascular disease [8,9]. Hence, the need for oral care in a systemic health regimen has been emphasized. Generally, dental plaques that have been deposited firmly as biofilms are removed mechanically while antibacterial mouthwashes are also effective in decreasing tooth surface plaque. In general, mouthwashes contain fluorides, alcohols, detergents or antibacterial substances. Ideal antibacterial substances must be effective against pathogenic or opportunistic pathogenic strains related to oral health, act rapidly, maintain activity at low concentrations, and have no side effects [10]. In this context, bacteriocins isolated from food related LAB could be used as alternative antimicrobials [11,12]. In the present study, the aim was to investigate the antimicrobial activity of LAB strains isolated from food products against oral pathogenic or opportunistic pathogenic bacteria. The inhibitory activity of food LAB strains was studied towards both Gram-negative and Gram-positive strains of bacteria associated with oral health. Moreover, the involvement of proteinaceous compound(s) to this Int. J. Mol. Sci. 2013, 14 4642 activity was examined. Finally, Fourier-transform infrared (FT-IR) spectroscopy was employed in order to unravel the changes at the cellular level caused by these antimicrobial substances on target bacteria. 2. Results and Discussion 2.1. Screening LAB Food Isolates for the Production of Bacteriocins Active against Oral Pathogens A total of 236 LAB strains of food origin (Table 1) were screened for antimicrobial activity against 10 target strains (3 Gram-negative and 7 Gram-positive). Table 1. Genus and species of the lactic acid bacteria used as potential producers of bacteriocins. Genus/species Number of strains Bacilli Lactobacillus acidipiscis 1 Lactobacillus acidophilus 3 Lactobacillus brevis 2 Lactobacillus delbrueckii subsp. bulgaricus 11 Lactobacillus delbrueckii subsp. delbrueckii 1 Lactobacillus delbrueckii subsp. lactis 4 Lactobacillus fermentum 3 Lactobacillus gasseri 6 Lactobacillus paracasei subsp. paracasei 17 Lactobacillus paracasei subsp. tolerans 1 Lactobacillus plantarum 52 Lactobacillus rennini 11 Lactobacillus rhamnosus 1 Lactobacillus sanfransinscensis 1 Lactobacillus sp. 25 Cocci Enterococcus faecalis 3 Enterococcus faecium 2 Lactococcus lactis 41 Leuconostoc lactis 2 Leuconostoc mesenteroides subsp. dextranicum 4 Pediococcus pentosaceus 7 Streptococcus bovis 1 Streptococcus macedonicus 5 Streptococcus thermophilus 32 Within the set of strains to be tested as bacteriocin-producers against oral bacteria, there were a number of strains that had been screened in our laboratory against food-related bacteria and were found to produce bacteriocins (Supplementary Table 1). From the total of 236 strains, approximately 140 had been previously screened and from those 41 were found positive based on the well diffusion assay (WDA, see Experimental Section). Thus, a conservative estimate of the percentage of bacteriocin-producing strains in the total of strains tested in the present study would be ≥17%. Different sets of target strains were used including common indicator strains employed by different Int. J. Mol. Sci. 2013, 14 4643 laboratories in similar studies (e.g., Lactococcus lactis CNRZ 117, Lactobacillus sakei LMG 2313, Listeria innocua BL86/26, etc.), as well as in-house indicator strains (e.g., Lactococcus lactis ACA-DC 49, Streptococcus thermophilus ACA-DC 4, Lactobacillus delbrueckii subsp. bulgaricus ACA-DC 84, etc.). Even though for some of the commonly used indicator strains their food origin is established (e.g., for Lactobacillus helveticus ATCC 15009 and L. lactis CNRZ 117, which are isolated from dairy products), for others (e.g., L. innocua BL86/26) the isolation source is not available in the literature or in the databases of culture collections. Our in-house indicator strains were all food isolates of dairy origin. In the current screening assay, in order to increase the possibility for bacteriocin production, all potential producers were grown in three different culture media, namely skim milk containing yeast extract, MRS and M17. The respective cell-free culture supernatants (CFCSs) were tested towards the target bacteria by the WDA. In case of positive results, the 10-fold concentrated CFCSs, obtained after ammonium sulfate precipitation, were also tested for antimicrobial activity against the sensitive bacteria by the WDA. None of the LAB strains was able to inhibit any of the Gram-negative strains used as target strains, apart from three strains, namely Lactococcus lactis ACA-DC 46 (Formaela cheese isolate), and E. faecium ACA-DC 3350 and ACA-DC 3359 (both Feta cheese isolates). More precisely, the milk CFCS of L. lactis, as well as the MRS CFCSs of both E. faecium strains gave a “borderline”