Characterization of the Tn916 Conjugative Transposon in a Food-Borne Strain of Lactobacillus paracasei

Food-borne antibiotic-resistant lactic acid bacteria have received growing attention in the past few years. We have recently identified tetracycline-resistant Lactobacillus paracasei in samples of milk and natural whey starter cultures employed in the manufacturing process of a typical Italian fermented dairy product, Mozzarella di Bufala Campana. In the present study, we have characterized at the molecular level the genetic context of tetracycline resistance determinants in these natural strains, which we have identified as tet(M). This gene was present in 21 independent isolates, whose fingerprinting profiles were distributed into eight different repetitive extragenic palindromic groups by cluster analysis. We provide evidence that the gene is associated with the broad-host, conjugative transposon Tn916, which had never before been described to occur in L. paracasei. PCR analysis of four independent isolates by use of specifically designed primer pairs detected the presence of a circular intermediate form of the transposon, carrying a coupling sequence (GGCAAA) located between the two termini of Tn916. This novel coupling sequence conferred low conjugation frequency in mating experiments with the recipient strain JH2-2 of Enterococcus faecalis.Several genetic determinants conferring tetracycline resistance have been described to occur in gram-positive, nonpathogenic bacteria (2, 20). Among them, tet(M), encoding a ribosomal protection protein, is most commonly found in lactic acid bacteria (LAB). The issue of antibiotic resistance spreading among commensal bacteria has received great interest in recent years, and the presence of antibiotic-resistant species in the environment, including food products, has been extensively reported (reviewed in references 2 and 20). Conjugative transposons represent important vehicles for dissemination of antimicrobial resistance within gram-positive and gram-negative bacteria (23). These elements can move from the genome of a donor bacterium to that of a recipient by conjugation (6). Tn916, an 18-kb element containing the genetic determinant for tetracycline resistance, was the first conjugative transposon to be identified. It carries the tet(M) gene and has a broad host range, comprising both gram-positive and gram-negative bacteria (7). Along with the tetracycline resistance gene, Tn916 carries the genes responsible for its own excision (xis) and integration (int) as well as the mob genes, which mediate conjugal transfer (4). The transposition process starts with excision of the transposon, mediated by the Int and Xis proteins, leading to the formation of a nonreplicative circular intermediate which is transferred to the recipient and integrates into a new target site. Excision represents the rate-limiting step and occurs through reciprocal, site-specific recombination between the nonhomologous regions located at the two termini of the integrated transposon, known as coupling sequences, which are retained in the circular intermediate (17).Lactobacillus paracasei belongs to the microbial group of LAB and represents, along with the closely related species Lactobacillus casei, one of the most common bacterial species employed in the food industry. It is naturally present in raw milk and in dairy products, such as typical cheeses obtained by traditional manufacturing procedures in different Mediterranean countries (1, 11, 18, 26). Moreover, due to its probiotic functions, it is also employed as food additive (3, 5). Among its beneficial properties for human health, a recent study suggested that L. paracasei can be considered a potential enhancer of systemic immunity (22). However, only a few studies analyzed antibiotic resistance in L. paracasei (15, 19).In the past few years, our studies have focused on the identification of genes responsible for antibiotic resistance in LAB isolated from traditional dairy foods manufactured without employing commercial starter cultures. Fermentation in such products is therefore carried out by natural starters, mostly reflecting the microbiological composition of raw milk, which is affected in turn by the environment in which the animals live. Moreover, selective pressure exerted by technological steps along the manufacturing procedure often has a deep impact on bacterial composition in the final product. The widespread use and misuse of antibiotics have applied strong selective pressure in the environment, favoring survival and spread of antibiotic-resistant species. It is therefore of special relevance to identify antibiotic resistance determinants in food-borne bacteria, their persistence along the production line of specific products, and their capability of horizontal transfer to those species that can colonize the human gut.In the present study, we have characterized at the molecular level a group of tetracycline-resistant L. paracasei isolates, previously identified in raw milk and natural whey starter cultures employed in the manufacture of the Italian traditional cheese Mozzarella di Bufala Campana (9). We provide evidence that in these isolates, tetracycline resistance is due to the presence of the conjugative transposon Tn916, carrying the tet(M) gene and capable of horizontal, interspecies transfer to the opportunistic pathogen Enterococcus faecalis via a circular intermediate containing a novel coupling sequence that confers a low-frequency-conjugation phenotype. Molecular analysis of the resulting primary E. faecalis transconjugants revealed the presence of a circular intermediate of Tn916 carrying the same coupling sequence found in the L. paracasei donor strains.