Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage

Abstract

The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc(r)) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)(5)-PCR DNA fingerprinting technique, the Tc(r) lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tc(r) lactic acid bacterial isolates displaying unique (GTG)(5)-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes.

Figures

FIG. 1.

Composite figure of (GTG)5-PCR fingerprints and inverted plasmid profiles of the 24 Tcr Lactobacillus isolates from FDS end products. The dendrogram was constructed after cluster analysis of the digitized (GTG)5-PCR fingerprints with the unweighted pair group method with arithmetic averages, with correlation levels expressed as a percentage of the Pearson correlation coefficient. Cophenetic correlations (shown on each branch of the dendrogram) indicate how faithfully the dendrogram represents the similarity matrix. A small triangle or circle indicates the place where the tet(M) or erm(B) probe, respectively, hybridized on the Southern blot of the plasmid DNA. Lactococcus lactis subsp. cremoris strain AC1 was used as a plasmid size marker (24). The tet(M)-1 and tet(M)-2 allele types correspond to the tet(M) genes found in Neisseria meningitidis (X75073) and Staphylococcus aureus MRSA 101 (M21136), respectively, based on restriction enzyme analysis (REA) with AccI and ScaI. Numerical designations indicate the fermented dry sausage type, and the letters A to D indicate different batches (type and batch are defined in Materials and Methods). tet(M) localization was determined by Southern blot analysis. The MIC of tetracycline was determined by Etest. Chr., chromosomal band.

FIG. 2.

Single most parsimonious tree (unrooted) for tet(M) gene relationships of the 24 Tcr lactic acid bacterial isolates and two reference strains, Neisseria meningitidis (X75073) and Staphylococcus aureus MRSA 101 (M21136). The recently published tet(M) gene of L. plantarum 5057 (AF440277) was included (8). The numbers of nucleotide substitutions are indicated on each branch. The bootstrap percentages (500 replicates) are indicated for the separation between the two homology groups.