Two different tetracycline resistance mechanisms, plasmid-carried tet(L) and chromosomally located transposon-associated tet(M), coexist in Lactobacillus sakei Rits 9

Abstract

Lactobacillus sakei is extensively used as functional starter culture in fermented meat products. One of the safety criteria of a starter culture is the absence of potentially transferable antibiotic resistance determinants. However, tetracycline-resistant L. sakei strains have already been observed. In this paper, we show that tetracycline resistance in L. sakei Rits 9, a strain isolated from Italian Sola cheese made from raw milk, is mediated by a transposon-associated tet(M) gene coding for a ribosomal protection protein and a plasmid-carried tet(L) gene coding for a tetracycline efflux pump. pLS55, the 5-kb plasmid carrying the tet(L) gene, is highly similar to the pMA67 plasmid recently described for Paenibacillus larvae, a species pathogenic to honeybees. pLS55 could be transferred by electroporation into the laboratory strain L. sakei 23K. While the L. sakei 23K transformant containing pLS55 displayed an intermediate tetracycline resistance level (MIC, <32 microg/ml), L. sakei Rits 9, containing both tetracycline-resistant determinants, had a MIC of <256 microg/ml, suggesting that Tet L and Tet M confer different levels of resistance in L. sakei. Remarkably, in the absence of tetracycline, a basal expression of both genes was detected for L. sakei Rits 9. In addition, subinhibitory concentrations of tetracycline affected the expression patterns of tet(M) and tet(L) in different ways: the expression of tet(M) was induced only at high tetracycline concentrations, whereas the expression of tet(L) was up-regulated at lower concentrations. This is the first time that two different mechanisms conferring resistance to tetracycline are characterized for the same strain of a lactic acid bacterium.

FIG. 1.

(A) Plasmid profile of L. sakei Rits 9 undigested (lane 1) and digested with PstI (lane 2). (B) PFGE analysis of total DNA from L. sakei Rits 9 undigested (lane 1) and digested with AscI (lane 2). (C) Southern blot analysis of the PFGE gel with the internal tet(M) probe. MWM, molecular weight marker.

FIG. 2.

Phylogenetic tree of homologs of the deduced Tet(L) and Tet(M) proteins (A and B, respectively). Protein accession numbers are given in brackets. Trees were constructed by the neighbor-joining algorithm and clustered by the unweighted-pair group method using average linkages, and bootstrap values (100 replicates) are given at the branch points. The distances refer to the percentages of different residues. Abbreviations: E. faecium, Enterococcus faecium; B. cereus, Bacillus cereus; M. haemolytica, Mannheimia haemolytica; A. pleuropneumoniae, Actinobacillus pleuropneumoniae; C. difficile, Clostridium difficile; E. coli, Escherichia coli; N. meningitidis, Neisseria meningitidis.

FIG. 3.

Genetic structure of the tet(L)-containing plasmid pLS55 (A) and L. sakei Rits 9 tet(M) and flanking regions (B). Arrows show the direction of transcription of the open reading frames. Relevant restriction sites and their locations are indicated. The genes which matched the highest homology scores and the homologies with the partial sequences of different transposons are indicated.

FIG. 4.

Relative expression levels of tet(L) and tet(M) in L. sakei Rits 9 grown in the presence of different tetracycline concentrations refereed to those obtained for the control culture (absence of antibiotic).