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Two Different Tetracycline Resistance Mechanisms, Plasmid-Carried tet(L) and Chromosomally Located Transposon-Associated tet(M), Coexist in Lactobacillus sakei Rits 9

Mohammed Salim Ammor,^1, Miguel Gueimonde,¹ Morten Danielsen,² Monique Zagorec,³ Angela H. A. M. van Hoek,⁴ Clara G. de los Reyes-Gavilán,¹ Baltasar Mayo,¹ and Abelardo Margolles^1*

Instituto de Productos Lácteos de Asturias (CSIC), Carretera de Infiesto s/n, 33300 Villaviciosa, Asturias, Spain,¹ Chr. Hansen A/S, Bøge Allé 10-12, 2970 Hørsholm, Denmark,² Unité Flore Lactique et Environnement Carné, UR309, INRA, Domaine de Vilvert, F-78350 Jouy-en-Josas, France,³ RIKILT - Institute of Food Safety, Wageningen UR, Bornsesteeg 45, Wageningen, The Netherlands⁴

Received 30 June 2007/ Accepted 26 December 2007

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 µg/ml), L. sakei Rits 9, containing both tetracycline-resistant determinants, had a MIC of <256 µg/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.

Published ahead of print on 11 January 2008.

Present address: Laboratory of Microbiology and Biotechnology of Foods, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece.