S6a-4

PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF
LACTOBACILLUS SAKEI ISOLATED FROM PORTUGUESE
FERMENTED MEAT PRODUCTS FOR TETRACYCLINE
RESISTANCE
Martins, A., Makita, M., Fernandes M.H., Fernandes M.J., Barreto A.S. and Fraqueza, 1Faculty of Veterinary Medicine, UTLisbon, Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Av. da Universidade Técnica, Pólo Universitário da Ajuda, 1300-477, Lisbon, Portugal Abstract –The resistance profile presented by 31 different Lactobacillus sakei starter strains was
investigated. These strains ability to function as resistant gene pools has granted an important
focus, not only but also, regarding tetracycline resistance. Antibiotic susceptibility testing was
performed by disc diffusion of the putative resistance responsible main genes, tet(K), tet(L) and tet
(M) detection was done by multiplex-PCR. We found that between 87% and 97% of strains were
resistant to vancomycin and trimethoprim–sulfamethoxazole. Also most strains were susceptible to
protein synthesis inhibitors such as erythromycin, dalfopristin-quinupristin both with 19%
resistance and chloramphenicol, and tetracycline with 29% resistant strains. For the
phenotypically identified tetracycline resistant strains only one was detected as carrier for the tet
(M) gene while 7 of the strains carried the tet(K). No strains were found to carry tet(L). Also,
genetic resistance determinants for macrolides, as major antibiotic therapeutic alternatives, should
also be evaluated for these same strains. Still, from our results, L. sakei can be selected as safe
starter strains to be used on Portuguese traditional meat fermented products.
Key Words – Antibiotic resistance, LAB, Tet(M)
The genus Lactobacillus belongs to the lactic acid group of bacteria (LAB), these are fastidious, gram positive bacteria found in different environments from plant material, through manure, till meat and dairy products [1]. Regarding Lactobacillus spp. in food the status of generally regarded as safe (GRAS) has been attributed to these microorganisms mainly due to their frequent used on a large-scale as starter cultures in food industries (e.g. in the production of fermented milk products or sausages) without imposing a health risk for the consumers or the environment [1, 2]. Although most species proved to be susceptible to the majority of tested antibiotics resistance to tetracycline and erythromycin has been occasionally detected [4]. Lactobacilli potential to pose as antibiotic resistance gene transfer vectors, capable of supplying antibiotic resistance genes to food-borne or enteric pathogens,associated with their broad distribution in various environments translates into a potentially major concern for public health safety [5, 7].
Tetracyclines are a group of broad-spectrum antibiotics whose general usefulness has been reduced with the onset of bacterial resistance. Tetracycline resistance (TcR) can be mainly due to two different mechanisms. Associated with the activity of ribosomal protection proteins, RPPs, linked to the expression of various tet genes (e.g. tet(M)) and also with efflux proteins as those encoded by tet(K) and tet(L) [5, 7, 8]. Both efflux and RPP encoding genes are commonly associated with mobile genetic elements and is mostly acquired and passed by horizontal gene transfer to other, either commensal or pathogenic, bacteria [5, 7, 8].
The Lactobacillus sakei tested strains used in this study belonged to the collection of the Laboratório de Tecnologia e Segurança dos Alimentos at the Faculty of Veterinary Medicine of Lisbon (UTL). L. sakei strains (n=31) were isolated from different Portuguese fermented/dry/smoked meat products (Linguiça. Paio, Chouriço de vinho, Chouriço de carne, Chourição and Salsichão grosso) originating from different industries on Alentejo region (South Portugal) and collected during 2011/12. Strains were grown on MRS agar for 48h at 30ºC under adequate anaerobic conditions. Antibiotic susceptibility testing by disc diffusion was performed as described in CLSI,[6] for 7 different antibiotics: vancomycin, 30µg; trimethoprim–sulfamethoxazole, 25µg; erythromycin, 15µg; tetracycline, 30µg; gentamicin, 10µg; chloramphenicol, 30µg; and dalfopristin-quinupristin, 15µg. Staphylococcus aureus ATCC 29213 and Enterococcusfaecalis ATCC 29212 were used as quality control strains. Strains were kept for 48h at 30ºC under adequate anaerobic conditions for posterior halo size readings.
Detection of TcR genes was performed by multiplex-PCR according to Gevers et al.[3] for 3 tetracycline resistance associated genes tet(K); tet(M) and tet(L). Control strains were kindly provided by Prof. Constança Pomba and included Staphylococcus aureus PR7/08, gene tet(M) and tet(K) carrying strain and Staphylococcus aureus 124.1, gene tet(M) and tet(L) carrying strain.
 RESULTS AND DISCUSSIONPhenotypic strains characterization regarding antibiotic susceptibility results are shown in Figure 1. As previously described heterofermentative Lactobacilli such as L. sakei are intrinsically resistant to vancomycin, as well as trimethoprim/sulfamethoxazole [7, 8]. Our results revealed that, 87% (n=27) and 97% (n=30) resistant strains, for both mentioned antibiotics respectively. Lactobacilli are typically susceptible to protein synthesis inhibitors. However was reported resistance for erythromycin, 19% (n=6); chloramphenicol, 29% (n=9); dalfopristin-quinupristin, 19% (n=6) and tetracycline, 29%, (n=9). Aminoglycosides presented an exception, which was also corroborated in the present study with 61% of resistance (n=19).
Figure 1. Disc susceptibility testing results for all tested L. sakei strains. V, vancomycin; STX, Trimethoprim/sulfamethoxazole; E, erythromycin; G, gentamicin; C, chloramphenicol; QD, dalfopristin-quinupristin and Te, tetracycline. R indicates resistance and S indicates Resistance to tetracycline has been mainly attributed to the existence of, not only, ribosomal protection proteins, encoded by one or more of tet(M), tet(O) and tet(W). But also, to tet efflux genes of which tet(K) and tet(L) have been primarily found in gram positive bacteria such as LAB [5, 8]. Both mentioned resistance mechanisms have been linked to mobile transferable genetic elements [5, 8]. In our study was found that none of the tested strains carried the tet(L) gene and although some strains, 7, carried the tet(K) gene; no correlation was assured between the occurrence of this efflux gene and the development of resistance due to the fact that the gene was only present in 2 of the 9 resistant strains and also present in phenotypically susceptible strains (n=5). On the other hand, tet(M) was described as the main gene attributed to tetracycline resistance, although from our preliminary results we were only able to detect one resistant strain carrying this gene further PCR protocol optimization it will be required before inferring an alternative resistance mechanism [5, 7, 8]. As previously described both tet(L) and tet(M) are, in different ways, mainly responsible for tetracycline resistance patterns. Of the tested 31 L. sakei strains only one carried both genes tet genes and expressed such resistance [5, 7, 8]. Lactic acid bacteria such as the fermented meat product starter L. sakei may act as relevant genetic reservoir for antibiotic resistance genes that are horizontally transferable to either commensal or pathogenic bacteria. Still, from our results, L. sakei can be selected as safe starter strains to be used on Portuguese traditional meat fermented products.
Tetracycline resistance was found for 29% of tested strains. None of the strains had the tet(L), 7 strains carried the tet(K) although 2 were phenotypically resistant. It was only possible to confirm the existence of tet(M) for one strain. This same strain was tet(K) positive and expressed TcR. Still, from our results, L. sakei can be selected as safe starter strains from all susceptible to antibiotics, and be used on Portuguese traditional meat fermented products.
 Ammor, M.S., Gueimonde, M., Danielsen, M., Zagorec, M., van Hoek, A.H., de Los Reyes- Gavilan, C.G., Mayo, B. & Margolles, A. (2008). Two different tetracycline resistance mechanisms, plasmid carried tet(L) and chromosomally located transposon-associated tet(M), coexist in Lactobacillus sakei Rits 9. Applied and Environmental Microbiology 74(5), 1394-1401.
 Gevers, D., Danielsen, M., Huys, G. & Swings, J. (2003a). Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage. Applied and Environmental Microbiology 69(2), 1270-1275.
 Gevers, D., Huys, G. & Swings, J. (2003b). In vitro conjugal transfer of tetracycline resistance from Lactobacillus isolates to other Gram positive bacteria. FEMS Microbiology Letters 225(1): 125-130.
 Teuber, M., Meile, L. & Schwarz, F. (1999). Acquired antibiotic resistance in lactic acid bacteria from food. Antonie Van Leeuwenhoek 76(1-4): 115-137.
 Danielsen, M. & Wind, A. (2003). Susceptibility of Lactobacillus spp. to antimicrobial agents. International Journal of Food Microbiology 82(1): 1-11.
 CLSI (2008). Clinical and Laboratory Standards Institute. 2008. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M07-A8. Clinical and Laboratory Standards Institute, Wayne, Pa  Egervärn, M., Danielsen, M., Roos, S., Lindmark, H. and Lindgren, S. (2007). Antibiotic susceptibility profiles of Lactobacillus reuteri and Lactobacillus fermentum. Journal of Food Protection 70(2):412-418.
 Egervärn, M., Roos, S. and Lindmark, H. (2009). Identification and characterisation of antibiotic resistance genes in Lactobacillus reuteri and Lactobacillus plantarum. Accepted for publication in Journal of Applied Microbiology.

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