Assessment of antibiotic susceptibility within lactic acid bacteria strains isolated from wine

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Abstract

Susceptibility to 12 antibiotics was tested in 75 unrelated lactic acid bacteria strains of wine origin of the following species: 38 Lactobacillus plantarum, 3 Lactobacillus hilgardii, 2 Lactobacillus paracasei, 1 Lactobacillus sp, 21 Oenococcus oeni, 4 Pediococcus pentosaceus, 2 Pediococcus parvulus, 1 Pediococcus acidilactici, and 3 Leuconostoc mesenteroides. The Minimal Inhibitory Concentrations of the different antibiotics that inhibited 50% of the strains of the Lactobacillus, Leuconostoc and Pediococcus genera were, respectively, the following ones: penicillin (2, ≤ 0.5, and ≤ 0.5 μg/ml), erythromycin (≤ 0.5 μg/ml), chloramphenicol (4 μg/ml), ciprofloxacin (64, 8, and 128 μg/ml), vancomycin (≥ 128 μg/ml), tetracycline (8, 2, and 8 μg/ml), streptomycin (256, 32, and 512 μg/ml), gentamicin (64, 4, and 128 μg/ml), kanamycin (256, 64, and 512 μg/ml), sulfamethoxazole (≥ 1024 μg/ml), and trimethoprim (16 μg/ml). All 21 O. oeni showed susceptibility to erythromycin, tetracycline, rifampicin and chloramphenicol, and exhibited resistance to aminoglycosides, vancomycin, sulfamethoxazole and trimethoprim, that could represent intrinsic resistance. Differences were observed among the O. oeni strains with respect to penicillin or ciprofloxacin susceptibility. Antibiotic resistance genes were studied by PCR and sequencing, and the following genes were detected: erm(B) (one P. acidilactici), tet(M) (one L. plantarum), tet(L) (one P. parvulus), aac(6′)-aph(2ʺ) (four L. plantarum, one P. parvulus, one P. pentosaceus and two O. oeni), ant(6) (one L. plantarum, and two P. parvulus), and aph(3′)-IIIa (one L. plantarum and one O. oeni). This is the first time, to our knowledge, that ant(6), aph(3′)-IIIa and tet(L) genes are found in Lactobacillus and Pediococcus strains and antimicrobial resistance genes are reported in O. oeni strains.

Introduction

Lactic acid bacteria (LAB) are common microorganisms in foods and also constitute the natural intestinal microbiota of humans and most animals (Tannock, 1995). LAB are involved in different fermentative processes, including wine malolactic fermentation (Liu, 2002). The four genera of LAB that usually can be found in grape must and wine are Lactobacillus, Leuconostoc, Pediococcus and Oenococcus (that only includes the Oenococcus oeni species) (Ribereau-Gayon et al., 1998a). During malolactic fermentation, O. oeni is maintained as the principal species that carries out this secondary wine fermentation. In some cases, Lactobacillus, Leuconostoc and Pediococcus isolates have been implicated in undesirable effects during wine production (Ribereau-Gayon et al., 1998a).

A large variety of antibiotics are currently being used in medicine and veterinary, and among them we can consider the β-lactams (penicillin), aminoglycosides (streptomycin, gentamicin and kanamycin), glycopeptides (vancomycin), macrolides (erythromycin), tetracycline, fluoroquinolones (ciprofloxacin), chloramphenicol, trimethoprim and sulfamethoxazole. Bacteria can develop numerous strategies to counteract the action of antibiotics, although most of them can be included in three types of resistance mechanisms (Walsh, 2003): 1) inactivation of the antibiotic by some enzyme(s); 2) active efflux pumps that extrude the molecule outside the cell; and 3) modification of the subcellular target where the antibiotics bind. Nowadays, there are lot of data about the prevalence of antibiotic resistance and the mechanisms implicated in clinical bacteria, but there is scarce information about the antibiotic susceptibility or the presence of antibiotic resistance genes in commensal bacteria such as LAB isolated from food (Teuber et al., 1999, Çataloluk and Gogebakan, 2004, Flórez et al., 2005, Zhou et al., 2005) and no data do exist about antibiotic susceptibility neither in O. oeni strains nor in LAB of oenological origin. The objective of this work was to assess bacterial susceptibility to twelve antibiotics in LAB strains isolated from wine and analyse the presence of different resistance genes involved in antibiotic resistance mechanisms.

Section snippets

Bacterial strains

Seventy five LAB strains, recovered from 75 wine samples during malolactic fermentation (vintages 1995–2002, in La Rioja, Spain), were included in this study (38 unrelated Lactobacillus plantarum, 3 Lactobacillus hilgardii, 2 Lactobacillus paracasei, 1 Lactobacillus sp, 21 unrelated O. oeni, 4 Pediococcus pentosaceus, 2 Pediococcus parvulus, 1 Pediococcus acidilactici and 3 Leuconostoc mesenteroides). Strains were initially identified by classical microbiological methods (gram staining, oxidase

Antimicrobial susceptibility in Lactobacillus, Leuconostoc and Pediococcus strains

Table 2 shows the MIC values obtained for the different antibiotics tested in our series of Lactobacillus, Leuconostoc, and Pediococcus strains.

Penicillin MIC values detected in the strains of these genera were low (MIC range from ≤ 0.5 to 4 μg/ml) and among them, L. plantarum showed slightly higher MICs (MIC50 2 μg/ml), than the other tested species (MIC50 0.5 μg/ml). Our observations are in accordance with the results of Danielsen and Wind (2003), who also reported low MICs of penicillin

Acknowledgements

This work was supported in part by a grant from the Ministry of Research and Science of Spain (AGL2002-2293) and FEDER of the European Community. B. Rojo-Bezares was supported by a doctorate grant from the Spanish Ministry of Education and Science (ref BI-UR-03/16586.840) and Y. Sáenz is a contractual technician supported by grant PTA-2003-02-00002 from the Spanish Ministry of Education and Science.

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