Susceptibility of Lactobacillus spp. to antimicrobial agents

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Abstract

Bacteria used as probiotics or in starter cultures may serve as hosts of antibiotic resistance genes, which can be transferred to pathogenic bacteria. Before launching a starter culture or a probiotic product into the market, it is therefore important to verify that the single bacterial isolates (strains) do not contain transferable resistance genes. A study has been undertaken to establish the levels of susceptibility of Lactobacillus spp. to various antimicrobial agents. This is a prerequisite for differentiating putative transferable resistance from natural resistance. A selection of 62 strains has been screened with the use of the Etest (ABBiodisk, Stockholm, Sweden) for their susceptibility to 25 antimicrobial agents. The strains belonged to the following species: Lactobacillus plantarum/pentosus, L. rhamnosus, L. paracasei, L. sakei, L. curvatus and species of the L. acidophilus group: L. johnsonii, L. crispatus, L. gasseri, and L. acidophilus.

The results from the Etests have shown that the level of susceptibility to the antimicrobial agents is species-dependent. For the following antimicrobial agents, susceptibility varied several folds between species: vancomycin, teicoplanin, tetracycline, norfloxacin, ciprofloxacin, fusidic acid, and clindamycin. The differences between the species were more subtle for the rest of the tested antimicrobial agents. On the basis of the result, it was possible to suggest minimal inhibition concentrations (MICs) for the individual Lactobacillus species to be used as a microbiological breakpoint when screening strains for transferable resistance genes.

Introduction

In recent years, increased focus has been given to food as potential vehicles of antibiotic resistance genes Perreten et al., 1997, Franz et al., 1999, Klein, 2000. It is of general belief that starter cultures have the potential to serve as a reservoir of such genes with the risk of transferring the genes to pathogenic bacteria. The presence of resistance genes in many lactic acid bacteria (LAB) and the transfer of plasmids and conjugative transposons to and from LAB have been reported as reviewed by Teuber et al. (1999).

Probiotics for use in the EU as additives in feedingstuffs must comply with the guidelines described in the EU council directive 87/153/EEC. In 1997 (updated 2001), a report by the Scientific Committee for Animal Nutrition (SCAN) recommended to the EU commission that the absence of transferable resistance genes should be an important prerequisite for approval (EC, 2001a). In an opinion from the SCAN on the 3rd of July 2001, it is explicitly told how to screen strains for the absence of transferable resistance genes (EC, 2001b). A number of microbiological breakpoints are given on the basis of two published articles Barrett and Jones, 1996, Zarazaga et al., 1999. Any level of susceptibility, which cannot be accounted for, is subject to further analysis by in vitro experiments to be proven intrinsic or mutational (EC, 2001b). The coming EU guidelines for transferable resistance genes in probiotics for animals will most likely be extended to probiotics for humans and starter cultures in the near future EC, 2000, EC, 2001b. Similar guidelines might also be implemented in the US as increased monitoring of antibiotic resistance to antimicrobial agents is planned (ITFAR, 2000).

Published information comparing MICs of individual Lactobacillus species and including a fair number of isolates, to our knowledge, is limited to the works of Felten et al. (1999) and Zarazaga et al. (1999). Both articles stress the need for differentiating between individual Lactobacillus species. Furthermore, data have been published on the screening of Lactobacillus delbrueckii subsp. bulgaricus and lactis (Katla et al., 2001) and L. rhamnosus (Charteris et al., 2001). Knowledge about natural resistance for individual Lactobacillus species will lessen the need for extensive experiments as suggested by SCAN and increase the safety of new products. This study has been carried out to make a contribution to the establishment of relevant microbiological breakpoints for a number of Lactobacillus species.

Section snippets

Bacterial strains

A selection of 62 strains from the Chr. Hansen Culture Collection was used for the survey. In total, 18 L. plantarum/pentosus, 13 L. paracasei, 9 L. rhamnosus, 5 L. sakei, 1 L. curvatus, and 16 from the L. acidophilus group (3 L. acidophilus, 3 L. johnsonii, 6 L. crispatus, and 4 L. gasseri) were used for the study. L. paracasei, L. rhamnosus, and L. crispatus have been identified to species level by 16S rRNA sequencing Mori et al., 1997, Pavlova et al., 2002. L. sakei, L. curvatus, L.

Results

In Table 1, Table 2, Table 3, Table 4, the results are grouped together on the basis of the mechanisms of action (Yao and Moellering, 1999). The β-lactams are presented in Table 1. In Table 2 are the non-β-lactam cell wall synthesis acting antimicrobial agents. The antimicrobial agents that inhibit protein synthesis or mRNA synthesis are presented in Table 3. The remaining antimicrobial agents are presented in Table 4. The results for L. sakei and L. curvatus are presented together as the

Discussion

A study of the surveys of antimicrobial agent resistance in lactobacilli showed that a variety of methods had been used (Etest: Croco et al., 1994, Herra et al., 1995, Felten et al., 1999, Charteris et al., 2001, Katla et al., 2001; agar dilution: Dutta and Devriese, 1981, Zarazaga et al., 1999, Goldstein et al., 2000; disk diffusion: Sozzi and Smiley, 1980, Charteris et al., 1998; and microbroth: Klein et al., 2000). In order to have as much information from the susceptibility testing as

Acknowledgements

We thank Kelli Doherty and Elise Pedersen for excellent technical assistance, Kim I. Sørensen for careful reading of the manuscript, and Elke Brockmann for the providing the data on the species identification. This work was supported by the Danish Academy of Technical Sciences grant EU 843.

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