Antibiotic resistance of Lactobacillus pentosus and Leuconostoc pseudomesenteroides isolated from naturally-fermented Aloreña table olives throughout fermentation process

https://doi.org/10.1016/j.ijfoodmicro.2013.11.025Get rights and content

Highlights

  • Aantibiotic resistance of LAB was shown to at least three antibiotics.

  • As deduced by PCA, the prevalence of antibiotic resistance in LAB was variable during fermentation.

  • The intrinsic resistance may be due in part to efflux pumps.

  • All LAB tested are safe because they lack transferable resistance-related genes.

Abstract

Antimicrobial resistance of Lactobacillus pentosus (n = 59) and Leuconostoc pseudomesenteroides (n = 13) isolated from Aloreña green table olives (which are naturally-fermented olives from Málaga, Spain) to 15 antibiotics was evaluated. Most Lb. pentosus (95%) and all Lc. pseudomesenteroides were resistant to at least three antibiotics. Principal component analysis determined that the prevalence of antibiotic resistance in LAB throughout the fermentation process was highly dependent on the fermenter where the fermentation took place. All Lb. pentosus and Lc. pseudomesenteroides strains were highly sensitive to amoxicillin and ampicillin (MIC  2 μg/ml), and also to chloramphenicol (MIC  4 μg/ml), gentamicin and erythromycin (MIC  16 μg/ml). However, they were phenotypically resistant to streptomycin (83–100%, MIC > 256 μg/ml), vancomycin and teicoplanin (70–100%, MIC > 128 μg/ml), trimethoprim (76% of Lb. pentosus and 15% of Lc. pseudomesenteroides, MIC > 128 μg/ml), trimethoprim/sulfomethoxazol (71–100%, MIC > 4–64 μg/ml) and cefuroxime (44% of Lb. pentosus and 85% of Lc. pseudomesenteroides, MIC > 32–128 μg/ml). Lb. pentosus was susceptible to tetracycline and clindamycin, while 46% of Lc. pseudomesenteroides strains were resistant to these antibiotics. Only Lb. pentosus strains were resistant to ciprofloxacin (70%, MIC > 4–64 μg/ml), although no mutations in the quinolone resistance determining regions of the genes encoding GyrA and ParC were found, thus indicating an intrinsic resistance. Similarly, no genes encoding possible transferable resistance determinants for the observed phenotypic resistance were detected by PCR. In some cases, a bimodal distribution of MICs was observed for some antibiotics to which both LAB species exhibited resistance. Nevertheless, such resistances resulted from an intrinsic mechanism, non-transferable or non-acquired resistance determinants which may in part be due to chromosomally encoded efflux pumps (NorA, MepA and MdeA). Results of the present study demonstrate that all Lb. pentosus and Lc. pseudomesenteroides strains lack transferable resistance-related genes (cat, bla, blaZ, ermA, ermB, ermC, msrA/B, ereA, ereB, mphA, mefA, tet(M), tet(O), tet(S), tet(W), tet(L), tet(K), aad(E), aac(6)-Ie-aph(2)-Ia, aph(2)-Ib, aph(2′)-Ic, aph(2′)-Id, aph(3′)-IIIa, ant(4′)-Ia, dfrA, dfrD, vanA, vanB, vanC and vanE) and should therefore, according to Qualified Presumption of Safety criteria, be considered safe for future application as starter cultures or as probiotics.

Introduction

Lactic acid bacteria (LAB) are widely consumed along with fermented foods and beverages because of their use as starter cultures in fermentation processes (Caplice and Fitzgerald, 1999, Leroy and De Vuyst, 2004, Wood and Holzapfel, 1995). They are also known for their role as protective cultures as they are involved in producing an arsenal of antimicrobial substances such as lactic acid (and other organic acids), hydrogen peroxide, diacetyl, acetoin, reuterin, reutericyclin, antifungal peptides, and bacteriocins (Holzapfel et al., 1995, Holtzel et al., 2000). In the last decades, LAB have been used as probiotics, with Bifidobacterium and Lactobacillus being the most commonly used genera (Servin, 2004). The application of LAB as probiotics has been prompted by their beneficial properties on general health of the consumers (Kechagia et al., 2013) and their “QPS” (Qualified Presumption of Safety) status based on a long history of safe use (Anadon et al., 2006, European Commission (SCAN), 2007). In this sense, international regulatory organizations recommended specific prerequisites for approval of a determined strain as feed additive. Accordingly, the European Scientific Committee on Animal Nutrition (European Commission, SCAN, 2005) and the European Food Safety Authority (EFSA, 2012) recommended that LAB strains consumed on a daily basis worldwide should lack acquired or transferable antimicrobial resistance genes prior to considering them safe for human and animal consumption and that any probiotic strain should have QPS status.

Recently, several foods have been considered as potential vehicles of antibiotic resistance genes (Bautista-Gallego et al., 2013, Duran and Marshall, 2005, Franz et al., 1999, Zhang et al., 2009) with fermented foods being one of the most important environments where several stresses (low pH, high salt concentration and antimicrobial compounds) and the high number of living bacteria may induce the exchange of such genes. Gene exchanges may enhance survival of LAB and pathogens and thus represent an important risk within the gastrointestinal tract for spread to other bacteria (Salyers et al., 2004, van Reenen and Dicks, 2011). The indiscriminate use of antibiotics in human medicine and animal husbandry during several decades has resulted in an important public health risk. Furthermore, the increasing use of biocides as disinfectants in hospitals and food industries has led to the emergence of cross-resistance phenotypes to clinically important antimicrobial compounds (Fraise, 2002) and to new resistance mechanisms, which impose an additional health risk for consumers or the environment. The development of antimicrobial resistance among bacteria introduced in the food chain is of great concern, thus the EFSA requires that bacteria which are to be introduced into the food chain lack acquired antimicrobial resistance determinants to prevent lateral spread of these (van Reenen and Dicks, 2011). In the present study, the susceptibility patterns and possible mechanisms determining resistance to several antibiotics in Lactobacillus pentosus and Leuconostoc pseudomesenteroides strains were investigated. These strains were isolated in a previous study (Abriouel et al., 2012) from Aloreña table olives (which are naturally-fermented olives manufactured by small and medium enterprises from Málaga, Spain), with the aim to select strains that lack acquired antimicrobial resistance genes for possible application as starter cultures or as probiotics. Furthermore, this study allowed us to detect the types and degrees of antibiotic resistance present among the LAB community in the natural olive fermentation environment, and also to determine the prevalence of such phenotypic resistance in LAB throughout the fermentation processes in different producing enterprises.

Section snippets

Bacterial strains and growth conditions

72 LAB strains (Abriouel et al., 2012) were obtained from Aloreña green table olives naturally-fermented by four small–medium enterprises (SMEs) from Málaga (Spain) and included lactobacilli (59 Lb. pentosus strains) and leuconostocs (13 Lc. pseudomesenteroides strains). These strains were routinely cultured at 30 ºC in de Man Rogosa and Sharpe (MRS) broth (Fluka, Madrid, Spain) or agar under aerobic conditions for 24–48 h. Strains were kept in 20% glycerol at − 80 ºC for long term storage.

Antibiotic susceptibility testing and MIC determination

MICs of

Antimicrobial susceptibility testing and MIC distribution profiles

MIC determination of the different antibiotics was performed with 59 Lb. pentosus and 13 Lc. pseudomesenteroides from Aloreña table olives (including selected LAB with potential probiotic features according to Abriouel et al. (2012). The results obtained (Table 1) indicated that the MICs of β-lactams (amoxicillin and ampicillin), chloramphenicol, gentamicin (aminoglycoside) and erythromycin (macrolide) did not exceed the ECOFF suggested in most cases by the European Food Safety Authority (EFSA,

Discussion

The use or rather misuse of antibiotics for decades in bacterial infection treatments, animal husbandry and agriculture (Wegener, 2003) has resulted in the increased emergence of resistant bacteria to modern antibiotics, leading to failure in therapy and causing also evolutionary and ecological problems as were reported by Gillings (2013) about the recruitment of more resistance genes into the resistome and mobilome.

Antibiotic-resistant bacteria represent a great challenge for the food

Conclusions

Almost all Lb. pentosus (95%) and all Lc. pseudomesenteroides strains isolated from naturally-fermented Aloreña green table olives can be regarded as safe because of the absence of acquired resistance determinants. The intrinsic resistance to more than three antibiotics will not become a problem in a medical setting, since they were also highly sensitive to other clinically relevant antibiotics. In this study, the occurrence of intrinsic multi-resistance in both LAB species was due in part to

Acknowledgments

This work was supported by grants AGL2009-08921, P08-AGR-4295, Plan propio de la Universidad de Jaén, and Campus de Excelencia Internacional Agroalimentario CeiA3. Leyre Lavilla Lerma was beneficiary of a fellowship from Spanish Ministry of Education and Science.

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