Antibiotic resistance of Lactobacillus pentosus and Leuconostoc pseudomesenteroides isolated from naturally-fermented Aloreña table olives throughout fermentation process
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.
References (66)
- et al.
Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers, and pigs in Denmark
Diagn. Microbiol. Infect. Dis.
(2000) - et al.
Culture-independent study of the diversity of microbial populations in brines during fermentation of naturally fermented Aloreña green table olives
Int. J. Food Microbiol.
(2011) - et al.
Characterization of lactic acid bacteria from naturally-fermented Manzanilla Aloreña green table olives
Food Microbiol.
(2012) - et al.
Antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacteria
Food Microbiol.
(2007) - et al.
Probiotics for animal nutrition in the European Union. Regulation and safety assessment
Regul. Toxicol. Pharmacol.
(2006) - et al.
Screening of lactic acid bacteria isolated from fermented table olives with probiotic potential
Food Res. Int.
(2013) - et al.
Food fermentations: role of microorganisms in food production and preservation
Int. J. Food Microbiol.
(1999) - et al.
Susceptibility of Lactobacillus spp. to antimicrobial agents
Int. J. Food Microbiol.
(2003) - et al.
Ready-to-eat shrimp as an international vehicle of antibiotic-resistant bacteria
J. Food Prot.
(2005) - et al.
Enterococci at the crossroad of food safety
Int. J. Food Microbiol.
(1999)
Lactic acid bacteria as functional starter cultures for the food fermentation industry
Trends Food Sci. Technol.
Antibiotic resistance of probiotic strains of lactic acid bacteria isolated from marketed foods and drugs
Biomed. Environ. Sci.
Incidence of antibiotic resistance and virulence determinants among Enterococcus italicus isolates from dairy products
Syst. Appl. Microbiol.
Antibiotic resistance in food lactic acid bacteria
Int. J. Food Microbiol.
Resistance of potential probiotic lactic acid bacteria and bifidobacteria of African and European origin to antimicrobials: determination and transferability of the resistance genes to other bacteria
Int. J. Food Microbiol.
Analysis of gyrA and parC mutations in enterococci from environmental samples with reduced susceptibility to ciprofloxacin
FEMS Microbiol. Lett.
Human intestinal bacteria as reservoirs for antibiotic resistance genes
Trends Microbiol.
Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens
FEMS Microbiol. Rev.
Characterisation and transferability of antibiotic resistance genes from lactic acid bacteria isolated from Irish pork and beef abattoirs
Res. Microbiol.
Antibiotics in animal feed and their role in resistance development
Curr. Opin. Microbiol.
Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins
Appl. Environ. Microbiol.
Plasmid-borne smr gene causes resistance to quaternary ammonium compounds in bovine Staphylococcus aureus
J. Clin. Microbiol.
Performance standards for antimicrobial susceptibility testing; Twenty-First Informational Supplement
Aerobic metabolism of lactic acid bacteria
Ir. J. Food Sci. Technol.
Antibiotic susceptibility of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese
Lait
Selectivity for d-lactate incorporation into the peptidoglycan precursors of Lactobacillus plantarum: role of Aad, a VanX-like d-alanyl-d-alanine dipeptidase
J. Bacteriol.
Detection of glycopeptides resistance genotypes and identification to the species level of clinically relevant enterococci by PCR
J. Clin. Microbiol.
Alterations in GyrA and ParC associated with fluoroquinolone resistance in Enterococcus faecium
Antimicrob. Agents Chemother.
Bile-mediated aminoglycoside sensitivity in Lactobacillus species likely results from increased membrane permeability attributable to cholic acid
Appl. Environ. Microbiol.
Opinion of the Scientific Committee on Animal Nutrition on the criteria for assessing the safety of microorganisms resistant to antibiotics of human clinical and veterinary importance
Opinion of the Scientific Committee on a request from EFSA on the introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA
EFSA J.
EFSA scientific colloquium summary report: QPS—qualified presumption of safety of microorganisms in food and feed
Scientific opinion of the panel on biological hazards on a request from the European Food Safety Authority on food borne antimicrobial resistance as a biological hazard
EFSA J.
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