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

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.