a. P. pentosaceus (16) 8 8 n.a. W. cibaria (15) 15 n.a. aMICs determined by a VetMIC test. The click here antibiotic dilution ranges were: 0.03-16 mg/L (ampicillin, clindamycin, penicillin and linezolid), 0.25-128 mg/L (vancomycin and
ciprofloxacin), 0.5-256 mg/L (gentamicin, streptomycin and neomycin), 2-1024 mg/L (kanamycin), 0.016-8 mg/L (erythromycin), 0.12-64 (tetracycline, chloramphenicol, rifampicin and trimethoprim). MICs which exceeded the upper or lower limit of the tested range are listed in the next dilution series. MICs higher than the EFSA breakpoints are indicated in bold. bLAB with MICs higher than the EFSA breakpoints are considered as resistant strains [15]. n.r., not required; n.a., not available. Detection of antibiotic resistance genes The non-enterococcal strains showing antibiotic resistances in the VetMIC assays (17 strains)
were further submitted to PCR in order to identify the presence of the respective antibiotic resistance genes. The tested strains were the following: Lb. selleck carnosus B43 (ampicillin resistant), P. pentosaceus TPP3 and SMF120 (tetracycline resistant), P. pentosaceus LPP32, LPM83 and B5 (clindamycin resistant), P. pentosaceus LPV57 and W. cibaria P50, P61, P64, P73, SDM381, SDM389, SMA14 and BCS50 (kanamycin resistant), and P. pentosaceus MAPK inhibitor LPM78 and W. cibaria SMA25 (kanamycin, erythromycin and clindamycin resistant). Acquired antibiotic resistances likely due to added genes were only found in strains within the genera Pediococcus (12.5%) and Weissella (6.7%). The genes involved in the horizontal transfer of resistance
to tetracycline [tet(K), tet(L) and tet(M)], kanamycin [aac(6´ )-Ie-aph(2´ ´ )-Ia] and erythromycin [erm(A), erm(B) and erm(C)] were not detected. However, P. pentosaceus LPM78 and W. cibaria SMA25 harboured the erythromycin resistance gene mef(A/E). The obtained amplicons were sequenced and found to have 99% homology with the macrolide-efflux protein (mefE) gene described for Streptococcus pneumoniae and other Streptococcus spp. Arachidonate 15-lipoxygenase Moreover, P. pentosaceus LPM78 and LPM83 harboured the lnu(A) gene encoding the lincosamide O-nucleotidyltransferase that inactivates lincomycin and clindamycin. Sequencing of both amplicons showed 97% and 93% homology with lincosamide nucleotidyltransferase [lnu(A)] gene described for Staphylococcus haemolyticus and S. aureus, respectively. Nevertheless, lnu(B) was not detected in any of the tested strains. With regard to E. faecium BNM58, which was phenotypically resistant to erythromycin, none of the respective genes [erm(A), erm(B), erm(C) and mef(A/E)] were detected.
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