Reasearch Of Linezolid Resistance Mechanism In Low Level Linezolid Resistant Enterococcus Faecalis

Objective: During the past few years, enterococci have emerged as important healthcare associated pathogens and most of them are susceptible to linezolid. However, resistance to linezolid has emerged following clinical treatments, and the main resistance mechanism were mutations in 23 S ribosomal RNA, mutations in ribosome proteins L3 and L4 and a multidrug resistance gene, named cfr, could also confer resistance to linezolid. And some low level linezolid enterococci(MICs ranging from 4~16 mg/L,) were lack of specific linezolid resistance mechanisms. This study aimed to characterize the resistance mechanisms in low level linezolid resistant Enterococcus faecalis in vitro.Methods: The in vitro development of increased linezolid resistance was assessed in 5 E. faecalis strains with the MIC ranging from from 8 to 16 mg/L and that lack specific resistance mechanisms to observe changes in the MICs of linezolid. After induction with linezolid, PCR and DNA sequencing were used to detect the mutation in the 23 S rRNA gene, as well as mutations in genes encoding the L3 and L4 ribosomal proteins with the quality control strain of E. faecalis ATCC29212. The G2576 T mutation and number of copies of the mutation was determined by restriction enzyme digestion and DNA sequencing. Furthermore, semi-quantitative biofilm assay was performed for 5 strains before and after induction. Accumulation of linezolid in resistant strains was quantified with HPLC/UV and MICs of linezolid were tested in the absence and presence of 4 common efflux pump inhibitors including reserpine, CCCP, verapamil and lansoprazole. Cell walls were measured with transmission electron microscopy(TEM).Results: After serial passages with linezolid in vitro, linezolid MIC for 5 E. faecalis isolates were successfully increased by 8 to 32 times, and the maximum MIC was more than 256 mg/L. The G2576 T mutation was detected in 3 of the 5 strains, while the other 2 isolates were negative for all possible mutations in the domain V region of the 23 S rRNA gene. No mutations were detected in genes encoding the L3 and L4 protein. Strains with the MIC of 8 mg/L were non-biofilm producers while with the MIC of 16 mg/L were weakly producers. And linezolid resistant strains had decreased uptake for linezolid when compared with the susceptible strain. MICs of linezolid were not altered at the presence of 4 efflux pump inhibitors. TEM results showed that cell wall thickness in resistant strains were significantly higher than that of in its parental strains in the presence of linezolid pressure.Conclusions: The results showed that high level resistance can be induced in vitro with linezolid in low level linezolid resistant E. faecalis, and the principal mechanism may be attributed to the G2576 T mutation in the domain V region of 23 S rRNA gene. The resistance mechanism of low level linezolid resistance in E. feacalis may relate to the thickened cell wall that could decrease the linezolid uptake.