| Objective: Enterococcus is an important pathogen of nosocomial infection.The main mechanisms of linezolid resistance in enterococci known to date include point mutations in 23 S rRNA and ribosomal proteins L3 and L4 as well as presence of methyltransferase gene cfr.However,these mechanisms cannot explain the low-level resistance to linezolid in some enterococci,which exhibit a minimum inhibitory concentration(MIC)of 4-16 mg/L.To perfect the mechanisms of linezolid resistance and find new drug resistance targets,we performed the Illumina HiSeq 4000 high-throughput transcriptome sequencing technology to screen out resistance-associated genes and pathways in low-level linezolid-resistant Enterococcus faecalis.Subsequently,the possible mechanisms and epidemiological characteristics of linezolid-resistant E.faecalis isolates from the First Affiliated Hospital of Chongqing Medical University from 2014 to 2017 were investigated.Method: 1.In order to explore the low-level linezolid-resistant E.faecalis resistance mechanism,whole-transcriptome profiling was performed on an E.faecalis strain P10748 with low-level linezolid resistance in comparison with a linezolid-susceptible strain 3138 and the standard control strain ATCC29212.The differential expressed genes(DEGs)were enriched by GO and KEGG,with some DEGs potentially involved in drug resistance were further confirmed by qRT-PCR.2.A total of 1120 non-duplicated E.faecalis isolates was performed by drug susceptibility testing,collected from Chongqing during August 2014 to June 2017.All linezolid-resistant E.faecalis(LRE)screened for optrA,cfr,mutation in 23 S rRNA and ribosomal proteins L3 and L4 by PCR amplification and sequencing.Multi-locus sequence typing(MLST)were used for epidemiological analysis.Results: 1.The results showed that a total of 3.57 Gb valid data were obtained by sequencing,1,920 unigenes were obtained by De novo,with a total length of 2,122,210 bp and an average length of 1,105 bp.A total of 150 DEGs(FDR≤0.001 and |log2Ratio| ≥1)were identified by differential gene pattern clustering,of which 141 were up-regulated and 9 were down-regulated.The significantly up-regulated DEGs,esp,optrA and fexA,predicted to be associated with drug resistance through active efflux pumps and biofilm formation.The GO functional annotation results showed that the catalytic activity,metabolic processes and cells are the most annotated terms.Pathway enrichment analysis revealed that peptidoglycan biosynthesis,valine leucine and isoleucine degradation and sulfur metabolism were significant enrichment pathways(p<0.05).QRT-PCR results were consistent with the sequencing results of the transcriptome.2.Of the 43 isolates were resistant to linezolid at a low level(MICs of 8-16mg/L).All of 43 LRE strains harbored optrA,while we failed to find the cfr and the 23 S rRNA mutations.OptrA protein with the novel amino acid substitutions at positions Glu60 Lys and Gly197 Asp.Novel mutations in the ribosomal proteins L3 and L4 including one deletion(Gln103del)and four substitutions(Ser113Leu,Thr35 Ala,Ile98Val,Asn79Asp).3.MLST analysis revealed that 43 LRE isolates belonged to 20 sequence types(STs),and ST16 was the most common type(14/43).The epidemiological investigation suggested that there was no clonal complex correlation between these isolates,which indicated that cases presented in our hospital was sporadically rather than outbreak.The present MLST results demonstrated the existence of eight novel sequence types(ST823 to ST830)and one allele(gki95),which first discovered in China.Conclusion: 1.It was speculated that membrane transporter and biofilm formation may play an important role in the mechanism of low-level linezolid resistance,which provided the reference drug target for the mechanism of low-level linezolid resistance in E.faecalis.2.Our results indicated that a high rate of optrA carriage among low-level linezolid resistant E.faecalis isolates in a China hospital.3.The optr A,may be as a useful marker for resistance screening,played an important role in linezolid resistance. |
