RamR Gene Mutations Involve In Klebsiella Pneumoniae Heteroresistant To Tigecycline

ObjectKlebsiella pneumonia(K.pneumonia)is one of the most important pathogen that causes several types of nosocomial infection.With widespread use of antibiotics agents,multi-drug resistant(MDR)or pan-drug resistant(PDR)K.pneumonia has emerged.Tigecycline is among the last-resort theraptic option for treating severe infections caused by MDR or PDR organisms.However,tigecycline-resistant K.pneumoniae is increasing gradually,which makes antimicrobial treatment more difficulty.It is speculated that heteroresistant to antibiotics agent is in the middle stage of bacterial resistance evolution.And it considered to be responsible for treatment failure.Therefore,the aim of current study is to screen tigecycline heteroresistance in K.pneumonia from clinical isolates,and to explore the underlying mechanism of tigecycline heteroresistance K.pneumonia(TGCHR-Kp).Methods1.The clinical isolates of tigecycline susceptible K.pneumoniae were collected.TGCHR-Kp were screened by K-B disk diffusion method and confirmed by population analysis profile(PAP).The subpopulation minimal inhibitory concentration(MIC)for tigecycline were detected by broth microdilution method after 14 d subcultures on antibiotic-free medium.Gene homology profiles of parental strains and subpopulation were investigated by ERIC-PCR.2.Efflux pump inhibition was performed with Phe-Argβ-naphthylamide dihydrochloride(PAβN),an efflux pump inhibitor,to assess the efflux activity.Relative expression acrA,acrB,tolC and ramA genes for parental strains and subpopulation were measured by real-time reverse transcription PCR(RT-PCR).3.The ramR gene was amplified by PCR and sequenced by Sanger method.The sequences were aligned with BlastN and DNAMAN software.RamR structure homology modeling was performed with SWISS-MODEL workspace.4.Eight single colony of tigecycline-susceptible K.pneumonia were randomly from ATCC13883 and two clinical isolates’ subculture,then inoculated in broth containing 2-fold increasing concentration of tigecycline by step.The resistant isolates were selected on the M-H plate containing 4mg/L tigecycline.The MIC of tigecycline-resistant isolates were measured by broth microdilution method.The efflux activatities were assessed and the relative expression of efflux pump genes were measured by RT-PCR.The ramR genes were sequenced and analysed.Results1.Of 334 clinical tigecycline-susceptible K.pneumonia,22 isolates were confirmed as TGCHR-Kp,with a positive rate of 6.6%.All the parental strains were tigecycline-susceptible,while all of the subpopulation were shown to be intermediate or resistant to tigecycline.The proportion of resistant subpopulation was ranged from 7.14×10-8 to 1.08×10-5.ERIC-PCR showed highly homologous between parental strain and subpopulation,respectively.2.Of 22 subpopulations,20 isolates had 2-fold to 8-fold reduction MICs for tigecycline by the effect of efflux pump inhibition,2 isolates remained unchangable MICs for tigecycline.The relative expression of acrA,acrB,tolC and ramA in subpopulation were significantly higher than those of in parent strains,which were associated with those of MICs of tigecycline.3.Of 22 parental strains,11 isolates ramR have non-synonymous nucleotide substitution,which mainly distributed among amino acid sequence after α6 helix,namely,the dimer-formation domain.Among 22 subpopulation,10 isolates ramR genes have non-synonymous nucleotide substitution,6 isolates have nucleotide insertion insertion or deletion.The mutations were predominately distributed in the DNA-binding domain.Compared with homologous parental strains,the other 6 isolates had the same ramR mutants,but they also have 2-fold to 16-fold increase of tigecycline MIC.4.All strains exposed to tigecycline could partly grown on M-H plates containing 4mg/L tigecycline and had a 4-fold to 8-fold increase than those of preexposure strains.Efflux pump inhibition showed 4-fold to 16-fold reductions in tigecycline MIC,which reversed the subpopulation antimicrobial susceptibility.AcrAB-Tolc efflux pump genes and ramA gene expression in tigecycline-exposed subpopulation were remarkable increased than those of pre-exposure strains.Various mutants of ramR were revealed among 7 subpopulation exposed to tigecycline,except for one subpopulation.ConclusionsIn the present study,we first reported TGCHR-Kp recovered from clinical tigecycline-sensitive isolates.The ramR gene non-synonymous nucleotide substitution,insertion or deletion may reduces its regulatory affinity on ramA,thus made AcrAB-TolC efflux pump genes overexpression,suggesting ramR gene mutation may play a key role in K.pneumoniae heteroresistant to tigecycline.During bacterial antibiotics-resistant evolution,tigecycline may plays an essential role in K.pneumoniae ramR gene mutation and in its developed to tigecycline resistance.The current study will help us to deepen understand the antibiotics resistance evolution in K.pneumoniae,and guides clinician to rational use of antimicrobial agents in practice.