Carbapenem-resistant Enterobacteriaceae:Colonized Status And In-host Evolution Strategy Of Acquired Resistance

The wide spread and prevalence of Carbapenem-resistant Enterobacteriaceae（CRE）has become a severe global issue,and China has also observed an increasing isolation rate in clinic,especially for Carbapenem-resistant Klebsiella pneumoniae（CRKP）.However,limited choices and poor prognosis make CRE infection treatment even more difficult.While in critically-ill inpatients,CRKP rectal carriers have a higher risk of infections and mortality.Nowadays,CRE surveillance has been recruited into the routine hospital infection control items in some regions,which prefers to screen rectal samples of swabs and feces.In this study,we hope to clarify the prevalence of colonizers and carbapenemases,illustrate the relationship between CRKP critically-ill colonizers and their subsequent CRKP infections as well as the in-host evolution strategy of CRKP with acquired resistance,which are significant to efficient infection control,appropriate prescription and improved prognosis.The first part recruited a total of 2404 double-rectal swabs from nonduplicate inpatients in four Chinese institutions during May 2018 to May 2019.One swab was cultured for Carbapenem-resistant organisms（CRO）and the other for Xpert Carba-R assay so as to clarify the status of CRE carriers and the efficiency of the rapid detection kit of carbapenemase genes.To further clarify the status of critically-ill carriers,the resistance profile and mechanism of colonized CRE strains in Hangzhou,78 CRE strains were conducted with the following tests,including antimicrobial susceptibility testing（AST）,polymerase chain reaction（PCR）and whole-genome sequencing（WGS）analysis.The results showed that 10.1%（362/2404）of inpatients were CRE colonizers.As the main subject for CRE screening,intensive care unit（ICU）showed that the colonized rate of 11.5%（172/1500）and 85.9%（240/256）of CRE strains carried with carbapenemases,of which K.pneumonia accounted for the most（62.5%）and followed by E.coli（11.3%）.Further analysis of 78 CRE strains from Hangzhou showed that most strains were colistin-susceptible（91%）;ST11 type was the main CRKP clone（76.8%）;KPC-2 was the major carbapenemase type（82.1%）.Besides,carriage of Amp C or/and ESBL genes might be the reason for carbapenem resistance in 11none-carbapenemase-producing strains.Mutations of regulator of efflux pump and two-component regulatory system were found in tigecycline-resistant and colistin-resistant strains,respectively.Carriage of MBLs genes mostly contributed to the ceftazidime/avibactam resistance.Furthermore,Xpert Carba-R assay provides reliable results for quick detection and differentiation of five carbapenemase genes in rectal swabs.The second part recruited a total of 818 rectal/nasopharyngeal swabs from 258 ICU inpatients in a Hangzhou institution during June to October in 2017.AST,pulsed-field gel electrophoresis（PFGE）and follow-up study were performed to clarify the status of CRKP colonization and risk factors for subsequent CRKP infections among carriers.The results showed that the CRKP isolation rate was 22.6%（185/818）,and most were colistin-susceptible（97.8%,181/185）.Among 258 ICU subjects,31%（80/258）were CRKP carriers and most were rectal carriage（72.5%,58/80）.Follow-up study showed that 16 of 53 CRKP carrier（without prior CRKP infections）had subsequent CRKP infections.There was a high relatedness between infected and colonized strains per person and most strains belonged to the same-PFGE cluster,which indicated the close relationship from prior CRKP colonization to infections,as well as the possibility of CRKP clone dismission in ICU.However,risk factors of subsequent infection of ICU carriers remained uncertain,which suggested to concern all ICU carriers.The third part focused CRKP in-host evolution of its mechanism of resistance to tigecycline and polymyxin under clinical therapy.A total of 11 CRKP strains were consecutively isolated from a male patient who suffered from continuous and multisite infections.Phenotypic testing,PCR,PFGE,Southern blotting,WGS and bioinformatic analysis as well as complementation experiments were performed to identify the phenotypic and molecular characteristics of the CRKP series strains,the effect of mutants to increased antimicrobial insusceptibility,and finally revealed the evolution strategy of acquired resistance.The results showed that all strains belonged to the CRKP ST11-KL64 clone with high relatedness,and bla _KPC-2was located on the100kb-sized plasmid.Phenotypic results showed seven hypermucoviscous strains and three tigecycline-resistant and four colistin-resistant strains.Diverse hypervirulence factors and resistance genes were identified on about three plasmids.Complementation with wide-type lon gene and three genes（pmr B,pho Q,mgr B）confirmed the specific mutation-mediated resistance to tigecycline and colistin,respectively.Therefore,ST11-KL64 clone is a potential threat to antiinfection treatment and is capable of rapid and diverse evolution of resistance during tigecycline and polymyxin treatment.