The Molecular Epidemiology Of Drug Resistance And Virulence In Klebsiella Pneumoniae From ICU Across China

Objective: A retrospective study was conducted to clarify the molecular characteristics and dynamical changes of carbapenem resistant Klebsiella pneumoniae(CRKP)isolates colonized in the gastrointestinal tract from three inpatients in Intensive Care Unit(ICU)by the drug resistance and virulence analysis.Furthermore,a multicenter cross-sectional surveillance study was conducted to characterize the molecular epidemiology of rectal and respiratory carriage of CRKP isolates in ICU patients in China nationwide,aiming at illustrating the disciplines and mechanisms underlying the rectal and respiratory carriage of CRKP for control and prevention measure.Methods: The CRKP rectal and clinical isolates from three ICU inpatients were identified and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS)and K-B method.The antimicrobial susceptibilities of the isolates were determined using a broth microdilution procedure.The presence of carbapenem-resistant genes(bla KPC,bla NDM,bla IMP,bla VIM and bla OXA-48-like)and virulence gene rmp A2 encoded on the virulence plasmid was screened using PCR.Multilocus Sequence Typing(MLST)and Pulsed-Field Gel Electrophoresis(PFGE)were performed to analyze the homology.S1-Nuclease PFGE(S1-PFGE)and Southern blot were conducted to acquire the genetic location of rmp A2.Whole genome sequencing analysis was conducted to understand the genetic characteristics.String test was performed to identify the hypermucoviscous phenotype.Galleria mellonella larva models were used to quantify the virulence.The rectal and respiratory samples of ICU patients were collected from 51 general hospitals located in 16 regions across China.The enrichment media supplemented with meropenem was used to isolate CRKP from rectal and respiratory samples.Similar experiments as described above were done to analyze the drug resistance and virulence of CRKP isolates.Results: CRKP isolates were continuously isolated from the gut of three ICU patients during their hospitalization,and two KPC-2-producing CRKP rectal isolates were identified with different plasmid profiles,drug resistance gene profiles and virulence gene profiles from the corresponding patient of three from different time during hospitalization.The major difference was that one KPC-2 producing CRKP rectal isolates acquired with a virulence plasmid carrying the virulence genes iuc ABCD and rmp A2 compared to the counterpart.bla KPC-2 was carried by three resistance plasmids with the sizes of ~80Kb,~90Kb and ~138Kb.Two patients were identified with same clone from gut and extraintestinal sites,while one of them was found to carry different clones from gut and extraintestinal sites when hospitalized in different wards.The CRKP isolates carrying the iuc ABCD and rmp A2-encoded virulence plasmid led to the higher mortality of infected Galleria mellonella larva than non-carriers.Among 898 patients hospitalized in ICUs of 51 general hospitals located in 16 regions in China,the CRKP carriage rates had highly diverse geographic distribution with the average rate of 28.5%。Several regions were in high risk of CRKP infection.ST11 bla KPC-2-bearing CRKP isolates were the predominant isolates with the prevalence rate of 64% and these isolates had been wide-spread among multiple regions.Several clones with rarely identified carbapenemase and sequence types(STs),including ST636 bla NDM-1,ST307 bla IMP-30,ST1779 bla IMP-4,ST2407 bla NDM-1,ST846 bla NDM-1,ST15 bla OXA-232 and ST2237 bla KPC-2,were endemic in several single centers in China.CRKP isolates from ICU inpatients exhibited multi-drug resistance with 85.8% detection rate of extended spectrum β-lactamases(ESBLs),and CTX-M-65 and SHV-12 were the main determinants for ESBLs.CRKP clones from intestinal and respiratory carriers had great diversity and were distinguished by 6 lineages and 37 STs.The virulence of CRKP isolates might be mainly associated with the virulence plasmids bearing iuc and rmp A2 virulence genes.KL64 CRKP isolates had the tendency to harbor more ESBLs-encoded genes and virulence genes than KL47 CRKP isolates,and the results of Galleria mellonella larva models showed the KL64 CRKP isolates had higher virulence.Additionally,CRKP rectal and respiratory isolates co-harbored by a carrier belonged to the same clone,which was identified in 86.5% of CRKP carriers.However,multiple clones were also identified with 13.5% in CRKP carriers with co-carriage of gut and respiratory tract.Conclusion: The human gastrointestinal tract as a major reservoir of CRKP strains,drug resistance and virulence genes,has a capacity to dynamically colonize multiple clones of CRKP strains.Additionally,the gut colonization might be the key risk factor for contributing to subsequent extraintestinal infection.bla KPC-2 gene could be incorporated into various plasmids that are transmissible among multiple bacterial hosts in the human gastrointestinal tract.ICUs in several regions across China were in high risk of CRKP infections.ST11 KPC-2-producing CRKP clones were the predominant with high prevalence rate and these clones had been wide-spread among multiple regions.KL64 ST11 KPC-2-producing CRKP isolates might be the high-risk clone mediating the multi-drug resistance and virulence.Several clones with rarely identified carbapenemase and STs were endemic in several single centers in China.Additionally,CRKP isolates from gut and respiratory in ICU inpatients were highly diverse,which indicated the positive communication of CRKP strains and genetic elements among human gut,respiratory and outer environment.Thus,close surveillance on CRKP carriage on admission and during hospitalization,together with effective infection prevention and control measures should be put into practice.