The Phenotypic And Molecular Characteristics Of OXA-232-producing Klebsiella Pneumoniae Recovered From ICU In Zhejiang Province

Objective:To performa cross-sectional surveillance of CRKP isolated from respiratory and intestinal samples of patients,and analyzed the phenotypic and molecular characteristics of blaOXA-232-carrying Kpneumoniae which were first screened in ICU in Zhejiang Province,and then assessed the possibility of prevalence of fblaOXA-48-like strains.Methods:Fresh sputum and feces samples from ICU patients in nine tertiary hospitals in Zhejiang Province were Collected from July 2017 to August 2018.After inoculated onto the culture medium,the VITEK-2 Compact bacteria identification instrument and matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)were used to identify Klebsiella pneumoniae,and then CRKPs were screened by the KB disc method.Expression of the hypermucoviscosity phenotype was deternined by the string test.The micro-broth dilution method were used to determine the common antimicrobial susceptibility.PCR was performed to detect ?-lactamases genes(blaKPC,blaNDM,blaVIM,blaIMP,blaOXA-48-like,blaCTX-M,blaTEM,blasSHV).The homology of CRKP was analyzed by pulsed-field gel electrophoresis(PFGE)and Multilocus sequence typing(MLST).Ability of the blaOXA-232-encoding genetic components to undergo transmission was determined by conjugation assay.Plasmid profiles of the blaOXA-232-encoding elementswere identified by S1-PFGE.Related genetic traits of isolates producing blaOXA-232 carbapenemase were analyzed by Genome sequencing.Finally,the G.mellonella larvae infection model and neutrophil bactericidal assay were used to analyze the virulence and anti-killing properties.Results:A total of 222 ICU patients from nine hospitals were recruited for this study.And 149 strains of Klebsiella pneumoniae were isolated,of which 65 showed a resistant phenotype to carbapenems,and the recovery rate of CRKP had reached 43.6%.The results of string test showed that only 2 strains were positive for the test.Antimicrobial susceptibility assay showed that most of the CRKP strains were resistant to multiple antibiotics except polymyxin B,tegacycline and ceftazidime/avibartan,and all of ten blaOXA-232-producing isolates were resistant to ertapenem but susceptible to meropenem and imipenem.PCR showed that 54 strains carried blaKPC-2,10 isolates produced blaOXA-232 and 1 strain carried WaNDM-5.Also,the detection rates of blaCTX-M-65 reached 55.4%.PFGE showed the clone transmission phenomenons were existed in the same hospital.According to the result of MLST,ST11(78%,43/55)was the most popular type among the blaKPC/NDM-producing isolates and ST 15 was the only type in these blaoxA-232 strains.Besides,Conjugation assay indicated that the blaOXA-232 gene was located on a non-conjugative plasmid,while S1-PFGE results indicated that these isolates harbored at least three or more plasmids with plasmid sizes of about 175 Kb,130 Kb and 78 Kb being most commonly detected.The genomes of the blaOXA-232-producing K.pneumoniae isolates ranged from 5.7?5.8 Mb in size,and pairwise SNP analysis based on their raw sequencing reads showed that their core genome differed only by a few SNPs(n?15).Moreover,the G.mellonella larvae infection model showed that blaKPC-producing CRKPs isolated from sputum exhibited a higher virulence level than strains recovered from fecal samples.Besides,despite harboring a pLVPK-like virulence plasmid,the mortality of G.mellonella caused by blaOXA-232-producing isolates was much lower than that caused by blaicpc-producing CRKPs under the same bacterial solution injection dose,and the neutrophil bactericidal assay also showed that it did not exhibit the high virulence potential.Conclusion:The CRKP isolates from parts of ICU in Zhejiang Province was highly prevalent,and the virulence of blaKPC/NDM-producingCRKP isolated from the respiratory tract was higher than that of CRKP in the intestinal tract.Meanwhile,the emergence and small-scale prevalence of blaOXA-232 K.pneumoniae strains indicated the potential of clonal dissemination for blaoxA-232-producing isolates,suggesting that the surveillance and detection of D-type ?-lactamase should be strengthened in clinical and laboratory to prevent the epidemic and transmission of the novel drug-resistant bacteria in China.