| Objective:1.To clarify the drug resistance of Klebsiella pneumoniae in recent years in our province,which based on the analysis of the susceptibility of Klebsiella pneumoniae clinical isolates to common antibiotics from 2017 to 2019 in a third-class hospital in Jiangxi province.2.To screen out Klebsiella pneumoniae resistant to polymyxin,and evaluate the operability and practicability of the rapid polymyxin susceptibility assay,so as to provide a basis for the subsequent rapid identification of polymyxin resistant Klebsiella pneumoniae.3.To explore the molecular mechanism of polymyxin resistant Klebsiella pneumoniae in the hospital through analyzing the whole genome of polymyxin resistant Klebsiella pneumoniae,and provided data support for subsequent molecular biological detection.Methods:1.The distribution and drug resistance rate of clinical Klebsiella pneumoniae in the third-class hospital were analyzed using WHONET5.6 analysis software recommended by the World Health Organization(WHO).2.The drug resistance of Klebsiella pneumoniae polymyxin was determined according to the method recommended by the Clinical and Laboratory Standards Institute(CLSI),the application of broth dilution method was to define the minimum inhibitory concentration(MIC),and the breakpoint was referred to the European Commission on Antimicrobial Susceptibility Testing(EUCAST).3.Seven housekeeping genes(gapA,infB,rpoB,mdh,tonB,pgiand phoE)of Klebsiella pneumoniae were amplified by PCR and sequenced by MLST.The results were submitted to the website(https://bigsdb.pasteur.fr/klebsiella/klebsiella.html)to compare the types of alleles,and then compareed the types of seven alleles to produce polymyxin ST type of Klebsiella pneumoniae.4.The whole genome of polymyxin resistant Klebsiella pneumoniae was extracted and sequenced by Illumina sequencing platform to complete the whole genome sequencing.Results:1.1482 strains of Klebsiella pneumoniae were isolated in 2017,2018 and 2019 in the hospital,and it’s 452 strains,468 strains and 562 strains respectively.It was found that the proportion of Klebsiella pneumoniae in Gram-negative bacilli elevated yearly,which was 18.23% in 2017,19.91% in 2018 and 21.69% in 2019,respectively.The maximum isolates of Klebsiella pneumoniae were 235 strains in the department of Neurosurgery(15.9%),210 strains from ICU(14.2%),152 strains from respiratory department(10.3%),136 strains from hepatobiliary Department(9.2%),102 strains from oncology department(8.6%),88 strains from urology department(5.9%)and 75 strains from cardiothoracic Department(5%).In the distribution of specimens,489(33%)were mainly blood samples,466(31.4%)were sputum samples,189(12.8%)were urine samples and 84(5.7%)were wound secretions.2.The 1482 clinical isolates of Klebsiella pneumoniae were collected in the past three years and analyzed the drug resistance rate of 13 commonly used antibiotics(piperacillin/tazobactam,cefazolin,ceftriaxone,cefepime,aztreonam,imipenem,amikacin,gentamicin,tobramycin,ciprofloxacin,levofloxacin,compound xinuoming and furantoin).The highest drug resistance rate was 60.2% for ceftriaxone,51.1% for ceftriaxone,40%-50% for aztreonam and furantoin,19.1% for imipenem,among which amikacin was the lowest,only 8.2%.Comparing the drug resistance rates of neurosurgery and ICU with the most distribution,we found that the drug resistance rates of the two departments were generally higher than the total drug resistance rates,especially in neurosurgery.The drug resistance rates of cefazolin,ceftriaxone and aztreonam were exceeded 60%,and the drug resistance rates of piperacillin / tazobactam,cefepime,ciprofloxacin,levofloxacin and furantoin were beyond 50%,the carbapenem resistant Klebsiella pneumoniae reached 44.3%,14.4% higher than 29.9% in ICU.3.We collected 721 clinical collected Klebsiella pneumoniae,and assayed the minimum inhibitory concentration by broth dilution method.We obtained 22 polymyxin resistant Klebsiella pneumoniae(PoLR-Kpn)strains referring to the polymyxin breakpoint established by European Commission on Antimicrobial Susceptibility Testing.After amplification of drug resistance genes,of the 22 drug-resistant strains,4 strains carried mcr associated resistance genes,including 3 of mcr-1,1 of mcr-8,18 strains were chromosome mediated resistance genes,10 of which were two-component system mutations of PmrAB,4 were mgrb gene mutations,and 4 were two-component system mutations of PhoPQ.Among the ST type of 17 polymyxin resistant Klebsiella pneumoniae strains,Kpn3,Kpn15,Kpn22 and Kpn23 are ST11 type,Kpn9 is ST22,Kpn4 is ST25 type,Kpn12 is ST105 type,Kpn19 is ST218 type,Kpn30 is ST294 type,Kpn8 is ST367 type,Kpn18 is ST378 type,Kpn11 is ST592 type,Kpn1,Kpn2 and Kpn21 are ST3918 type,while the ST type of Kpn7 and Kpn17 was not detected.4.The rapid polymyxin resistance assay indicated that 20 of 22 strains of polymyxin resistant Klebsiella pneumoniae were positive and 2 were negative,while 12 strains of polymyxin susceptibility Klebsiella pneumoniae were all negative.The improved rapid polymyxin assay showed that all three strains of Klebsiella pneumoniae carrying mcr-1 were negative,and one strain of Klebsiella pneumoniae carrying mcr-8 resistance gene showed a susceptible phenotype.5.Whole-genome sequencing of Klebsiella pneumoniae resistant to polymyxin,we found 3 strains carrying mcr-1 resistance genes(Kpn4,Kpn18 and Kpn30),and 1 strain carrying mcr-8 resistance genes(Kpn23).Further bioinformatics analysis found that the mcr-1 of Kpn4 and Kpn30 strains may be present in the plasmid,and the mcr-1 resistance gene and phoP gene mutations of Kpn30 strain were both present.Conclusions:1.Klebsiella pneumoniae is an important conditional pathogen of nosocomial infection,the rate of resistance is increasing yearly,especially rate of carbapenem resistance,Klebsiella pneumoniae isolates have the highest resistance rate to carbapenem antibiotics in neurosurgery and ICU departments.2.Among the 721 strains of Klebsiella pneumoniae screened by broth dilution method,22 strains were resistant to polymyxin,three of them were resistant to mcr-1,one of them was resistant to mcr-8.It was indicated that it could screen the polymyxin resistant Klebsiella pneumoniae and provide a primary screening method for the detection of polymyxin resistant Klebsiella pneumoniae by evaluating the rapid polymyxin resistance test.3.The whole genome of Klebsiella pneumoniae with polymyxin resistance was sequenced and bioinformatics analysis showed that mcr-1 was the main gene of polymyxin resistance mediated by plasmids in this hospital.Therefore,we should take corresponding screening measures as soon as possible. |
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