| ObjectiveIn this study,clinical isolates of CRKP from many hospitals in Hunan Province were collected for identification,drug sensitivity analysis and drug resistance gene screening to understand the prevalent genotypes of CRKP and the resistance of common antibiotics in Hunan Province,and to further analyze and explore the possible drug resistance mechanism of CRKP,so as to provide a theoretical basis for clinicians to use antibiotics rationally and control nosocomial infection.Methods1.From May 2019 to November 2020,96 strains of CRKP were collected from Hengyang,Changsha,Xiangtan and Huaihua regions of Hunan Province,and the distribution of departments,sex and age of CRKP strains were statistically analyzed.2.The hypermucoviscosity phenotype of CRKP strains of 96 CRKP strains were screened by string test,and the preliminary identification and analysis of CRKP strains were conducted.3.The drug sensitivity analysis and strain identification were carried out by using VITEK 2 COMPACT microbial analysis system,and the MIC of common antimicrobials was determined by broth dilution method.KB method was used to verify the drug sensitivity of 96 strains of CRKP.4.96 strains of CRKP confirmed carbapenem phenotype by mCIM combined with eCIM test.PCR molecular method was used to detect the related drug resistance genes of CRKP,including carbapenem resistance genes(KPC,NDM,VIM,OXA-48),polymyxin resistance genes(MCR-1)and outer membrane porin genes(Ompk35 and Ompk36),and the detected drug resistance genes were identified by sequencing.Results1.The 96 strains of CRKP collected from hospitals were mainly isolated from sputum(55.21%),urine(14.58%)and blood samples(11.46%).The distribution of clinical departments were mainly from ICU(40.63%),neurosurgery(18.75%)and neurology(8.33%).Among the patients from which CRKP strains came,the isolation rate of male was 67.71%(65/96)higher than that of female 32.29%(31/96).The age distribution is mainly concentrated between 21 and 90 years old,and the highest was 61-70 years old,accounting for 21.88%(21/96).2.The positive rate of 96 CRKP strains with high myxoid phenotype detected by string test was 14.43%(14/96).3.The resistance of 96 CRKP strains to 26 kinds of commonly used antimicrobials was analyzed.The results showed that CRKP strains of resistance rates to carbapenem antibiotics--imipenem and ertapenem were 100%(96/96).The resistance rate to 26 antimicrobial agents was more than 50%.Among them,the resistance rate to penicillin antibiotics--ampicillin,mezlocillin,amoxicillin,carbenicillin--and lactamase combined inhibitors--ampicillin / sulbactam and amoxicillin / clavulanic acid was100%(96/96).The resistance rate to a variety of cephalosporins such as ceftriaxone,cefuroxime,cefotetan,ceftazidime and cefepime was more than 90%,while the resistance rate to aminoglycoside amikacin,gentamicin and tobramycin was relatively low,which were 61.46%(59/96),68.75%(66/96)and62.5%(60/96),respectively.4.After the combined test of mCIM and eCIM,the positive rate of mCIM test alone was 79.17%(76/96),and the positive rate of mCIM and eCIM test was 16.67%(16/96),suggesting that the CRKP strains in this study mainly produce serine enzymes.5.The drug resistance genes of CRKP strains were detected by PCR technology.And the results showed that KPC gene was detected in 95 strains(98.96%)of 96 CRKP strains,17 strains(17.71%)of NDM,9 strains(9.38%)of VIM,18 strains(18.75%)of OXA-48.16 strains(16.67%)of KPC and NDM genes were simultaneously detected,9 strains(9.38%)of KPC and VIM genes,17 strains(17.71%)of KPC and OXA-48 genes,3 strains(3.12%)of NDM and VIM genes,5 strains(5.21%)of NDM and OXA-48 genes,2 strains(2.08%)of VIM and OXA-48 genes.And 3 strains(3.12%)of KPC,NDM and VIM genes,4 strains(4.17%)of KPC,NDM and OXA-48 genes,1 strain(1.04%)with NDM,VIM and OXA-48 genes,and there were only 1 strain of KPC,NDM,VIM,and OXA-48 genotypes were detected.All the detected genes were confirmed as target genes by gene sequencing.6.The PCR results of MCR-1 gene resistance of polymyxin B showed that there were 34 strains(35.42%)of CRKP of MCR-1 gene detected in this study.All the detected genes were confirmed as target genes by gene sequencing.7.The 96 strains of CRKP were all detected the outer membrane pore protein Ompk35 gene,91 CRKP strains with Ompk36 genes were detected,and only 5 strains(5.21%)had outer membrane pore protein Ompk36 gene deletion.All the detected genes were confirmed as target genes by gene sequencing.Conclusions1.From the 96 strains of CRKP collected from several hospitals in Hunan Province,the strains are mainly isolated from sputum samples,and the clinical departments are mainly distributed in ICU,males were higher than that of females in clinical departments.The positive rate of Hypermucoviscous K.pneumoniae was also higher.Clinical surveillance of key departments and key groups should be strengthened to control nosocomial infection.2.The CRKP strains collected in this study mainly produce serine enzymes and are resistant to a variety of commonly used antimicrobials.The main mechanism of drug resistance is to produce KPC resistance gene,at the same time,the deletion of Ompk36 gene,and the positive rate of MCR-1 gene of polymyxin B resistance is also high. |
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