| Objectives:1.In order to guide clinical medication,we are going to detect the sensitivity of NDM-1 positive CRKP to various common anti-bacterial drugs and analyze the characteristics for clinical infected patients.In addition,for analyzing the epidemiological characteristics of NDM-1 positive CRKP and confirming the situation of strain cloning,Multilocus sequence typing(MLST)will be used to analyze strains'ST typing.2.Study the virulence phenotype and virulence gene carrying status of NDM-1 positive CRKP strains.And lay the foundation for the molecular mechanism of blaNDM-1 resistance and pathogenicity.Methods:1.310 non-repeating CRKP isolated strains from clinical specimens of the First Affiliated Hospital of Kunming Medical University from January 2018 to June 2019 were collected.PCR method was used to determine blaNDM-1 strains.Vitek-2 Compact automatic microbiological analysis system combined with the disk diffusion method(KB method)and E-test method was used for drug sensitivity test.Then we retrospectively analyzed the clinical infection characteristics,including patient age,gender,department,specimen type,invasive procedures and antibiotic use.Multilocus sequence typing(MLST)was used to analyze the epidemiological characteristics for 26 NDM-1 positive CRKP strains.2.30 NDM-1 negative CRKP strains and 30 NDM-1 negative CSKP strains were collected at the same time as a control group.All strains were initially evaluated the virulence phenotype by drawing experiment and crystal violet semi-quantitative bio-film formation experiment.PCR combined gene sequencing was used to detect common High virulence capsule genes(K1,K2,K5,K54,K57,K20)and common virulence related genes(entB,ybtS,ureA,ycf,WabG,FimH,uge,iutA,KfuB,aero,rmpA,magA,Alls,IrnN and VatD).Results:1.The positive rate of NDM-1 positive CRKP strain was 5.16%.26 infected patients were mainly neonates and elderly patients.The separation rate of sputum was the highest in specimen types,and they were mainly from the ICU.The length of hospital stay varied from 9 to 100 days,and the average hospital stay was 36 days.17 of 26 Patients had performed invasive procedures.All patients used antibiotics,of which 16 patients used carbapenem antibiotics.Drug sensitivity results showed high resistance to commonly used antibacterial drugs.And to compound sinomine and amikacin,the resistance rates were relatively low,34.60%and 57.69%,respectively.At the same time,26 strains were sensitive to tigecycline and polymyxin B.26 NDM-1 positive CRKP were divided into 6 ST types by MLST detection,of which 14 strains were ST11 type,accounting for 54%,4 strains of ST105,3 strains of ST36 and ST37,1 strain of ST433 and ST1137.9 of 12 strains isolated from the NICU were ST11 type,indicating that there might be small-scale clonal dissemination in the hospital2.Virulence phenotype experiment,high virulence capsule genes and common virulence related genes carrying situation:?The drawing tests in 26 NDM-1 positive CRKP strains and 30 NDM-1 negative CRKP strains were negative.However,21 of 30 CSKP strains were positive.?According to strong,medium and weak classification,the proportion of biofilm formation in NDM-1 positive CRKP was 67%,24%and 9%,respectively.For NDM-1 negative CRKP,it was 63%,24%and 13%,respectively.For CSKP,it was 57%,27%and 16%.The biofilm formation ability in NDM-1 positive CRKP strains and NDM-1 negative CRKP strains were 0.5766±0.085 and 0.6178±0.1072,respectively.And there was no statistical difference between them(t=0.817,P=0.966).For CRKP and CSKP strains,the biofilm formation ability were 0.6008±0.0966 and 0.6273±0.1557.There was no statistical difference between them(t=0.460,P=0.849).?We cannot detect K1,K2,K5,K20,K54,K57 capsular serotypes in NDM-1 positive CRKP strains and NDM-1 negative CRKP strains.in CSKP strains,there were 15 strains of K1,7 strains of K2,1 strains of K5 and 2 strains of K57.?The virulence related genes of NDM-1 positive CRKP strains were most widely distributed with entB,ureA,ybtS,FimH and ycf,with positive rates of 100%,96%,96%,77%,and 88%,respectively.According to statistical analysis,the p value of 15 types of virulence genes in NDM-1 positive CRKP group and NDM-1 negative CRKP group were more than 0.005,and the difference was no statistically significant.We did not detect virulence genes of rmpA,Alls,vatD and IroN in the CRKP group.In CSKP group,14 types of virulence genes were detected except VatD.After statistical analysis,the P value in the virulence genes of iutA,kfuB,aero,rmpA,magA,Alls,and FimH was less than 0.005.There was a statistical difference.?In the NDM-1 positive CRKP group and the NDM-1 negative CRKP group,carrying 6 or 7 types of virulence genes were the most common types,with a maximum of 8 types of virulence genes,while the CSKP group mainly carried 9-11 types of virulence genes.Conclusions:1.NDM-1 positive CRKP is severely drug-resistant.And the molecular typing is mainly ST11.Small clonal transmission exists in NICU.2.the invasive ability of CRKP is weaker than CSKP.No high virulence CRKP carrying blaNDM-1 is found in this study. |
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