Molecular Characteristics And The Relationship Between Drug Resistance And Virulence Of BlaNDM-1 Gene Positive Enterobacter Cloacae

Objectives:1 Carbapenem-resistant Enterobacter cloacae(CR-ECL)with blaNDM-1-positive were studied by MLST to understand its molecular prevalence characteristics and determine whether there is clonal dissemination.2 Analyze the sensitivity of blaNDM-1 positive CR-ECL to common antibacterial drugs,and guide the clinical rational use of drugs;study the relationship of Enterobacter cloacae(ECL)carrying blaNDM-1 resistance genes and virulence genes,biofilm formation ability,to clarify the correlation between bacterial resistance and virulence.Methods:1 The non-repetitive CR-ECL strains were collected from January 2015 to December 2020 in the First Affiliated Hospital of Kunming Medical Universit and collect patient medical records;PCR amplifies blaNDM-1 resistance genes and MLST analyzes the epidemiological characteristics molecules of blaNDM-1 positive CR-ECL.2 The antibacterial drug sensitivity experiment was completed by the VITEK2 Compact automatic bacterial susceptibility identification system combined with K-B method and E-test method;the blaNDM-1 negative CR-ECL strains and CS-ECL strains isolated at the same time were collected as control group to study the relationship between drug resistance and virulence of Enterobacter cloacae.All strains used PCR method to amplify 28 pairs of virulence genes,including Yersin virulence island genes(irp-2,fyuA),siderophore genes(fhuA,sodB,sltA),and type III secretion system(T3SS)Genes(escV,nleB),type V secretion system(T5SS)genes(pic,pet),type VI secretion system(T6SS)genes(clpB,icmf,VasD/Lip),lipopolysaccharide genes(WaaL,WaaG),type Ⅰ Fimbriae gene(fimH),type III fimbriae gene(mrkD),P fimbriae(papC),S fimbriae(sfaD),biofilm extracellular polysaccharide genes(wcaA,wcaM,wza),AcrAB-TolC efflux pump Genes(acrA,tolC),Shiga enterotoxin gene(shET1),cytotoxic necrosis factor gene(cnf1),hemolysin gene(hlyA),secretory autotransporter toxin gene(sat),heat stable enterotoxin gene(astA),to understand the distribution of virulence genes;The crystal violet experiment was used to preliminarily evaluate the biofilm forming ability of different groups of strains,and select 3 strains with strong biofilm forming ability in each group,the plate count method was used to determine the total number of viable bacteria in the biofilm,the soft agar plate method was used to determine the cell migration ability,Phenol-sulfuric acid method was used to determine the content of extracellular polysaccharides in biofilms,and laser confocal microscopy(CLSM)to observe the thickness of biofilms.Results:1 A total of 104 strains of Enterobacter cloacae were collected,67 strains of CR-ECL and 35 strains of CS-ECL respectively.Among 67 CR-ECL strains,5 blaNDM-1 positive strains were collected by the research group in the early stage,and 30 of the remaining 62 CR-ECL strains were detected with blaNDM-1 resistance gene,the detection rate was 48.4%.The 35 blaNDM-1 positive strains were mainly isolated from NICU and surgical wards.The specimens were mainly urine and blood;MLST typing results showed that ST74 is the most common clonal type of blaNDM-1 positive strains in this area(11/35),followed by ST114(10/35),ST78(3/35),ST336(3/35),etc.There are 7 cases of blaNDM-1 positive strains isolated from the NICU ward in a short time,all of which are the same clonotype ST 114,present a small-scale outbreak.2 Drug susceptibility test results,virulence gene carrying status and biofilm formation ability2.1 Drug susceptibility results showed that 35 strains of blaNDM-1 positive CR-ECL all showed multi-drug resistance,among which the resistance rate to amikacin was low at 2.9%,and they were all sensitive to tigecycline and polymyxin;2.2 Among all 104 ECL strains,the detection rates of AcrAB-TolC efflux pump and biofilm extracellular polysaccharide-encoding genes acrA,tolC,wcaA,wcaM and wza have higher detection rates,which are 79.8%,90.4%,87.5%,73.1%,92.3%respectively.the detection rate of T6SS coding genes clpB,icmf,VasD/Lip were all above 60%,T3SS coding genes(escV,nleB),T5SS coding genes(pet),P fimbriae(papC),S fimbriae(sfaD),hemolysin gene(hlyA),secretory autotransport protein toxin gene(sat),heat stable enterotoxin gene(astA)were not detected.Statistical analysis showed that the detection rate of virulence genes clpB,icmf,VasD/Lip and acrA in the blaNDM-1 positive CR-ECL group was significantly higher than that in the blaNDM-1 negative CR-ECL group,and the difference was statistically significant(P<0.05).The detection rate of virulence genes clpB,icmf,VasD/Lip in CR-ECL group was significantly higher than that in CS-ECL group,and the difference was statistically significant(P<0.05).2.3 ① The semi-quantitative crystal violet experiment showed that 104 ECL strains formed biofilms to varying degrees.The OD590nm measured values of CR-ECL biofilm formation in the blaNDM-1 positive and negative groups were 0.2655(0.1515)and 0.2157(0.1609),respectively.Statistics analysis showed that there was no significant difference between the two groups(P>0.05);the measured values of OD590nm of biofilm formation in the CR-ECL group and the CS-ECL group were 0.2317(0.1557)and 0.2157(0.0922),respectively,and there was no statistical difference between the two(P>0.05).②The median total number of viable bacteria in the biofilm of blaNDM-1 positive and negative CR-ECL strains were 56×105 CFU/ml and 62×105 CFU/ml,respectively,there was no statistical difference between them(P>0.05);The total number of viable bacteria in the biofilm of the CS-ECL group and the CS-ECL group were 58×105 CFU/ml and 72×105 CFU/ml,respectively,and there was no statistical difference between the two(P>0.05).③Bacterial swimming ability,the median diameter of the bacterial circle in the blaNDM-1 positive and negative CR-ECL groups were 26.5mm and 18.25mm,respectively,and statistical analysis showed no difference(P>0.05);CR-ECL group and CS-ECL group was 21.88mm and 29.00mm,and there was no difference in statistical analysis(P>0.05).④The extracellular polysaccharide content of biofilms,blaNDM-1 positive and negative CR-ECL were 45.96μg/ml and 46.48μg/ml,respectively,CR-ECL group and CS-ECL group were 46.22μg/ml and 44.73μg/ml,there was no statistical difference between the groups.⑤ The results of CLSM observation of the biofilm thickness showed that the CR-ECL biofilm thickness of the blaNDM-1 positive group and the negative group were 8.2 μm and 8.87 μm,respectively,and there was no statistical difference between the two;the CR-ECL group and the CS-ECL group were 8.35 μm,7.66μm,statistical analysis showed no difference between the two(P>0.05).Conclusions:1 The MLST types of blaNDM-1 positive CR-ECL strains are mainly ST74 and ST114.ST74 is scattered in various departments.ST114 may have caused a small-scale outbreak and spread in the NICU ward of the hospital.2 The blaNDM-1 positive CR-ECL resistance situation is severe,and it also increases the carrying rate of certain virulence genes,but has no effect on the formation of biofilms.