Analysis Of Drug Resistance And In Vivo And In Vitro Combined Drug Susceptibility Study Of Carbapenem-resistant Enterobacter Cloacae

Part I:Antimicrobial resistance analysis of clinical isolates of Enterobacter cloacaeObjective:To investigate the prevalence of isolated Enterobacter cloacae and antimicrobial resistance in anhui provinceMaterials and methods:525 strains of non-repeat Enterobacter cloacae were collected from 34 hospitals in anhui from September 2013 to September 2017.Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC 27853 were used as drug sensitive quality control strains.Agar dilution method was used to test minimal inhibitory concentrations(MIC)of 525 strains of Enterobacter cloacae against 25 kinds of antimicrobial agents.Results:From 2014 to 2017,there was an increasing trend of clinical isolates of Enterobacter cloacae in anhui province.And since 2015,the proportion of Enterobacter cloacae in Enterobacteriaceae has been on the rise for three consecutive years.Among the patients with clinical isolates of Enterobacter cloacae,the proportion of male patients was significantly higher than that of female patients.Enterobacter cloacae was originated from patients of all ages,and the majority of the infected patients were>61-year-old patients,accounting for 57.5%.Sputum(56.7%)was the most common source of specimens,followed by urine(17.09%).There were no regional differences in the distribution of clinical isolates of Enterobacter cloacae in anhui province.Enterobacter cloacae showed different degrees of resistance to common clinical antibiotics,with the highest resistance rate of cefazolin(93.6%),followed by ampicillin(86.2%).The drug resistance rate of imipenem and meropenem was less than 10%.Amikacin was also sensitive,with a drug resistance rate of 10.8%.Furantoin was the most sensitive,with a drug resistance rate of only 3.9%.46 strains of imipenem-resistant cloacae were resistant to almost all antimicrobial agents.The most common drug-resistant phenotype was aztreonam-ciprofloxacin-gatifloxacin-chloramphenicol-piperacillin/tazobatan-gentamicin,accounting for 20%.Conclusion:1.The proportion of clinical isolated Enterobacter cloacae in Enterobacteriaceae increased year by year in anhui province.>61-year-old patients accounted for 57.5%of all patients.56.7%specimens were isolated from sputum.2.Clinical isolates of Enterobacter cloacae in anhui province showed different degrees of resistance to common antibiotics.The drug resistance rate of imipenem and meropenem was less than 10%.Furantoin was the most sensitive,with a drug resistance rate of only 3.9%.3.The most common multidrug resistant phenotype of clinical isolates of carbapenem-resistant Enterobacter cloacae in anhui province was aztreonam-ciprofloxacin-gatifloxacin-chloramphenicol-piperacillin/tazobatan-gentamicin.Part?:Analysis of drug resistance and homology of clinical isolates ofcarbapenem-resistant Enterobacter cloacaeObjective:To investigate the phenotype,drug resistance genes and homology analysis of imipenem-resistant Enterobacter cloacae isolates in anhui province.Materials and methods:The resistant phenotypes of 5 years imipenem-resistant Enterobacter cloacae in anhui province were analyzed.In 2017,10 strains of Enterobacter cloacae resistant to imipenem were isolated from clinical samples of various hospitals in anhui province and sequenced by PCR amplification for carbapenemase,Extended-Spectrum?-Lactamases,cephalosporin resistance gene and quinolone resistance gene.mCIM and eCIM experiments were performed to verify carbapenem enzyme and metallocapenem enzyme.J53 plasmid conjugation was performed on Enterobacter cloacae with positive NDM gene to verify whether NDM was on the transfer plasmid.Finally,MLST and PFGE were used to determine whether imipene-resistant Enterobacter cloacae in anhui province were monocloned.Results:A total of 46 strains of imipene-resistant Enterobacter cloacae were found in 525non-repeat Enterobacter cloacae specimens.The drug resistance rate of 46 strains of imipenem cloacae to ampicillin was as high as 100%,83.3%to ampicillin,70%to third generation cephalosporins,60-70%to quinolones and 35%to amikacin.In 2017,8strains of 10 strains of carbapenem-resistant Enterobacter cloacae were tested positive for class B metallocapenemase.Among the 8 strains,7 were positive for NDM,all of which were NDM-1 type by sequencing.One strain was positive for IMP,and was identified by sequencing as IMP-4 type.KPC and other carbapenemases such as VIM,GES,SME and OXA-48 were negative.mCIM and eCIM experiments showed that 7 of8 double positive strains were positive for NDM gene and 1 was positive for IMP gene.E.coli J53 plasmid conjugation with sodium azide resistance was successfully conjugated in 6 of 7 strains of NDM positive Enterobacter cloacae.In addition to ST754 in GN0576 and GN2968,the other 8 strains were all of different ST types.PFGE also confirmed that the 10 strains were not homologousConclusion:1.The drug resistance rate of carbapenems resistant Enterobacter cloacae in anhui province to the second and third generation cephalosporins and aztreonam is up to over70%.Resistance to quinolones ranged from 60 to 70%.They were sensitive to cefepime,compound antibiotics with?-lactamase inhibitors and amikacin.2.Carbapenems resistant Enterobacter cloacae in anhui province are mostly NDM-1enzyme producing strains,and no KPC enzyme producing strains were found.3.MLST showed that Enterobacter cloacae resistant to carbapenems in anhui province was dispersed rather than cloned.Part?:In vitro and in vivo combined drug susceptibility research of clinicalisolates of carbapenem-resistant Enterobacter cloacaeObjective:The antibacterial activity of imipenem combined with polymyxin E was studied by checkerboard assay and time-kill assay in vitro,and the in vivo effects of imipenem combined with polymyxin E were evaluated by the larval infection model.Materials and methods:Two strains of clinically isolated Enterobacter cloacae(imipenem resistant GN0791,imipenem sensitive GN1059)were screened out.In vitro,the combination of imipenem and polymyxin E dilution checkerboard assay was used to calculate the partial inhibitory concentration index and sensitive break-point index in order to analyze whether the combination of the two drugs has synergistic effect.In vitro,imipenem and polymyxin E were used as single agents and combined in dynamic time-kill assay,and the method of calculating the number of colonies was used to determine whether there was synergistic effect and bactericidal effect.An animal model using the larva of Galleria mellonella infected by Enterobacter cloacae was established.The survival curve of larva was made according to the survival number of larva after injecting the bacterial solution into larva,and the single drug and the combination of antibacterial drugs were used to observe whether there was a synergistic effect between imipenem and polymyxin E in vivo.Results:Microbroth dilution method was used to determine the minimum inhibitory concentration of polymyxin E against Enterobacter cloacae.MIC of strain GN0791against polymyxin E was 1mg/L,and MIC of strain GN1059 against polymyxin E was0.5mg/L.It was found in the in vitro checkerboard assay combined drug susceptibility test that,regardless of imipenem resistance,polymyxin E could significantly reduce the MIC value of imipenem in the combined treatment of Enterobacter cloacae.By calculating partial inhibitory concentration index and sensitive break-pointindex,it can be concluded that the combination of the two drugs has synergistic effect in vitro.In vitro bactericidal experiments,both 1×MIC and 2×MIC in the combination group of imipenem and polymyxin E showed sustained and rapid bactericidal effect on Enterobacter cloacae,and there was a synergistic effect between the combination group and the single drug group.Optimal concentration(80%mortality of larva)of Enterobacter cloacae infection was between 10~5~10~6 CFU/larva,the optimal temperature of incubation was at 37?.The survival rate of the larvae was only related to the living bacteria,and the death of the bacteria or the metabolites after the death of the bacteria could not lead to the death of the larva of Galleria mellonella.The larva dose was calculated based on the average body weight and simulated in vivo experiment,and the survival curve was made based on the number of larvae surviving every 24 hours.It was confirmed that imipenem combined with polymyxin E could significantly improve the survival rate of larvae infected by imipenem resistant Enterobacter cloacae,which was consistent with the results of combined drug sensitivity in vitro.Conclusion:1.The checkerboard assay combined with time-kill assay confirmed that imipenem combined with polymyxin E had synergistic effect on Enterobacter cloacae in vitro.2.The survival curve of the larva of Galleria mellonella confirmed that imipenem combined with polymyxin E had a synergistic effect on Enterobacter cloacae in vivo.3.In vivo and in vitro experiments further verified the efficacy of imipenem combined with polymyxin E in the treatment of imipenem-resistant Enterobacter cloacae.