Molecular Characteristics Of Carbapenem-resistant Enterobacteriaceae Isolated From Clinical Infection And Fecal Survey Samples

Background and objectiveCarbapenems are often used to treat the infections caused by bacteria which can product extended spectrum β-lactamase β-lactamases(ESBLs).The positive rate of carbapenem-resistant Enterobacteriaceae(CRE)has gradually increased due to the extensive and irregular use of carbapenem antibiotics in recent years,especially carbapenem-resistant Klebsiella pneumoniae(CRKP).If patients are infected with CRE,it will be at high risk of death due to lack of appropriate antibiotics and high potential to cause outbreaks in healthcare settings.The human intestinal tract forms a major reservoir of various bacteria,including multidrug resistant(MDR)bacteria,and has an important role in the transmission of resistance genes.In recent years,it has been reported that CRE carrying one or more carbapenemase genes or colistin-resistant genes isolated from human feces.CRE intestinal colonization of some patients or people without any underlying disease,which will greatly increase the risk of clinical infection caused by CRE.The aim of this study was to compare the molecular characteristics of carbapenem-resistant enterobacteriaceae isolated clinical infection samples and fecal survey samples,and provide information for CRE infection prevention and control.Methods1.Collection of samples We collected CRE isolated from various clinical infection samples submitted by our hospital from July 2016 to June 2019.Moreover,2000 fecal survey samples were collected from July 2016 to June 2019,followed by incubation on MacConkey agar plates with 2μg/mL meropenem.And collecting gram-negative bacilli which were not sensitive to meropenem.2.Species identification and antimicrobial susceptibility testing Analysis of susceptibility to antimicrobial compounds was performed using the automated BD Phoenix 100 Microbiology System(Becton Dickinson and Co.,Franklin Lakes,NJ,USA)according to the guidelines of the Clinical and Laboratory Standards Institute(CLSI)M100-S29 criteria.Minimum inhibitory concentration(MIC)breakpoints of susceptibility to tigecycline were performed by E-test according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing(EUCAST).3.PCR detection of carbapenemase genes Detecting common carbapenemase genes,including blaKPC、blaNDM、blaVIM、blaIMP、blaSPM、blaOXA-48 in CRE isolated from clinical infection samples and fecal survey samples by PCR,and confirm positive PCR products for sequencing.4.PCR detection of ESBL genes PCR detection of common ESBL genes,including blaCTX-M、blaTEM and blaSHV in CRE isolated from clinical infection samples and fecal survey samples,and confirm positive PCR products for sequencing.5.PCR detection of colistin-resistant genes PCR detection of colistin-resistant genes,including mcr-1、mcr-2、mcr-3、mcr-4 and mcr-5 in CRE isolated from clinical infection samples and fecal survey samples.The positive PCR products were sequenced to confirm.6.PCR detection of class Ⅰ,Ⅱ,Ⅲ integrons and gene cassettes Detection of class Ⅰ,Ⅱ,and Ⅲ integrase genes in CRE isolated from clinical infection samples and fecal survey samples,and screened strains carring class Ⅰ,Ⅱ,and Ⅲ integrators.Then,the variable regions were further amplified and sequenced after RFLP analysis.7.PCR detection of ISCR1 and the gene cassettes The ISCR1 gene in the CRE isolated from clinical infection samples and fecal survey samples was screened,and strains carrying the ISCR1 gene was screened,and the variable region was further amplified,followed by RFLP analysis and sequencing.8.Plasmid conjugation and transfer Strains that need to be tested for plasmid conjugation are selected.EJ53 is used as the recipient for plasmid conjugation and transfer test,which were inoculated on a screening plate containing 2ug/mL meropenem,and drug susceptibility tests and drug resistance genes were tested for identifing the conjugation test.9.ERIC-PCR The genotyping and electrophoresis cluster analysis of ERIC-PCR was performed for the three species with the highest proportion in clinical CRE and fecal CRE,to study the clonal correlation and molecular epidemiological between the strains.10.The whole genome sequencing and evolutionary analysis Two strains were selected from clinical CRE and fecal CRE for whole-genome sequencing,and genomic analysis was performed based on the sequencing results.Results1.Collection of samples 99 CRE strains were isolated from clinical infection samples,mainly from the hematology department(37.4%)and the Intensive Care Unit(22.2%).30 CRE were screened out of 2000 fecal survey samples.2.Species identification and antimicrobial susceptibility testing CRKP is the dominant species in clinical CRE and fecal CRE,accounting for 66.67%of all CRE,followed by Enterobacter cloacae(17.05%)and E.coli(8.53%).Most of strains were resistant to cephalosporin antibiotics,and most of them were sensitive to tigecycline and colistin.3.Carbapenemase genes Strains carring carbapenemase genes were accounting for 97.8%in all CRE.The blaKPC-2(93.8%)was dominant carbapenemase gene,followed by blaNDM(52.7%),and blaSPM and blaOXA-48 were not detected.4.ESBL genes Strains carring carbapenemase genes were accounting for 97.8%in all CRE.blaCYX-M、blaTEM and blaSHV were detected in 24.2%,78.8%and 87.9%strains isolated from clinical samples,respectively;In fecal CRE blaCTX-M、blaTEM and blaSHV were detected in 30.0%、90.0%and 86.7%,respectively.5.Colistin-resistant genes Two strains were positive for mcr-1 gene in fecal CRE,and mcr-2、mcr-3、mcr-4 and mcr-5 were negative in all strains.6.Class Ⅰ,Ⅱ,and Ⅲ integrons and gene cassette In all strains,class Ⅰ and Ⅱintegrons were detected in 78.3%and 4.7%，the cassettes of class Ⅰ and Ⅱ integrons were detected in 28.7%and 50.0%,respectively.Class Ⅱ integrons were not detected.In additions,two novel gene cassettes of class Ⅱ integron were detected.7.ISCR1 and the gene cassette ISCR1 was detected in 69.8%in all strains,and the gene cassettes of ISCR1 were detected in 4.4%.ISCR1 in clinical CRE was detected in 71.7%,and ISCR1 in fecal CRE was detected in 63.3%.8.Plasmid conjugation and transfer The positive conjugators can express the characteristics of resistant to carbapenems and carry resistant genes,which indicates that the drug-resistant genes are located in the plasmid and have the potential for horizontal transfer.9.Genotyping of ERIC-PCR The similarity of Klebsiella pneumoniae strains isolated from clinical CRE and fecal CRE showed more than 90%,while Enterobacter cloacae and E.coli showed a high genetic diversity.10.The whole genome sequencing and analysis The comparative genomics analysis of two CRKP isolated from clincal samples and fecal samples,respectively,showed that two strains had the highest similarity.It indicated that clincal CRKP may be closely related to fecal CRKP.Analysis the differences of two CRKP showed that MarR family transcriptional regulator gene and mutations in the soxR gene may be related to the sensitivity of Klebsiella pneumoniae to tigecycline.Conclusion1.In this study,the molecular characteristics of 99 CRE isolated from clinical infection samples and 30 CRE isolated from 2000 fecal survey samples,mainly including the mechanism of resistant to antibiotic(carbapenem,colistin and tigecycline),mobile genetic elements(MGE),ERIC genotying and the whole genome sequencing(WGS),which indicates that fecal CRE is closely related to clinical CRE.Therefore,strengthening the monitoring and management of feces is essential for the prevention and control of CRE.2.MarR family transcriptional regulator gene and the mutation of soxR gene may participate in resistance to tigecycline of K.pneumoniae.