| BackgroundThe bacteria and its subspecies of Enterobacter cloacae complex(ECC)exist and spread widely in nature.As important clinically conditional pathogens,they can cause a variety of local or systemic infections,including nosocomial infections of the respiratory tract,blood,urethra,skin and soft tissues,biliary tract and central nervous system.They were also common pathogens in clinical infections caused by intravenous catheters.In addition,ECC strains have relatively complex and powerful genetic elements.They can colonize and survive in a variety of metabolic environments.It is more difficult to prevent and control such bacteria clinically than other pathogenic bacteria.At present,due to the pressure of selection of antibacterial drugs,the widely use of invasive operations in clinical and the patients with serious underlying diseases,the detection rate of ESBL-producing strains and even carbapenem-resistant ECC strains(CREC)have been rised gradually all over the world.The reports of CHINET showed that the carbapenem-resistant rate of Enterobacter cloacae in 2007 was less than 1.0%.But in 2017,the drug resistance rates to imipenem,meropenem and ertapenem of Enterobacter cloacae had increased rapidly to 6.9%,7.0%and 8.2%.In view of the mult-drug resistance of CREC strains,the infection caused by CREC strains will significantly increase medical costs,prolong the time of hospitalization,and lead to higher mortality rates.This poses threat to public health security.So far,a lot of research has been done on ECC strains at home and abroad.But both natural mutations and external human interventions will cause adaptive mutations in strains and generate new drug-resistant genes.Drug-resistant genes can be transferred in bacteria through various movable elements such as plasmids,insert sequences and transposons by conjugation and transformation.In addition,there is less research on whether there is a certain relationship between the ECC strains of intestinal tract and the strains that cause bloodstream infections.Based on the above,it has become one of the research focuses in the field of clinical microbiology to identify the transmission sources and routes of ECC strains and to explore the pathogenicity and drug resistance mechanism of the strains.With the rapid development of bioinformatics and high-throughput sequencing,it is possible to study the epidemiological and genomic characteristics,drug resistance,and transmission mechanisms of ECC strains.We collected 80 ECC strains from different sources.Vitro susceptibility test can be used to understand the resistance characterization of all strains.We used PCR,S1 nuclease pulsed field gel electrophoresis and southern blotting to determine the distribution of drug-resistant genes and locate them,and preliminary understand the mechanism of dissemination in ECC.We sequenced the whole genome by the second generation high-throughput sequencing to clearly identify strains and analyze their epidemiological characteristics,resistance genes and virulence genes.And we generate the genetic environment of drug resistance genes through RAST and Easyfig.The phylogenetic tree of ECC strains can be constructed based on SNP of the whole genome sequence and analyze the genetic relationship and evolution of the strains.Finally,our research can provide laboratory support for clinical treatment and prevention and control of ECC infections in hospital.Method1.ECC strains were screened and purified by Mac medium containing 2μg/ml cefotaxime,and identified by mass spectrometry.2.The drug sensitivity test was carried out by micro broth dilution method and agar dilution method to determine the resistance characterization.3.PCR and sequencing techniques were used to screen and identify the common ESBL and carbapenem resistance genes,and S1 nuclease pulsed field gel electrophoresis and southern blotting were used to locate the resistance genes.4.The second generation high-throughput sequencing technology was used to sequence the whole genome of all strains;the Average Nucleotide Identity(ANI)analysis was used to identify all strains;the MLST results were obtained by the whole genome sequence and the STs were assigned using the MLST database;RAST and Easyfig were used to understand and analyze the gene environment around the drug-resistant gene;the drug-resistant genes and virulence genes were carried out by CARD,VFDB,Resfinder and other bioinformatics software;TreeBeST was used to construct the phylogenetic tree based on the SNP of the whole genome to analyze the genetic relationship and evolution between strains.Results1.80 ECC isolates were screened from 2558 samples from various sources.The 80 isolates were identified by ANI analysis as E.hormaechei(n=69),E.cloacae(n=5),E.roggenkampii(n=2),E.ludwigii(n=2)and E.asburiae(n=2).2.The drug sensitivity test results showed that all isolates exhibited low susceptibility rates to Amoxicillin/clavulanate(2.47%),cefotaxime(4.94%),ceftazidime(32.10%),aztreonam(43.21%)and cefpirome(48.15%).Multidrug-resistant and even pan-resistant strains carry multiple resistance genes,including β lactams,carbapenems,macrolides,quinolones,aminoglycosides,tetracyclines,sulfonamides,rifampicin,trimethoprim and phenicols,which are basically consistent with their drug sensitivity results.3.PCR and sequencing results showed that a total of 30 ECC strains carried blaCTX-M gene,including blaCTX-M-9(n=15),blaCTX-M-15(n=6),blaCTX-M-3(n=4),blaCTX-M14(n=4),blaCTX-M-55(n=1)and blaCTX-M-64(n=1).In addition,7 ECC strains were detected to carry blaNDM-1 gene.4.MLST assigned all isolates to 46 STs(including 39 novel STs as of March 2020).The most prevalent clone of all isolates was ST78(n=8),followed by ST116(n=5),ST50(n=3)and ST97(n=2).The most common clone of the strains carrying the blaCTX-M gene was also ST78(n=6),followed by ST51,ST93 and ST116.The clone of the strains carrying the blaNDM-1 gene was all inconsistent.5.The blaCTX-M of 18 isolates were located in plasmids ranging from～50kb to～3 10kb,including blaCTX-M-9(n=11),blaCTX-M-3(n=2),blaCTX-M-14(n=2),blaCTX-M-27(n=2)and blaCTX-M-64(n=1).Eight different types of genetic environments of blaCTX-M-9 were observed in 11 isolates,and IS3000-blaCTX-M-9 was found in nine isolates.6.The blaNDM-1 gene of six strains was located on plasmids,and the surrounding genetic contexts of blaNDM-1 were different.BlaNDM1-ble-trpF-dsbD existed in all strains except SKLX53287.7.The phylogenetic tree showed that there was no significant clustering between ECC strains isolated from stool and blood.And some strains have high homology.8.All strains contain many more putative virulence factors,including IlpA、sodB、bioB、hemL、hemE、clpP、pgi、orfM、lpxA、galE、kdsA、wbjD/wecB,some of which were clade specific.Conclusion1.The STs of all the isolates perform a high genetic diversity.ST78 is one of the important factors to promote the spread of CTX-M gene in our hospital.The genetic environment of blaCTX-M-9 is diverse,and there is multiple genetic elements involved metastasis.2.The genetic environment of blaNDM-1 is diverse,blaNDM-1-ble-trpF-dsbD presents in most strains(5/6).3.ECC strains that cause bloodstream infections can come from intestinal colonies... |
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