The Distribution, Transmission Mechanism And Genetic Environment Of CTX-M-65 Type Extended Spectrum Beta-lactamases

BackgroundIn 1989,the Extended spectrum beta-lactamases was first discovered in Germany.In recent years,due to the resistance selection pressure caused by the wide application of the third,fourth generation cephalosporins and new broad-spectrum beta-lactamases,the clinical detection rate of CTX-M type of ESBL is increasing,which has replaced SHV type and TEM type.It has become the most common ESBL type in the acquired infection of the hospital and the community.The CTX-M type of ESBL has rapidly spread globally.At present,CTX-M type ESBL has been detected in at least 26 strains of bacteria such as Escherichia coli and Klebsiella pneumoniae.The CTX-M-65 gene belongs to the CTX-M-9 group.Compared with CTX-M-14,there are two mutations in the CTX-M-65 gene,namely,Ala（GCC）80?Val（GTC）and Ser（AGC）275?Arg（CGC）.Since the first discovery of CTX-M-65 ESBL in a C.freundii isolate in a hospital in China in 2007,bla_CTX-M-65TX-M-65 has been rapidly spread in China,South Korea,the United States,and other countries in the past decade.The host bacteria involved various species of E.coli,Klebsiella pneumoniae,and Salmonella,and the infected host includes humans,poultry,wildlife,and water sources.It exists in various fields of human and animal daily life and poses a serious threat to the public health and safety,hinder the development of animal husbandry.Therefore,the study of CTX-M-65 gene can effectively control its metastasis,thereby curbing its diffusion and spread,and its clinical significance and social value are significant.Bla_CTX-MTX-M gene mostly located on the plasmid,a small part o^[6f]them present on the chromosomes.The location of drug _M^CTr^Xe^-sistance genes is related to the type of strains.The bla_CTX-MTX-M gene is located on plasmids with poor genetic stability.Conversely,when it is present on chromosomes,it has good genetic stability.The bla_CTX-MTX-M gene is transmitted horizontally by the plasmid.The conjugative plasmid can mediate the transfer and spread of the bla_CTX-MTX-M gene between bacteria of the same or different genus,leading to the drastic increase of the drug-resistant bacterial strain and the multiple drug-resistant or even pan-resistant strains outbreak and epidemic.The surrounding gene environment of bla_CTX-MTX-M gene,such as ISEcpI,IS26 and IS903,ORF477 and so on,which are inserted upstream of bla_CTX-M,play a very important regulatory role in its expression and metastasis.The above-mentioned movable element alone or at the same time involved in the regulation of drug resistance gene expression.In our previous study,bla _CTX-M-65TX-M-65 ESBL was detected in Klebsiella pneumoniae for the first time in Henan Province.However,whether bla_CTX-M-65TX-M-65 was prevailing in other local strains in this region.It is unclear whether the upstream and downstream gene structures of bla_CTX-M-65TX-M-65 and the ability to disseminate,and whether there are associations among the epidemic strains in other regions of the country.To solve the above problems,we entered the study of the subject,to be collected through clinical analysis of clinical specimens,a clear Henan area with bla_CTX-M-65TX-M-65 strain distribution,prevalence;gene mapping by bla _CTX-M-65TX-M-65 and high-throughput sequencing and probing the gene environment.The mechanism of bla_CTX-M-65TX-M-65 transmission was analyzed by plasmid transfer and ligation experiments.The genetic heterogeneity of bla _CTX-M-65TX-M-65 strain was studied by multilocus sequence typing clinical control with bla_CTX-M-65TX-M-65 strains provide scientific guidance.Method1.Strain identification and drug susceptibility analysisCollection from September 2016 to January 2017 in the bacterial room of the laboratory of the First Affiliated Hospital of Zhengzhou University.It was derived from sputum（including bronchoalveolar lavage fluid）,secretions（including pus）,blood,urine,and feces.Various drainage fluids and other specimens were resistant to the third-generation cephalosporin-resistant gram-negative bacilli.All the experimental strains were identified using a VITEK II COMPACT automatic microorganism identification analyzer.Drug susceptibility results were interpreted according to the 2016 CLSI standard..2.ESBL screening and phenotypic confirmatory testsThe ESBL and phenotypic confirmatory tests of suspicious strains were performed in strict accordance with the CLSI 2014 standard.3.Localization of bla_CTX-M-65TX-M-65 in bacteria and analysis of gene environmentThe bla_CTX-M-9TX-M-9 gene was amplified by PCR,and the amplified product was subjected to agarose gel electrophoresis.The amplified product was subjected to UVP gel imaging analysis and primary screening of bla_CTX-M-65TX-M-65 positive strains.The plasmid gene of CTX-M-65 primary screening positive strain was extracted and amplified by PCR.The gene sequence of bla_CTX-M-65TX-M-65 gene was determined and the gene environment of bla_CTX-M-65TX-M-65 was determined.4.Conjugation experimentThe transferability of bla_CTX-M-65TX-M-65 gene plasmids was analyzed by Conjugation experiment.5.Homologous analysis of strainsHomology analysis of the strains carrying the bla_CTX-M-65TX-M-65 gene was performed by multilocus sequence typing（MLST）technique.Result1.Distribution of CTX-M-65 ESBL in gram-negative bacilliAmong the 369strains of Gram-negative bacilli resistant to the third generation cephalosporins,PCR screening results showed that 12 strains of bla_CTX-M-65TX-M-65 were found in 279 strains of Enterobacteriaceae,of which 10 strains were Klebsiella pneumoniae,2 strains of E.coli.No bla_CTX-M-65TX-M-65 gene was detected in 90 non-fermenting bacteria.2.Gene Mapping and gene environment analysis of bla_CTX-M-65TX-M-65 The experimental results show that bla_CTX-M-65TX-M-65 gene of all strains is located on the plasmid.The upstream and downstream gene components of bla_CTX-M-65TX-M-65 gene were amplified and sequenced.The results showed that the upstream of bla_CTX-M-65TX-M-65 gene was mainly inserted into ISEcp1 and IS26 and the downstream was IS903.A total of four different types of genetic components were detected.Element I:IS26-?ISEcp1-472bp-bla_CTX-M-65-?IS903;Element II:IS26-ΔISEcp1-42bp-bla_CTX-M-65-ΔIS903;Element III:IS26-ΔISEcp1-66bp-bla_CTX-M-65-ΔIS903;Element IV:bla_CTX-M-65-ΔIS903.3.Conjugal transfer of bla_CTX-M-65TX-M-65 gene8 double anti-MacConkey flat conjugate growth,donor bacteria,recipient bacteria are not growing.By extracting the zygote plasmid DNA,the result of PCR amplification was used to perform local sequence alignment BLAST.The similarity of bla_CTX-M-65TX-M-65 gene sequence was 100%.4.Genetic relatednessIn this study,four different MLST types were obtained.MLST typing of 10 strains of Klebsiella pneumoniae resulted in ST11 and ST258,mainly ST11（8/10）.Two strains of Escherichia coli MLST genotyping results ST117 and ST4360,respectively,from Respiratory and Neonatology.Conclusion1.The CTX-M-65 ESBL in Henan Province has a high prevalence among gram-negative bacilli,and the bla_CTX-M-65TX-M-65 strain is a multi-drug resistant strain.2.The isolated strain carries the bla_CTX-M-65TX-M-65 gene located on the plasmid.The upstream of the bla_CTX-M-65TX-M-65 gene is mainly the insertion sequences ISEcp1 and IS26,and the downstream is the insertion sequence IS903.3.The plasable plasmid and inserts around bla_CTX-M-65,such as ISEcp1 and IS903,are involved in the widespread dissemination and metastasis of the bla_CTX-M-65TX-M-65 gene.