The Location And Genetic Environment Of CTX-M-55-producing Extended Spectrum Beta-lactamases In Gram Negative Bacteria And Dissemination Mechanism Of Bla_CTX-M-55 Genes

BackgroundThe detection rate of CTX-M-type extended spectrum beta-lactamases increasing yearly,which spread and diffusion rapidly in the world because of the abuse of broad spectrum beta-lactamases antimicrobial agents and the third generation cephalosporins.Since CTX-M-type extended-spectrum ?-lactamases were firstly reported in 1989,at least 26 bacterial species spreading around the world,which have become one of the most prevalence ESBLs type amongcommunity and nosocomial isolates.More than 150 different variants of CTX-M have been reported,of which CTX-M-55 belongs to CTX-M-1cluster,firstly reported in 2004 in Thailand and has been identified in E.coli,K.pneumoniae,S.marcescens and other ten species bacteria.More ten countries has isolated CTX-M-55,such as in China,South Korea,India,Japan,the United States,Britain,Russia and so on,and the host of CTX-M-55-producing bacteria contained various fields of human survival,included homo sapiens,poultry,livestock,water,vegetables,wild animals and plants,which serious threatened the health of public.The investigate of the dissemination mechanism of bla_CTX-M-55 genes can curb its spread,control its transfer,which has a significant social and clinical values.The bla_CTX-M often located on the plasmid,but some times it also can exist in the chromosome,which may related to strain type and the mechanism of which is to strengthen the drug-resistant genetic stability.Conjugative plasmid is one of the most important mechanisms for the emergence and spread of bla_CTX-M,which can make horizontal transfer to other pathogenic bacterial strains or even cross species barrier.Insertion sequences?ISs?are the smallest transposable element?<2.5Kb?capable of independent transposition in an organism,thereby causing insertion mutations and genome rearrangements and greatly increasing the opportunity that a resistant determinant becomes transferable.Various types of genetic platforms are associated with bla_CTX-M genes,of which ISEcp1 is frequently detected upstream of the bla_CTX-M.ISEcp1 can mobilize the downstream-located bla_CTX-M gene and provide a promoter to enhance its expression.In addition,other insertion sequences,including IS26,IS903,ORF477,are also frequently detected surrounding the bla_CTX-M.The dissemination mechanism of bla_CTX-M-55 genes is sophisticated.Our previous study have detected bla_CTX-M-55 in klebsiella pneumoniae and further research shows that the CTX-M-55 plays an important role in the process of the drug resistance of CTX-M.But whether the CTX-M-55 has a special ability to spread and has a unique genetic environment structure in Henan Province.If there is a correlation between domestic and foreign popular strains has not clearly.The puepose of the study is to research the gene localization,the environment of bla_CTX-M-55 ESBLs and way of transfer between resistant genes in bacteria by gene location detection analysis,methods of environmental research and plasmid combining test will use in this project.Thus it could help to clarify the evolvement mechanism of the enzyme activity of resistant,the alternative ways of genetic evolution and the rule of transferred between bacteria,and ultimately provide a laboratory guideline to clinical treatment of infectious diseases and the use of antimicrobial agents,as well as an important reference to delay or prevent the spread of antibiotic resistance.MethodsBacterial isolatesA total of 357 non-duplicate and resistance third and fourth generation cephalosporins gram negative bacteria were obtained from the First Affiliated Hospital of Zhengzhou University in Central China from July 2015 to December 2015.All isolates were identified by using Vitek 2?bioMérieux,France?.Antimicrobial susceptibility testing and ESBL confirmationAntimicrobial susceptibility for the bla_CTX-M-55-positive isolates and transconjugants were initially tested by using Vitek 2?bioMérieux,France?and then followed by the measurement of the MIC through the broth microdilution method.The standard microbroth and agar dilution methods were established in accordance with Clinical and Laboratory Standards Institute?CLSI?standard.The MIC results were interpreted according to the 2014 CLSI criteria.Location of bla_CTX-M-55 resistance genes.Plasmid DNA and chromosome DNA purification and ues PCR methods detected bla_CTX-M-55 respectively.Purified PCR products were directly sequenced from both ends.The genetic contexts of bla_CTX-M-55.Conjugation experiments and genetic environment detected to analyze the dissemination mechanism of bla_CTX-M-55 genes.The genetic environment of bla_CTX-M-55 detected through PCR methods and sequenced.Phylogenetic group determination and multilocus sequence typing?MLST?.MLST was used to analyze the homology of antibiotic resistance isolates.Results1.Identification of bla_CTX-M-55-positive isolates,antimicrobial susceptibility and resistance determinants.Based on the results of this study,13 Enterobacteriaceae were identified as bla_CTX-M-55 positive,including 7 E.coli,3 K.pneumoniae,1 C.freundii,1 M.morganii,1 S.marcescens,which were detained from specimens of blood?n=6?,urine?n=3?,and sputum?n=3?.2.Conjugal transfer of bla_CTX-M-55 gene.Conjugative assays revealed that all of the bla_CTX-M-55 plasmids were successfully transferred to E.coli C600 from the 13 donors by conjugation.3.Genetic contexts of bla_CTX-M-55.The flanking region of bla_CTX-M-55 is shown in Fig.1.Four different architectures [type??9 isolates?,type??2 isolates?,type ??1 isolate?,type ??1 isolate?] were identified for the genetic contexts of the plasmid-mediated bla_CTX-M-55 genes.Type? architecture(ISEcp1?-bla_CTX-M-55-?ORF477)was the most common and found in 9?69.23%?of 13 bla_CTX-M-55-positive isolates;type ?(IS26-?ISEcp1-bla_CTX-M-55-?ORF477)and type ? architecture(ISEcp1?-IS1294-?ISEcp1-bla_CTX-M-55-?ORF477)were similar to type? architecture with the exception that ISEcp1 was disrupted by IS26 in type ? and by IS1294 in type ?.Type ?(ISEcp1?-bla_CTX-M-55-?IS903)was a novel arrangement,which was characterized by the presence of IS903,located downstream of bla_CTX-M-55.Moreover,among 13 isolates carrying bla_CTX-M-55,5 isolates contained blaTEM and 2 isolates had both blaTEM and blaSHV4.MLSTMLST was performed for all of the bla_CTX-M-55-positive E.coli and K.pneumoniae isolates.Nine types of MLST were identified among the 7 E.coli isolates?ST156,ST305,ST182,ST381,ST446 and ST2?and 3 K.pneumoniae isolates?ST148,ST269 and ST37?,two E.coli isolates?EC32 and EC45?shared the same ST type?ST305?.Conclusions1.CTX-M-55 ESBLs has diffusion between different spices bacteria,especially in enterobacteriaceae.The clinical should be raise vigilance,to prevent bla_CTX-M-55 prevalence in nosocomial.2.The plasmid conjugation and the genetic context of bla_CTX-M-55,especially ISEcp1,plays an important role in mobilization,transfer and expression of bla_CTX-M-55 drug-resistant gene in our region.Clinical should strengthen the monitoring,to prevent the the local outbreak popularity.of cloning plants and nosocomial infection.