Molecular Mechanism Of Resistance For Enterobacteriaceae Clinical Isolates Producing ?-lactamases

BackgroundThe Enterobacteriaceae,especially Escherichia coli and Klebsiella pneumoniae,are common causes of nosocomial and community infections among humans,and?-lactams are most commonly used antibiotics for their infection.The epidemiological features of ?-lactam-resistant Enterobacteriaceae are always diverse in different countries and regions.Investigation of the clinical features and resistance mechanism provides important basis for hospital infection control.ObjectivesTo investigate the clinical properties and molecular mechanism of resistance for?-lactam-resistant Enterobacteriaceae strains in our hospital and to provide experimental evidence for antimicrobial chemotherapy of related infections.MethodsClinical Enterobacteriaceae strains were collected from The First Affiliated Hospital of Guangzhou Medical University between 2010 and 2015.Clinical data was analyzed by the SPSS software.Multiplex PCR was applied to detect thep-lactamase genes 'and plasmid types.ESBL genes in K.pneumoniae KP01 was detected by the double-disc synergy test and multiplex PCR.Conjugation and electro-transformation were performed to transfer plasmids.Total DNA of K.pneumoniae KP01 and E.coli was extracted by bacterial genomic DNA extraction kits,and Illumina Miseq was used for high-throughput sequencing.Sequencing data was analyzed by bioinformatic tools,including Edena,RAST,ResFinder,PlasmidFinder,MLST and NCBI Blast.ResultsA total of 4474 ?-lactam-resistant strains among 5829 Enterobacteriaceae isolates collected between 2010 and 2015 were mainly from urine(50.8%)and sputum(20.9%).The ward source included urology(27.6%)and ICU(11.9%),and E.coli(45.5%)and K.pneumoniae(35.6%)were the major species.The susceptibility rates of these strains to ampicilin,ceftriaxone,cefepime,imipenem and ertapenem were 0%,36.7%,71.9%,96.4%and 95.2%,respectively.They were also resistant in different extent to non-?-lactam antibiotics.blaTEM?blaCTX-M and blaSHV were found to be the major?-lactamase gene types,and IncF plasmid were the major plasmid types.K.pneumoniae KP01 was isolated from an old patient with severe pneumoniae,and was multidrug resistant.PCR detection revealed that KP01 carried blaCTX-M-24,blaSHV-11Hand blaGES-5 genes.This was the first report of blaGES-5-producing Enterobacteriaceae in China.The gene blaGES-5 could only be transferred by electro-transformation,suggesting that blaGES-5 was located on non-conjugative plasmids.Transformant carrying blaGES-5 showed significant MICs to?-lactam antibiotics.High throughput sequencing obtained a 167.7-fold-deep data with 99 contigs more than 500 bp length.Resistance genes analysis identified 3 for?-lactams(blaGES-5,blaSHV-11 and blaCTX-M-24),3 for aminoglycosides(aadA2,strA and strB),1 for sulfonamides(sul2),1 for trimethoprim(dfrA12)and 1 for tetracyclines(tetA).MLST analysis identified KP01 as ST895.blaGES-5 was found to be localized on a 28.5-kb plasmid pGES-GZ,and the replication-related genes share high sequence similarity(99%)with plasmids from non-fermenting bacteria.Analysis of gene environment revealed a novel class 1 integron without 3' conserve region(3'CS).Three gene cassettes,blaGES-5 and two identical orf28 genes,were identified within the integron.E.coli HX43 was isolated from a pediatric patient with severe pnemoniae,and was resistant to all antibiotics tested except carbapenems.High throughput sequencing obtained a 142-fold-deep data with 169 contigs more than 500 bp length.Resistance genes analysis identified 3 for?-lactams(blaCMY-42,blaCTX-M-14 and blaOXA-30),5 for aminoglycosides(aac(3)-IIa,aadA5,strA,strB and aac(6')-Ib-cr),1 for quinolones(aac(6')-Ib-cr),two for macrolides(erm(B)and mph(A)),2 for chloramphenicol(catB3 ? cm1A1),2 for sulfonamides(sull and sul2),1 for trimethoprim(dfrA12)and 1 for tetracyclines(tet(B)).blaCMY-42 was rare in China,and it was localized downstream of a truncated ISEcpl element,with outer membrane lipo-protein gene blc and disinfectant resistance gene sugE downstream.Plasmid analysis identified IncI1,Col,IncFIA,IncFIB,and IncFII plasmid types.MLST analysis found the HX43 belonged to ST3835.ConclusionsThe main ?-lactam-resistant Enterobacteriaceae strains were E.coli and K.pneumoniae in our hospital,with multiple P-lactamase genes and plasmids.The current study reported blaGES-5-producing Enterobacteriaceae in China for the first time.E.coli producing blaCMY-42 carried different ?-lactamase genes and plasmids.