Detcetion Of Extended-spectrum β-lactamase Genotypes In Gram-negative Bacteriods Isolated From Clinical Specimens By PCR

Application of antibiotics has an important role in the controlling infectious diseases,however the emergence of resistant strains of bacteria brings a lot of problems in clinical practice.It has been estimated that enterobacteria and other species of gram-negative bacteria cause up to 20-30%nosocomial bacterial infections in developed countries,and their colonization in hospitalized patients appears to be associated with the use of antibiotics.Recently,the prevalence of multiple-drug-resistant bacterial strains has significantly restricted the availability of antibiotics for effective treatment of the bacterial infections. Production of extende-spectrumβ-lactamases(ESBLs)is a major cause of resistance to oxyimino-β-lactams among clinical Enterobacteriaceae.Of the various distinc ESBL type that have been described so far,the most widely spread are the SHV,TEM and CTX-M.In order to determine the genetic basis of the extende-spectrumβ-lactamase phenotype,PCR assays aimed at the ESBLs were performed.[Objective]To illustrate the genetic basis of the extende-spectrumβ-lactamase phenotypes of gram-negative bacteria isolates from several hospitals in Changchun,Jilin,Cnina.[Method]All isolates(49 Esherichia coli strains,13 Enterobacter cloace strains,18 Serratia marcescens strains,12 Burkholderia cepacia starins,10 Enterobacter aerogens strains,24 Klebsiella pneumoniae strains and 39 Pseudomonas aeruginosa starins)were identified by conventional test scheme.Drug-resistant genes associated with ESBLs in gram-negative bacterial isolates were amplified by PCR.[Results]1.32(65.3%)of the 49 Esherichia coli strains,10(76.9%)of the 13 Enterobacter cloace strains,4(22.2%)of the 18 Serratia marcescens strains,3(25%)of the 12 Burkholderia cepacia starins,4(40%)of the 10 Enterobacter aerogens strains,14(58.3%)of the 24 Klebsiella pneumoniae strains and 3(7.6%)of the 39 Pseudomonas aeruginosa starins carried drug resistant genes of ESBLs.2.TEM-1,CTX-M-1,CTX-M-9,TEM+CTX-M-1,TEM+CTX-M-9, CTX-M-1+CTX-M-9 and TEM+CTX-M-1+CTX-M-9 of ESBLs were found in Esherichia coli isolates,the positive rates of thses resistant genes are 12.2%,8.2%,8.2%,8.2%,2.0%,8.2%,and 18.4%,repectively.SHV, TEM,CTX-M-1,CTX-M-9,TEM+CTX-M-1,TEM+CTX-M-9, TEM+CTX-M-1+CTX-M-9 and SHV+TEM+CTX-M-1+CTX-M-9 of ESBLs are positive in Klebsiella pneumoniae isolates,the positive rates of these resistant genes are 12.5%,20.8%,4.2%,4.2%,4.2%,4.2%,4.2% and 4.2%,repactively.SHV,TEM+CTX-M-9 and CTX-M+CTX-M-9 of ESBLs were found in Pseudomonas aeruginosa isolates,the positive rates of thses genes are 2.6%,2.6%and 2.6%,respectively.SHV(11.1%)and CTX-M-1(11.1%)of ESBLs only were found in Serratia marcescens isolates.TEM(20%)and TEM+CTX-M-1+CTX-M-1(20%)of ESBLs are positive in Enterobacter aerogens isolates.TEM(25%)of ESBLs is found only in Burkholderia cepacia isolates.SHV(7.7%),TEM(30.8%), CTX-M-1(7.7%),SHV+TEM(7.7%),TEM+CTX-M-1(7.7%), TEM+CTX-M-1+CTX-M-9(7.7%)and SHV+TEM+CTX-M-1+CTX-M-9(7.7%)of ESBLs are positive in Enterobacter cloace isolates.3.Sequencing and analysis reveal that PCR product of SHV genotype in this study harbored 711 bp which match SHV genes in the GenBank database, named as SHV-11.TEM gene containing 789 bp is recognized as TEM-1. PCR product of CTX-M-1 genotype,which have 448 bp,was identified as CTX-M-3 and CTX-M-15 subtype according to DNA sequence and on the basis of GenBank database.CTX-M-4 genotype,which habored 787 bp, was finally determined as CTX-M-14 and CTX-M-18 subtpye. Furthermore,We also find that a point mutation occurs in CTX-M-14 gene (EMBL registration number:EF158299)presented in a strain of Klebsiella pneumoniae isolates.[Conclusion]1.In this experimental studies,PCR test for detection of ESBL genotypes in all isolates was performed,the results show a higher positive rate of ESBLs in all isolates in compared with other domestic hospitals.It suggestes that drug-resisteant strains of bacterial isolates have resulted in an increase in the incidence of infections that are caused by these organisms and that cannot be treated with currently avaailable antimicrobial agents.2.Drug-resistant genes in Esherichia coli isolates include TEM,CTX-M and multiple drug-resistant genes,such as TEM+CTX-M-1,TEM+CTX-M-9 and TEM+CTX-M-1+CTX-M-9,SHV was not found.In Klebsiella pneumoniae isolates,SHV and TEM genes are predominat,and multiple-drug resistant genes include TEM+CTX-M-1,TEM+CTX-M-9 and TEM+CTX-M-1+CTX-M-9.In Serratia marcescens isolates,SHV and CTX-M-1 are positive.In Enterobacter aerogens isolates,the positive drug resistant gegnes are TEM and TEM+CTX-M-1+CTX-M-9.In Burkholderia cepacia isolates,ESBL genes are negative except TEM genotype.In Enterobacter cloace isolates,we have found SHV,TEM, CTX-1,SHV+TEM,TEM+CTX-M-1,TEM+CTX-M-1+CTX-M-9 and SHV+TEM+CTX-M-1+CTX-M-9.In Pseudomonas aeruginosa isolates, drug-resistant genes include SHV,TEM+CTX-M-9 and CTX-M-1+CTX-M-9.3.The PCR has been extensively applied in medical diagnosis.Identification to the gene level of drug-resistance in gram-negative bacteria is important, in particularly for species that are intrinsically resistant to antibiotics.PCR assay provides a specific and rapid alternative for detection of genotypes of ESBLs in bacterial isolates.4.From our data,it should be ponited out here that in gram-negative bacterial isolates exhibit TEM-1 subtype,CTX-M-14 subtype,CTX-M-18 subtype,SHV-11 and multiple-drug resistant genes in Changchun area. Multiple-drug resistant genes both in Esherichia coli isolates and in Enterobacter cloace isolates should be emphasized.