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Objective To investigate the distribution of high pathogenicity island(HPI)in multiple-drug-resistance gram-negative bacilli and analyze the protein sequence.Methods To amplify thefyuA-irp2 gene cluster of the 84 isolates by multiple polymerase chain reaction(PCR),the product was subsequently sequenced.Results The positive rate ofirpl,irp2,irp3,irp4 and fyuA was 40.48％,41,67％,5.95％,O％and 16.67％,respectively.Theamino sequence offyuA comefromEC06748,Kp7151 and PAE7 was usedto compare with AL590842,there are 100％identities.Amino sequence ofirp2 come from Kp49 and Kp51 have 99％identities with AAA27636.1,but amino sequence of irp2 come from EC04 and EC07 only have 90％identities with 1176840.The GenBank accession number is FJ211852 and FJ211851.Amino sequence ofirpl come fromKp 10,Kp49 and Kp51 have 99％identities with AL590842。and amino sequence ofirp3 come from EC03,Kp51,Kp10 and Kp49 have 97％identities with CAA73128.There are the same mutation among the same species,and different mutation among different species.Conclusion There was different extant mutant lost in thefy～t-i,v2 gene cluster in multiple-drug-resistanee gram-negative bacilli.
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Objective To investigate the distribution of the CTX-M- extended spectrum beta-lactamase (ESBLs) producing Esche- richia coli(ECO) and the molecular mechanism of dissemination. Methods To analyze the drug resistance of the 43 isolates, Kirby-Bauer susceptibility method was used. Multiple polymeraso chain reaction (PCR) was used to amplify the gene of ESBLs, AmpC, full length of blaCTX-M-like gene, insertion sequence (IS) ISEcp1B, IS903 , IS26 and integron I. NEST-PCR was used to detect if the beta-lactamase gene lo- cated in the integron I. The product of full length of bla-CTX-M like gone amplified by PCR was sequenced. Results Susceptibility test showed the resistance from high to low in turn was Ampicillin (97.68%), Coftriaxone (67.44 % ), piperacillin(65.12 % ), Cefotaxime (62.79 % ) ,Coftasidime(58.14% ), Cofasolin(55.81% ), Cofepime (53.49%), Cefexitin(51.16%), ciprofloxacin (44. 19% ), Aztreo- nam(41.86% ), Cefoperasone/Sulbactam ( 20.93% ), Amikacin (0% ), Imipenem (0% ), respectively. ECO was susceptive to Imipenem. CTX-M-G1 was found in 25 strains of ECO , TEM, SHV, CTX-M-G1, ISEcp1B, and integron I were found in the nine isolates. IS903 were found in ECO 3 and 5, and IS26 was found in ECO 3. In ECO 3 and 5, blaCTX-M-like was flanked upstream by ISEcp1B element that provided -35 and -10 promoter sequences and a right inverted repeat (IRR) recognized by transposase, downstream by IS903 provided an inverted re- peat, ISEcp1 B and IS903 composed the complex transpeson. Conclusion ISEcplB may drive the expression and dissemination of blaCTX-M-like gene at a high level.
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OBJECTIVE To investigate the phenotypical and genotypical characteristics of the ?-lactamase in the 302 strains of Gram-negative bacilli,and assess the characteristics of the drug resistance.METHODS The phenotypes of ?-lactamase were detected in the Gram-negative bacilli by disc diffusion screen test,which were further ascertained by disc diffusion confirmatory test,a cefoxitin three dimensional test,and metallo ?-lactamases double-disc synergy test.The ?-lactamases were detected by multiple PCR amplification,then DNA product was sequenced.Antibiotics susceptibility was detected by K-B method.RESULTS Among the clinical Gram-negative isolates,a total of 26.49%(80/302)strains′ ?-lactamases phenotypes were positive,ESBLs,ESBLs combined with AmpC enzyme,ESBLs combined with AmpC enzyme and MBL,and AmpC enzyme producing strains were found in 16.56%,6.62%,1.66% and 0.66%,respectively.A total of 24.83% strains′?-lactamases phenotypes were positive,strains produced ESBLs,ESBLs combined with AmpC enzyme,ESBLs combined with AmpC enzyme and MBL,and AmpC enzyme producing strains were found in 17.55%,6.95%,0.33%,and 0.33%,respectively.Drug-sensitivity test showed multi-resistant Gram-negative isolates were more sensitive to amikacin(18.75%),and heavier resistant to ampicillin(97.50%).CONCLUSIONS ?-Lactamase producing Gram-negative bacilli are resistant to multi-antibiotics.
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Objective To investigate the main genotypes and the epidemic characteristics of TEM-type and SHV-type extended-spectrum beta-lactamases (ESBLs) of the gram-negative bacilli in Guangzhou.Methods The phenotype of ESBLs from 3 500 clinical isolates were primary screened and confirmed by the NCCLS confirmatory test according to guidelines of NCCLS(2001). PCR were performed on 3 500 clinical isolates using primers specific for blaTEM and blaSHV, respectively, the PCR product were purified and sequenced by ABI prism 377 DNA sequencer.Results Total of 3 500 un-replicated and consecutive Gram-negative bacilli were isolated from 13 hospitals in Guangzhou in the past two years, and the prevalence of ESBLs-producing clinical Gram-negative isolates was 31.0%(1 084/3 500). The positive rates of PCR results for blaTEM, blaSHV, and both of them in all the clinical Gram-negative isolates were 24.0%(840/3 500),10.8%(378/3 500),and 3.7%(128/3 500), respectively. All PCR products for blaTEM were further identified as TEM-1-type non-ESBLs gene, including TEM-1,TEM-1B,TEM-1D,and TEM-1F,by DNA sequencing analyses. However, almost all of the blaSHV gene were further identified as SHV-type ESBLs gene(the prevalence of SHV-1 is 7.2% only).The prevalence of SHV-12/5a accounted for highest(50.0%,152/304) in Guangzhou. Our test also showed that 53%(340/641) of blaTEM-producers [JP2] were Escherichia coli, and 57.9%(176/304) of blaSHV-producers were Klebsiella pneumoniae.Conclusions [JP]We could conclude from above results that SHV-type ESBLs, especial SHV-12/5a, was the prevalent genotype of ESBLs of clinical gram-negative bacilli in Guangzhou. TEM-type ESBLs did not exist in our city. In addition, SHV-12/5a-producing strains probably were main epidemic strains in Guangzhou. As for detection of ESBLs with regular phenotype methods, there was possibility of false negative and false positive.
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Objective To investigate the antibiotic resistance and molecular epidemiology character of ?-lactamase producers among clinical isolates of Citrobacter freundii.Methods Four strains of Citrobacter freundii were detected by K-B susceptibility method and three-dimensional test.The ?-lactamase gene was identified by polymerase chain reaction(PCR) and sequencing.Results The No.29 strain of Citrobacter friundii was multiple-drug-resistant and proved to produce CMY,DHA type AmpC,TEM and SHV type of extended-spectrum ?-lactamases(ESBLs).The sequencing of corresponding DNA revealed 97% identities of the CMY-2 amino acid sequence.It was a new type of cephalosporinase.Conclusion A novel strain of Citrobacter friundii with CMY type AmpC,DHA type AmpC,TEM and SHV type ESBLs has been found in southern China and it is multiple-drug-resistant.