| Extended-spectrum P -lactamases(ESBLs) are defined as 3 -lactamases capable of hydrolyzing oxyimino-cephalosporins that are inhibited by clavulanic acid (CA).These enzymes belong to Ambler's molecular class A and are placed into functional group 2be. The latest characterization of ESBLs are 5 types: SHV, TEM, CTX-M, OXA and other ESBLs.The major ESBL-producing bacteria are Enterobacteriaceae, especially Klebsiella pneumoniae and Eschrichia coli . The prevalence of ESBLs' genotypes varies greatly from country to country, region to region. Because different genotype has different susceptibility and biochemical characteristis,so it is important to strenghthen monitoring ESBLs' producing and understanding genotypes' distribution on our region.The purpose of this study was to investigated the prevalence of the genobtypes of ESBLs in Klebsiella pneumoniae and Eschrichia coli isolated in Hangzhou City in 2000-2001.1. Materials and MethodsA total of 198 isolates of E. coil and K. pneumoniae were isolated in December 2000 to June 2001 from urine,sputum,purulent secretion ,blood and et al on five hospitals in Hangzhou city.ESBLs-producing isolates were detected by inhibitor -potentiated disc diffusion test. (1) Initial screen test :Antimicrobial disk concentration: cefotaxime(CTX) 30ug, ceftazide (CAZ) 30ng, aztreonam(ATM) SO^g, ceftriaxone(CRO) 30(ig,Drugs sensitivity test was operated according to Kirby-Bauer method.The results' judge was on the basis of NCCLs 1999 standards. (2) Phenotypic confirmatory test: CTX, CAZ ,CTX / CA (CD3,30/10), CAZ /CA (CD2, 30/10). disces were OXOID program produce.An increase of 5mm in the zone of inhabiton in a disc containing clavulanic acid (CD2,CD3) compared to the drugs (CAZ,CTX) alone was considered a postive testQC recommendations were E.coli ATCC 25922.Agar dilution method was to determine the minimum inhibitory concentrations(MICs) of cefotaxime and ceftazidime.ESBLs genes analysised by isoelectric focusing(IEF) and Polymerase chain reaction (PCR) initially. IEF was performed by applying the supernatant of a crude sonic extract to Phast gels(pH gradient of 3-9) in a Phast-System apparatus. Base on SHV-1,TEM-1,OXA-10, OXA-2, CTX-M-3,CTX-M-5,CTX-M-8,CTX-M-9 DNA template,we designed and synthersised specific primers for the coding sequence.Sequnecing was performed with a big DyeDeoxyTerminator cycle sequencing kit (Perkin-Elmer).Finally, genotypes were determined by ammo acid sequence analysis.2. ResultsTotal of 51 isolates(25.76%)of 198 E. coil and K. pneumoniae strains,In ESBLs producers.there were 31.49%(40/127) of E.coli and 15.49%(11/71) of K.pneumoniae.The MICs results showed that incidence of cefotaxime resistance was 64.75%(33/51), the incidence of ceftazidime resistance was 9.8%(5/51). IEF analysis showed these ESBLs-producing strains expressed 1~6 kinds 3 -lactamases, pi 5.4, 8.0 and 7.6, 8.4were the major. PCR results showed that incidences of SHV, TEM, CTX-M type ESBLs were 17.65%,84.31% and 80.65%. Amino acid sequence analysis showed that genotypes were: SHV-11 ,12; TEM-1; CTX-M-3 and CTX-M-3-like, CTX-M-13, 14.3. Conclusions3.1 There were prevalence of ESBLs-production among E. coil and K.pneumoniae in December 2000 to June 2001 in Hangzhou city,the ESBLs'incidence was 25.76%.3.2 CTX-M-type ESBLs: CTX-M-3, CTX-M-13,CTX-M-14 were themajor genotypes of ESBLs in K. pneumoniae and Eschrichia coli isolated inHangzhou city.3.3 The first select in treatment to ESBLs-production bacteria inHangzhou city is imipenem ,then 3 -lactama/ 3 -lactamase inhibitor .Tazobactam is the best inhibitor.
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