摘要目的 了解初筛试验疑似产超广谱β内酰胺酶(ESBLs)但确证试验未能确认,而对头孢吡肟敏感的大肠埃希菌和克雷伯菌属中的ESBLs和质粒介导的AmpC酶.方法 纸片扩散法检测18株细菌对常用抗菌药物的敏感性;PCR及多重PCR法检测细菌中的ESBLs和质粒AmpC酶基因;质粒转移接合试验检测耐药质粒的可传递性;细菌基因间重复一致序列(ERIC)-PCR法检测供体大肠埃希菌和受体E.coli J53及其接合子的同源性;脉冲场凝胶电泳(PFGE)对11株大肠埃希菌和6株肺炎克雷伯菌进行同源性分析.结果 18株细菌均为2005年1月至12月期间上海华山医院临床分离的菌株,其中大肠埃希菌11株,肺炎克雷伯菌6株,产酸克雷伯菌1株,按常规方法对细菌进行重新鉴定和药敏试验.18株细菌经美国临床和实验室标准协会(CLSI)推荐的ESBLs初筛试验结果均为ESBLs产生可疑菌株,但确证试验未能确认;所有菌株的头孢吡肟抑菌圈直径均在18 mm以上,显示敏感.PCR检测结果显示,11株大肠埃希菌中有9株产CIT型质粒AmpC酶,DNA测序及序列比对结果证实为CMY-2型AmpC酶,未发现TEM、SHV、CTX-M、PER、VEB、SFO等广谱或ESBLs;6株肺炎克雷伯菌中有5株产DHA型质粒AmpC酶,DNA测序及序列比对结果证实为DHA-1型AmpC酶;5株产DHA-1型AmpC酶的肺炎克雷伯菌株中,4株同时伴有广谱或ESBLs:其中2株产SHV-11型广谱酶,另2株分别产CTX-M-14型ESBLs和SHV-62型ESBLs;1株产酸克雷伯菌亦单产DHA-1型AmpC酶;质粒转移接合试验结果表明,携带耐药基因的质粒可从供体菌转移至敏感细胞中;PFGE结果显示,6株肺炎克雷伯菌的谱型各不相同,而11株大肠埃希菌可分为5种谱型,其中B型包含7株细菌,这7株细菌均产生质粒介导的CMY-2型AmpC酶,并分离自外科病房,提示可能存在克隆菌株的流行传播.结论在确证试验未能确认的疑似产ESBLs中,对头孢吡肟敏感的大肠埃希菌和克雷伯菌属细菌主要产生质粒介导的AmpC酶,但尚有少数菌株同时伴有产ESBLs.对同时产生ESBLs和AmpC酶的菌株,临床微生物实验室必须报告这些菌株对头孢吡肟耐药.

abstractsObjective To study dIe ESBLs and plasmid-mediated AmpC enzymes in E.Coli and Klebsiella spp. with CLSI ESBL-screening test positive,confirmation test negative but cefepime susceptible.Methods Antimierobial susceptibility testing were performed by Kirby-Bauer(K-B)method.The genes encoding ESBLs and plasmid-mediated AmpC enzymes were detected by PCR Transfer of ESBLs or plagmid-mediated AmpC resistance was studied by conjugation experiments.The homology of donor (E.coli),recipient(E.coli J53)and their transconjugants were analyzed by ERIC-PCR DNA fingerprints of E.coli and Klebsiella pneumoniae were analyzed by PFGE as recommended bv PulseNet protocoL Results Of 18 isolates from Huashan Hospital,11 were E.coli.6 were Klebsiella pneumoniae and 1 was Klebsiella oxytoca.Antimicrobial susceptibility testing indicated all of 18 isolates were positive on the CLSI ESBL screening test but negative on the confirmation test.and all of isolates were susceptible to cefepime(a zoneof-inhibition diameter of≥18 mm wag considered to indicate susceptible).PCR results indicated that 9 of the 11 E.coli isolates predued CMY-2 AmpC enzyme.TEM,SHV,CTX-M,PER,VEB or SFO type β-lactamages were not identified.Of 6 Klebsiella pneumoniae isolates.5 were DHA-1 AmpC-producing strains.4 of the 5 DHA-1 AmpC-producing strains were coexistence of broad-speetrumβ-lactamaae or extended-spectrumβ-lactamase.including two producing SHV-11 and two producing CTX-M-14 and SHV-62 type ESBL respectively.One Klebsiella oxytoca wag also DHA-1 AmpC producing strain.Conjugation experiments indicated that both ESBLs and AmpC enzymes could be transfefred from donor to recipient.PFGE indicated that the DNA fingerprints of K.pneumoniae were difierent but seven CMY-2 AmpC-producing E.coli isolates from general surgieal ward were similar.Concluslons The main mechanism of antibiotic resistance in CLSI ESBLs-screening test-positive but eefepime.susceptible E.coli and KIebsiellaspp.is production of plagmid-mediated AmpC enzymes.Some strains produce both AmpC enzyme and ESBLs.Such strains should be reported as resistant to cefepime.The results suggest that laboratories should routinely conduct research on the ESBLs and plnsmid.mediated AmpC enzymes in Enterobacteriaceae in order to report antimicrobial susceptibility testing results more correcdy.