老年患者大肠埃希菌产质粒AmpC酶基因型特点及耐药性分析

1、老年患者大肠埃希菌产质粒AmpC酶基因型特点及耐药性分析         11-01-12 16:03:00     编辑：studa20                   作者：张淑芳 吴多荣 张应爱 黄会 韩小胜【摘要】  目的 了解临床分离老年患者大肠埃希菌产质粒AmpC酶的基因型

2、及其耐药(DR)性特点，为临床合理选用抗生素提供依据。方法 收集2009年度老年患者临床不重复分离大肠埃希菌233株，用三维试验和确证试验分别进行羟氨苄青霉素(AmpC)酶和超广谱内酰胺酶(ESBLs)检测，用接合试验证实酶基因型的转移，通过PCR检测及序列分析进行质粒AmpC酶基因型确定，采用药敏纸片扩散法(KB法)进行药敏试验。结果 233株大肠埃希菌中，头孢西丁抑菌环直径17 mm的有53株，占22.7%，酶提取物三维试验检测7株阳性，4株(1.7%)PCR扩增出CMY2型质粒AmpC酶，有1株接合试验阳性;ESBLs阳性占68.7%(160/233)。药敏结果显示产质粒AmpC酶大肠埃

3、希菌对二、三代头孢菌素、氨曲南、头孢西丁和含酶抑制剂的DR率明显高于非产酶株(P<0.05)，所有大肠埃希菌对亚胺培南和美罗培南均100%敏感。结论 从老年患者中成功分离出产CMY2型质粒AmpC酶大肠埃希菌，产质粒AmpC酶菌株对内酰胺类抗生素的耐药性明显高于非产酶株。 【关键词】  质粒AmpC酶;大肠埃希菌;基因型;耐药性【Abstract】 Objective To understand the genotype characteristics and drug resistance (DR) in Escherichia coli producing plasmidm

4、ediated AmpC lactamases in the elderly patients so as to guide the rational use of antibiotics in clinical.Methods 233 strains of E.coli isolated from the elderly patients in the 8 hospitals in 2009 were collected.Threedimensional test and certified test were used to detect AmpC and ESBLs;conjugatio

5、n experiment were used to verify the transitivity of genotype.PCR amplification test and screening analysis were performed to confirm the genotype of plasmidmediated AmpC lactamases;Disk diffusion susceptibility (KB method) was adopted to detect drug sensitive test.Results Among 233 Escherichia stra

6、ins，there were 53 Escherichia strains of which Cefoxitin inhibition zone was equal to or below 17 mm (22.7%)，7 positive strains of threedimensional test from the enzymic extraction of which there were 4 strains producing CMY2 type AmpC(1.7%);the drugsensitivity data showed that the resistance to the

7、 second and third generation cephalosporins，aztreonam，cefoxitin and containing enzyme inhibitors were significantly higher than those of the no enzymic strains(P<0.05).All the strains of Escherichia colis were sensitive to meropenem and imipenem.DR rate to 12 antibiotics of these strains producin

8、g enzyme (AmpC alone，ESBLs only，both plasmid AmpC and ESBLs) or not had statistically significant differences (P<0.05).Nonlactamase producing strains remained at a high sensitivity rate to 15 kinds of antibiotics;DR rate of the AmpCproducing strains to the second and third generation cephalospori

9、ns，aztreonam and cefoxitin was up to 100%.However，these strains showed a high sensitivity rate to cefepime and carbapenems;ESBLsproducing strains also showed high resistance to the third and fourth generation cephalosporins and aztreonam and other antibiotics，but they were mostly sensitive to carbap

10、enem，enzyme inhibitors and cefoxitin.Both AmpC and ESBLs producing strains increased DR rate to other antibiotics except meropenem and imipenem (sensitive) significantly.Conclusions The resistance of Escherichia coli to lactam antibiotics mainly includes the production of ESBLs and AmpC，the enzyme p

11、roduction strains can be selected carbapenem on the basis of clinical experience.【Key words】 Escherichia coli;Plasmidmediated AmpC;ESBLs;Drug resistance大肠埃希菌是最常见的医院内感染的细菌之一，其严重耐药(DR)问题已引起临床上的广泛关注，其中产质粒AmpC酶和ESBLs是其DR的主要原因，尤其产质粒AmpC酶的大肠埃希菌耐药性更为严重，它对除第四代头孢菌素和碳青霉烯类之外的所有内酰胺类抗生素DR1，此类菌株的流行传播对临床治疗和控制医院感染造

12、成极大困难，目前质粒介导的AmpC酶已经发现了数十种，但国内报道的质粒AmpC酶主要为DHA1、ACT1和CIT1型等2。为了解我院老年患者临床分离大肠埃希菌产质粒介导AmpC酶的基因型和耐药性情况，现对2009年度从我院老年患者各类标本分离的231株大肠埃希菌进行分析报道如下。1 材料与方法1.1 菌株来源、仪器及PCR引物 收集2009年度老年(6098岁)患者临床标本不重复分离大肠埃希菌株233株，其中：尿102份、痰81份、分泌物16份、血液14份、胆汁10份、脓液5份、其他5份。所有菌株用VITEK32细菌分析仪鉴定到种。质控菌株：阴沟肠杆菌(029M)作为AmpC酶的阳性对照菌株(

13、北京协和医院检验科惠赠);肺炎克雷伯菌(ATCC700603)为ESBLs 阳性对照。药敏质控菌为大肠埃希菌(ATCC25922)、铜绿假单胞菌(ATCC27853)、金黄色葡萄球菌(ATCC25923)。VITEK32细菌分析仪及GNI+鉴定卡购自法国梅里埃公司;PCRC1000扩增仪(美国BioRad)，PCR用引物由上海生工生物公司合成。阿莫西林/克拉维酸(AMC)、哌拉西林/他唑巴坦(TZP)、亚胺培南(IPM)、美洛培南(MEM)、头孢噻肟(CTX)、头孢他啶(CAZ)、头孢吡肟(FEP)、氨曲南(ATM)、庆大霉素(CN10)、妥布霉素(TOB)、阿米卡星(AK)、头孢西丁(FOX

14、)、左氧氟沙星(LEV)、头孢呋辛(CXM)、MH(MuellreHintn)培养基和肉汤均为英国Oxoid公司产品。1.2 药敏试验 采用CLSI推荐的纸片扩散法(KirbyBauer)进行，根据CLSI2009年颁布的判断标准进行药敏结果判断3。1.3 ESBLs检测 参照CLSI20091推荐的筛选试验和确证试验进行。1.4 AmpC酶检测 AmpC酶表型筛选试验：用纸片扩散(KB)法测FOX抑菌环17 mm者，提示FOX中介或DR，估测其可能产AmpC酶。AmpC酶三维试验：参照赵虎等4报道的三维试验方法(冻融法)进行。阴沟肠杆菌029M为持续高产AmpC酶阳性对照，大肠埃希菌ATCC25922作为阴性对照。接合试验参照文献5进行。质粒AmpC基因型的鉴定：根据AmpC基因的分群设计多个特异性引物序列，采用PCR扩增法对鉴定菌进行基因型鉴定。PCR引物：分析GenBank数据库收录的质粒AmpC基因序列，将质粒AmpC基因按相似性分为6组，见参考文献6(引物序列见表1)。表1 质粒AmpC基因型检测引物1.5 细菌总DNA提取 采用煮沸法提取细菌总DNA。1.6 PCR扩增 反应体系为25 l：2×PCR 混合物12.5 l，上下游引物(10 pmol/L)各1 l，模板1 l，用dd H2O补充体积至25 l，矿物油覆盖，放入C1000扩增仪