老年患者大腸埃希菌產(chǎn)質(zhì)粒AmpC酶基因型特點(diǎn)及耐藥性分析

1、老年患者大腸埃希菌產(chǎn)質(zhì)粒AmpC酶基因型特點(diǎn)及耐藥性分析         11-01-12 16:03:00     編輯：studa20                   作者：張淑芳 吳多榮 張應(yīng)愛(ài) 黃會(huì) 韓小勝【摘要】  目的 了解臨床分離老年患者大腸埃希菌產(chǎn)質(zhì)粒AmpC酶的基因型

2、及其耐藥(DR)性特點(diǎn)，為臨床合理選用抗生素提供依據(jù)。方法 收集2009年度老年患者臨床不重復(fù)分離大腸埃希菌233株，用三維試驗(yàn)和確證試驗(yàn)分別進(jìn)行羥氨芐青霉素(AmpC)酶和超廣譜內(nèi)酰胺酶(ESBLs)檢測(cè)，用接合試驗(yàn)證實(shí)酶基因型的轉(zhuǎn)移，通過(guò)PCR檢測(cè)及序列分析進(jìn)行質(zhì)粒AmpC酶基因型確定，采用藥敏紙片擴(kuò)散法(KB法)進(jìn)行藥敏試驗(yàn)。結(jié)果 233株大腸埃希菌中，頭孢西丁抑菌環(huán)直徑17 mm的有53株，占22.7%，酶提取物三維試驗(yàn)檢測(cè)7株陽(yáng)性，4株(1.7%)PCR擴(kuò)增出CMY2型質(zhì)粒AmpC酶，有1株接合試驗(yàn)陽(yáng)性;ESBLs陽(yáng)性占68.7%(160/233)。藥敏結(jié)果顯示產(chǎn)質(zhì)粒AmpC酶大腸埃

3、希菌對(duì)二、三代頭孢菌素、氨曲南、頭孢西丁和含酶抑制劑的DR率明顯高于非產(chǎn)酶株(P<0.05)，所有大腸埃希菌對(duì)亞胺培南和美羅培南均100%敏感。結(jié)論 從老年患者中成功分離出產(chǎn)CMY2型質(zhì)粒AmpC酶大腸埃希菌，產(chǎn)質(zhì)粒AmpC酶菌株對(duì)內(nèi)酰胺類(lèi)抗生素的耐藥性明顯高于非產(chǎn)酶株。 【關(guān)鍵詞】  質(zhì)粒AmpC酶;大腸埃希菌;基因型;耐藥性【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大腸埃希菌是最常見(jiàn)的醫(yī)院內(nèi)感染的細(xì)菌之一，其嚴(yán)重耐藥(DR)問(wèn)題已引起臨床上的廣泛關(guān)注，其中產(chǎn)質(zhì)粒AmpC酶和ESBLs是其DR的主要原因，尤其產(chǎn)質(zhì)粒AmpC酶的大腸埃希菌耐藥性更為嚴(yán)重，它對(duì)除第四代頭孢菌素和碳青霉烯類(lèi)之外的所有內(nèi)酰胺類(lèi)抗生素DR1，此類(lèi)菌株的流行傳播對(duì)臨床治療和控制醫(yī)院感染造

12、成極大困難，目前質(zhì)粒介導(dǎo)的AmpC酶已經(jīng)發(fā)現(xiàn)了數(shù)十種，但國(guó)內(nèi)報(bào)道的質(zhì)粒AmpC酶主要為DHA1、ACT1和CIT1型等2。為了解我院老年患者臨床分離大腸埃希菌產(chǎn)質(zhì)粒介導(dǎo)AmpC酶的基因型和耐藥性情況，現(xiàn)對(duì)2009年度從我院老年患者各類(lèi)標(biāo)本分離的231株大腸埃希菌進(jìn)行分析報(bào)道如下。1 材料與方法1.1 菌株來(lái)源、儀器及PCR引物 收集2009年度老年(6098歲)患者臨床標(biāo)本不重復(fù)分離大腸埃希菌株233株，其中：尿102份、痰81份、分泌物16份、血液14份、膽汁10份、膿液5份、其他5份。所有菌株用VITEK32細(xì)菌分析儀鑒定到種。質(zhì)控菌株：陰溝腸桿菌(029M)作為AmpC酶的陽(yáng)性對(duì)照菌株(

13、北京協(xié)和醫(yī)院檢驗(yàn)科惠贈(zèng));肺炎克雷伯菌(ATCC700603)為ESBLs 陽(yáng)性對(duì)照。藥敏質(zhì)控菌為大腸埃希菌(ATCC25922)、銅綠假單胞菌(ATCC27853)、金黃色葡萄球菌(ATCC25923)。VITEK32細(xì)菌分析儀及GNI+鑒定卡購(gòu)自法國(guó)梅里埃公司;PCRC1000擴(kuò)增儀(美國(guó)BioRad)，PCR用引物由上海生工生物公司合成。阿莫西林/克拉維酸(AMC)、哌拉西林/他唑巴坦(TZP)、亞胺培南(IPM)、美洛培南(MEM)、頭孢噻肟(CTX)、頭孢他啶(CAZ)、頭孢吡肟(FEP)、氨曲南(ATM)、慶大霉素(CN10)、妥布霉素(TOB)、阿米卡星(AK)、頭孢西丁(FOX

14、)、左氧氟沙星(LEV)、頭孢呋辛(CXM)、MH(MuellreHintn)培養(yǎng)基和肉湯均為英國(guó)Oxoid公司產(chǎn)品。1.2 藥敏試驗(yàn) 采用CLSI推薦的紙片擴(kuò)散法(KirbyBauer)進(jìn)行，根據(jù)CLSI2009年頒布的判斷標(biāo)準(zhǔn)進(jìn)行藥敏結(jié)果判斷3。1.3 ESBLs檢測(cè) 參照CLSI20091推薦的篩選試驗(yàn)和確證試驗(yàn)進(jìn)行。1.4 AmpC酶檢測(cè) AmpC酶表型篩選試驗(yàn)：用紙片擴(kuò)散(KB)法測(cè)FOX抑菌環(huán)17 mm者，提示FOX中介或DR，估測(cè)其可能產(chǎn)AmpC酶。AmpC酶三維試驗(yàn)：參照趙虎等4報(bào)道的三維試驗(yàn)方法(凍融法)進(jìn)行。陰溝腸桿菌029M為持續(xù)高產(chǎn)AmpC酶陽(yáng)性對(duì)照，大腸埃希菌ATCC25922作為陰性對(duì)照。接合試驗(yàn)參照文獻(xiàn)5進(jìn)行。質(zhì)粒AmpC基因型的鑒定：根據(jù)AmpC基因的分群設(shè)計(jì)多個(gè)特異性引物序列，采用PCR擴(kuò)增法對(duì)鑒定菌進(jìn)行基因型鑒定。PCR引物：分析GenBank數(shù)據(jù)庫(kù)收錄的質(zhì)粒AmpC基因序列，將質(zhì)粒AmpC基因按相似性分為6組，見(jiàn)參考文獻(xiàn)6(引物序列見(jiàn)表1)。表1 質(zhì)粒AmpC基因型檢測(cè)引物1.5 細(xì)菌總DNA提取 采用煮沸法提取細(xì)菌總DNA。1.6 PCR擴(kuò)增 反應(yīng)體系為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補(bǔ)充體積至25 l，礦物油覆蓋，放入C1000擴(kuò)增儀