三黃片對(duì)大腸桿菌耐藥質(zhì)粒消除作用的研究

1、    三黃片對(duì)大腸桿菌耐藥質(zhì)粒消除作用的研究*        提要用三黃片作為質(zhì)粒消除劑，對(duì)48株院內(nèi)感染大腸桿菌多重耐藥株采用連續(xù)稀釋培養(yǎng)法進(jìn)行質(zhì)粒消除試驗(yàn)，并與傳統(tǒng)SDS法進(jìn)行了比較。經(jīng)試驗(yàn)前后藥敏試驗(yàn)及質(zhì)粒指紋譜分析，結(jié)果前法質(zhì)粒消除率為31%(15/48)，其耐藥性也隨之部分消失；后法質(zhì)粒消除率為27%(13/48；經(jīng)X2檢驗(yàn)，兩法無(wú)顯著性差異(P0.05)；研究表明，三黃片對(duì)耐藥質(zhì)粒有較強(qiáng)的消除作用。關(guān)鍵詞三黃片醫(yī)院內(nèi)感染大腸桿菌質(zhì)粒消除 STUDY ON PLA

2、SMID ELIMINATION OF SAN HUANG TABLETS TO NOSOCOMIAL E.coli*Kang MeiChen ZhixingXu XiuchengPan Peigen(The First University Hospital, West China University of Medical SciencesChengdu 610041)ABSTRACTSan Huang Tablet, which is a kind of traditional Chinese medicine, was chosen as a curing agent to elimi

3、nate plasmid of 48 multiply antibiotic-resistant nosocomial E.coli strains by series dilution,and was compared with another traditional curing agent-SDS. The result demonstrated that the curing percentage of San Huang tablet is 31%(15/48) and 27% of SDS(13/48).No significant difference was found bet

4、ween San Huang tablet and SDS in effect on plasmid elimination (P0.05). It suggested that San Huang tablet is a effective plasmid curing agent to E. coli in hospital infection and has important significance for therapying and controlling hospital infection.Key WordsSan Huang tabletNosocomial Infecti

5、on E.coliCuring of plasmidMultiply antibiotic-resistance用消除質(zhì)粒的方法解決細(xì)菌耐藥問(wèn)題，國(guó)外采用的消質(zhì)劑多為對(duì)人體有害的化學(xué)物質(zhì)或方法本身不適用于人體13，而有關(guān)臨床藥物的質(zhì)粒消除作用，國(guó)內(nèi)外文獻(xiàn)中多為單一化學(xué)抗菌藥物，草藥和中成藥僅偶有報(bào)道，并且消除率均很低?，F(xiàn)選用臨床常用價(jià)廉易得的復(fù)方中成藥三黃片作為消質(zhì)劑與傳統(tǒng)消質(zhì)劑SDS作對(duì)比研究。1材料、方法和結(jié)果1.1材料1.1.1菌株大腸桿菌多重耐藥株(來(lái)源于1997年4月1998年11月本院檢驗(yàn)科細(xì)菌室分離鑒定的臨床菌株，耐兩種或兩種以上作用機(jī)制不同的抗生素)；藥敏質(zhì)控對(duì)照菌株ATCC

6、25922大腸桿菌(衛(wèi)生部臨床檢驗(yàn)中心)；標(biāo)準(zhǔn)分子量質(zhì)粒菌株V5174分子量54.2,7.3,5.6,5.2,3.9,3.0,2.7,2.1 kb(華西醫(yī)科大學(xué)公共衛(wèi)生學(xué)院衛(wèi)生檢驗(yàn)教研室)。1.1.2培養(yǎng)基Mueller-Hinton(MH)瓊脂平板(浙江軍區(qū)后勤部，批號(hào)960109)；Luria-Bertani(L-B)肉湯(1.0%胰蛋白胨，0.5%酵母浸汁，1.0%NaCl,pH7.47.6);0.015%SDS-LB肉湯1000 ml LB肉湯中加入5 ml 3%SDS(十二烷基磺酸鈉)；三黃片LB肉湯LB肉湯中加入三黃片粉末使其終濃度為三黃片的亞抑菌藥濃度(120 mg/ml)。1.

7、1.3抗生素紙片青霉素(P)、氨芐青霉素(AMP)、慶大霉素(GTM)、頭孢唑啉(CZL)、頭孢哌酮(CPZ)、丁胺卡那霉素(AMK)、氟哌酸(FOX)、頭孢噻甲羧肟(CAZ)、頭孢呋新(CXM)、氧哌嗪青霉素(PIP)等藥敏紙片由北京天壇藥物生物技術(shù)開發(fā)部提供，批號(hào)9612。1.1.4試劑與試藥質(zhì)粒提取用試劑按文獻(xiàn)5。三黃片(河南省百泉制藥廠批號(hào)970521)主要成份為大黃、黃芩總苷、鹽酸黃連素，其比例為0.30.30.005。1.2方法質(zhì)粒消除前后，48株多重耐藥株藥敏試驗(yàn)采用K-B法6；從48株菌中小量提取質(zhì)粒DNA，采用Takahhashi改良法操作5。1.2.1消質(zhì)劑亞抑菌濃度的選擇

8、傳統(tǒng)消質(zhì)劑SDS亞抑菌濃度的確定見(jiàn)文獻(xiàn)8，經(jīng)計(jì)算得0.015%SDS-LB肉湯；中藥消質(zhì)劑三黃片亞抑菌濃度的確定見(jiàn)文獻(xiàn)7，8，結(jié)合文獻(xiàn)3方法計(jì)算得120 mg/ml三黃片LB肉湯。1.2.2質(zhì)粒消除試驗(yàn)參照文獻(xiàn)1方法，將試驗(yàn)菌株轉(zhuǎn)種MH平板復(fù)蘇后，轉(zhuǎn)種2 ml LB肉湯，37振搖培養(yǎng)1618 h，取5 l加入2 ml含亞抑菌濃度消質(zhì)劑的LB肉湯中，37振搖培養(yǎng)18 h后取5 l加入2 ml消質(zhì)劑LB肉湯中重復(fù)操作，作用4854 h后，用營(yíng)養(yǎng)肉湯作質(zhì)粒提取和藥敏試驗(yàn)。1.3結(jié)果1.3.1用三黃片作為消質(zhì)劑進(jìn)行質(zhì)粒消除前后質(zhì)粒譜的比較見(jiàn)1，消除試驗(yàn)后，有15株大腸桿菌丟失了一至數(shù)條質(zhì)粒帶，質(zhì)粒消除

9、率為31%(15/48)；用SDS作為消質(zhì)劑進(jìn)行質(zhì)粒消除前后質(zhì)粒譜的比較見(jiàn)2，消除試驗(yàn)后，有13株大腸桿菌丟失了一至數(shù)條質(zhì)粒帶，質(zhì)粒消除率為27%(13/48)。Fig 1Plasmid profiles of some strains before and after curing plasmid by San Huang tabletA-EPlasmid profiles of E.coli V517 and strain No.2298， No.2202 No.2314 No.2410 before curing plasmidA-EPlasmid profiles of E.coli

10、V 517 and strain No.2298 No.2202 No.2314 No.2410 after curing plasmidFig 2Plasmid profiles of some strains before and afer curing plasmid by SDSA-EPlasmid profiles of E.coli V517 and strain No.2193 No.2337 No.2201 No.2410 before curing plasmidA-EPlasmid profiles of E.coli V517 and strain No.2193 No.

11、2337 No.2201 No.2410 after curing plasmid1.3.2兩種消質(zhì)劑對(duì)質(zhì)粒進(jìn)行消除的結(jié)果比較見(jiàn)表1，經(jīng)X2檢驗(yàn)P0.05，兩法無(wú)顯著性差異；消除質(zhì)粒前后受試菌藥敏結(jié)果比較見(jiàn)表2、表3。Table 1Comparison of antibiotics resistance before and after two curing methodsMethod forcuring plasmidAfter curing plasmidTotalNo. of strainslost plasmidNo. of strainslost no palsmidSan Huan

12、g tablet351348SDS3315482 testP0.05Table 2Antibiotics resistance before and after curing plasmids in some E.coli strainsStrainNo.Antibiotic resistant profilebefore curing plasmidcuring plasmid by San Huangcuring plasmid by SDS143P.AMP.PIP.NOR.CXMP.NORP.AMP.CXM.NOR156P.AMP.PIP.GM.NOR.CXMP.AMPP.AMP267P

13、.AMP.PIP.GM.NOR.CZLP.AMP.PIP.GM.CZLP.AMP.PIP.GM.CZL.NORA1011P.AMP.PIP.GM.CZL.CPZ.NOR.CXMP.AMP.PIP.GM.CFP.NORP.AMP.PIP.GM.CFP.NOR2190P.AMP.PIP.GM.CZLP.AMP.PIP.GMP.AMP.PIP.GM.CZL2193P.AMP.PIP.CZL.NOR.CXMP.AMP.PIP.NORP.AMP.PIP.NOR2201P.AMP.PIP.GMP.AMP.GMP.AMP.PIP.CXM2206P.AMP.PIP.GM.CZL.CPZ.AMK.NORP.AM

14、P.CFP.NORP.AMP.CFP.NOR2258P.AMP.PIP.GM.CZL.CPZ.NOR.CXMP.AMP.PIP.NORP.AMP.PIP.GM.NOR.CXM2260P.PIPPP.PIP2410P.AMP.CZLPP5152P.AMP.PIP.GM.NORP.AMP.GM.NORP.AMP.GM.NOR5167P.AMP.PIP.NOR.CXMP.AMP.NORP.AMP.NORTable 3Comparison of antibiotics resistance before and afer two curing methodsMethod forcuring plasm

15、idAfter curing plasmidNo. of strains*No. of strains*San Huang tablet3811SDS3513* Become susceptible to at least one kind of antibiotics after curing plasmid；* No change in antibiotics resistant profile after curing plasmid ；2 testP0.052討論以上實(shí)驗(yàn)選用價(jià)廉易得，毒副作用小的大黃、黃芩、黃連三種中藥的復(fù)方制劑三黃片與傳統(tǒng)消質(zhì)劑SDS進(jìn)行對(duì)比研究，三黃片中的三種主要

16、成分不僅有一定的抑菌作用9，而且有一定的質(zhì)粒消除作用1012。研究結(jié)果表明，三黃片對(duì)耐藥菌株有明顯的消質(zhì)作用，提示臨床抗感染治療中三黃片與抗生素聯(lián)合用藥可防止菌株產(chǎn)生耐藥性。而三黃片的消質(zhì)作用是否比單一用藥的文獻(xiàn)報(bào)道的質(zhì)粒消除使用更強(qiáng)，尚待進(jìn)一步的對(duì)比研究。在實(shí)驗(yàn)中還發(fā)現(xiàn)部分菌株經(jīng)三黃片與SDS作用后，藥敏變化很明顯，但質(zhì)粒譜卻無(wú)改變，推測(cè)三黃片或SDS作用后并未真正消除質(zhì)粒但可能滅活該耐藥質(zhì)粒或抑制了質(zhì)粒介導(dǎo)的耐藥性表達(dá)；這部分菌株的耐藥系染色體介導(dǎo)而非質(zhì)粒介導(dǎo)，消質(zhì)劑可能部分改變了染色體的結(jié)構(gòu)，造成染色體上耐藥基因的失治或丟失等。此推測(cè)是否確證待進(jìn)一步實(shí)驗(yàn)。以上三黃片與SDS兩種方法消質(zhì)效

17、果相當(dāng)。這為探討中藥抗菌機(jī)理；采用中西醫(yī)結(jié)合的治療方法來(lái)對(duì)付難治性感染以控制院內(nèi)感染；對(duì)開發(fā)中醫(yī)藥資源具有重要的理論意義和廣闊的應(yīng)用前景。* 美國(guó)中華醫(yī)學(xué)基金會(huì)CMB92-558資助課題作者單位：華西醫(yī)科大學(xué)附一院檢驗(yàn)科醫(yī)學(xué)分子生物學(xué)實(shí)驗(yàn)室*成都 610041參考文獻(xiàn)1劉正友，王其南，杜繼昭等.革蘭氏陰性桿菌院內(nèi)感染株菌的質(zhì)粒分析.中國(guó)抗生素雜志，1991，16(5)3442Asheshov EH. Loss of antibiotic Resistance in staphylococcus aureas resulting from growth at high temperature.

18、J Gen microbiol, 1996, 42(3)4033Bouanchand DH, Scavizzi MR, Chabbert YA. Elimination by ethidium bromide of antibiotic resistance in enterobaoteria and stanhyldocci. J Gen microbiol, 1969,54(12)4174Macrina FL. A multiple plasmid-containing escherich coli strain:Convenient source of size reference plasmid moleculars. Plasmid, 1978, 1(1)4175Takahashi,Nagano. Rapid procedure for isolation of plasmid DNA and application to epidemioligical analysis. J Clin microliol, 1984, 20(4)6086WHO Guidelines for antimicrobiol susceptibility testin