【作者】 段金菊；

【导师】 刘卓拉；

【作者基本信息】 山西医科大学 ， 呼吸内科学， 2007， 博士

【摘要】 革兰阴性杆细菌是导致医院内感染最为常见的病原菌。革兰阴性细菌继产超广谱β-内酰胺酶(ESBLs)后,又产生了新型的AmpCβ-内酰胺酶(AmpC beta-lactamases,AmpC酶),AmpC酶多数由染色体介导;但近年来陆续发现了数十种质粒介导的AmpC酶,由于质粒介导ampC耐药基因可在同种或不同种属细菌间广泛播散,给临床抗菌治疗带来困难,已引起广泛重视。为了解太原地区部分医院革兰阴性杆菌耐药与抗生素的使用情况以及它们之间的关系、革兰阴性杆菌高产AmpC酶的发生水平、质粒介导AmpC酶的基因型分布、流行情况、产酶菌的耐药性以及头孢吡肟对质粒介导的AmpCβ-内酰胺酶大肠埃希菌的接种效应等进行了本课题的研究。材料与方法1、1998-2006年山医二院抗生素使用情况及细菌耐药性分析资料来源:抗生素的消耗数据来自山西医科大学第二医院1998年1月至2006年12月9年的药品出库数量,按抗生素的化学名分别统计其规格、单价、DDD值、销售金额及用药频度DDDs,然后分类汇总。试验用菌株:来源于同一医院1998年1月至2006年12月的临床分离菌株,标本来源包括痰、分泌物、咽拭子、血液、尿液、胸腔积液、腹腔积液等。分析方法:(1)采用世界卫生组织(WHO)推荐的限定日剂量法分析各种抗菌药的DDDs。各种药物的DDD值据《中国药典》2005年版二部及《新编药物学》15版中成人常用日剂量,新药采用药品说明书推荐的常规剂量。DDDs=某抗菌药年消耗量/该药的DDD值。(2)用V1TEK2系统对临床分离茵株进行鉴定,采用K-B纸片扩散法及VITEK2系统和GNI细菌鉴定卡对所分离的革兰阴性杆菌进行药敏试验,按2004年版CLSI/NCCL标准对结果进行判定,用WHONET5.3软件对数据进行统计学分析。(3)用SPSS统计软件进行耐药率与用药量的相关性分析,细菌耐药率与抗生素使用频度DDDs之间的关系,用偏相关分析方法分析。2、革兰阴性菌高产AmpC酶的检测受试菌株:为2004年9月至2006年8月间,太原地区四家医院感染对青霉素类、第一、二代及一种以上第三代头孢菌素耐药的无重复革兰阴性菌株。产AmpC酶菌株的初筛与确证试验:采用K-B法以头孢西丁纸片检测受试菌株,抑菌圈直径≤17mm者为疑产AmpC酶株。对疑产株通过AmpC酶提取物做头孢西丁三维试验检测AmpC酶。同时产AmpC酶和ESBIs菌株的检测:对产AmpC酶菌株的酶提取物进行头孢曲松三维试验检测ESBLs。AmpC酶表型筛选:采用IPM,FEP,CD02,CD03,CTX五种纸片进行筛选产AmpC酶细菌。3、革兰阴性菌产质粒介导AmpCβ-内酰胺酶细菌的基因型及耐药性研究受试菌株:2004年9月至2006年8月间,山西太原地区四家医院内感染无重复临床分离菌株。采用V1TEK2鉴定系统和GNI细菌鉴定卡进行鉴定。质粒DNA模板的制备:采用B型质粒小样抽提试剂盒提取三维实验AmpC酶阳性的临床分离株细菌质粒。引物设计:根据各亚型AmpC酶核昔酸序列的同源性,将其分为5组:DHA组、ACT组、FOX-1-6组、CMY-G1组、CMY-G2组,分别设计通用引物。将各亚型ESBLs核昔酸序列分为9组:TEM组、SHV组、CTX-M1组、CTX-M9组、TeTA组、TeTB组、gyrA组、gyrB组、PArC组,分别设计通用引物。PCR扩增ampC以及同产的ESBLs基因、产物分析与测序:ampC和ESBLs基因PCR扩增后,产物在含0.5ug/ml溴乙啶的1.2%琼脂糖凝胶中电泳,用凝胶成像系统观察结果并照相后切胶回收纯化试剂盒纯化后,送往测序公司测序,结果与GenBank提供的质粒型AmpC酶和ESBLs基因核苷酸序列进行比对以确定其基因亚型。质粒接合转移实验:耐叠氮钠的大肠埃希菌J53Azi~R为受体菌,以PCR电泳ampC基因阳性条带菌株为供体菌。4、产质粒介导AmpC酶细菌的耐药性及其分子流行病学研究受试菌株:临床分离株为2004年9月至2006年8月间,太原地区四家医院临床分离产质粒介导AmpC酶革兰阴性菌株。细菌染色体DNA的制备:采用煮沸法提取菌体DNA.菌株DNA同源性分析:采用肠杆菌科基因组内重复一致序列聚合酶链反应(ERIC-PCR)进行产酶菌克隆株的DNA分型。PCR产物分析:采用1.5%琼脂糖凝胶电泳。DNA同源性判定标准:PCR扩增条带完全相同,为同一基因型;相差1条者为基因型密切相关;相差2-3条者为基因型可能相关,不满足上述条件者为不同的基因型。重复性检测:大肠埃希菌ATCC25922以及从80株临床分离株中随机抽取2株,在三天内各培养18小时后,分别提取其菌体DNA进行PCR扩增。5、头孢吡肟对质粒介导的AmpCβ-内酰胺酶的大肠埃希菌的接种效应受试菌株:质粒产AmpC酶的大肠埃希菌株。受试药物:头孢吡肟、头孢噻肟、头孢他啶、头孢哌酮/舒巴坦和亚胺培喃,将其配成浓度依次为512、256、128、64、32、16、8、4、2、1、0.5μg/ml。药物敏感实验:用两种不同的细菌浓度,即标准浓度5*10~5 cfu/mL和高浓度5*10~7 cfu/mL,分别测定其MIC值。接种效应阳性判断标准:在5*10~7 cfu/mL细菌接种浓度下测得的MIC值≥8倍的标准浓度下测得的MIC值,为有接种效应。结果1.1998-2006年山医二院抗生素使用情况及细菌耐药性分析结果(1)革兰阴性菌的分离株数呈上升趋势,尤其鲍曼不动杆菌的上升速度自2004年明显。(2)非发酵菌的耐药率普遍较高,其中鲍曼不动杆菌的耐药率最高,除对亚胺培南仍保持着较好的敏感性外(耐药率≤10%),对其它抗生素2004年以后耐药率几乎都超过了50%,铜绿假单胞菌对头孢他啶的耐药率保持在20%左右,略高于对亚胺培南的耐药率。(3)肠杆科细菌对亚胺培南的耐药率都很低,几乎为0,耐药最严重的为阴沟肠杆菌,除对亚胺培南外,对其它抗生素几乎都超过了50%。(4)抗生素的总用药频度DDDs,2003年达到高峰,以后有下降趋势;销售金额逐年上升,但占年总药物销售金额的比例2003年达高峰,占42.8%,以后逐年下降,2006年占27.4%;头孢霉素类在9年间的DDDs没有太大的变化,但加酶抑制剂复合制剂在2003年后DDDs上升幅度很大。亚胺培南的DDDs9年来一直保持DDDs较低水平。相关性分析结果:以下相关为正相关,相关性具有统计学意义(P＜0.05):(1)铜绿假单胞菌对亚胺培喃的耐药率与亚胺培喃的DDDs之间,以及对头孢哌酮/舒巴坦的耐药率与头孢哌酮/舒巴坦DDDs之间;(2)鲍曼不动杆菌对哌拉西林/他唑巴坦的耐药率与哌拉西林/他唑巴坦的DDDs,以及与加酶抑制剂类的DDDs之间;对头孢他啶的耐药率与头孢他啶的DDDs之间;对头孢吡肟的耐药率与头孢吡肟的DDDs之间;以及对头孢哌酮/舒巴坦的耐药率与加酶抑制剂类的DDDs之间;(3)阴沟肠杆菌对头孢哌酮/舒巴坦的耐药率与头孢哌酮/舒巴坦的DDDs,以及与加酶抑制剂的DDDs;(4)肺炎克雷伯菌对哌拉西林的耐药率与哌拉西林的DDDs之间;(5)大肠埃希氏菌对头孢吡肟的耐药率与头孢吡肟的DDDs,同时,对头孢吡肟的耐药率、CTX的耐药率、及哌拉西林的耐药率均与加酶抑制剂的DDDs之间;(6)在对革兰阴性菌总的耐药率与抗生素使用之间的相关性分析中,G~-菌对头孢吡肟的的耐药率与其DDDs、对头孢他啶的耐药率与其DDDs之间;同时,对头孢哌酮/舒巴坦的耐药率与加酶抑制剂类的DDDs、哌拉西林的耐药率与加酶抑制剂类DDDs之间。2、革兰阴性菌产AmpC酶的检测结果867株革兰阴性耐药菌中,三维试验阳性菌株共237株,总检出率为27.3%(237/867),其中单产AmpC酶菌株123株,检出率为14.2%(123/867),同时产AmpC酶和ESBLs菌株114株,检出率为13.1%(114/867)。表型筛选结果与三维试验结果比较:867株革兰阴性菌从药敏表型筛选结果分析,表现为高产AmpC酶的菌株共277株,其中237株三维试验阳性,符合率为85.5%。3、革兰阴性菌产质粒介导AmpCβ-内酰胺酶的基因型及耐药性研究结果酶粗提物头孢西丁三维实验阳性率为7.5%(237/3159),对237株阳性临床分离株进行5组质粒介导AmpC酶PCR检测,其中95株临床分离株有阳性结果,基因型为ACT-1、DHA-1及CMY-G2。检出率:DHA-1组:大肠埃希菌0.9%(7/789),铜绿假单胞菌0.8%(5/628),阴沟肠杆菌1.5%(6/390),鲍曼不动杆菌2.4%(10/423);ACT-1组:大肠埃希菌1.6%(13/789),肺炎克雷伯菌2.5%(11/439),铜绿假单胞菌1.0%(6/628),阴沟肠杆菌2.3%(9/390),鲍曼不动杆菌2.1%(9/423),沙雷菌属2.8%(7/246)、枸椽酸杆菌属1.6%(2/124);CMY-G2组:铜绿假单胞菌0.3%(2/628)同时产DHA-1和ACT-1组:阴沟肠杆菌0.8%(3/390),鲍曼不动杆菌1.2%(5/423)。在95株质粒介导AmpC酶的阳性菌株中,同时产ESBLs有45株,大肠埃希菌11株,铜绿假单胞菌8株,阴沟肠杆菌3株,克雷伯菌属10株,鲍曼不动杆菌7株,沙雷菌属5株,枸橼酸杆菌属1株。在TEM、CTX-M1及gyrA、gyrB、PArC组有阳性条带。对产AmpC酶的阳性菌株进行测序,测得的DHA-1型基因序列与GenBank中基因的EF406115(blaDHA-1)相符率99%(719/722);测得的ACT-1型基因序列与EF125014(blaACT-1)相符率98%(760/769);测得的CMY-G2型基因序列与AF373218(blaCMY-G2)相符率99%(711/714)。产ESBLs酶的阳性菌株进行测序,测得的TEM型基因序列与GenBank中基因的X56095相符率99%(776/777),但与blaTEM只有20%的相符率;测得的CTX-M1型基因序列与DQ915953(blaCTX-M1)相符率99%(849/850)。测得的gyrA型基因序列与X57174(blagyrA)相符率93%(452/528)。测得的gyrB型基因序列与AB083954(blagyrB)相符率99%(404/405)。测得的PArC型基因序列与AB083954(blaPhrC)相符率99%(381/382)。在95株质粒型AmpC酶的临床分离菌中,经质粒接合转移实验有80株成功(84.2%)转移,接合后较接合前其耐药表型保留了对头孢西丁,对大多数三代头孢的耐药性,对亚胺培喃敏感性与接合转移前相似,都有高的敏感性;对四代头孢头孢吡肟的耐药性有所下降,其耐药率经SPSS统计软件进行X~2统计,差异有统计学意义(P＜0.05),接合前后耐药率差异有统计学意义的还有对头孢噻肟、头孢哌酮、头孢哌酮/舒巴坦及哌拉西林/他唑巴坦(P＜0.05)。4、产质粒介导AmpC酶细菌的耐药性及其分子流行病学研究结果产质粒介导AmpC酶革兰阴性菌对头霉素中的头孢西丁(FOX)及对三代头孢菌素类抗生素显著耐药,其耐药率都在80%以上,但对第四代头孢菌素头孢吡肟(FEP)的耐药率为44.2%。但对亚胺培南(IPM)仍保持良好的敏感性,只有铜绿假单胞菌出现了3株耐药菌,其它革兰阴性杆菌对亚胺培南的敏感性为100%。95株产质粒介导AmpC酶的ERIC-PCR结果,所扩增出的条带1-8条不等,扩增片段的大小在100-2000bp之间,在57株单产ACT-I的菌株中,大肠埃希菌的32,34,36,37,38,与不动杆菌属的41,42,43,44,46,47,48,49,50,为同一克隆,肺炎克雷伯的21,22,24,26,27,28为同一克隆,铜绿假单胞菌的1,6,8,9与阴沟肠杆菌的36,37,38及沙雷克雷伯菌属的51,52为同一克隆。在单产DHA-1的28菌株中,大肠埃希菌的73,77,79,80,与阴沟肠杆菌的67,71为同一克隆,铜绿假单胞菌的63,70为同一克隆,不动杆菌属的64,65,74,75为同一克隆。CMY-G2的两株铜绿假单胞菌相差一条片段。同产ACT-1+DHA-1的8株菌株片段各不相同。同一克隆的菌株多来自同一家医院,也有来自四家医院,四家医院有不少为密切相关。大肠埃希菌ATCC25922及从95株中随机挑选两株,ERIC-PCR后电泳结果显示:所测的3株细菌的条带只是亮度上有些差异,重现性实验结果一致。5、头孢吡肟对质粒介导的AmpCβ-内酰胺酶的大肠埃希菌的接种效应五种药物对19例质粒介导的大肠埃希菌在标准接种浓度下,亚胺培南的敏感率为100%,头孢吡肟也有84.2%的高敏感率,头孢噻肟、头孢他啶、头孢哌酮/舒巴坦的敏感率分别为0、0、5.3%:在10~7 cfu/ml高的接种浓度下,亚胺培南仍能保持高的敏感性,而头孢吡肟的敏感性大幅度降低至0%。头孢吡肟具有强的接种效应,在10~5 cfu/ml标准接种浓度和10~ 7cfu/ml接种浓度下,头孢吡肟的MIC值增加几十倍到上百倍;亚胺培南几乎没有接种效应。结论1、革兰阴性菌的耐药率呈上升趋势,抗生素使用频度呈下降趋势,销售金额呈上升趋势,抗生素的使用档次越来越高。2、革兰阴性菌特别是肠杆科细菌对亚胺培南仍保持非常好的敏感性,对喹诺酮类药物耐药性严重。3、细菌对很多抗生素的耐药率都与加酶抑制剂的使用有正相关。4、医院感染产AmpC酶革兰阴性菌中,基本上约50%的比例单产AmpC酶,另约50%为同产AmpC酶和ESBLs酶细菌。5、三维试验可作为一种实验室用来鉴别常见临床分离革兰阴性杆菌耐药株是否产AmpC酶、ESBLs和金属酶的方法。表型筛选试验操作简单、快速,可以作为临床实验室初选AmpC酶的筛选方法。6、太原地区分离到DHA-1、ACT-1及CMY-G2三种基因型的质粒AmpC酶。铜绿假单胞菌中发现了CMY-G2基因,国内未见相关报道。7、三种质粒产AmpC酶的基因型均可通过质粒接合传递实验将耐药质粒传给受体菌。8、同产ESBLs的基因型以CTX-M1为主,同时还有gyrA、gyrB及ParC型基因,PCR的TEM型基因的阳性检测结果,经测序为非TEM型。9、接合子保留了对头霉素及大多数第三代头孢菌素的耐药性,对第四代头孢菌素、加酶抑制剂的β-内酰胺类抗生素的耐药率有所下降,对碳青霉烯类药物敏感。10、对产AmpC酶细菌感染,首选碳青霉素烯类如亚胺培南,第四代头孢菌素如头孢吡肟有一定疗效。11、产ACT-1、DHA-1及CMY-G2的革兰阴性菌尽管存在多克隆,但是不同医院间有很大的相似性。提示中小城市可能更容易造成携带耐药质粒基因的传播和流行。12、对于产质粒介导的AmpCβ-内酰胺酶的大肠埃希菌,头孢吡肟有强的接种效应,而亚胺培南几乎没有接种效应。虽然,接种效应实验为体外实验,头孢吡肟仍不推荐为临床治疗由质粒介导的AmpCβ-内酰胺酶大肠埃希菌引起的严重感染的药物。更多还原

【Abstract】 Gram-negative bacteria are the present most prevalent pathogens causing nosocomial infection. Selected for by pressure from the spectrum antibiotics,drug—resistance pathogens are more and more increasing by means of mutation and selection as well as the spread of resistance gene.AmpC beta-lactamases produced by gram-negative bacteria are the important cause resulting in resistance to many new extended-spectrum beta-lactam antibiotics.AmpC beta-lactamases are mainly mediated by chromosome,but dozens of plasmid encoded AmpC beta-lactamases were found in recent years,which can transmit horizontally widely among the same or different species,and could lead to outbreaks of nosocomial infection which cause the difficulty of therapy.In order to investigate the gram-negative bacteria resistance and the consumption of antibiotic,the occurrence of AmpC beta-lactamases and the distribution,epidemic state of plasmidmediated AmpC beta-lactamases from part hospitals of Taiyuan in China.We carry out series of this research,and the susceptibility test of the AmpC beta-lactamases produced bacteria are studied as well.Otherwise we also study the inoculum effect of Cefepime with bloodstream infections of Escherichia coli producing plasmid-mediated AmpC-type beta-lactamase.Materials and Methods1,Analysis of the resistance of gram-negative bacteria and the consumption of antibiotics from the year of 1998 to 2006 in one hospitalData origin:consumption of antibiotic drugs comes from the drugstore of one hospital from the year of 1998 to 2006.Consumption of an antibiotic drug was expressed as the number of defined daily dosages(DDD)per calendar year according to the WHO—recommended DDD method to probe into medication frequency(DDDs).Detected strains:The Detected strains were collected from clinical samples,including sputum,urine, pleural fluid,abdominal fluid,swab,and blood,between January 1998 and December 2006.They were identified by VITEK2.Antibiotic susceptive test was done by Kirby—Bauer method and VITEK2.The result was judged by the CLSI standard of 2004’ S edition and the data were analyzed by WHONET5.3 software.The partial correlation between the resistance of gram-negative bacteria and the consumption of antibiotics is analyzed by SPSS statistic software. 2,Detection of AmpC beta-lactamase producing gram-negative bacteriaDetected strains:Clinical isolates of no repetitive gram-negative bacteria resistance to penicillin’s, first-,second- and at least one third-generation cephalosporins were celected between September 2004 and August 2006 from four hospitals in Taiyaun of China.Species identification was performed by VITEK2Screening and confirmatory tests for clinical isolates producing AmpC beta-lactamases:Strains were detected with cefoxitin disks by Kirby-Bauer disk diffusion method.Those with cefoxitin zone diameter≤17 mm were suspicious of producing AmpC beta-lactamases.Cefoxitin three-dimensional extract tests were performed for suspicious strains to confirm whether produce AmpC beta-lactamases or not.Detection of clinical isolates producing AmpC beta-lactamases and ESBLs:ESBLs were determined further by ceftriaxone three-dimensional extract tests for those strains producing AmpC beta-lactamases.Phenotype screening test for AmpC beta-lactamases:Phenotype screening test is performed by five disks namely IPM.FEP,CD02,CD03,CTX.3,Study on susceptibility and genotype of plasmid-mediated AmpC beta-lactamases in gram-negative bacteriaDetected strains:Clinical isolates of no repetitive gram-negative bacteria of nosocomial infection were collected from four hospitals in Taiyaun of China between September 2004 and August 2006.Plasmid conjugation tests:Using E.coli J53Azi~R as the recipient strains.Using clinical isolates producing AmpC beta-lactamases as supply strains.Primer designation:The information ofplasmid-encoded ampC gene was collected from GenBank and reference s.Universal primers for ampC genes were designed by aligning their conserved encoding sequences.Five groups of universal primers were designed to amplify ampC genes:DHA group,ACT group,CMY-G1 group,CMY-G2 group,FOX groups.Likewise,nine groups of universal primers were designed to amplify ESBL genes.They are TEM group,SHV group, CTX-M1 group,CTX-M9 group,TeTA group,TeTB group,gyrA group,gyrB group and PArC group.Preparation of plasmids DNA:Plasmids DNA from clinical isolates of gram-negative bacteria producing AmpC enzyme were extracted using plasmid mini—preps kit as templates for polymerase chain reaction(PCR).Amplification ofampC and ESBLs genes in producing AmpC beta-lactamases strains and sequencing:ampC and ESBLs genes on plasmids were amplified by PCR,the products were separated by electrophoresis on an 1.2%agarose gel with 0.5 u g/ml ethidium bromide and purified by gel extraction kit after photos.Nucleic acid sequence of the purified products were analyzed by Sanger dideoxy chain of termination method,and the results were compared with nucleic acid sequence of ampC genes provided by GenBank in order to identify their sub-genotype.4,Study on susceptibility and molecular epidemiology of gram-negative bacteria producing plasmid-mediated AmpC beta-lactamasesDetected strains:Plasmid-mediated AmpC beta-lactamases producing isolates were obtained from four hospitals in Taiyuan of China between September 2004 and August 2006.Preparation of chromosome DNA of bacteria:Chromosome DNA was extracted by boiling. Homology analysis of DNA:Enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR)was used for typing of cloning strains.Analysis of PCR products:The products were separated by electrophoresis on 1.5%agarose gel. Criterion of homology:The identical amplification bands of PCR was thought to be the same genotype;one band different was close correlate;2 or 3 bands different was possible correlate; exceeding 3 bands different was the different genotypes.Reproducibility tests:Reproducibility was examined by comparing patterns of E.coli 25922 and 2 strains randomly selected from 95 plasmid-mediated AmpC beta-lactamases producing isolates obtained on 3 different days.5,the inoculum effect of Cefepime in tests with Escherichia coli producing plasmid-mediated AmpC-type beta-lactamaseDetected strains:Plasmid-mediated AmpC beta-lactamases producing isolates were obtained from two teaching hospitals between September 2004 and August 2006.Detected antibiotics:Cefotaxime,ceftazidime,cefepime,sulperazon and imipenem were as detected drugs and their concentrations were adjusted to 512,256,128,64,32,16,8,4,2,1 and 0.5μg/ml.Antimicrobial susceptibility tests:The MIC of 10~5 and 10~7 cfu/mL inoculum concentration of five antibiotics was determined using a broth microdilution MIC method.The definition of a positive inoculum effect was an eight-fold or greater increase in MIC on testing with the higher inoculum.Results1,Analysis results to the resistances of gram-negative bacteria and the consumption of antibiotics from the year of 1998 to 2006 in one hospital.(1)There is an increasing tide of gram-negative bacteria isolates from the year of 1998 to of 2006 in one hospital,and an obviously increasing in A.baumannii strains.(2)The resistances to antibiotics were generally higher in No fermenting strains than in Enterrobact-eriaecae.strains the resistance rates of A.baumannii strains to almost antibiotics except imipenem exceeded 50%from the year of 2004.The resistance rates of P.Aeruginosa to ceftazidime kept about 20%,almost similar to imipenem.(3)There were very low resistance rates to imipenem in Enterrobact-eriaecae strains,almost to zero.The serious resistance is E.cloacae among Enterrobact-eriaecaes.Strains which resistance rates to most antibiotics exceeded 50%except imipenem.(4)During the 9 years of 1998-2006,the DDDs reached a peak in 2003 with a decrease afterwards.The total distributed sum of antibiotic has been rising during 1998-2006,but the highest component ratio of antibiotic to total medicine every year was in 2003.The total medication frequencies of Cephalosporins maintained a few change,but the total medication frequencies of the compound preparations ofβ-lactamase inhibitors sharply increased after 2003.There were small medication frequencies of Imipenem in the 9 years.The results of partial correlation by SPSS statistics software:There were positive correlations between the following resistances of gram-negative bacteria and the DDDs of antibiotics,and the following resistances of gram-negative bacteria were significantly related to the following consumption of antibiotics(p＜0.05).(1)The resistance rates of P.aeruginosa to Imipenem related to the DDDs of Imipenem and sulperazon.(2)The resistance rates of A.Baumannii to Piperacillin/Tazobactam related to it’s DDDs and the total DDDs of Antibiotic/beta-lactamase inhibitor preparations.The resistance rates of A.Baumannii to ceftazidime and cefepime related to their DDDs severally.The resistance rates of A.Baumannii to sulperazon related to the total DDDs of Antibiotic/beta-lactamase inhibitor preparations.(3)The resistance rates of E.cloacae to sulperazon related to it’s DDDs and the total DDDs of Antibiotic/beta-lactamase inhibitor preparations.(4)The resistance rates of K.pneumonia to Piperacillin related to it’s DDDs.(5)The resistance rates of E.coli to ceftazidime related to it’s DDDs,and resistance rates of E.coli to ceftazidime,Cefotaxime and Piperacillin fully related to the total DDDs of Antibiotic/beta-lactamase inhibitor preparations.(6)At the analysis of total resistance of gram-negative bacteria to antibiotics,we found that the total resistance rates of gram-negative to ceftazidime and ceftazidime related to their DDDs,and the total resistance rates of gram-negative to Piperacillin and sulperazon related to the total DDDs of Antibiotic/beta-lactamase inhibitor preparations.2,Results of detection of AmpC beta-lactamases producing gram-negative bacteria237 isolates were positive in three-dimensional tests in a total 867 clinical isolates,which the positive rate was 27.3%(237/867).Among 237 positive isolates,123 isolates were AmpC beta-lactamase producers only,114 isolates produce both AmpC beta-lactamase and ESBLs,which the positive rate was 14.2%and 13.1%,respectively.Comparison of phenotype screening test and three-dimensional test:Analysis by phenotype screening test,277 out of 867 isolates were detected to be high level AmpC beta-lactamases producing strains,which compared with three-dimensional test,the coincidence rate was 85.5%.3,Results of susceptibility and genotypes of plasmid-mediated AmpC beta-laetamase producing bacteriaThe positive rate of FOX three-dimensional test was 7.5%(237/3159).Amplified the above 237 clinical isolates by PCR with specific primers and then sequenced,95 strains were identified to produce plasmid-mediated AmpC beta-lactamases.The specific distribution among species was:for DHA-1:E.coli 0.9%(7/789),P.aeruginosa 0.8%(5/628),E.cloacae 1.5%(6/390),A.baumannii 2.4%(10/423);for AC-1:E.coli 1.6%(13/789),Klebsiella 2.5%(11/439),P.aeruginosa strains 1.0%(6/628),E.cloacae strains2.3%(9/390),A.baumannii 2.1%(9/423),Serratia 2.8%(7/246), freundii 1.6%(2/124);for CMY-G2:P.aeruginosa 0.3%(2/628).In addition,among 95 producing plasmid-mediated AmpC positive strains,45strains also produced ESBLs including E.coli 11 strains, P.aeruginosa 8 strains,beta-lactamases modified cefoxitin three-dimensional test suggested that 3 strains of E.coli,2 strains,E.cloacae 3 strains,Klebsiella10 strains,A.baumannii 7 strains,Serratia 5 strains and freundii 1 strains.There were posotive PCR results in TEM,CTX-M1,gyrA,gyrB and ParC.Nucleic acid sequences of ampC genes were compared with provided by GenBank.There were 99%(719/722)between DHA-1 gene and EF406115(blaDHA-1),98%(760/769)between ACT-1 gene and EF125014(blaACT-1),and 99%(711/714)consistency of nucleic acid sequences between CMY-G2 gene and AF373218(blaCMY-G2)in GenBank.Nucleic acid sequences of ESBL genes were compared with provided by GenBank.There were 99%(776/777)consistency between TEM gene and X56095,but only 20%consistency compared to blaTEM in GenBank.There were 99%((849/850))between CTX-M1 gene and DQ915953(bla CTX-M1),93%(452/528)between gyrA gene and X57174(blagyrA),99%(404/405)between gyrB gene and AB083954(blagyrB) and 99%(381/382)consistency between PArC gene and AB083954(blaPArC)in GenBank.Of 95 clinical ampC genes positive isolates tested,80(84.2%)strains succeeded to be transconjugants.Kirby-Bauer disk diffusion test was used for identifying the susceptibility 80 transconjugants,and compared their susceptibilities to susceptibilities before to be transconjugants. The transconjugants remained resistance to cefoxitin and most susceptible to extended-spectrum third-generation cephalosporins.And,transconjugants and most of clinical isolates were susceptible to imipenem.By analyzing the differences between transconjugants and their clinical isolates by SPSS software,the differences of Cefepime,Cefotaxime,Cefoperazon,sulperazon and Piperacillin/Tazobactam were significant(p＜0.05).4,Results of susceptibility and molecular epidemiology of gramnegative bacteria producing plasmid-mediated AmpC beta-lactamasesStains with plasmid-mediated AmpC were consistently resistant to cefoxitin of cephamycin and above 80%resistant to the third generation cephalosporins,but resistance to the fourth generation cephalosporins cefepime was much lower(44.2%).The enzymes also provided different resistant to aztreonam,ofloxacin,amikacinl,but all stains susceptible to imipenem except 3 strains of Pseudomonas Aeruginosa.Amplified bands of ERIC-PCR ranged from 1 to 8 bands,and the size of bands ranged from 100 to 3000 bp.Of the 57 isolates of ACT-1 producing strains,E.coli32,34,36,37,38,and A.Baumannii 41,42,43,44,46,47,48,49and 50 were the same clone.P.aeruginosa 1,6,8,9,and E. cloacae 36,37,38 and Serratia 51 and 52 were the same clone.K pneumoniae 21,22,24,26, 27and 28 were the same clone.Of the 28 isolates of DHA -1 producing strains,E.coli 73,77,79, 80 and E.cloacae 67 and 71 were the same clone.P.aeruginosa 63 and 70 was the same clone,and A.Baumannii 64,65,74 and 75 was the same clone.Of the 2 isolates of CMY-G2 were similar genes with one difference.Of the 8 isolates ACT-1 and DHA-1 were different clones.The above same clone strains were isolated from the same hospital and also from the different hospitals.Reproducibility tests demonstrated that only brightness of the electrophoresis bands showed a little different in 3 strains detected,the reproducibility result was good.5,Results of inoculum effect of Cefepime in tests with Escherichia eoli producing plasmid-mediated AmpC-type beta-lactamaseOf 19 strains of E.coli blood isolates producing plasmid-mediated AmpC beta-lactamases, antimicrobial susceptibility of Cefotaxime,ceftazidime,sulperazon,cefepime and imipenem were 0,0,5.3%,84.2%and 100%respectively at Standard inoculum.Cefepime had strong inoculum effect.Because its MIC of had above 100 times in the higher inoculum tests than in the standard inoculum tests.However,the inoculum effect was least frequently detected with imipenem.Conclusions1,There was a increasing tide of gram-negative bacteria isolates,a decreasing tide of DDDs of antibiotics,and a increasing tide of consumption money,and the ranks of antibiotics were more and more advanced during nine years. 2,Imipenem remained good susceptibility against gram-negative bacteria especially Enterobacter. Fluoroquinolones had badly resistance to gram-negative bacteria especially Enterobacter.3,Resistances of gram-negative bacteria to many antibiotics were related to the total DDDs of Antibiotic/beta-lactamase inhibitor preparations.4,Of nosocomial infection bacteria producing AmpC beta-lactamases,about half are producers only, and another half are AmpC and ESBL enzyme producers.5,Three-dimensional test can be used to identify clinical Gram-negative isolates whether they produce AmpC enzyme,ESBIs or Metallo-beta-lactamases.Phenotype screening test is easily operated and can be the use as the primary screening method to detect AmpC beta-lactamases.6,Three genotypes of plasmid-mediated AmpC beta-lactamases were identified that is DHA-1,ACT -1 and CMY-G2 in Taiyuan.There have been not reports which we investigated on P.aeruginosa of producing CMY-G2 in China.7,Three genotypes are transferable plasmids to recipient strains.8,Of AmpC and ESBL enzyme,genotypes of ESBL enzyme mostly are CTX-M1,and also are gyrA, gyrB and ParC.Positive TEM gene by PCR is not the sequences of TEM by nucleic acid sequencing.9,Transconjugants remain resistance to extended-spectrum third-generation cephalosporins,but show decreasing resistance to fourth-generation cephalosporins and Antibiotic/beta-lactamase inhibitor preparations.All transconjugants are susceptible to imipenem.10,Carbapenems could be better choice for treatment of infections caused by AmpC enzyme producers,and could also choose fourth-generation cephalosporins as secondary antibiotic to treat the infection of AmpC enzyme producers on the basis of susceptibility tests.11,Gram-negative bacteria producing ACT 1,DHA-1 and CMY-G2 demonstrate the multi-cloning patterns,however,there are high similar among them.Evidences show that there is more possibility of diffusion and prevalence with producing plasmid-mediated AmpC gram-negative bacteria in medium and small cities.12 In higher inoculum tests,cefepime was dramatically affected.However,the inoculum effect was least frequently detected with imipenem.Although the inoculum effect is an in vitro laboratory phenomenon,these results suggest that cefepime may be a less than reliable agent for therapy in cases of high inoculum infections caused by AmpC[beta]-lactamase-producing Escherichia coli.更多还原