Purification of beta-lactamases and enzyme kinetic studies on aztreonam.

Standard beta-lactamases K1, P99, TEM-1, SHV-1 and beta-lactamase K-CAZ purified using FPLC through ion-exchange chromatography and filtration chromatography, were identified by determination of isoelectric points, molecular weights and substrate profiles. The results showed that the beta-lactamase stability of domestic aztreonam was very similar to that of cefotaxime, ceftazidime and much better than that of cefoperazone. Aztreonam showed a high affinity to chromosomal-mediated cephalosporinase P99, its Ki for inhibition of P99 with cephaloridine as substrate was 0.0159 microns. Aztreonam and the three third generation cephalosporins tested were not stable to beta-lactamase K-CAZ, which hydrolysed them in different degrees.

     

耐头孢酮的的E.coliHX88108株的β—内酰胺酶研究

近年国外报道许多对第三代头孢菌素耐药的细菌是由于产生新的β－内酰胺酶所致，为探讨耐头孢哌酮的Ｅ．ｃｏｌｉＨＸ８８１０８及其４株转化菌ＰＦＣ，ＰＦＴ１ＰＦＴ２ＰＦＴ３的β－内酰胺酶性质，进行了β－内酰胺酶底物轮廓测定，酶抑制剂棒酸和舒巴坦敏感性研究以及薄层分析性等电聚焦电泳。     

AmpC酶和超广谱β-内酰胺酶在阴沟肠杆菌中的表达及对其耐药性的影响

目的：研究AmpC酶和超广谱β-内酰胺酶（ESBL）在阴沟肠杆菌中的流行情况及对其耐药性的影响。方法：琼脂稀释法测定最低抑菌浓度（MIC）,三维试验、等电聚焦电泳、接合试验及聚合酶链反应分析耐药菌的产酶情况。结果：106株阴沟肠杆菌中,单纯高产AmpC酶者、单纯产ESBL者、高产AmpC酶并产ESBL者分别占16.0％、10.4％、13.2％。CTX-M型和SHV型ESBL的检出率分别为17.0％（18/106）和6.6％（7/106）。碳青霉烯类抗生素对阴沟肠杆菌均有很好的抗菌活性(MIC90≤2цg/ml)。单纯高产AmpC酶菌对头孢吡肟、阿米卡星、庆大霉素和环丙沙星的敏感率均超过70％;单纯产ESBL菌对哌拉西林/他唑巴坦、头孢哌酮/舒巴坦、环丙沙星和阿米卡星的敏感率均超过60％;高产AmpC酶并产ESBL菌对除碳青霉烯类外的其他抗生素的敏感率均低于25％。结论：CTX-M型ESBL已经在阴沟肠杆菌中流行。产ESBL和高产AmpC酶同样是导致阴沟肠杆菌对第3代头孢菌素耐药的重要原因。ESBL在高产AmpC酶菌株中的流行增强了此类菌株的耐药性。     

阴沟肠杆菌AmpC酶和金属酶的表达及其对耐药性的影响

目的:研究AmpC酶和金属酶在阴沟肠杆菌中的表达状况及这两种酶对阴沟肠杆菌耐药性的影响,为临床合理使用抗菌药物提供依据。方法:采用三维试验,EDTA-亚胺培南纸片法测定AmpC酶和金属酶,用纸片扩散(K-B)法检测阴沟肠杆菌对13种抗生素的耐药性。结果:120株阴沟肠杆菌中,AmpC酶和金属酶检出率分别为32.5%(39/120)和5%(6/120)。AmpC酶阳性菌株对头孢曲松、头孢噻肟、头孢西丁耐药率高于AmpC酶阴性菌株,两者差异具有统计学意义(P<0.05)。金属酶阳性菌株对头孢曲松、头孢噻肟、头孢他啶、哌拉西林/他唑巴坦、亚胺培南耐药率明显高于阴性菌株,两者差异具有统计学意义(P<0.05)。结论:AmpC酶和金属酶在阴沟肠杆菌中流行分别是对第三代头孢菌素、头霉素类抗生素及碳青霉烯类抗生素耐药的主要原因,临床应根据药敏结果合理选用抗菌药物,以减少耐药菌株的产生和流行。     

12种β—内酰胺类抗生素对β—内酰胺酶的稳定性研究

实验采用紫外分光光度法测定了(1)11株国际标准产酶菌的β—内酰胺酶活性;(2)11种β—内酰胺酶对12种β—内酰胺类抗生素的相对水解率;(3)CP—45899对11种酶的抑酶率。结果表明:酶K—1和TEM—2活性分别为699和319单位外,其余酶活性在22—72单位之间。12种抗生素中,第三代头孢菌素(除CPZ外)的酶稳定性最好;第二代头孢菌素中CXM的酶稳定性明显优于CMD和CPZ(P<0.01)。第一代头孢菌素和青霉素类易被酶水解。CP—458991ug/ml对多种β—内酰胺酶显示抑制作用,对CRD、CMD具有保护作用。     

头孢菌素类与青霉素类抗生素交叉过敏反应的临床观察

目的：观察头孢菌素类与青霉素类的交叉过敏反应。方法：选择注射使用头孢唑啉、头孢拉定、头孢哌酮、头孢曲松、头孢噻肟等5种头孢菌素类药物的住院病人4672例，记录其药物及食物过敏史和过敏反应发生情况。结果：在总用药人群中过敏反应84例(1．80％)，各药物及性别和各年龄组过敏发生率无显著差异。青霉素过敏史阳性、其它物质过敏史阳性和无过敏史者分别为316例、161例和4195例，头孢菌素类用药后过敏反应例数分别为44例(13．92％)、8例(4．97％)和32例(0．76％)，三组人群头孢菌素类过敏发生率差异极其显著，P＜0．0l。结论：头孢菌素类与青霉素之间存在共同过敏和交叉过敏现象，交叉过敏反应发生率约8％。     

Epidemiological and antibiotic resistant study on extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Zhejiang Province

Objective To investigate the epidemiological status of extended-spectrum β-lactamase (ESBL) producing Escherichia coli (E.coli) and Klebsiella pneumoniae (K.pneumoniae) and the drug resistance profiles of such organisms .Methods A total of 282 clinical isolates of E .coli and 180 of K .pneumoniae were collected from different districts of Zhejiang Province .Inhibitor potentiated broth dilution tests were performed for detecting extended-spectrum β-lactamases .Etests were performed to detect the drug resistance of these strains against nine commonly used antibiotics Results The prevalence of extended-spectrum β-lactamases in E .coli and K .pneumoniae was 34.0% and 38.3%, respectively .The average prevalence of extended-spectrum β-lactamases in E .coli and K .pneumoniae was 35.7% .The resistance prevalence of extended spectrum β-lactamase producing strains to ceftazidime and cefotaxime was 40% and 26% respectively, so were those to cefepime, cefoxitin, piperacillin-tazobactam, cefoperazone-sulbactam, amikacin and ciprofloxacin .All these strains were sensitive to imipenem .Conclusion The results in this study showed that the prevalence of extended-spectrum β-lactamases was high, while extended-spectrum β-lactamase producing strains were resistant to most antimicrobial agents except imipenem .     

2012至2015年昆明医科大学第一附属医院流感嗜血杆菌耐药性监测

目的 了解昆明医科大学第一附属医院临床分离的流感嗜血杆菌对常用抗菌药物的耐药情况, 为临床合理使用抗生素提供依据.方法 收集2012年1月至2015年12月184株临床分离的流感嗜血杆菌, 采用纸片扩散法 (K-B法) 进行抗菌药物敏感性试验, Nitrocefin纸片检测定β-内酰胺酶, 用WHONET5.6软件对流感嗜血杆菌耐药率进行统计分析.结果 184株临床分离的流感嗜血杆菌对氨苄西林耐药率最高, 达46.7%, 其次对氯霉素、氨苄西林/舒巴坦耐药率分别为23.2%、13%.该菌对阿奇霉素、环丙沙星、美洛培南、头孢呋辛、头孢噻肟的耐药率均在10%以下.儿童分离株对氨苄西林的耐药率 (56.9%) 高于成人分离株 (42.1%) ;除环丙沙星、美洛培南和氯霉素外, 儿童株对其他测试抗菌药物的耐药率均较成人株高.产β-内酰胺酶株总检出率为52.8%, 其中儿童分离株产酶检出率为64.3%高于成人分离株产酶检出率47.6%.共检出氨苄西林耐药而β-内酰胺酶阴性菌株1株, 占0.5%.结论 流感嗜血杆菌主要分离自住院患者呼吸道分泌物标本.虽然该菌对头孢菌素类、大环内酯类、酶抑制剂复方制剂、喹诺酮类、碳青霉烯酶类抗菌药物保持良好的抗菌活性, 但是产β内酰胺酶仍是流感嗜血杆菌的重要耐药机制, 表现为氨苄西林耐药率较高, 因此不建议作为流感嗜血杆菌感染的经验性治疗.     

大肠杆菌、金葡菌和铜绿假单胞菌对喹诺酮类药物的耐药性研究

目的 研究大肠埃希氏杆菌（大肠杆菌）、金黄色葡萄球菌（金葡菌）和铜绿假单胞菌分别暴露于喹诺酮类药物、染料、紫外线后耐药性产生情况及不同浓度喹诺酮类药物作用于这三种细菌后其生长和形态特征的改变。方法 采用琼脂平板表面涂菌和试验双倍稀释法。结果 引起细菌耐喹诺酮类药物的主要原因是低浓度喹诺酮类产生耐药性。细菌经不同浓度环丙沙星作用后,产生不同程度的数量减少和形态改变。结论 大肠杆菌最容易产生喹诺酮耐药性,在环丙沙星作用后量易发生形态和耐药性改变,而不易被喹诺酮类药物杀灭。     

三维实验法检测耐亚胺培南的铜绿假单胞菌产β-内酰胺酶

[目的]了解耐亚胺培南的铜绿假单胞菌产β-内酰胺酶的分布及分离率。[方法]采用K-B法进行药物敏感试验,筛选耐亚胺培南的铜绿假单胞菌;再进行改良三维实验,对菌株所产β-内酰胺酶进行分析。[结果]临床分离耐亚胺培南的铜绿假单胞菌40株中,单产超广谱β-内酰胺酶（ESBLs）9株（22.5%）,单产高产染色体头孢菌素酶（AmpC）2株（5.0%）,5株为超超广谱β-内酰胺酶（SSBL）（12.5%）,3株产酶量过高或其他原因（7.5%）,21株无酶活性（52.5%）。[结论]产β-内酰胺酶是耐亚胺培南铜绿假单胞菌的主要耐药机制之一。     

医院内肠杆菌科细菌耐药性监测结果分析

目的:调查医院内肠杆菌科细菌常见临床分离株的耐药性.方法:采用K-B方法,对中国医科大学附属第一医院1999年1月～2003年12月临床分离的肠杆菌科细菌进行药敏试验.根据NCCLS推荐方法检测大肠埃希菌和肺炎克雷伯菌产超广谱β内酰胺酶(ESBLs)的情况.结果:5年间,最常见的肠杆菌科细菌为大肠埃希菌、肺炎克雷伯菌、阴沟肠杆菌、变形杆菌属、沙雷菌属.耐药分析显示,亚胺培南一直对所有常见肠杆菌科细菌保持良好的抗菌活性,其次为头孢哌酮/舒巴坦和头孢吡肟,耐药率均＜20%.氟喹诺酮类药物对除大肠埃希菌以外的其它肠杆菌细菌抗菌活性好.大肠埃希菌和肺炎克雷伯菌产ESBLs的比例逐年增高,分别从1999年的9.7%、18.4%上升到2003年的26.3%和33.9%(均P＜0.05).产ESBLs菌株对头孢噻肟和氨曲南的耐药率明显高于头孢他啶.结论:院内细菌耐药性监测不仅能指导临床合理使用抗菌药物,还能延缓耐药性的产生和蔓延.     

肺炎克雷伯杆菌β-内酰胺酶的分子特性

目的 探讨头孢哌酮（MIC≥8mg/L）加舒巴坦后头孢哌酮的最低抑菌浓度（MIC）降低2倍以上的肺炎克雷伯杆菌β－内酰胺酶分子特性。方法 用PCR扩增和DNA测序方法,测定分子量及等电点以及酶动力。结果 4株为克雷伯杆菌含有TEM型基因,2株肺炎克雷伯杆菌TEM型酶的氨基酸序列与TEM－1相比在117位及118位发生了氨基酸变异。4株肺炎克雷伯杆菌都产生2种以上的酶,等电点从5.4-9.3,相对分子量从23000－34000。酶动力学研究表明,肺炎克雷伯杆菌中99592及99607产生的酶能够水解头孢他啶、头孢噻肟及头孢曲松,并且99592的酶活性大于99607的酶活性。结论 肺炎克雷伯杆菌可能产生新的抑制剂敏感的TEM型超广谱酶。     

Antibiotic sensitivity pattern of bacterial pathogens in the intensive care unit of Fatmawati Hospital,Indonesia

Objective

To evaluate the sensitivity pattern of bacterial pathogens in the intensive care unit (ICU) of a tertiary care of Fatmawati Hospital Jakarta Indonesia.

Methods

A cross sectional retrospective study of bacterial pathogen was carried out on a total of 722 patients that were admitted to the ICU of Fatmawati Hospital Jakarta Indonesia during January 2009 to March 2010. All bacteria were identified by standard microbiologic methods, and their antibiotic susceptibility testing was performed using disk diffusion method.

Results

Specimens were collected from 385 patients who were given antimicrobial treatment, of which 249 (64.68%) were cultured positive and 136 (35.32%) were negative. The most predominant isolate was Pseudomonas aeruginosa (P. aeruginosa) (26.5%) followed by Klebsiella pneumoniae (K. pneumoniae) (15.3%) and Staphylococcus epidermidis (14.9%). P. aeruginosa isolates showed high rate of resistance to cephalexin (95.3%), cefotaxime (64.1%), and ceftriaxone (60.9%). Amikacin was the most effective (84.4%) antibiotic against P. aeruginosa followed by imipenem (81.2%), and meropenem (75.0%). K. pneumoniae showed resistance to cephalexin (86.5%), ceftriaxone (75.7%), ceftazidime (73.0%), cefpirome (73.0%) and cefotaxime (67.9%), respectively.

Conclusions

Most bacteria isolated from ICU of Fatmawati Hospital Jakarta Indonesia were resistant to the third generation of cephalosporins, and quinolone antibiotics. Regular surveillance of antibiotic susceptibility patterns is very important for setting orders to guide the clinician in choosing empirical or directed therapy of infected patients.     

外科监护室耐药大肠杆菌和肺炎克雷伯杆菌产超广谱β-内酰胺酶的基因型研究

目的:了解外科监护室产生超广谱β-内酰胺酶(extended-spectram β-lactamases,ESBLs)的大肠杆菌和肺炎克雷伯杆菌的耐药性和基因型.方法:琼脂稀释法测定临床分离株的最小抑菌浓度(minimal inhibitory concentration,MIC)值,分别用TEM、SHV和CTX-M的特异性引物扩增临床菌株和接合子的超广谱β-内酰胺酶基因.接合试验提取接合子质粒进行聚合酶链反应(polymerase chin reaction,PCR),产物测序,确定基因类型.结果:93.5%的菌株含CTX-M基因,38.7%的菌株含SHV基因,87.1%的菌株含TEM基因.测序明确了TEM-1,CTX-M-1,3,14,22等基因类型.结论:外科监护室大肠杆菌和肺炎克雷伯杆菌产超广谱β-内酰胺酶的基因以CTX-M为主.     

多重PCR检测临床产ESBLs大肠埃希菌的耐药基因型研究

目的了解产超广谱β-内酰胺酶(ESBLs)大肠埃希菌的耐药性、产ESBLs的阳性率及耐药基因之间的关系。方法采用Kirby-Bauer(K-B)琼脂扩散法检测120株临床分离的大肠埃希菌对18种抗生素的耐药性,用酶抑制剂增强实验纸片法检测产ESBLs菌株,运用多重PCR技术检测相关耐药基因,用DNA序列分析确认基因亚型。结果120株大肠埃希菌产ESBLs的有51株,阳性率为42.5%。120株大肠埃希菌对亚胺培南全部敏感,产ESBLs的大肠埃希菌对头孢唑啉、头孢噻肟、头孢曲松、头孢哌酮、头孢他啶等头孢类抗菌药物的耐药率高达100%;51株产ESBLs菌株其中46株扩增到CTX-M型耐药基因,44株扩增到TEM型基因,2株扩增到SHV基因,1株OXA型基因,未检出PER和VEB型。结论大肠埃希菌产ESBLs较高,耐药显著,碳青酶烯类抗生素是目前治疗产ESBLs大肠埃希菌最有效的药物;产ESBLs大肠埃希菌的主要基因型别为CTX-M型和TEM型。     

尖锐湿疣Fas、bcl-2、Ki-67的表达与细胞凋亡的关系

目的　探讨尖锐湿疣 ( CA)细胞凋亡与调节和增殖的关系及其意义。方法　采用 TUNEL 法对2 8例 CA患者的角质形成细胞凋亡进行了原位检测 ,并用免疫组化法观察了 Fas、bcl- 2及 Ki- 6 7的阳性表达。结果　本组病例中的 2 4例 ( 85 .7% )有凋亡细胞检出 ,2 8例 ( 10 0 % )有 Fas和 Ki- 6 7阳性表达 ,但 bcl- 2仅 8例 ( 2 8.6 % )有中、低度的表达。其凋亡指数 ( AI)、Fas、bcl- 2及 Ki- 6 7阳性表达与病情严重度之间无明显关系 ( P>0 .0 5 )。AI与Fas表达呈显著正相关 ( r=0 .86 6 ,P=0 .0 0 0 ) ,与 bcl- 2及 Ki- 6 7呈显著负相关 ( r=- 0 .416 ,P=0 .0 18;r=- 0 .475 ,P=0 .0 0 6 )。结论　 CA中角朊细胞的凋亡可能在限制病毒的繁殖上起重要作用 ,Fas对凋亡具上调作用 ,bcl- 2有抑制作用 ,Ki- 6 7过度表达可能与细胞周期加速有关     

A STUDY OF ANTIBACTERIAL ACTIVITY OF FURBENICILLIN AGAINST (3-LACTAMASE PRODUCING GRAM-NEGATlIVE ORGANISMS

The in vitro activity of furbenicillin and ampicillin against 383 strains isolated by clinical laboratories in Beijing was studied. B-lactamases were prepared from 62 resistant gram-negative organisms selected from clinics. The f3-lactamase extracts were identified by the method of iso- electric focusing for studying enzyme stability of furbenicillin, ampicillin, cefazolin, cefaman- dole, and cefotaxime in a biological system. The results showed that the antibaoterial activity of furbenicillin against Escherichia coli, Klebsiella spp, Pseudomonas aeruginosa and Enterobacteria cloacae was better than that of ampiciIlin. Cefotaxime was most stable com- pound to the p-lactamases among all of the anti- biotics examined. The stability of furbenicillin and ampicillin to the B-lactamase extracts in the presence of clavulanic acid was similar to that of cefataxime. The synergism of furbenicillin and clavulanic acid against the resistant gram- negative bacilli was better than that of ampicil- Iin combined with clavulanic acid. The results of the identification of B-lactamases used were similar to those reported in the literature, TEM-l enzyme type being the most common type of the f3-lactamases extracted from enzyme producing resistant gram-negative bacilli.     

Prevalence of resistance of N. gonorrhoeae to penicillin and three other antibiotics in a rural area in Kenya.

Of 90 isolates of Neisseria gonorrhoeae in a rural area in Kenya, 44 (48.9%) produced beta-lactamase (penicillinase). Testing for susceptibility of 35 penicillinase producing N. gonorrhoeae (PPNG) strains to four antibiotics yielded the following results: 16 (45.7%) showed a decreased susceptibility to tetracycline; six (17.1%) showed resistance, probably plasmid mediated; 10 (28.6%) had intermediate susceptibility to gentamicin; one (2.9%) was resistant; and two (5.7%) isolates were resistant to cefotaxime. 16 (57.1%) of 28 non PPNG strains showed a decreased susceptibility to penicillin; 10 (35.7%) were resistant. Nine (32.1%) of 28 non-PPNG isolates showed intermediate susceptibility to tetracycline; one (3.6%) was resistant. Eight of non PPNG isolates (28.6%) showed decreased susceptibility to gentamicin. These results imply that penicillin and tetracycline should be abandoned as primary therapy. For the time being, thiamphenicol and spectinomycin seem to be good alternatives. The observation of cefotaxime resistance in N. gonorrhoeae in a rural area implies a warning concerning future possibilities for use of third generation cephalosporins.     

某院肺炎克雷伯菌产质粒AmpC酶基因型及耐药性研究

目的调查该院临床分离肺炎克雷伯菌产质粒AmpC酶表型和基因型携带情况及其对常用抗生素的耐药性,为临床合理用药提供参考。方法采用Vitek2-compact全自动细菌鉴定药敏系统对临床分离菌株进行鉴定及药物敏感试验,采用头孢西丁三维试验检测AmpC酶表型,采用多重PCR方法检测AmpC基因并经DNA测序确定其基因型别。结果 2010年7月至2011年6月共分离到116株肺炎克雷伯菌,产AmpC酶肺炎克雷伯菌占18.1%(21/116)。产AmpC酶肺炎克雷伯菌多重耐药情况严重,对氨基糖甙类、喹诺酮类及大多数头孢菌素类抗菌药物的耐药率在57.1%～100%,对加酶抑制剂复合药氨苄西林/舒巴坦、阿莫西林/克拉维酸、头孢哌酮/舒巴坦及哌拉西林/他唑巴坦的耐药率分别为90.5%、85.7%、42.9%、38.1%,所有菌株对碳青霉烯类抗生素亚安培南和美罗培南仍全部敏感。产AmpC酶菌株对头孢西丁、第3代头孢菌素及氨苄西林/舒巴坦、阿莫西林/克拉维酸等抗生素的耐药率明显高于不产酶株(P<0.05)。21株产酶菌株均携带DHA-1型AmpC酶基因。结论该院肺炎克雷伯菌产AmpC酶株检出率较高,产酶菌株多重耐药情况严重。临床应根据药敏结果合理选用抗菌药物,以减少耐药菌株的产生。     

产ESBLs大肠埃希菌和肺炎克雷伯菌的耐药性分析

目的了解产超广谱β-内酰胺酶（ESBLs）大肠埃希菌和肺炎克雷伯菌的检出率及其耐药率，为临床合理使用抗生素提供依据。方法收集2007年1～12月海口市人民医院临床各类标本中分离出（非重复）的大肠埃希菌和肺炎克雷伯菌，采用CLSI推荐的K—B法和表型确证试验分别对其进行药敏试验和ESBLs检测。采用WHO推荐的Whonct5．4软件进行耐药率统计分析。结果2007年海口市人民医院共分离出278株大肠埃希菌和220株肺炎克雷伯菌，其中产ESBLs的大肠埃希菌和肺炎克雷伯菌的检出率分别为69．1％（192／278）和45．9％（101／220），ESBLs阳性的菌株对同一类抗生素的耐药率均高于ESBLs阴性的菌株（P〈0．05），其中产ESBLs的大肠埃希菌和肺炎克雷伯菌对头孢噻肟的耐药率分别为81．7％和64．4％，对头孢西丁的耐药率分别为39．6％和31．8％，未发现对碳青酶烯类药物（亚胺培南、美洛培南）耐药的菌株。结论开展检出菌产ESBLs的检测，以便临床根据药敏试验结果合理使用抗生素，对预防和控制ESBk的产生及传播十分重要。