鲍曼不动杆菌超广谱β-内酰胺酶检测及耐药性分析

为了解厦门中山医院鲍曼不动杆菌产超广谱β 内酰胺酶(ESBLs)情况及耐药现状,指导临床用药,采用美国临床实验室标准化委员会(NCCLS)推荐的纸片扩散法,测定 2002 ~2003年临床分离的 211株鲍曼不动杆菌产ESBLs的比例及其对头孢噻肟、阿米卡星等 13种抗菌药物耐药性特征。结果表明 211株鲍曼不动杆菌中ESBLs阳性 27株(占 12. 8% ),产ESBLs菌株对 13种抗菌药物的耐药率明显高于非产ESBLs株。鲍曼不动杆菌的产ESBLs株有较高的交叉耐药性,采用纸片确证实验检出ESBLs对于临床用药有一定的指导意义。     

肺炎克雷伯菌超广谱β-内酰胺酶和AmpC酶基因检测及耐药性分析

目的 了解下呼吸道感染肺炎克雷伯菌超广谱β-内酰胺酶（ESBLs）和质粒AmpC酶的产生情况及耐药性,研究AmpC酶的基因型别.方法 用纸片扩散确证法检测ESBLs;用酶提取三维试验检测AmpC酶,聚合酶链反应（PCR）扩增AmpC酶的基因,DNA序列测定检测AmpC酶的基因型;K-B法检测细菌耐药性.结果 58株下呼吸道感染肺炎克雷伯菌中ESBLs阳性21株,AmpC酶阳性5株,其中3株ESBLs和AmpC酶均阳性,5株AmpC酶阳性菌中,4株扩增出DHA基因,经测序均为DHA-1,1株扩增出MIR基因.产酶菌株的耐药性明显高于非产酶株.结论 肺炎克雷伯菌中ESBL＊s和AmpC酶均有较高的检出率,AmpC酶以DHA基因型为主.产ESBLs和AmpC酶是肺炎克雷伯菌耐药的主要原因.     

革兰阴性杆菌同产超广谱β-内酰胺酶和AmpC酶的耐药性分析

目的了解革兰阴性杆菌同时表达超广谱β-内酰胺酶(ESBLs)和AmpC酶(AmpCs)情况及耐药性。方法采用ESBLs确认试验检测ESBLs,头孢西丁三维试验检测AmpCs,用KB法进行药敏试验。结果4098株革兰阴性杆菌中,检出同产ESBLs+AmpCs株739株,占18.0%;其中ESBLs+高产AmpCs508株,占12.4%,主要见于不动杆菌、大肠埃希菌和肺炎克雷伯菌,检出率分别为50.8%、44.1%和42.2%;ESBLs+诱导AmpCs231株,占5.6%,粘质沙雷菌、不动杆菌检出率分别为35.1%、27.7%;产ESBLs+高产AmpCs菌株对亚胺培南(IMP)、头孢哌酮-他唑巴坦(CSL)和头孢吡肟(FEP)的耐药率分别为3.54%、6.30%和51.8%,均显著低于其余抗生素的耐药率(均P<0.001);产ESBLs+诱导AmpCs菌株对亚胺培南、头孢哌酮-他唑巴坦和头孢吡肟的耐药率分别为1.30%、6.06%和38.5%,均显著低于其余抗生素的耐药率(均P<0.001)结论同时产生超广谱β-内酰胺酶和AmpCs菌株对多重药物耐药,IMP、CSL和FEP对其具有良好的抗菌活性。     

细菌超广谱β-内酰胺酶提取方法研究进展

超广谱β-内酰胺酶(ESBLs)可水解β-内酰胺类抗生素,使产ESBLs细菌对目前常用的头孢菌素和青霉素类等抗生素产生多耐药性和高耐药性,由此使得产ESBLs细菌感染的诊断和治疗成为临床抗感染诊疗工作的重点。目前细菌的ESBLs提取主要包括超声波破碎法、溶菌酶法和反复冻融法等。本文对现有文献中此三种方法的提取原理、提取步骤和影响因素进行了综述,同时对各种提取方法的优缺点进行了分析比较,并对采用反复冻融法联合超声法和溶菌酶法联合超声法等联合提取法提高酶提取效率的展望予以了简要分析,以期为产酶多重耐药菌的控制提供参考。     

非产超广谱β-内酰胺酶肺炎克雷伯杆菌的耐药性分析

目的了解临床分离肺炎克雷伯杆菌中非产超广谱β-内酰胺酶（ESBLs）菌株对18种常见抗菌药物的耐药性。方法CLSI表型确证试验-纸片增强法检测非产ESBLs肺炎克雷伯菌,K-B法测定非产ESBLs肺炎克雷伯杆菌对18种常见抗菌药物的敏感性。结果非产ESBLs肺炎克雷伯杆菌株对头孢唑啉、头孢呋辛的耐药率〉50．0％,对其余抗生素的耐药率均低于25．0％,对亚胺培南、美罗培南非常敏感,耐药率分别为2．3％和2．0％;痰标本的分离株对头孢唑啉、头孢呋辛的耐药率明显高于血、尿液标本分离株,差异有统计学意义（P〈0．05）。结论非产ESBLs肺炎克雷伯杆菌对第一、二代头孢菌素耐药显著,对第三代头孢菌素、亚胺培南、美罗培南等抗生素比较敏感。     

革兰氏阴性杆菌产超广谱β-内酰胺酶菌的耐药分析

目的了解ESBLs的发生率和耐药特点,为临床合理用药提供依据.方法从住院及门诊病人的尿、粪、痰、血、分泌物等标本中分离的菌株共284株,使用法国梅里埃公司的细菌分析仪进行细菌鉴定和药敏检测及纸片扩散法确证试验.结果检出ESBLs菌株81株,检出率28.52%;产ESBLs的菌株主要是大肠埃希氏菌、肺炎克雷伯氏菌、铜绿假单胞菌;以阿莫西林和复方新诺明耐药率最高,达90%以上.结论对ESBLs细菌感染应根据药敏结果选择碳青霉烯类、喹诺酮类及β-内酰胺类/酶抑制剂等抗生素治疗.     

产超广谱β-内酰胺酶细菌感染临床流行病学

目的:了解产超广谱β-内酰胺酶(ESBLs)肺炎克雷伯菌(Kpn)和大肠埃希菌(Eco)感染的影响因素.方法:根据我院实验室登记的各种标本中细菌培养的阳性报告,回顾调查了1999年2月～2000年12月在我院住院的201份病历.结果:201份病例送检的标本中,检出产ESBLs细菌76株,检出率37.8%(其中Kpn 25株,Eco 51株).非产ESBLs细菌为125株(其中Kpn 24株,Eco 101株).多因素分析结果表明侵袭性操作、第三代头孢菌素及其他β-内酰胺类药物的应用、医院感染为产生ESBLs肺炎克雷伯菌和大肠埃希菌感染的危险因素(OR值分别为2.314、3.433、2.276、2.665);而基础疾病、免疫抑制剂、喹诺酮类药物的应用,与产ESBLs肺炎克雷伯菌和大肠埃希菌感染无统计学意义(P＞0.05).结论:影响产ESBLs菌感染的临床危险因素为第三代头孢菌素及其他β-内酰胺类药物的应用、医院感染、侵袭性操作.     

超广谱β-内酰胺酶菌种间分布及耐药性分析

目的：了解质粒介导超广谱β-内酰胺酶（ESBL）的产生及菌种间分布。方法：将初筛的可疑菌用E-test法确诊,对细菌进行ESBL检测,药物敏感试验用K-B纸片法进行。结果：717株革兰阴性菌中,检出ESBL61株,其中大肠埃希菌16株,肺炎克雷伯菌12株,阴沟杆菌9株,弗劳地枸椽酸杆菌6株,类产碱杆菌5株,黄杆菌,施氏假单胞菌各3株,聚团肠杆菌2株,液化沙雷菌2株,铜绿假单胞菌,不动杆菌,鼠伤寒沙门菌各1株,对亚胺培南,丁胺卡那,环内沙星,呋喃妥因和头孢他啶的敏感率分别为100%,30.2%,26.6%,71.5%和37.7%,结论：ESBL易借助耐药质粒传播,在细菌间分布广泛,具有较高的检出阳性率。     

产β-内酰胺酶鲍曼不动杆菌基因型研究

目的了解临床科室分离的鲍曼不动杆菌耐药状况并分析多重耐药株产β-内酰胺酶基因型,为有效的临床治疗和医院感染控制提供实验室依据。方法采用纸片扩散（K-B）法测定临床分离的312株鲍曼不动杆菌对13种抗菌药物的敏感性,对其中120株多重耐药株用聚合酶链反应（PCR）扩增β-内酰胺酶基因,并对其扩增产物进行基因测序。结果鲍曼不动杆菌对美洛培南、亚胺培南、头孢哌酮/舒巴坦和替卡西林/克拉维酸的耐药率分别为0.0%、1.0%、30.8%和31.4%,其余抗菌药物耐药率在38.5%-80.1%;120株多重耐药菌株中,β-内酰胺酶基因分布AmpC型基因阳性率为66.7%（80/120）、SHV-12型基因阳性率为14.2%（17/120）,PER-1型基因阳性率为16.7%（20/120）,CTX-M-9型基因阳性率为8.2%（10/120）,TEM-1型基因阳性率为9.2%（11/120）;VER-1,CTX-M-1,CTX-M-2,OXA型均阴性。同时携带2种基因型有16株,携带3种基因型有14株。结论临床分离的鲍曼不动杆菌流行株主要是产AmpC酶,且呈多重耐药。     

产超广谱β-内酰胺酶阳性菌检测及其耐药性分析

目的 了解新乡市中心医院近年来产超广谱β-内酰胺酶（ESBLs）阳性菌的种类、分布及对11种常用抗菌药物的耐药情况,为控制其扩散、暴发流行和指导临床治疗提供科学依据。方法 对该院2003年1月～2004年1月从临床各类标本中分离的革兰阴性菌用双纸片协同法筛选产超广谱β-内酰胺酶阳性菌株,采用K-B纸片扩散法比较亚胺培南等11常用抗菌药物对产ESBLs耐药菌体外抗菌作用。结果 产ESBLs耐药株占全部革兰阴性菌的22.82%,克雷伯菌属、肠杆菌属、埃希菌属、假单胞菌属和沙雷菌属ESBLs的流行较严重,检出率分别为31.22%、29.02%、27.23%、25.19%和23.08%,在11种常用抗菌药物中,亚胺堵南的体外抗菌活性最好,敏感率为100%。结论 革兰阴性菌中,ESBLs阳性菌的发生率较高,同时ESBLs阳性菌有较高的耐药性。实验室应注重ES-BLs的检测和报告,指导临床合理应用抗生索,有效地控制ESBLs菌株在医院内的传播和流行。     

Purification and characterization of an extracellular beta-lactamase produced by Acinetobacter calcoaceticus.

A beta-lactamase was purified 430-fold from the culture supernatant of Acinetobacter calcoaceticus by ion exchange chromatography on CM-Sephadex and affinity chromatography on phenylboronic-acid-agarose. The purified enzyme was homogeneous as judged by SDS-PAGE, and was characterized with respect to molecular mass (38 and 41 kDa by gel filtration on Sephadex G-75 and SDS-PAGE, respectively), pH optimum (pH 7.0), temperature optimum (45 degrees C) and isoelectric point (9.3). The beta-lactamase showed mainly cephalosporinase activity. It was inhibited by cloxacillin, carbenicillin, penicillanic acid sulphone (sulbactam) and aztreonam. It was not inhibited by clavulanic acid up to a concentration of 0.25 mM. Neither EDTA nor p-chlormercuribenzoate, up to concentrations of 1 or 100 mM, respectively, affected activity. According to these characteristics, it is a typical CEP-N cephalosporinase.     

Characterization of conjugative plasmids carrying antibiotic resistance genes encoding 16S rRNA methylase, extended-spectrum beta-lactamase, and/or plasmid-mediated AmpC beta-lactamase

In this study, we identified extended-spectrum β-lactamase (ESBL) and plasmid-mediated AmpC β-lactamase which were associated with 16S rRNA methylase gene on the conjugative plasmid. Among 82 clinical isolates of Enterobacteriaceae that carry 16S rRNA methylase gene (64 strains, armA, and 18 strains, rmtB), bla _SHV-12 was detected either alone or combined with bla _DHA-1, bla _CTX-M-3, and bla _CTX-M-14 in 30 strains carrying armA and 6 strains carrying rmtB. The bla _CTX-M-3 was detected in 13 of 64 strains carrying armA but no strains carrying rmtB. Whereas bla _CTX-M-14 was detected in 15 of 18 strains carrying rmtB but only 2 of 64 strains carrying armA. Overall, bla _SHV-12 and bla _CTX-M-14 was the most common ESBL gene which was associated with armA and rmtB, respectively. In addition, we found that bla _CTX-M-3 localized with armA on the same IncL/M plasmid and bla _CTX-M-14 localized with rmtB on the same IncA/C plasmid. Restriction fragment length polymorphism of conjugative plasmids and pulsed-field gel electrophoresis of genomic DNAs revealed that intercellular horizontal transfer of conjugative plasmid and clonal transmission have been occurred at the same time.     

Susceptibility, phenotypes of resistance, and extended-spectrum β-lactamases in Acinetobacter baumannii strains

Acinetobacter baumannii plays an increasing role in the pathogenesis of infections in humans. The bacilli are frequently isolated from patients treated in intensive care units. A growing resistance to antibiotics is leading to the emergence of strains that are multidrug-resistant and resistant to all available agents. The objective of this study was to assess susceptibility to antibiotics and to determine the presence and current level of the extended-spectrum β-lactamases (ESBLs) and attempt to isolate the Acinetobacter baumannii strain carrying the blaPER gene. A total of 51 strains of A. baumannii identified by phenotypic features were examined. That the strains belonged to the species was confirmed by the presence of the blaOXA-51-like; gene. A broth microdilution method was used for antibacterial susceptibility testing. The occurrence of ESBLs was determined using phenotypic double-disk synergy tests. The PCR technique was used to confirm the presence of the blaPER-1; gene encoding ESBL. The most active antibiotics were meropenem, cefepime and ampicillin/sulbactam, with susceptibility shown by 76.5%, 60.8% and 56.9% of the strains, respectively. The strains exhibited the highest resistance (> 75%) to piperacillin, tetracycline, ciprofloxacin and cefotaxime. Phenotypic tests revealed ESBL mechanism of resistance in approximately 20% of Acinetobacter baumannii isolates. However, the PCR technique did not confirm the presence of the blaPER-1; gene in any of the Acinetobacter baumannii strains examined in our hospital. Acinetobacter baumannii strains demonstrate considerable resistance to many groups of antibiotics. Our findings indicate the involvement of enzymes belonging to families other than PER β-lactamase in resistance to β-lactams in A. baumannii.     

Phenotypic and molecular characterization of TEM-116 extended-spectrum beta-lactamase produced by a Shigella flexneri clinical isolate from chickens.

Extended-spectrum beta-lactamases (ESBLs) produced by a clinical isolate of Shigella flexneri from chickens were detected with confirmatory phenotypic tests of the Clinical and Laboratory Standards Institute, and minimum inhibitory concentrations of several antibacterial drugs against the isolate were determined by the twofold dilution method. The genotype and subtype of the ESBL-producing S. flexneri isolate were identified by PCR amplifying of ESBL genes and DNA sequencing analysis. The results revealed that the isolate was able to produce ESBLs. They were resistant to third-generation cephalosporins such as ceftiofur and ceftriaxone and showed characteristics of multidrug resistance. The ESBL gene from the S. flexneri isolate was of the TEM type. Sequence analysis indicated that the TEM-type gene had 99.1% and 99.2% identity to TEM-1D ESBL and TEM-1 beta-lactamase, respectively, at the nucleotide level. The amino acid sequence inferred from the TEM-type gene revealed three substitutions compared with the TEM-1 and TEM-1D enzymes: Ser51Gly, Val82Ila and Ala182Val. When it was compared with TEM-116 (99.8% identity), there were only two mutations (A(151)G and T(403)C) in the TEM-type gene, resulting in the substitution of Ser to Gly at position 51 in the amino acid sequence. The TEM type was a TEM-116 derivative.     

Resistance pattern of extended-spectrum beta-lactamase producing Enterobacteriaceae isolates

Gram-negative pathogens harboring extended-spectrum beta-lactamases (ESBL) are becoming an increasing therapeutic problem in many wards. The aim of our work was to study ESBL production by Enterobacteriaceae strains from Eastern Romania and their antimicrobial resistance. We selected 54 clinical isolates among 1068 enterobacteria according to their susceptibility spectrum (National Committee for Clinical Laboratory Standards, 1999). Antimicrobial susceptibility tests were performed using the Rapid ATB E gallery of mini API system (BioMérieux) and by a macrodilution method in Mueller-Hinton agar following standard procedure of the National Committee for Clinical Laboratory Standards (NCCLS). ESBL production was established by using both double disk synergy test (DDT) and Expert computer program of mini API. The isoelectric point (pI) was determined by isoelectric focusing in polyacrylamide gel and revealed by nitrocefin. As references we used beta-lactamases with known pI. The Expert computer program of mini API confirms the positive DDT test for all selected strains. Almost all strains displayed resistance to ampicillin, ampicillin/sulbactam or third generation cephalosporins and aztreonam. By IEF we identified 51 strains which have a unique enzyme. IEF pattern showed presence of two enzymes in three Escherichia coli strains. According to our results, the ESBL TEM-type are the most common for the studied isolates. The production of extended-spectrum beta-lactamases and the presence of the multiresistant of antimicrobial agents reflect, probably, the over use of third generation cephalosporins in Eastern Romania.     

Functional analyses of AmpC beta-lactamase through differential stability.

Despite decades of intense study, the complementarity of beta-lactams for beta-lactamases and penicillin binding proteins is poorly understood. For most of these enzymes, beta-lactam binding involves rapid formation of a covalent intermediate. This makes measuring the equilibrium between bound and free beta-lactam difficult, effectively precluding measurement of the interaction energy between the ligand and the enzyme. Here, we explore the energetic complementarity of beta-lactams for the beta-lactamase AmpC through reversible denaturation of adducts of the enzyme with beta-lactams. AmpC from Escherichia coli was reversibly denatured by temperature in a two-state manner with a temperature of melting (Tm) of 54.6 degrees C and a van't Hoff enthalpy of unfolding (deltaH(VH)) of 182 kcal/mol. Solvent denaturation gave a Gibbs free energy of unfolding in the absence of denaturant (deltaG(u)H2O) of 14.0 kcal/mol. Ligand binding perturbed the stability of the enzyme. The penicillin cloxacillin stabilized AmpC by 3.2 kcal/mol (deltaTm = +5.8 degrees C); the monobactam aztreonam stabilized the enzyme by 2.7 kcal/mol (deltaTm = +4.9 degrees C). Both acylating inhibitors complement the active site. Surprisingly, the oxacephem moxalactam and the carbapenem imipenem both destabilized AmpC, by 1.8 kcal/mol (deltaTm = -3.2 degrees C) and 0.7 kcal/mol (deltaTm = -1.2 degrees C), respectively. These beta-lactams, which share nonhydrogen substituents in the 6(7)alpha position of the beta-lactam ring, make unfavorable noncovalent interactions with the enzyme. Complexes of AmpC with transition state analog inhibitors were also reversibly denatured; both benzo(b)thiophene-2-boronic acid (BZBTH2B) and p-nitrophenyl phenylphosphonate (PNPP) stabilized AmpC. Finally, a catalytically inactive mutant of AmpC, Y150F, was reversibly denatured. It was 0.7 kcal/mol (deltaTm = -1.3 degrees C) less stable than wild-type (WT) by thermal denaturation. Both the cloxacillin and the moxalactam adducts with Y150F were significantly destabilized relative to their WT counterparts, suggesting that this residue plays a role in recognizing the acylated intermediate of the beta-lactamase reaction. Reversible denaturation allows for energetic analyses of the complementarity of AmpC for beta-lactams, through ligand binding, and for itself, through residue substitution. Reversible denaturation may be a useful way to study ligand complementarity to other beta-lactam binding proteins as well.     

Structure-based discovery of a novel,noncovalent inhibitor of AmpC beta-lactamase

beta-lactamases are the most widespread resistance mechanisms to beta-lactam antibiotics, and there is a pressing need for novel, non-beta-lactam drugs. A database of over 200,000 compounds was docked to the active site of AmpC beta-lactamase to identify potential inhibitors. Fifty-six compounds were tested, and three had K(i) values of 650 microM or better. The best of these, 3-[(4-chloroanilino)sulfonyl]thiophene-2-carboxylic acid, was a competitive noncovalent inhibitor (K(i) = 26 microM), which also reversed resistance to beta-lactams in bacteria expressing AmpC. The structure of AmpC in complex with this compound was determined by X-ray crystallography to 1.94 A and reveals that the inhibitor interacts with key active-site residues in sites targeted in the docking calculation. Indeed, the experimentally determined conformation of the inhibitor closely resembles the prediction. The structure of the enzyme-inhibitor complex presents an opportunity to improve binding affinity in a novel series of inhibitors discovered by structure-based methods.     

Characterization of a beta-lactamase produced by Pseudomonas paucimobilis.

A novel beta-lactamase enzyme produced by a strain of Pseudomonas paucimobilis is described. The enzyme differs from other recorded beta-lactamases from Gram-negative aerobic bacteria. It was constitutive, and had the characteristics of a penicillinase. One single band of beta-lactamase activity at pI 4.6 was seen on iso-electric focusing. The enzyme had a molecular mass of 30 kDa. The beta-lactamase was strongly inhibited by tazobactam, sulbactam and clavulanic acid but not by the thiol residue inhibitors p-chloromercuribenzoate and p-chloromercuriphenylsulphonic acid, or by metallo-enzyme inhibitors. Plasmid DNA was not demonstrable, suggesting that the enzyme was chromosomally encoded.