鲍曼不动杆菌16S rRNA甲基化酶与氨基糖苷修饰酶基因检测研究

目的了解高水平耐氨基糖苷类鲍曼不动杆菌16S rRNA甲基化酶、氨基糖苷修饰酶基因的流行情况。方法从2008年9月至2011年1月收集的110株鲍曼不动杆菌,琼脂二倍稀释法测定其对6种氨基糖苷类抗生素的药物敏感性,并筛选出对阿米卡星MIC≥256μg/mL的60株鲍曼不动杆菌,PCR法检测7种甲基化酶基因(armA、rmtA-rmtE、NpmA)和3种氨基糖苷修饰酶基因(aac(6′)-Ib、ant(3″)-Ia、aph(3′)-I)。结果鲍曼不动杆菌对氨基糖苷类高水平耐药率较高(46.4%～65.4%),armA、aac(6′)-Ib、ant(3″)-Ia、aph(3′)-I的基因检出率分别为66.7%(40株)、51.7%(31株)、81,7%(49株)、58.3%(35株),其余基因未检出。结论鲍曼不动杆菌对氨基糖苷类抗生素的高度耐药性与16S rRNA甲基化酶基因及氨基糖苷修饰酶基因有关。     

鲍曼不动杆菌临床分离株16S rRNA甲基化酶基因研究

目的 了解临床分离鲍曼不动杆菌(ABA)氨基糖苷类药物耐药性与16S rRNA甲基化酶基因存在的关系,探讨多药耐药机制.方法 收集临床标本20株鲍曼不动杆菌,采用K-B法测定细菌对卡那霉素、阿米卡星、妥布霉素、庆大霉素、奈替米星的敏感性;采用PCR法检测armA、rmtA、rmtB、rmtC和rmtD 5种16S rRNA甲基化酶基因.结果 19株鲍曼不动杆菌对上述5种氨基糖苷类药物全部耐药,1株对阿米卡星敏感,对另外4种表现为中度耐药.基因检测显示armA阳性率为90％,且armA基因存在变异,未检测到rmtA、rmtB、rmtC和rmtD基因.结论 鲍曼不动杆菌对氨基糖苷类药物耐药情况严重,16S rRNA甲基化酶基因armA为本次临床分离菌株耐药的主要原因,且armA基因存在变异.     

多重耐药鲍曼不动杆菌16S rRNA甲基化酶基因研究

目的 调查临床分离多重耐药鲍曼不动杆菌中介导氨基糖苷类高水平耐药的16S rRNA甲基化酶基因armA、rmtA、rmtB的流行情况.方法 收集台州市中心医院、台州市立医院两家多重耐药鲍曼不动杆菌128抹,用K-B法测定18种抗菌药物;采用琼脂稀释法测定4种氨基糖苷类药物MIC值;PCR检测armA、rmtA、rmtB...     

氨基糖苷类修饰酶基因aac(2 ')-Ⅰb型在耐药鲍曼不动杆菌中流行

目的 调查一组耐药鲍曼不动杆菌对氨基糖苷类的耐药机制.方法 收集2013年1至3月江苏省淮安市第一人民医院住院患者标本中分离到的鲍曼不动杆菌共32株,用分子鉴定法确认为鲍曼不动杆菌,再用聚合酶链反应(PCR)方法分析9种氨基糖苷类修饰酶基因与7种16S rRNA甲基化酶基因.结果 本组32株耐药鲍曼不动杆菌共检出5种氨基糖苷类修饰酶基因:aac(2')-Ⅰb 32株(100.0％)、aac(3)-Ⅰ 15株(46.9％)、aac(6')-Ⅰb 19株(59.4％)、ant(3")-Ⅰ 20株(62.5％)、aph(3')-Ⅰ 19株(594％),与1种16S rRNA甲基化酶基因armA 25株(78.1％).阳性基因共分为7种检测模式,其中以aac(2')-Ⅰb+ aac(3)-Ⅰ+ aac(6')-Ⅰb+ ant(3")-Ⅰ+ aph(3')-l+armA等6种基因均阳性的模式最高,为12株(37.5％).结论 产5种氨基糖苷类修饰酶基因(aac(2')-Ⅰb、aac(3)-Ⅰ、aac(6')-Ⅰb、ant(3")-Ⅰ、aph(3)-Ⅰ)与1种16S rRNA甲基化酶基因armA是本组耐药鲍曼不动杆菌对氨基糖苷类耐药的重要原因.在鲍曼不动杆菌临床分离株检出aac(2')-Ⅰb型氨基糖苷类修饰酶基因为国内首次报道.     

多重耐药大肠埃希菌16S rRNA甲基化酶、氨基糖苷类修饰酶基因研究

目的了解多重耐药大肠埃希菌(ECO)氨基糖苷类药物耐药机制。方法采用PCR检测20株多重耐药ECO菌11种16S rRNA甲基化酶和氨基糖苷类修饰酶基因。结果1株检出16S rRNA甲基化酶基因(5.0%),并为新亚型;16株检出氨基糖苷类修饰酶基因(80.0%)。15号株rmtB基因测得序列翻译成氨基酸序列与美国核酸库(GenBank)已登录的rmtB氨基酸不同,为新亚型。结论本组多重耐药ECO菌氨基糖苷类耐药机制与产16S rRNA甲基化酶和产氨基糖苷类修饰酶相关。多重耐药ECO菌存在新的氨基糖苷类药物耐药机制。     

氨基糖苷类药物双圈耐药型鲍曼不动杆菌16S rRNA甲基化酶基因研究

目的 探讨在K-B法药物敏感试验中对氨基糖苷类药物双圈耐药的鲍曼不动杆菌16S rRNA甲基化酶基因携带状况以及药物诱导对该基因mRNA表达量的影响.方法 收集42株鲍曼不动杆菌,K-B法测定其药敏情况;PCR法扩增三种16S rRNA甲基化酶基因armA、rmtB及rmtC;用RT-PCR的方法分析阿米卡星诱导前后耐药基因表达量.结果 90.4％(38/42)的菌株阿米卡星药敏纸片周围呈现双圈耐药,且均检测到armA,未检测到rmtB及rmtC,其余3株为敏感,1株为普通耐药,且PCR检测耐药基因为阴性.RT-PCR结果显示经阿米卡星诱导后细菌armA基因mRNA表达量显著上升.结论 双圈耐药现象与armA基因诱导型表达有关.     

儿科患者鲍曼不动杆菌氨基糖苷类修饰酶基因的表达

目的 研究从儿科肺炎患者中分离的鲍曼不动杆菌对氨基糖苷类抗生素的耐药性和常见的7种氨基糖苷类修饰酶基因特征.方法 56株鲍曼不动杆菌(AB)收集自2006年分离的儿科临床肺炎患儿的深部痰培养标本,均采用 VITEK-32全自动微生物鉴定仪GNI和GNS卡进行细菌鉴定和药敏试验.氨基糖苷类修饰酶基因检测采用聚合酶链式反应(PCR)方法.结果 检测的56株鲍曼不动杆菌菌有8株呈多重耐药性,阳性率14.29%,8株多重耐药菌对氨基糖苷类药物阿米卡星、妥布霉素和庆大霉素均耐药,其余菌株对氨基糖苷类药物均敏感,氨基糖苷类药物的耐药率14.29%.除呋喃妥因及β-内酰胺类抗生素氨苄西林、头孢唑林和头孢曲松外其它抗生素的耐药率均在15%以下.7种氨基糖苷类修饰酶基因中检出2株aac(3)-Ⅱ(3.57%),2株aac(6')-Ib(3.57%),4株aac(6')-Ⅱ(7.14%),6株ant(3")-Ⅰ(10.71%);aac(3)-Ⅰ、ant(2")-Ⅰ和aac(6')-Ⅰad均阴性.对3种氨基糖苷类抗生素耐药的菌株均检出了氨基糖苷类修饰酶基因.结论 (1)儿科患者虽然极少应用氨基糖苷类抗生素,但由于氨基糖苷类修饰酶基因可借助于整合子、转座子和质粒等在同种和异种细菌间传播,使其对氨基糖苷类抗生素也产生了一定的耐药性.(2)鲍曼不动杆菌儿科患者分离株对阿米卡星、庆大霉素和妥布霉素的耐药与aac(3)-Ⅱ、aac(6')-Ib、aac(6')-Ⅱ和ant(3")-Ⅰ四种基因有关.不同地区细菌的修饰酶基因有很大差异,而儿童与成人患者亦有不同,因此要重视儿科患者鲍曼不动杆菌氨基糖苷类抗生素耐药性和耐药基因研究.     

鲍曼不动杆菌老年患者分离株中发现16S rRNA甲基化酶基因新亚型

目的了解鲍曼不动杆菌老年患者分离株16SrRNA甲基化酶和氨基糖苷类修饰酶基因型。方法采用PCR检测20株鲍曼不动杆菌老年患者分离株12种16SrRNA甲基化酶和氨基糖苷类修饰酶基因。结果7株检出16SrRNA甲基化酶基因（armA），19株检出氨基糖苷类修饰酶基因[其中aac（3）-I阳性率为95％，ant（3″）-I为95％，aac（3）-II为40％，aac（δ′）-Ib为15％）]。6号株armA基因测得序列翻译成氨基酸序列与美国核酸库（GenBank）已登录的armA氨基酸不同，为新亚型。结论本组鲍曼不动杆菌老年患者分离株95％携带16SrRNA甲基化酶和氨基糖苷类修饰酶基因。     

鲍曼不动杆菌质粒上氨基糖苷类耐药基因的研究

目的探讨鲍曼不动杆菌(Acinotobacter baumannii)对氨基糖苷类抗生素的耐药性及其耐药机制。方法对71株MDR A.baumanni用VITEK分析仪进行药敏分析,通过PCR和测序探索质粒上的相关耐药基因(aaaCl,aacC2,aacC3,aacA4和armA)。结果 71株A.baumanni对庆大霉素、妥布霉素和阿米卡星的耐药率分别为100%、73%和20%,其中20%的菌株对3种氨基糖苷类抗生素完全耐药,且aaaCl、aacA4和armA基因阳性率分别为100%、100%和84.5%。结论质粒上广泛携带aacCl、aacA4和armA基因是鲍曼不动杆菌对氨基糖苷类抗生素多重耐药的主要机制之一。     

aac(6')-II和qacE△1-sull基因在非发酵革兰阴性杆菌烧伤分离株中流行

目的 研究非发酵革兰阴性杆菌烧伤分离株携带的氨基糖苷类耐药基因和整合子、转座子遗传标记.方法:测定分离自烧伤患者的铜绿假单胞菌(Pa)和鲍曼不动杆菌(Ab)各20株对20种抗菌药物的敏感性,PCR检M95种氨基糖苷类修饰酶基因(aac(3)-II,aac(6')-Ib,aac(6')-II,ant(3"),-I、ant(2")-I)、3种16S rRNA甲基化酶基因(rmtA、rmtB、rmtD),1种I类整合子遗传标记(gacEA l-sull)和1种转座子遗传标记(merA).结果 40株非发酵菌对阿米卡星、庆大霉素及妥布霉素等氨基糖苷类抗生素耐药率达到95%-100%,38株(95%)非发酵菌携带aac(6)-II基因,39株(97.5%)非发酵菌携带gacEA l-sull基因.20株铜绿假单胞菌检出rmtB基因.结论 本组非发酵菌烧伤分离株对氨基糖苷类抗生素耐药严重,aac(6')-II和qacEAI-sull基因在非发酵菌烧伤分离株中流行.     

Effects of intestinal flora on the expression of cytochrome P450 3A in the liver

Living organisms eliminate foreign low-antigenic substances, such as drugs and environmental pollutants, by detoxification mediated by metabolizing cytochrome P450 (CYP). We have examined the possible regulation of CYP expression by enteric bacteria. Cyp mRNA expression levels, Cyp3a protein expression level, and the activity of Cyp3a in hepatic microsomal fractions were compared in germ-free (GF) and specific pathogen-free (SPF) mice. We evaluated hepatic Cyp3a11 mRNA expression levels and Cyp3a metabolic activity in GF and SPF mice after five days of antibiotic administration. The fecal levels of lithocholic acid (LCA)-producing bacteria and hepatic taurolithocholic acid (TLCA) were also measured. Cyp mRNA expression levels, Cyp3a protein expression level, and the activity of Cyp3a in SPF mice were higher than those in GF mice, indicating that enteric bacteria increases hepatic Cyp3a expression. The effects of enteric bacteria-reducing antibiotics on Cyp3a expression were examined. We observed that decreasing enteric bacteria with antibiotics in SPF mice caused a significant decrease in the hepatic Cyp3a11 mRNA expression, TLCA, and fecal LCA-producing bacteria compared to the group that did not receive antibiotics. No change in Cyp3a11 expression was observed in GF mice that were treated with antibiotics. Administration of LCA to GF mice showed an increase in Cyp3a11 expression similar to that of SPF mice. The enzymes of the enteric bacteria are believed to metabolize and detoxify drugs by either reduction or hydrolysis. The results of this study indicate that changes in enteric bacteria may alter the expression and activity of hepatic drug metabolizing enzymes and pharmacokinetics. Therefore, enteric bacteria should be closely monitored to ensure the safe use of drugs.     

The Development of β-Lactam Antibiotics in Response to the Evolution of β-Lactamases

beta-Lactam antibiotics, viz., penicillin, penicillin derivatives, cephalosporins, cephamycins, carbapenems, monobactams. and monocarbams, are the most widely used of all antimicrobial classes by virtue of their high efficacy and specificity and the availability of several derivatives. The expression of one or several beta-lactamases (beta-lactam antibiotic-inactivating enzymes) represents the most widespread and the most clinically relevant resistance mechanism to these antibiotics. The development of beta-lactam antibiotics has thus been a continuous battle of the design of new compounds to withstand inactivation by the ever-increasing diversity of beta-lactamases. This article traces antibiotic development in response to the evolution of beta-lactamases.     

Suppression of inducible nitric oxide synthase expression and nitric oxide production by macrolide antibiotics in sulfur mustard-exposed airway epithelial cells

Sulfur mustard, a vesicant chemical warfare agent, causes airway injury due to massive release of destructive enzymes and mediators of inflammation. Nitric oxide plays an important yet controversial role in inflammation. An impressive number of reports suggest that excessive amount of nitric oxide may promote inflammation-induced cell injury and death. Overproduction of nitric oxide is catalysed by up-regulated expression of the inducible isoform of nitric oxide synthase (iNOS). In this study, we used quantum dot-mediated immunocytochemistry to analyse iNOS expression and flow cytometry to analyse the intracellular nitric oxide production in sulfur mustard-exposed normal human small airway epithelial cells and bronchial/tracheal epithelial cells and studied the effect of four US Food and Drug Administration-approved macrolide antibiotics, namely, azithromycin, clarithromycin, erythromycin and roxithromycin. Exposure to 100 microM sulfur mustard significantly up-regulated iNOS expression and resulted in overproduction of nitric oxide in these cells. Addition of macrolide antibiotics to 100 microM in the medium reduced both iNOS expression and nitric oxide production to near normal level. Thus, the current study provides in vitro evidence of the immunomodulatory effects of macrolide antibiotics in sulfur mustard-exposed airway epithelial cells. These results suggest that macrolide antibiotics may serve as potential vesicant respiratory therapeutics through mechanisms independent of their antibacterial activity.     

Emergence of resistance in gram-negative bacteria: a risk of broad-spectrum beta-lactam use

A number of new beta-lactam antibiotics have been developed to overcome bacterial resistance to older agents. Such resistance usually is caused by plasmid-mediated, constituently produced beta-lactamases. Second- and third-generation cephalosporins, ureidopenicillins, acylamino penicillins, and monobactams generally are resistant to hydrolysis by these enzymes. However, inducible beta-lactamases may confer resistance to these antibiotics. This induction may occur spontaneously or in response to cefoxitin or other beta-lactam agents. The mechanisms by which inducible enzymes produce this resistance are reviewed and implications for the prophylactic and therapeutic use of newer beta-lactams are considered.     

New aminocoumarin antibiotics from genetically engineered Streptomyces strains

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A(1) are produced by different Streptomyces strains and are potent inhibitors of DNA gyrase. The biosynthetic gene clusters of all three antibiotics have been cloned and sequenced, and the function of most genes contained therein has been elucidated. In the last years, a number of "unnatural" aminocoumarins could be generated using the genetic information for the biosynthesis of these antibiotics. The investigated enzymes of aminocoumarin biosynthesis have less-than-perfect substrate specificity, facilitating the production of new antibiotics by various methods. Several new aminocoumarins could be produced by targeted genetic manipulation in the natural producers, but also in heterologous host Streptomyces coelicolor after expression of the respective gene cluster. Mutasynthesis experiments, i. e. generation of a cloQ-defective mutant of the clorobiocin producer and feeding of 13 different structural analogs of 3-dimethylallyl-4-hydroxybenzoic acid to this mutant, allowed the isolation of 32 new aminocoumarins. These compounds contained, instead of the genuine 3-dimethylallyl-4-hydroxybenzoyl moiety, the externally added analogs as the acyl components in their structures. Production of new aminocoumarins was also achieved by chemoenzymatic synthesis in vitro. Several biosynthetic enzymes have been heterologously expressed, purified und used for chemoenzymatic synthesis. The structures of the new aminocoumarins were elucidated by NMR and mass spectroscopy. Their inhibitory activity on gyrase in vitro as well as their antibacterial activity was determined. These results give further insight into the structure-activity relationships of aminocoumarins.     

Combinatorial biosynthesis, metabolic engineering and mutasynthesis for the generation of new aminocoumarin antibiotics

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A1 are produced by different Streptomyces strains. They are potent inhibitors of bacterial gyrase and topoisomerase IV, and novobiocin has been licensed as antibiotic for clinical use (Albamycin). They also have potential applications in oncology. The biosynthetic gene clusters of all three antibiotics have been cloned and sequenced, and the function of nearly all genes contained therein has been elucidated. Rapid and versatile methods have been developed for the heterologous expression of these biosynthetic gene clusters, and in Streptomyces coelicolor M512 as heterologous host these antibiotics were produced in yields comparable to those in the natural producer strains. lambda RED-mediated homologous recombination was used for genetic modification of the gene clusters in Escherichia coli. The phage PhiC31 attachment site and integrase functions were introduced into the cosmid backbones and employed for stable integration of the clusters into the genome of the heterologous hosts. Modification of the clusters by single or multiple gene replacements or gene deletions resulted in the formation of numerous new aminocoumarin derivatives, providing an efficient tool for the rational generation of antibiotics with modified structure. Additionally, many new antibiotics were generated by mutasynthesis experiments, i.e. the targeted deletion of genes required for the biosynthesis of a certain structural moiety of the antibiotic, and the replacement of this moiety by structural analogs which were added to the culture broth. The diversity of new structures obtained by this approach could be expanded by further genetic modifications of the gene deletion mutants, especially by expression of heterologous biosynthetic enzymes with appropriate substrate specificity.     

重症监护病房中革兰阴性菌耐药机制及抗菌药物应用对策

革兰阴性菌(G^-菌)主要耐药机制有以下三方面：使药物失活、阻止药物到达作用靶、药物作用靶位改变。G菌感染用药对策包括：尽量减少抗生素不适当应用，尽早应用有针对性的敏感抗生素。本通过分析重症监护病房中G菌耐药机制及抗菌药物应用对策，为临床合理用药提供参考。     

Structural and mechanistic aspects of evolution of beta-lactamases and penicillin-binding proteins.

Penicillin-binding proteins (PBPs) and b-lactamases are related enzymes, the former are the targets for b-lactam antibiotics and the latter are resistance enzyme to these antibiotics. The two families of enzymes share structural topologies and certain mechanistic features. However, these classes of enzymes have diversified substantially and have broadened the reaction repertoire for their catalytic properties. This report addresses the issues of the evolution of function of these proteins.     

Structure-activity relationships in the beta-lactam family: an impossible dream

The difficulty of establishing structure-activity relationships in the beta-lactam family of antibiotics stems from the fact that: (1) The targets in various bacteria exhibit widely different sensitivities. (2) Some bacteria produce beta-lactamases, enzymes capable of destroying the antibiotics. The rates of the reactions with the beta-lactamases and the target enzymes are not necessarily related. (3) In Gram-negative bacteria, the diffusion rate through the outer membrane varies independently from the two other factors.     

Recent developments in carbapenems

Carbapenems are β-lactam antibiotics characterised by the presence of a β-lactam ring with a carbon instead of sulfone in the 4-position of the thyazolidinic moiety. The first carbapenem to be utilised in therapy was imipenem, the N-formimidoyl derivative of thienamycin. Imipenem is coadministered with cilastatin, an inhibitor of human renal dehydropeptidase I, as imipenem is hydrolysed by this enzyme. Meropenem was the first carbapenem with a 1-β-methyl group and 2-thio pyrrolidinyl moiety, which renders this antibiotic stable to renal dehydropeptidase I. Other carbapenems for parenteral administration later discovered include biapenem, panipenem, ertapenem, lenapenem, E-1010, S-4661 and BMS-181139. Carbapenems which are orally administered include sanfetrinem, DZ-2640, CS-834 and GV-129606. Carbapenems have an ultra-broad spectrum of antibacterial activity and stability to almost all clinically relevant β-lactamases. This differentiates them from all other currently available classes of β-lactam antibiotics. However, Class B β-lactamases, along with some rare Class A and D enzymes, are able to hydrolyse these antibiotics. Although Class B enzymes are generally chromosomally-encoded (isolated from Stenotrophomonas maltophilia, Aeromonas spp., Bacillus cereus, Bacteroides fragilis, Flavobacterium spp. and Legionella gormanii), plasmid-metallo-β-lactamases now are appearing in B. fragilis, Pseudomonas aeruginosa, Acinetobacter baumannii and members of Enterobacteriaceae such as Serratia marcescens and Klebsiella pneumoniae. The number of these enzymes compared to the number of other β-lactamase types is still low, however, it is likely that they will spread due to the increased selective pressure of carbapenem use. The very broad spectrum of antimicrobial activity associated with a good clinical efficacy and a favourable safety profile makes the carbapenems valuable as ‘first-line’ antibiotics in initial empirical therapy for the treatment of severe infections.