鲍曼不动杆菌耐药性及碳青霉烯酶检测

目的了解临床分离鲍曼不动杆菌对临床常用抗菌药物的耐药性及其产碳青霉烯酶情况,为临床抗感染治疗提供依据。方法采用MicroScan WalkAway-40全自动微生物分析仪对240株临床分离的鲍曼不动杆菌进行鉴定和药敏试验,改良的Hodge试验检测耐碳青霉烯类鲍曼不动杆菌产生的碳青霉烯酶,并采用SPSS 13.0软件进行统计分析。结果 240株鲍曼不动杆菌对亚胺培南的耐药率最低(20.8%),对青霉素类、头孢菌素类、喹诺酮类、复方新诺明的耐药率均大于50%;耐碳青霉烯类鲍曼不动杆菌碳青霉烯酶的检出率为64.0%。结论临床分离鲍曼不动杆菌耐药情况严重,碳青霉烯酶是鲍曼不动杆菌对碳青霉烯类抗菌药物耐药的主要原因。     

质粒介导碳青霉类抗生素耐药性产生的研究进展

碳青霉烯类抗生素是治疗β-内酰胺酶类耐药革兰氏阴性杆菌感染的最有效手段,也是目前对抗耐药细菌最有效的抗菌药物之一。但随着碳青霉烯类耐药菌的出现,无疑给临床抗菌药物选择带来严峻的考验。临床上发现的碳青霉烯类耐药菌常含碳青霉烯酶,可分为A、B、D三类：A类为肺炎克雷伯菌碳青霉烯酶(Klebsiella pneumoniae carbapenemases,KPCs),B类为是新德里金属酶(new Delhi metalloenzyme,NDM)、亚胺培南酶(imipenemase,IMP)和维罗纳整合子金属β-内酰胺酶(verona integronencoded metallo-β-Lactamases,VIM),D类为是苯唑西林酶(Oxacillinase,OXA)。目前发现编码这些酶的基因大都位于质粒上,使得这些耐药基因能够在细菌之间进行水平或垂直传播。该文将介绍近年编码不同碳青霉烯酶的基因通过质粒的介导在革兰氏阴性杆菌中的分布、表达与传播,以期为科学防控碳青霉烯类耐药菌及合理使用抗菌药物提供理论依据。     

耐碳青霉烯类鲍曼不动杆菌OXA和NDM-1耐药基因的研究

目的检测江苏盛泽医院耐碳青霉烯类抗生素鲍曼不动杆菌的OXA和NDM-1耐药基因,分析耐碳青霉烯类抗菌药物的耐药机制。方法采用改良Hodge试验检测30株耐碳青霉烯类抗生素鲍曼不动杆菌产酶情况;用PCR的方法检测OXA-23、OXA-24、VIM、IMP和NDM-1碳青霉烯酶耐药基因。结果 30株分离菌中25株菌改良Hodge试验阳性,22株携带OXA-23型碳青霉烯酶耐药基因,未扩增出NDM-1碳青霉烯酶耐药基因。结论本院耐碳青霉烯类抗生素鲍曼不动杆菌的耐药机制主要是携带OXA-23型碳青霉烯酶基因。     

肺炎克雷伯菌产碳青霉烯酶特性与耐药趋势研究

目的探讨临床分离的耐碳青霉烯类肺炎克雷伯菌(Klebsiella pneumoniae carbapenem-resistant,CRKP)的耐药机制及耐药趋势,为抗感染治疗提供依据。方法收集2008-2013年分离的CRKP 178株,分别运用VITEK-2 Compact全自动细菌分析仪和K-B纸片法检测菌株对药物的敏感性,改良Hodge试验确定是否产碳青霉烯酶以及EDTA抑制试验确定是否产金属酶,用PCR扩增和基因测序技术检测耐药基因。结果临床分离出肺炎克雷伯菌(Klebsiella pneumonia,Kpn)中CRKP的比例从2008年0.1%升至2013年2.2%,呈明显上升趋势;药敏结果显示,其对碳青霉烯类高度耐药外,对头孢曲松、氨曲南、环丙沙星等多种抗菌药物呈现出多重耐药;改良Hodge试验阳性170株(阳性率为95.5%);EDTA双纸片协同试验阳性1株(阳性率为0.6%)。PCR共检出150株携带KPC-2酶基因(blaKPC-2),阳性率为84.3%(150/178),1株肺炎克雷伯菌同时携带blaKPC-2和blaIMP-4,占0.6%(1/178)。结论台州医院分离的CRKP比例逐年升高,对多种抗菌药物高度耐药,产KPC-2型碳青霉烯酶是分离菌株对碳青霉烯类抗菌药物耐药主要原因。     

阴沟肠杆菌耐碳青霉烯类抗生素的耐药机制研究进展

阴沟肠杆菌是肠杆菌科中常见的院内感染细菌,碳青霉烯类抗生素由于其抗菌谱广、抗菌力强,成为治疗产ESBLs和AmpC酶革兰阴性杆菌感染的有效抗菌药物.但随着碳青霉烯类抗生素的广泛应用,临床上出现很多耐碳青霉烯类抗生素的阴沟肠杆菌(carbapenem-resistant Enterobacter cloacae,CREL),本研究就其耐药机制,从产碳青霉烯酶和非产碳青霉烯酶两方面做一综述.     

碳青霉烯类耐药大肠埃希菌耐药机制研究

目的研究碳青霉烯类耐药大肠埃希菌的的耐药机制。方法收集本院2011年8月至2012年8月的碳青霉烯类耐药大肠埃希菌,采用琼脂稀释法进行药敏试验;改良Hodge试验筛查菌株是否产碳青霉烯酶;利用聚合酶链反应扩增KPC、IMP、VIM、NDM、SHV、TEM、CTX-M-1组、CTX-M-9组耐药基因,并对扩增产物进行测序。结果 21株实验菌均为多重耐药菌,对17种抗菌药物中耐药率60%的有11种,其中耐药率90%的有5种,分别为头孢他啶、头孢噻肟、头孢西丁、环丙沙星和氨曲南。耐药率最低的为多粘菌素B,均表现为敏感。9株改良Hodge试验阳性。6株携带碳青霉烯类耐药基因(3株NDM-1阳性、3株IMP阳性)。共有18株检出β-内酰胺类耐药基因。结论该院碳青霉烯类耐药大肠埃希菌携带的碳青霉烯酶基因以NDM-1和IMP基因较常见。     

耐碳青霉烯类肠杆菌科细菌耐药基因传播机制研究现状

耐碳青霉烯类肠杆菌科细菌(Carbapenem-resistant enterobacteriaceae,CRE)在临床中的分离率越来越高,在抗菌药物的选择性压力下,碳青霉烯酶耐药基因可位于不同的移动元件,在不同种属和同种属细菌之间播散,给临床抗感染治疗带来了极大的困难。对碳青霉烯酶耐药基因可移动元件进行了简要综述,旨在阐明CRE耐药基因传播机制,为CRE感染的预防、治疗及感染的控制提供了参考依据。     

奇异变形杆菌耐药性的4年监测及碳青霉烯类耐药株的耐药机制研究

目的了解本地区2007年到2010年奇异变形杆菌的临床分布与常用抗菌药物的耐药情况,了解碳青霉烯类耐药菌株可能存在的机制。方法回顾分析2007年到2010年临床分离奇异变形杆菌的资料及整体耐药情况;对保存的耐亚胺培南（IPM）、美罗培南（MEM）或厄他培南（ETP）的菌株进行复苏,并做Hoage试验进行产碳青霉烯酶的确认,同时对试验菌株进行耐药基因的PCR扩增检测。结果2007年到2010年,奇异变形杆菌在临床各送检样本中以痰液分离率最高：51．1％、34．4％、22．1％和35．4％,其次为尿液：14．3％、28．O％、34．9％和33．6％;耐药监测分析显示,4年间对喹诺酮类、青霉素类、头孢菌素类及氨基糖苷类耐药率相对较高且较为稳定;对碳青霉烯类耐药最低但增加明显,亚胺培南从2007年的1．8％升到2010年的16．1％,美罗培南从2007年的1．7％升到2010年的16．8％。15株耐碳青霉烯类菌株中,Hoage试验阳性7株,6fn。基因阳性11株,blaCTX-M基因阳性13株。结论本地区奇异变形杆菌对临床常用的抗菌药物均有较高的耐药性,对碳青霉烯类药物的耐药率最低,但增加明显。位于质粒上的blaKPc基因所产生的碳青霉烯酶和6如cTx-M基因所产生的超广谱β-内酰胺酶是本菌对β-内酰胺类抗菌药物耐药的主要原因,临床应引起高度重视。     

多重耐药鲍曼不动杆菌同源性分析

目的研究台州医院中心重症监护病房（ICU）及脑外重症监护病房（SICU）2006年1月出现的多重耐药鲍曼不动杆菌（MDR-AB）的耐药性、碳青霉烯酶基因型及同源性。方法运用VITEK-60全自动微生物仪对分离自2006年1月ICU和SICU患者多重耐药鲍曼不动杆菌8株进行菌种的重新鉴定,采用微量板稀释法测定β-内酰胺类、氨基糖苷类、氟喹喏酮类等17种抗菌药物的体外最低抑菌浓度（MIC）（其中头孢哌酮／舒巴坦、美洛配能采用K-B法测定耐药性）。采用PCR对8株菌株进行OXA型碳青霉烯酶基因型检测,运用脉冲场凝胶电泳分析菌株的同源性。结果8株菌株仅对头孢哌酮／舒巴坦敏感,对头孢菌素类、氨基糖苷类、氟喹喏酮类和碳青霉烯类等抗菌药物均显示出较高水平的耐药;8株菌株均产OXA-23型碳青霉烯酶;脉冲场凝胶电泳证实其为同一克隆。结论本组鲍曼不动杆菌为多重耐药株,同一克隆株在不同感染个体的相互传播,导致了这次院内感染的流行。     

头孢地尔:新型铁载体头孢菌素抗多重耐药革兰阴性杆菌感染北大核心CSCD

抗菌药物耐药是目前全世界面临的重要公共卫生问题之一,亟需开发有效的广谱抗菌药物以应对多重耐药革兰阴性杆菌的感染。头孢地尔是日本Shionogi公司开发的新型铁载体头孢菌素类抗菌药物。该药物对碳青霉烯耐药的肠杆菌目细菌(carbapenem resistant Enterobacterales,CRE)、铜绿假单胞菌、鲍曼不动杆菌和嗜麦芽窄食单胞菌等具有广谱强效的抗菌活性。该药物克服了革兰阴性杆菌因外膜孔道蛋白表达量下调和主动外排泵表达量上调产生的耐药性,并对丝氨酸酶和金属碳青霉烯酶具有较好的稳定性。该药可用于治疗由革兰阴性杆菌引起的复杂性尿路感染(包括肾盂肾炎)、院内获得性肺炎和呼吸机相关性肺炎。文中通过汇总头孢地尔的化学结构、抗菌作用机制、体外抗菌活性、药代动力学、药效学和临床治疗等信息,展现头孢地尔作为新型铁载体头孢菌素在治疗多重耐药革兰阴性杆菌感染中的应用前景。     

Antimicrobial susceptibility profile of Acinetobacter species isolated from blood cultures in two Japanese university hospitals

Carbapenem‐resistant Acinetobacter baumannii has rapidly spread worldwide. This study investigated antibiotic susceptibility and genotypic resistance of 123 consecutive blood culture isolates of Acinetobacter species collected between 2003 and 2011 in two Japanese hospitals. The isolates were assigned to 13 species. Carbapenem resistance was detected in four isolates. Only one A. baumannii isolate had bla_OXA‐23 together with ISAba1; the remaining three isolates had IMP‐1 metallo‐β‐lactamase. Quinolone resistance was detected in five isolates that had point mutations in the quinolone resistance‐determining region. The predominance of various non‐A. baumannii species and low prevalence of carbapenem resistance among blood culture isolates of Acinetobacter species in two Japanese hospitals were confirmed.     

摩根摩根菌β-内酰胺酶检测及耐药性分析

目的了解摩根摩根菌临床分离株产超广谱β-内酰胺酶（ESBLs）、头孢菌素酶（AmpC）、金属酶（MBLs）、碳青霉烯酶（KPC）情况,并分析其对17种常见抗菌药物的耐药性。方法 ESBLs和AmpC及MBLs采用三维试验检测,碳青霉烯酶采用改良Hodge试验进行检测,并以K-B法测定17种常见抗菌药物的耐药性。结果 102株摩根摩根菌单产ESBLs 15株,检出率为14.71%;单产AmpC 8株,检出率为7.84%;单产金属β-内酰胺酶（MBLs）3株,检出率为2.94%;所有菌株中未检出碳青霉烯酶（KPC）;同产ESBLs及AmpC 6株,检出率为5.88%;未发现其他双产酶菌株。摩根摩根菌非产酶分离株对17种抗生素的耐药率均低于50.0%;摩根摩根菌产酶分离株对亚胺培南、美罗培南培南的耐药率低于15.0%,与非产酶菌株相比,差异无统计学意义（P〉0.05）;对其余抗生素的耐药率均明显高于非产酶菌株（P〈0.05）。同产ESBLs＋AmpC与耐亚胺培南摩根摩根菌分离株呈多重耐药。结论我院摩根摩根菌分离株产生多种β-内酰胺酶,且对常用抗生素耐药性比较严重,建议临床医师合理使用抗生素,以免耐药菌株的产生。     

Class III β-tubulin and the cytoskeletal gateway for drug resistance in ovarian cancer

The Class III β-tubulin isotype (βIII-tubulin) is a predictive biomarker in ovarian cancer and other solid tumor malignancies. We discovered that βIII-tubulin function is linked to two GTPases: guanylate-binding protein 1 (GBP1), which activates its function, and GNAI1, which inhibits it. This finding was demonstrated in a panel of ovarian cancer cells resistant to several chemotherapeutic agents. Using a protein microarray, we identified PIM1 as the downstream partner of GBP1, recruited into the cytoskeleton under hypoxic conditions. The clinical value of these observations was tested by performing an archive study of 98 ovarian cancer patients, which demonstrated that the βIII-tubulin -/PIM1- cohort responded to treatment, exhibiting long overall survival (OS), while βIII-tubulin +/PIM+ patients experienced poor outcomes and OS times similar to patients receiving palliation alone. βIII-tubulin expression is commonly believed responsible for paclitaxel resistance due to its enhancement of the dynamic instability of microtubules, which counteracts the activity of taxanes. In contrast, our research reveals that βIII-tubulin behaves as a gateway for prosurvival signals, such as PIM1, to move into the cytoskeleton. When cells are exposed to microenvironmental stressors, they activate this pathway by telling the cytoskeleton to incorporate PIM1 through GBP1 and βIII-tubulin, which ultimately leads to drug resistance. This discovery reveals that βIII-tubulin does not act alone but requires partners to play its role. The discovery of such protein:protein interactions underlying this prosurvival cascade makes feasible the development of therapeutic approaches using novel compounds that are capable of inhibiting the transmission of prosurvival signals into the cytoskeleton.     

β‐1,3‐Glucanases in wheat and resistance to the Russian wheat aphid

The Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko) is one of the most destructive insect pests of cereals world-wide. Although resistant cultivars have been bred, the biochemical mechanism of resistance is unknown. The aim of this work was to gain information on the mechanism of resistance which could contribute to more directed breeding of resistant cultivars in the future. The effect of RWA infestation on the inter- and intracellular β-1,3-glucanase activities was studied in different resistant wheat (Triticum aestivum L.) cultivars containing the Dn-1 gene for RWA resistance and corresponding near-isogenic susceptible cultivars. The activity was determined spectrophotometrically by measuring the release of glucose from laminarin. Infestation differentially induced the intra- and intercellular activities to much higher levels in resistant than susceptible cultivars within 48 h. According to immunological studies induced enzyme activities were due to increased protein levels. The intracellular β-1,3-glucanase contained about 8% exo-activity. The exo-activity made an insignificant contribution to the intercellular activity. The genetic background into which the resistance gene was bred did affect the level of activity that corresponded to the resistance performance. Seven apoplastic isoforms of β-1,3-glucanase, varying from acidic to basic, were resolved by isoelectric focusing. All isoenzymes were equally induced and no specific one could be linked to resistance or susceptibility. The RWA induced β-1,3-glucanase activity in resistant cultivars closely resembles defence responses during pathogenesis and seems to be part of a general defence response like the hypersensitive reaction (HR), which confers resistance to the RWA. This knowledge might be helpful in future to identify genes for RWA resistance. The increased β-1,3-glucanase activity after RWA infestation might serve as an additional measure to biochemically trace resistance in crosses during breeding.     

肺炎克雷伯菌碳青霉烯酶检测方法的研究进展

碳青霉烯类抗生素是治疗产超广谱β-内酰胺酶以及高产头孢菌素酶肠杆菌科细菌感染最有效的药物.但近年来,出现越来越多对其耐药的细菌,其中肺炎克雷伯菌碳青霉烯酶(Klebsilla pneumoniae carbapenemase,KPC)是引起肠杆菌科细菌对碳青霉烯类耐药的主要机制,目前产KPC细菌呈世界范围流行趋势,且其感染死亡率高,故需快速、灵敏的检测方法,以提高感染患者治愈率.     

178 Targeting “Beta lactamase-C” in development of novel anti-tuberculosis therapeutics

Tuberculosis is a common, and in many cases lethal, infectious disease caused by various strains of Mycobacterium, usually Mycobacterium tuberculosis. (Kumar et al., 2007) In addition, co-infection with Mycobacterium tuberculosis and HIV (TB/HIV), especially in Africa, and multidrug resistant and extensively drug-resistant tuberculosis in all regions, (WHO, 2010) makes it important to develop novel therapeutics against this bacterium. Penicillin like β-Lactam antibiotics are among the most clinically prescribed drugs for anti-bacterial therapeutics. The general mechanism of action involved the inhibition of enzyme d,d-transpeptidases, which takes part in the biosynthesis of the bacterial cell wall (Heesemann, 1993). A major strategy of bacterial resistance to β-lactams is the production of β-lactamases that catalyze the hydrolysis of β-lactams, leading to the inactivation of the antibiotics. β-Lactams have not been used in clinical practice to treat TB infections, because an active penicillinase was reported in M. tuberculosis (Lessel, 1996). BlaC is a class A β-lactamase that contains a nucleophilic serine residue (Ser70) and shares sequence homology with the penicillin-binding protein domain of the ancestral d,d-transpeptidases. Recent studies including our lab show that β-lactam drugs like Clavulanate, Carbapenem, and Meropenem are used primarily against this type of resistant bacteria (Hugonnet et al., 2009). β-lactamase induces the same acetylating reaction with all of these drugs but cannot induce deacetylation. As a result, those drugs remain attached with β-lactamase even after the distortion of their β-lactam ring. At this time, secondary treatment has been done by applying previously used potent penicillin like β-lactam drugs with this primarily treated β-lactamase. In current study, we conducted kinetic and mass spectrometric analysis of different BlaC inhibitors, like NXL104 (Xu et al., 2012) and showed that how they quantitatively inactivates BlaC by forming a carbamyl linkage with the enzyme. In addition, we determined the three-dimensional structures of the different reactive forms of these drugs for better understanding the undergoing mechanisms involved in this inhibition process. Based on our understanding, we are trying to develop novel small molecules with better inhibitory process.     

miR-335-5p induces insulin resistance and pancreatic islet β-cell secretion in gestational diabetes mellitus mice through VASH1-mediated TGF-β signaling pathway

Multiple studies have reported different methods in treating gestational diabetes mellitus (GDM); however, the relationship between miR-335-5p and GDM still remains unclear. Here, this study explores the effect of miR-335-5p on insulin resistance and pancreatic islet β-cell secretion via activation of the TGFβ signaling pathway by downregulating VASH1 expression in GDM mice. The GDM mouse model was established and mainly treated with miR-335-5p mimic, miR-335-5p inhibitor, si-VASH1, and miR-335-5p inhibitor + si-VASH1. Oral glucose tolerance test (OGTT) was conducted to detect fasting blood glucose (FBG) fasting insulin (FINS). The OGTT was also used to calculate a homeostasis model assessment of insulin resistance (HOMA-IR). A hyperglycemic clamp was performed to measure the glucose infusion rate (GIR), which estimated β-cell function. Expressions of miR-335-5p, VASH1, TGF-β1, and c-Myc in pancreatic islet β-cells were determined by RT-qPCR, western blot analysis, and insulin release by ELISA. The miR-335-5p mimic and si-VASH1 groups showed elevated blood glucose levels, glucose area under the curve (GAUC), and HOMA-IR, but a reduced GIR and positive expression of VASH1. Overexpression of miR-335-5p and inhibition of VASH1 contributed to activated TGFβ1 pathway, higher c-Myc, and lower VASH1 expressions, in addition to downregulated insulin and insulin release levels. These findings provided evidence that miR-335-5p enhanced insulin resistance and suppressed pancreatic islet β-cell secretion by inhibiting VASH1, eventually activating the TGF-β pathway in GDM mice, which provides more clinical insight on the GDM treatment.     

β1 integrins mediate resistance to ionizing radiation in vivo by inhibiting c‐Jun amino terminal kinase 1

This study was carried out to dissect the mechanism by which β₁ integrins promote resistance to radiation. For this purpose, we conditionally ablated β₁ integrins in the prostatic epithelium of transgenic adenocarcinoma of mouse prostate (TRAMP) mice. The ability of β₁ to promote resistance to radiation was also analyzed by using an inhibitory antibody to β₁, AIIB2, in a xenograft model. The role of β₁ integrins and of a β₁ downstream target, c‐Jun amino‐terminal kinase 1 (JNK1), in regulating radiation‐induced apoptosis in vivo and in vitro was studied. We show that β₁ integrins promote prostate cancer (PrCa) progression and resistance to radiation in vivo. Mechanistically, β₁ integrins are shown here to suppress activation of JNK1 and, consequently apoptosis, in response to irradiation. Downregulation of JNK1 is necessary to preserve the effect of β₁ on resistance to radiation in vitro and in vivo. Finally, given the established crosstalk between β₁ integrins and type1 insulin‐like growth factor receptor (IGF‐IR), we analyzed the ability of IGF‐IR to modulate β₁ integrin levels. We report that IGF‐IR regulates the expression of β₁ integrins, which in turn confer resistance to radiation in PrCa cells. In conclusion, this study demonstrates that β₁ integrins mediate resistance to ionizing radiation through inhibition of JNK1 activation. J. Cell. Physiol. 228: 1601–1609, 2013. © 2013 Wiley Periodicals, Inc.     

Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of β-lactamase

In order to understand how TEM-1 β-lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions. The 161–170 region includes a portion of an omega loop structure, which is involved in the formation of the active-site pocket. The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended-spectrum cephalosporin ceftazidime was determined. It was found that the sequence requirements for wild-type levels of ampicillin resistance are much more stringent than the sequence requirements for ceftazidime resistance. Surprisingly, more than 50% of all amino acid substitutions in the 161-170 region result in levels of ceftazidime resistance at least three times greater than wild type. In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100-fold increase in ceftazidime resistance were identified. Characterization of altered β-lactamase enzymes indicated that while their catalytic efficiency (K_cat/K_m) for ceftazidime hydrolysis is higher, the enzymes are poorly expressed relative to wild-type TEM-1 β-lactamase.