新型超级细菌NDM-1可能全球蔓延

<正>抗生素由某些微生物在生活过程中产生的,对某些其它病原微生物具有抑制或杀灭作用的一类化学物质。它也是人类抵御细菌感染类疾病的主要武器。但最近研究人员已经发现一种名为新德里金属β内酰胺     

产NDM-1肠杆菌科细菌的研究进展

产新德里金属β-内酰胺酶-1(New delhimetallol-β-lactamase 1,NDM-1)肠杆菌科细菌对碳青霉烯类抗生素耐药,只对多黏菌素和替加环素敏感,接近于泛耐药菌株。此酶的blaNDM-1基因在肠杆菌科细菌间高效率的转移和人为因素(如旅游、卫生和食品的生产和制备)的影响,使其在全球迅速传播。目前有关其治疗的临床研究较少,为减缓NDM-1肠杆菌科细菌的传播,应加速开发诊断和治疗的相应措施。现就产NDM-1肠杆菌科细菌的分子生物学特点、诊断、治疗、新药研发和预防作一综述。     

NDM-1结构功能及抑制剂的抑制机理

在过去的10年中,以新德里金属β-内酰胺酶-1(NDM-1)为代表的金属β-内酰胺酶在全球范围内广泛传播,对公共卫生安全产生了较大的威胁.尤其是近些年这些酶的突变体的出现使得耐药菌给人类健康造成了更加复杂和困难的挑战.目前,临床上仍然缺乏有效的治疗药物和手段.研发有效广谱的抑制剂成为解决此问题的重点.因此本文将针对ND...     

NDM-1的同源建模及其模型活性位点分析

近期研究发现的超级病原菌耐药的原因,是其含有一种特殊的金属内酰胺酶—NDM-1。采用计算生物学技术,通过分子建模、使NDM-1与其它金属β-内酰胺酶类相比对,探索NDM-1对于β-内酰胺类抗生素具有广谱的水解能力的分子机理,为相关新药的开发提供理论依据。将NDM-1序列用BLAST进行同源性搜索,挑选一些相似性较高的序列进行多序列比对的同源建模,对所得的模型进行评估。根据已知的VIM-2晶体结构,使计算的NDM-1模型活性位点与已知VIM-2金属酶结构进行比较分析;结果发现NDM-1在锌离子结合位点的几个关键的氨基酸残基,与VIM-2金属酶较为相似。同时活性位点附近的氨基酸残基的立体折叠结构也与VIM-2存在相似性。NDM-1水解谱的广泛性,可能在于活性位点附近一些氨基酸残基的差异,后者可通过改变空间结构,从而增加了NDM-1的水解活性。     

Mechanistic and spectroscopic studies of metallo-β-lactamase NDM-1

In an effort to biochemically characterize metallo-β-lactamase NDM-1, we cloned, overexpressed, purified, and characterized several maltose binding protein (MBP)-NDM-1 fusion proteins with different N-termini (full-length, Δ6, Δ21, and Δ36). All MBP-NDM-1 fusion proteins were soluble; however, only one, MBP-NDM-1Δ36, exhibited high activity and bound 2 equiv of Zn(II). Thrombin cleavage of this fusion protein resulted in the truncated NDM-1Δ36 variant, which exhibited a k(cat) of 16 s(-1) and a K(m) of 1.1 μM when using nitrocefin as a substrate, bound 2 equiv of Zn(II), and was monomeric in solution. Extended X-ray absorption fine structure studies of the NDM-1Δ36 variant indicate the average metal binding site for Zn(II) in this variant consists of four N/O donors (two of which are histidines) and 0.5 sulfur donor per zinc, with a Zn-Zn distance of 3.38 ?. This metal binding site is very similar to those of other metallo-β-lactamases that belong to the B1 subclass. Pre-steady-state kinetic studies using nitrocefin and chromacef and the NDM-1Δ36 variant indicate that the enzyme utilizes a kinetic mechanism similar to that used by metallo-β-lactamases L1 and CcrA, in which a reactive nitrogen anion is stabilized and its protonation is rate-limiting. While they are very different in terms of amino acid sequence, these studies demonstrate that NDM-1 is structurally and mechanistically very similar to metallo-β-lactamase CcrA.     

NDM-1对阴沟肠杆菌的致病性及适应性的影响

产生新德里金属β-内酰胺酶-1 (New delhi metallol-β-lactamase 1,NDM-1)的肠杆菌科细菌对大部分β-内酰胺抗生素耐药,其导致的感染难以治疗。细菌中编码NDM-1基因的获得可能会影响它的致病性和适应性,这可能使产NDM-1的细菌的感染治疗和传播更加复杂。产NDM-1阴沟肠杆菌流行率高,现对阴沟肠杆菌的致病过程以及NDM-1对其致病性和适应性的影响作一概述,为产NDM-1阴沟肠杆菌的感染治疗和传播控制提供应对策略。     

产NDM-5大肠埃希菌的鉴定及特征分析

对分离自血液标本的1株碳青霉烯类耐药大肠埃希菌(Escherichia coli)SCNJ06进行特征分析,以期为临床耐药菌株感染的防治提供理论参考。采用全基因组测序以及生物信息学分析,该菌株属于序列型167(ST167),含有11种耐药基因,分别是rmtB、aph(3″)-Ib、aph(6)-Id、bla_(NDM-5)、bla_(TEM-1B)、bla_(CTX-M-55)、fosA3、floR、sul2、tet(A)和mdf(A)。其中,bla_(NDM-5)位于IncX3型质粒pNDM5_SCNJ06上,mdf(A)位于染色体上,其余耐药基因位于IncFII型质粒prmtB_SCNJ06上。接合试验显示,pNDM5_SCNJ06和prmtB_SCNJ06均能够发生接合转移。应加强抗菌药物临床应用管理和医院感染防控措施,重视细菌耐药监测工作。     

细菌间bla_(NDM-1)传播规律的研究进展

新德里金属β-内酰胺酶-1 (New Delhi metallo-β-lactamase 1,NDM-1)导致革兰氏阴性细菌对几乎所有的β-内酰胺类抗生素(β-lactam antibiotic)产生抗性。携带NDM-1编码基因(bla_(NDM-1))的肠杆菌科(Enterobacteriaceae)和不动杆菌属(Acinetobacterspp.)细菌在世界各地广泛流行,严重威胁着公共健康。现对NDM-1的结构、突变亚型和传播规律进行综述,为预防和控制NDM-1携带菌提供策略。     

泛耐药基因NDM-1在大肠杆菌中的克隆表达及耐药性检测

目的:构建bla(NDM-1)基因重组质粒,表达新德里金属β内酰胺酶1(NDM-1),并检测携带bla(NDM-1)基因重组质粒的大肠杆菌的耐药状况。方法:PCR扩增编码NDM-1的基因bla(NDM-1),构建表达载体pGEX4T-1-NDM-1,并转化至大肠杆菌,转化子经PCR后测序,以确认构建和转化成功;用Western印迹验证重组蛋白的表达;用药敏纸片法检测含重组质粒pGEX4T-1-NDM-1的大肠杆菌的耐药谱;用E-test法测定其最低抑菌浓度(MIC)。结果:PCR及测序结果显示载体构建和转化成功;含重组质粒pGEX4T-1-NDM-1的大肠杆菌在37℃时,经1 mmol/LIPTG诱导5 h后,SDS-PAGE可见目的条带;除对替加环素和粘菌素敏感外,该重组子对多种碳青霉烯类抗生素耐药,E-test检测其对亚胺培南的MIC为64μg/mL。结论:构建了含泛耐药基因bla(NDM-1)的重组质粒,转入大肠杆菌后表达了融合蛋白,并对多种碳青霉烯类抗生素耐药。为进一步研究bla(NDM-1)基因和蛋白的功能奠定了基础。     

人类与病原菌的军备竞赛:NDM-1耐药基因与超级细菌

在人类历史上,每一次诸如鼠疫和肺结核病等瘟疫的大流行,都曾给人类的生存带来巨大的威胁。抗生素的应用使人类掌握了抵抗细菌感染的锐利"武器",但同时病原菌也通过突变和水平基因转移等方式产生了诸多耐药基因,从而获得了应对抗生素杀伤的坚固"盾牌";于是人类又不断地开发新式抗生素"武器"来破解病原菌的耐药"盾牌"——一场"军备竞赛"愈演愈烈。近来研究发现,携带编码NDM-1基因的耐药质粒不仅可以在细菌间转移,而且能使所在宿主菌成为可以耐受几乎全部抗生素的超级细菌。但是,凭借着日益进步的科技和医学,以及科学的用药策略,我们一定可以再次战胜超级细菌。     

泛耐药细菌NDM-1基困Taqman PCR快速检测方法的建立

产NDM-1(New Delhi Metallo-β-lactamase 1,Ⅰ型新德里金属β-内酰胺酶)细菌是新近报道的一种泛耐药细菌,由于对绝大多数常用抗生素均耐药,又被称为超级细菌.目的:建立一种可快速检测泛耐药细菌NDM-1基因的Taqman探针实时荧光定量PCR法.方法:根据NDM-1基因序列,设计引物和Ta...     

快速检测NDM-1基因的环介导恒温扩增技术的建立与评价

基于DNA环介导恒温扩增技术 (Loop-mediated isothermal amplification,LAMP),探索建立一种应用于NDM-1基因 (New Metallo-β-Lactamase-1 Gene,NDM-1) 的快速检测方法,以适应临床实验室等的检测需求。利用LAMP技术,以NDM-1基因为靶序列,设计4组LAMP引物,并筛选最优引物组,建立LAMP反应体系与条件,进行灵敏度和特异性实验。结果表明整个检测过程仅需1 h,即可通过肉眼直接目测实验结果。在灵敏度试验中,NDM-1基因的最低检测限为6 拷贝/反应。在特异性实验中,以4株病原菌 (肺炎克雷伯氏菌、大肠埃希氏菌、金黄色葡萄球菌、肺炎链球菌) 以及肠道菌群元基因组DNA、土壤菌群元基因组DNA为模板对NDM-1基因进行检测,结果显示均没有发生非特异性扩增反应。文中建立的LAMP检测方法能够快速检测NDM-1基因,且可直接观察到实验结果,实现了检测结果的可视化。具有操作简单安全、检测灵敏度高、特异性高的特点,能够满足基层实验室、应急检测或现场监测等方面的使用需求,具有良好的应用价值。     

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。     

Structure of apo- and monometalated forms of NDM-1--a highly potent carbapenem-hydrolyzing metallo-β-lactamase

The New Delhi Metallo-β-lactamase (NDM-1) gene makes multiple pathogenic microorganisms resistant to all known β-lactam antibiotics. The rapid emergence of NDM-1 has been linked to mobile plasmids that move between different strains resulting in world-wide dissemination. Biochemical studies revealed that NDM-1 is capable of efficiently hydrolyzing a wide range of β-lactams, including many carbapenems considered as "last resort" antibiotics. The crystal structures of metal-free apo- and monozinc forms of NDM-1 presented here revealed an enlarged and flexible active site of class B1 metallo-β-lactamase. This site is capable of accommodating many β-lactam substrates by having many of the catalytic residues on flexible loops, which explains the observed extended spectrum activity of this zinc dependent β-lactamase. Indeed, five loops contribute "keg" residues in the active site including side chains involved in metal binding. Loop 1 in particular, shows conformational flexibility, apparently related to the acceptance and positioning of substrates for cleavage by a zinc-activated water molecule.     

Discovery of a novel covalent non-β-lactam inhibitor of the metallo-β-lactamase NDM-1

The inhibition of metallo-β-lactamases (MBL) can prevent the hydrolysis of β-lactam antibiotics and hence is a promising strategy for the treatment of antibiotic resistant infections. In this study, we present a novel reversible covalent inhibitor of the clinically relevant MBL New Delhi metallo-β-lactamase 1 (NDM-1). Electrospray ionization-mass spectrometry (ESI-MS) and single site directed mutagenesis were used to show that the inhibitor forms a covalent bond with Lys224 in the active site of NDM-1. The inhibitor was further characterized using an enzyme inhibition assay, a surface plasmon resonance (SPR) based biosensor assay and covalent docking. The determined inhibition constant (K_I¹) was 580 nM and the inhibition constant for the initial complex (K_I) was 76 μM. To our knowledge, this inhibitor is the first example for a reversible covalent non-β-lactam inhibitor targeting NDM-1 and a promising starting point for the design of potent covalent inhibitors.     

肺炎克雷伯菌CS309耐药基因检测和NDM-1基因的克隆及原核表达

目的 检测产NDM-1肺炎克雷伯菌CS309是否同时携带产IMP、VIM型金属β-内酰胺酶或KPC型碳青霉烯酶的耐药基因,同时构建NDM-1基因原核表达质粒,并在大肠埃希菌中进行表达。方法 采用聚合酶链反应（PCR）扩增IMP、VIM和KPC耐药基因;以产NDM-1肺炎克雷伯菌CS309为DNA模板,PCR扩增NDM-1全长,并将其与pGEM-T克隆载体连接后转化至大肠埃希菌DH5α,继而对阳性克隆进行双酶切,将酶切片段与pET-28a(+)表达载体连接,并转化大肠埃希菌BL21,再用异丙基-β-D-硫代半乳糖苷（IPTG）诱导蛋白表达,并采用SDS-PAGE和Western blot技术验证NDM-1蛋白。结果 经PCR和测序证实,该菌同时携带NDM-1和IMP-4两种金属酶基因,未扩增出VIM、KPC耐药基因。经双酶切和测序证实,原核表达质粒pET-28a(+)-NDM-1构建成功。SDS-PAGE发现,重组菌株经诱导后在28 kDa附近有明显条带,与预期蛋白大小27.9 kDa一致。Western blot表明诱导产生的融合蛋白可与NDM-1抗体特异性结合。结论 肺炎克雷伯菌CS309同时携带NDM-1和IMP-4两种金属酶基因;成功构建了NDM-1基因的原核表达质粒,该质粒在大肠埃希菌BL21中高效融合表达。     

High Prevalence of New Delhi Metallo-β-Lactamase-1 (NDM-1) Producers among Carbapenem-Resistant Enterobacteriaceae in Kuwait

The aim of the study was to determine the prevalence of New Delhi metallo-β lactamase-1 (NDM-1) producing Enterobacteriaceae in Kuwait over a one year period. Consecutive Enterobacteriaceae isolates with reduced susceptibility to carbapenems were collected from four government hospitals in Kuwait from January–December 2014. Their susceptibility to 18 antibiotics was performed by determining the minimum inhibitory concentration. Isolates resistant to carbapenems were tested by PCR for resistant genes. Finger printing of the positive isolates was done by DiversiLab^®. Clinical data of patients harboring NDM-1 positive isolates were analyzed. A total of 764 clinically significant Enterobacteriaceae isolates were studied. Of these, 61 (8%) were carbapenem-resistant. Twenty one out of these 61 (34.4%) were NDM-1-producers. All patients positive for NDM-1-carrying bacteria were hospitalized. About half were females (11/21 [52.3%]), average age was 53.3 years and the majority were Kuwaitis (14/21 [66.6%]). Six patients (28.5%) gave a history of travel or healthcare contact in an endemic area. Mortality rate was relatively high (28.6%). The predominant organism was Klebsiella pneumoniae (14 [66.6%]) followed by E. coli (4 [19%]). All NDM-1-positive isolates were resistant to meropenem, ertapenem, cefotaxime, cefoxitin and ampicillin, while 95.2% were resistant to imipenem, cefepime, and piperacillin-tazobactam. They were multidrug resistant including resistance to tigecycline, but 90% remained susceptible to colistin. About two-thirds of isolates (61.9%) co-produced-extended spectrum β-lactamases. During the study period, an outbreak of NDM-1 positive K. pneumoniae occurred in one hospital involving 3 patients confirmed by DiversiLab^® analysis. In conclusion, NDM-1-producing Enterobacteriaceae is a growing healthcare problem with increasing prevalence in Kuwait, especially in hospitalized patients, leaving few therapeutic options. A high prevalence of NDM-1 necessitates the implementation of strict infection control to prevent the spread of these organisms.     

Identification of New Delhi metallo-β-lactamase gene (NDM-1) from a clinical isolate of Acinetobacter junii in China

New Delhi metallo-β-lactamase-1 (NDM-1) is a novel type of metallo-β-lactamase (MBL) responsible for bacterial resistance to β-lactam antibiotics. Acinetobacter junii was previously shown to possess a MBL phenotype; however, the genes responsible for this phenotype were not identified. In this study, we reported the identification of NDM-1 gene in a clinical isolate of A. junii from a child patient in China, which was resistant to all β-lactams except aztreonam but sensitive to aminoglycosides and quinolones. The cloned NDM-1 gene contained an open reading frame of 813 bp and had a nucleotide sequence 99.9% identical (812/813) to reported NDM-1 genes carried by Acinetobacter baumannii , Enterococcus faecium , Escherichia coli , and Klebsiella pneumoniae . Recombinant NDM-1 protein was successfully expressed in E.?coli BL21, and antibiotic sensitivities of the NDM-1-producing E.?coli were largely similar to the A.?junii 1454 isolate. The findings of this study raise attention to the emergence and spread of NDM-1-carrying bacteria in China.