Molecular characterization of a carbapenem-hydrolyzing beta-lactamase from Chryseobacterium (Flavobacterium) indologenes

Chryseobacterium (Flavobacterium) indologenes 001 clinical strain was resistant to several beta-lactam classes including carbapenems. Shotgun cloning experiments of Sau3AI restricted genomic DNA of C. indologenes 001 into pBKCMV cloning vector followed by transformation into Escherichia coli DH10B gave one recombinant plasmid possessing a 4.2-kb DNA insert. It encoded a pI 7.2 beta-lactamase of 239 amino acids (IND-1) which is a metallo-enzyme with a broad spectrum beta-lactam hydrolysis profile. This class B carbapenem-hydrolyzing beta-lactamase shares the highest identity (43%) with BlaB from C. meningosepticum, thus showing heterogeneity of carbapenem-hydrolyzing beta-lactamases in Chryseobacterium spp.     

Exploring the Role of a Conserved Class A Residue in the {Omega}-Loop of KPC-2 β-Lactamase: A MECHANISM FOR CEFTAZIDIME HYDROLYSIS

Gram-negative bacteria harboring KPC-2, a class A β-lactamase, are resistant to all β-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A β-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32→128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative β-lactamase for more detailed analysis, we observed only a modest 25% increase in k(cat)/K(m) for ceftazidime (0.015→0.019 μm(-1) s(-1)). Employing pre-steady-state kinetics and mass spectrometry, we determined that acylation is rate-limiting for ceftazidime hydrolysis by KPC-2, whereas deacylation is rate-limiting in the R164S variant, leading to accumulation of acyl-enzyme at steady-state. CD spectroscopy revealed that a conformational change occurred in the turnover of ceftazidime by KPC-2, but not the R164S variant, providing evidence for a different form of the enzyme at steady state. Molecular models constructed to explain these findings suggest that ceftazidime adopts a unique conformation, despite preservation of Ω-loop structure. We propose that the R164S substitution in KPC-2 enhances ceftazidime resistance by proceeding through "covalent trapping" of the substrate by a deacylation impaired enzyme with a lower K(m). Future antibiotic design must consider the distinctive behavior of the Ω-loop of KPC-2.     

Structure activity relationship studies on rhodanines and derived enethiol inhibitors of metallo-β-lactamases

Metallo-β-lactamases (MBLs) enable bacterial resistance to almost all classes of β-lactam antibiotics. We report studies on enethiol containing MBL inhibitors, which were prepared by rhodanine hydrolysis. The enethiols inhibit MBLs from different subclasses. Crystallographic analyses reveal that the enethiol sulphur displaces the di-Zn(II) ion bridging ‘hydrolytic’ water. In some, but not all, cases biophysical analyses provide evidence that rhodanine/enethiol inhibition involves formation of a ternary MBL enethiol rhodanine complex. The results demonstrate how low molecular weight active site Zn(II) chelating compounds can inhibit a range of clinically relevant MBLs and provide additional evidence for the potential of rhodanines to be hydrolysed to potent inhibitors of MBL protein fold and, maybe, other metallo-enzymes, perhaps contributing to the complex biological effects of rhodanines. The results imply that any medicinal chemistry studies employing rhodanines (and related scaffolds) as inhibitors should as a matter of course include testing of their hydrolysis products.     

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。     

29株耐亚胺培南和/或美罗培南鲍曼不动杆菌产碳青霉烯酶基因型检测

目的 了解临床分离耐亚胺培南和/或耐美罗培南鲍曼不动杆菌中产碳青霉烯酶的基因型别.方法 采用聚合酶链反应扩增IMP、VIM、OXA型碳青霉烯酶基因并测序.结果 29株对碳青霉烯类耐药的鲍曼不动杆菌中,以产OXA-24型和IMP型酶菌株最多,二者均占51.7％ (15/29).产OXA-24+ IMP型5株、OXA-24+ OXA-51+IMP型4株、VIM型4株、OXA-24+ OXA-58+ IMP型、OXA-23+ OXA-24+ IMP型各2株,OXA-23+IMP型、OXA-51+OXA-24型、OXA-24型、IMP型各1株,8株细菌PCR检测结果为阴性.结论 耐亚胺培南和/或美罗培南鲍曼不动杆菌主要产OXA-24型和IMP型碳青霉烯酶,部分菌株可同时产2种或以上碳青霉烯酶.     

耐碳青霉烯类肺炎克雷伯菌的耐药机制研究

目的了解碳青霉烯类耐药肺炎克雷伯菌及耐药机制。方法对2012-2013年临床分离的耐碳青霉烯类肺炎克雷伯菌共计12株进行分析,药敏采用MIC方法检测,用WHONET 5.6软件进行分析,KPC表型检测采用改良Hodge试验,基因检测采用PCR方法。结果 12株碳青霉烯类耐药肺炎克雷伯菌改良Hodge试验阴性,基因测序为KPC-2型。结论 KPC-2基因是引起本院肺炎克雷伯菌耐药的主要原因。     

Comparison of phenotypic and PCR methods for detection of carbapenemases production by Enterobacteriaceae

Dissemination of carbapenem resistance via Enterobacteriaceae, particularly among Klebsiella pneumoniae and Escherichia coli, is a major public health concern. Rapid methods for determining antimicrobial susceptibility are important to ensure adequate and appropriate use of antimicrobial agents and to limit the spread of these bacteria. In the current study, we compared the rapidity, sensitivity and specificity of traditional methods and molecular-based Xpert Carba-R PCR assay to identify sixty isolates, (26 E. coli and 34 K. pneumoniae). The specificity of MicroScan was 100% while sensitivity to ertapenem (ERT), imipenem (IMI), and meropenem (MER) was 93%, 68.9%, and 55.17%, respectively. For the modified Hodge test, the specificity was 96.77% and sensitivity was 89.65%. Although some results of phenotypic assays matched with the definite PCR identification, some results were misleading. Out of the 29 positive PCR samples, three samples of K. pneumoniae were negative for the MHT and one E. coli sample was MHT positive but negative for the PCR. Nine samples were positive for the PCR but were determined as carbapenem sensitive by MicroScan. While MicroScan and MHT requires several hours and multi-steps to obtain results, Xpert Carba-R PCR assay takes less than an hour. Therefore, we recommend using Gene xpert Carba-R assay for the optimal carbapenemnase detection with reducing material, manpower and cost. Also it is important to know the type of carbapenemase is present.     

碳青霉烯酶在碳青霉烯类耐药肺炎克雷伯杆菌的分布及分子流行病学

目的了解碳青霉烯酶在碳青霉烯类抗生素耐药肺炎克雷伯杆菌的分布及其分子流行病学。方法收集我院2010年1月至2012年12月分离的碳青霉烯类耐药肺炎克雷伯杆菌120株,采用改良Hodge试验及EDTA协同试验进行碳青霉烯酶表型的确认,用PCR方法扩增碳青霉烯酶基因(KPC、IMP、NDM、VIM、OXA-48),经电泳检测扩增产物后纯化测序。通过MLST及ERIC-PCR进行分子流行病学分析。结果120株碳青霉烯类耐药肺炎克雷伯杆菌中,95株CRKP菌株改良Hodge试验阳性,64株菌株EDTA协同试验阳性。经PCR及测序确认,83株主要携带KPC-2型碳青霉烯酶,占69.2%;其次为IMP-4型金属酶,占28.3%(34/120),NDM-1型金属酶15株,占12.5%,未发现VIM和OXA-48碳青霉烯酶。ERICPCR及MLST分型显示出20个基因分型,其中ST395-A型和ST11-B型主要流行于产KPC-2型菌株中,占43.88%;ST263-B型和ST15-C型主要流行于产NDM-1菌株中,占13.27%;而ST11-B型主要分布于产IMP-4和KPC-2菌株中,占24.49%。结论我院碳青霉烯酶耐药肺炎克雷伯杆菌存在多种碳青霉烯酶,主要以KPC-2为主,产不同种类碳青霉烯酶菌株存在不同的流行基因型。     

mCIM试验检测临床产碳青霉烯酶革兰阴性杆菌的应用价值

目的评估改良碳青霉烯灭活试验(mCIM)在检测临床产碳青霉烯酶革兰阴性杆菌中的应用价值。方法采用mCIM、改良Hodge(MHT)及Carba NP试验分别检测106株碳青霉烯酶基因阳性的革兰阴性杆菌(36株肠杆菌科细菌、26株铜绿假单胞菌及44株鲍曼不动杆菌),并比较差异。同时,收集湘雅医院2016年1月-12月临床分离的非重复性耐碳青霉烯类革兰阴性杆菌106株,同期随机选取100株分离的碳青霉烯类敏感菌株作为对照组,mCIM试验检测碳青霉烯酶,PCR检测碳青霉烯酶基因,分析其敏感性及特异性。结果 (1)106株碳青霉烯酶基因阳性菌株,mCIM试验:肠杆菌科细菌的敏感性、特异性为88.9%(32/36),铜绿假单胞菌均为阴性。MHT试验:肠杆菌科细菌的敏感性、特异性为77.8%(28/36),铜绿假单胞菌的敏感性、特异性为69.2%(18/26)。Carba NP试验:肠杆菌科细菌的敏感性、特异性为97.2%(35/36),铜绿假单胞菌的敏感性、特异性为57.7%(15/26)。鲍曼不动杆菌3种方法均为阴性。肠杆菌科细菌中,MHT与Carba NP试验差异有统计学意义(χ~2=6.222,P=0.028),MHT与mCIM试验差异无统计学意义(χ~2=1.600,P=0.343),Carba NP与mCIM试验差异无统计学意义(χ~2=1.934,P=0.357);铜绿假单胞菌中,MHT与Carba NP试验阳性,二者差异无统计学意义(χ~2=0.746,P=0.565)。(2)144株临床分离肺炎克雷伯菌(碳青霉烯类耐药及敏感菌株分别为44株、100株),采用美罗培南和亚胺培南分别做mCIM试验,其敏感性和特异性均为100%,且与PCR的结果一致。结论 mCIM试验在肠杆菌科细菌中敏感性高、特异性强,操作简单,结果易于判断,具有良好的临床应用价值。     

MHT和mCIM试验检测肠杆菌科金属碳青霉烯酶效能的评价

目的 探讨改良Hodge试验（MHT）及改良碳青霉烯灭活试验（mCIM）检测肠杆菌科细菌金属碳青霉烯酶的应用价值。方法 VITEK 2 Compact全自动细菌鉴定及药敏系统进行细菌鉴定和药敏试验,筛选2015-2017年非重复临床分离的碳青霉烯类耐药的肠杆菌科细菌,MHT及mCIM进行产碳青霉烯酶表型确证试验,PCR检测常见的金属碳青霉烯酶IMP-4、IMP-8、VIM-1、VIM-2、NDM基因。比较MHT及mCIM对肠杆菌科金属碳青霉烯酶的检测效能。结果 本实验共收集40株临床分离菌株,MHT阳性36株,阳性率90.0%。mCIM阳性39株,阳性率为97.5%。PCR产物测序Blast比对证实4株为产IMP酶菌株,5株产NDM型菌株,未检测到VIM基因。MHT试验检测IMP酶的灵敏度、特异性、阳性预测值、阴性预测值分别为100.0%、11.1%、11.1%、100.0%,MHT检测NDM酶分别为40.0%、2.9%、5.6%、25.0%。mCIM检测IMP酶的灵敏度、特异性、阳性预测值、阴性预测值分别100.0%、2.8%、10.3%、100.0%,mCIM检测NDM型分别为100.0%、2.9%、12.8%、100.0%。结论 MHT及mCIM检测肠杆菌科细菌产金属碳青霉烯酶具有良好的灵敏度,但特异性偏低,应结合分子生物学方法进行检测,为感染控制提供保障。