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目的探讨一种快速、准确检测产超广谱β-内酰胺酶(ESBLs)革兰阴性菌的ESBLs基因分型方法。方法双纸片法确定产ESBLs的临床分离菌,聚合酶链反应(PCR)扩增ESBLs的SHV基因片段,用焦磷酸测序技术对29株成都市区临床分离的产ESBLs肺炎克雷伯菌和大肠埃希菌进行SHV基因分型研究,检测SHV基因片段中编码35位氨基酸和编码43位氨基酸位点的基因多态性。同时,采用纸片扩散法进行药物敏感性试验。结果焦磷酸测序发现,本地区分离出的29株产ESBLs临床分离菌有21株扩增出SHV基因片段,且在43位氨基酸密码子均没有多态性,35位密码子有基因多态性,核苷酸由T突变为A,亮氨酸变为谷氨酰胺,突变发生率达到42.9%(9/21)。29株产ESBLs的菌株对亚胺培南全部敏感;对头孢西丁、头孢吡肟、头孢他啶耐药率分别为:大肠埃希菌29.4%、11.8%、41.2%;肺炎克雷伯菌50.0%、8.3%、33.3%;对氨苄西林、哌拉西林、头孢唑啉、头孢呋辛和复方磺胺甲口恶唑的耐药性较高,均达到75%以上,对其他药物均有不同程度的耐药性。结论焦磷酸测序技术可快速对临床分离菌产生的ESBLs耐药基因分型,具有准确、快速、实时和高通量等优点。  相似文献   
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The aim of this study was to characterize by a multi-level approach extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates other than E. coli from Portuguese hospitals. Eighty-eight ESBL-producing clinical isolates (69 Klebsiella pneumoniae, 13 Enterobacter cloacae complex, 3 Klebsiella oxytoca, 1 Enterobacter asburiae, 1 Proteus mirabilis and 1 Serratia marcescens) recovered from hospitals located in the North (A) or Centre (B, C) regions during two time periods (2006–7 and 2010) were analyzed. Standard methods were used for bacterial identification, antibiotic susceptibility testing, ESBL characterization, clonal (PFGE, MLST) and plasmid (S1-PFGE, I-CeuI-PFGE, replicon typing, hybridization) analysis. Isolates produced mostly CTX-M-15 (47%) or SHV-12 (30%), and less frequently other SHV- (15%; SHV-2, -5, -28, -55, -106) or TEM- (9%; TEM-10, -24, -199)-types, with marked local and temporal variations. The increase of CTX-M-15 and diverse SHV ESBL-types observed in Hospital A was associated with the amplification of multidrug-resistant (MDR) K. pneumoniae epidemic clones (ST15, ST147, ST336). SHV-12 and TEM-type ESBLs were mostly identified in diverse isolates of different Enterobacteriaceae species in Hospitals B and C in 2006–7. Particular plasmid types were linked to blaCTX-M-15 (IncR or non-typeable plasmids), blaSHV-12 (IncR or IncHI2), blaSHV-28/-55/-106 (IncFIIK1 or IncFIIK5), blaTEM-10 (IncL/M) or blaTEM-24 (IncA/C), mostly in epidemic clones. In our country, the amplification of CTX-M-15 and diverse SHV-type ESBL among non-E. coli Enterobacteriaceae is linked to international MDR K. pneumoniae clones (ST15, ST147, ST336) and plasmid types (IncR, IncFIIK). Furthermore, we highlight the potential of IncFIIK plasmids (here firstly associated with blaSHV-2/-28/-55/-106) to disseminate as antibiotic resistance plasmids.  相似文献   
4.
The many and diverse β-lactamases produced by bacteria, particularly by Gram-negative pathogens, are increasingly posing a serious threat to the clinical utility of β-lactams. First-generation inhibitors (clavulanic acid, sulbactam, tazobactam) focus on Ambler class A enzymes. However, recent structural upgrades of class A β-lactamases (e.g. TEM, SHV) have extended their spectrum (extended-spectrum β-lactamases and carbapenemases [Sme, NMC-A, IMI-1]) and have brought about the possibility of β-lactamase-inhibitor resistance. Furthermore, the mobilisation and spread of originally chromosomal class C enzymes (CMY, MIR), the growing clinical importance of class B enzymes (IMP, VIM), the emergence of inhibitor-resistant, broad spectrum class D (OXA) enzymes and the co-existence of different classes of β-lactamases in the same pathogen have spurred research toward universal inhibitors. A complicating issue is target accessibility in Gram-negative bacteria, particularly in Enterobacter, Acinetobacter, Pseudomonas, Stenotrophomonas and other organisms, which is necessary in order for the inhibitor to synergise with vulnerable β-lactam antibiotics. Several new, broad-spectrum inhibitors have emerged: cephem sulfones and oxapenems are upgrades of penam sulfones and oxapenams, respectively, with cephem sulfones possibly extending their inhibition to class B metallo-enzymes; and boronates and phosphonates are designed de novo, based on common structural and mechanistic features of serine β-lactamases.  相似文献   
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张哲  许德义  蒋晓飞 《现代实用医学》2009,21(8):799-801,807,F0002
目的建立变性梯度凝胶电泳(DGGE)检测超广谱β内酰歧酶(ESBL)基因中单核苷酸多态性(SNP)的方法,并获得鲍曼不动杆菌中SHV丛因SNP的资料。方法采用DGGE并结合DNA序列测定,检测了24株SHV基闳刚性的鲍曼不动杆菌SHV基因SNP。结果对这些菌株SHV基因的PCR产物直接进行DGGE电泳时,24例标本均呈现出单一条带,但条带的位置明显不同,可分为A、B两组;对两组标本分别用含GC后夹的引物进行SHV基因PCR扩增后再行DGGE,结果显示各产物均为单一条带,而且每组条带均处于同一位置。测序结果表明两组产物分别为SHV-1b.Like、SHV一18-Like。结论目前该地区鲍曼不动杆菌中SHV基因存在两种型别。通过DGGE,不仅能简便、迅速地获得某种基因的SNP资料,而且有利于高效和经济地筛选出新的核苷酸突变。  相似文献   
7.
Extended-spectrum beta-lactamases (ESBLs) are often mediated by (bla-)SHV, (bla)TEM and (bla)CTX-M genes in Enterobacteriaceae and other Gram-negative bacteria. Numerous molecular typing methods, including PCR-based assays, have been developed for their identification. To reduce the number of PCR amplifications needed we have developed a multiplex PCR assay which detects and discriminates between (bla-)SHV, (bla)TEM and (bla)CTX-M PCR amplicons of 747, 445 and 593 bp, respectively. This multiplex PCR assay allowed the identification of (bla-)SHV, (bla)TEM and (bla)CTX-M genes in a series of clinical isolates of Enterobacteriaceae with previously characterised ESBL phenotype. The presence of (bla)SHV, (bla)TEM and (bla)CTX-M genes was confirmed by partial DNA sequence analysis. Apparently, the universal well-established CTX-M primer pair used here to reveal plasmid-encoded (bla)CTX-M genes would also amplify the chromosomally located K-1 enzyme gene in all Klebsiella oxytoca strains included in the study.  相似文献   
8.
目的观察化黏活精方治疗精液黏稠度增高症(SHV)的临床疗效。方法 98例SHV症患者随机分为治疗组和对照组。治疗组口服化黏活精方,对照组则口服维生素C、维生素E及锌硒宝片,4周为一个疗程,共治疗3个疗程。治疗前后采用玻璃棒法检测精液黏稠度,计算机辅助精子分析技术(CASA)分析测定精子密度、a级精子百分率、a+b级精子百分率、精子活率、轨迹速度(VCL)、直线运动速度(VSL)、平均路径速度(VAP);D iff-Qu ik法染色检测畸形精子率。结果治疗前两组间基线特征均衡性良好(P〉0.05),具有可比性。治疗后两组精液黏稠度均有好转,与本组治疗前比较,差异有统计学意义(P〈0.05);且治疗组改善程度明显优于对照组,两组比较,差异有统计学意义(P〈0.05)。治疗后治疗组精液质量、精子畸形率等各项指标均较治疗前有所好转(P〈0.05),与对照组治疗后比较,差异有统计学意义(P〈0.05)。结论化黏活精方治疗精液黏稠度增高症能有效降低患者精液黏稠度,同时对精子的运动参数有不同程度的正向作用。  相似文献   
9.
Background  The extended spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are the major pathogens causing pneumonia and have a significant impact on the clinical course. Limited data exist on molecular characterization of ESBL-producing E. coli and K. pneumoniae that cause pneumonia. The aim of this study was to investigate the comprehensive multilevel characteristics of E. coli and K. pneumoniae causing pneumonia in China for the first time.
Methods  E. coli (17) and K. pneumoniae (21) isolates responsible for pneumonia were isolated from 1270 specimens collected in a prospective multi-center study in eight teaching hospitals in China from June to December in 2007. The susceptibilities, ESBL confirmation, sequence typing, blaCTX-M and blaSHV genes, their genetic environment and plasmid Inc/rep types were determined.
Results  Sixteen E. coli (94.1%) and eleven K. pneumoniae (52.4%) isolates were ESBL producers. About 77.8% and 66.7% of them were resistance to ciprofloxacin and levofloxacin, and 100% were susceptible to imipenem. The most prevalent ESBL gene was CTX-M-14, followed by SHV-2, CTX-M-15, CTX-M-3, CTX-M-65, SHV-12, SHV-26 and SHV-28. SHV-1 and SHV-11 were also detected and coexisted with blaCTX-Ms in five strains, and three strains contained only SHV-1. All CTX-M-14 were detected ISEcp1 upstream and nine were found IS903 downstream and the majority of them (64.3%) were carried by IncF plasmids. All blaSHV were flanked by recF and deoR, located on IncF, IncN, IncX and IncH plasmids. Two SHV-2, one SHV-1 and the only SHV-28 were further preceded by IS26. Genes lacY and lacZ were detected at further upstream of two blaSHV-1. The K. pneumoniae carrying SHV-28 was susceptible to β-lactams, and no mutations or deletions in gene or promoter sequences were identified to account for susceptibility. Multilocus sequence typing experiments showed the ESBL-producing strains were genetically diverse.
Conclusions  The rate of occurrence of blaESBL in E. coli and K. pneumoniae causing pneumonia was high, and blaCTX-M-14 was dominant and probably mobilized by ISEcp1 mainly on IncF plasmids. Importantly, unexpressed blaESBL genes may occur in susceptible isolates and hence may have clinical implications.
  相似文献   
10.
While the main era of β-lactam discovery programs is over, these agents continue to be the most widely prescribed antimicrobials in both community and hospital settings. This has led to considerable β-lactam pressure on pathogens, resulting in a literal explosion of new β-lactamase variants of existing enzyme classes. Recent advances in the molecular tools used to detect and characterize β-lactamases and their genes has, in part, fueled the large increase in communications identifying novel β-lactamases, particularly in Gram-negative bacilli. It now seems clear that the β-lactams themselves have shaped the field of new enzymes, and the evolution of key amino acid substitutions around the active sites of β-lactamases continues to drive resistance. Over 130 variants of TEM β-lactamase now exist, and more are reported in the scientific literature each month. The most disturbing current trend is that many bla structural genes normally limited to the chromosome are now mobilized on plasmids and integrons, broadening the spread of resistance to include carbapenems and cephamycins. Furthermore, in some Enterobacteriaceae, concomitant loss of outer membrane porins act in concert with these transmissible β-lactamase genes to confer resistance to the most potent β-lactams and inhibitor combinations available. Continued reviews of the literature are necessary in order to keep abreast of the ingenuity with which bacteria are changing the current genetic landscape to confer resistance to this important class of antimicrobials.  相似文献   
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