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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   
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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   
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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   
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目的:通过焦磷酸测序技术检测产超广谱β-内酰胺酶(ESBLs)铜绿假单胞菌的SHV基因点突变,探讨一种快速、准确的ESBLs基因分型方法。方法:双纸片法确定产ESBLs的铜绿假单胞菌,纸片扩散法(K-B法)进行药敏试验,聚合酶链反应(PCR)法扩增ESBLs的SHV基因片段,用焦磷酸测序法检测16株产ESBLs铜绿假单胞菌的SHV基因35位和43位密码子点突变。结果:焦磷酸测序发现,本地区分离出的16株产ESBLs铜绿假单胞菌有10株扩增出SHV基因片段,且在43位密码子基因没有突变,35位密码子有基因突变,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70%(7/10)。16株产ESBLs的菌株对亚胺培南全部敏感。结论:焦磷酸测序技术可快速检测产ESBLs铜绿假单胞菌的SHV基因点突变,具有准确、快速、实时和高通量等优点,可应用于产ESBLs菌株的ESBLs基因分型。  相似文献   
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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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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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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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