大肠埃希菌耐药性水平传播实验研究

目的研究重症监护病房（ICU）患者标本中分离的大肠埃希菌的耐药情况以及耐药性水平传播的实验研究。方法采取双纸片法（K-B）检测细菌的耐药性;产超广谱β-内酰胺酶（ESBLs）大肠埃希菌为供体菌,耐利福平大肠埃希菌（对其他抗生素敏感）作为受体菌进行接合实验;采用聚合酶链反应（PCR）技术扩增整合子和耐药基因。结果30株大肠埃希菌中产ESBLs菌株检出率为46．7％;接合培养后,接合菌携带23kb和25kb大质粒,而无供体菌中一系列小质粒;供体菌和接合菌均携带I型整合子。结论大肠埃希菌耐药性严重,且呈多重耐药性;产ESBLs菌株可通过质粒和整合子将耐药基因转移给敏感菌,导致耐药性传播。     

产超广谱β-内酰胺酶大肠埃希菌Ⅰ类整合子检测与耐药性关系

目的了解I类整合子在产ESBLs和非产ESBLs大肠埃希菌中分布状况,分析I类整合子在细菌多重耐药中的作用。方法用PCR方法扩增I类整合酶基因,经电泳后检测扩增产物。用2χ检验进行统计学分析,P<0.05为差异有显著性。结果105株大肠埃希菌检出I类整合子46株,检出率为43.8%。I类整合子在产ESBLs菌的检出率为53.4%,明显高于非产ESBLs菌(31.9%),2χ检验,P<0.01。I类整合子阳性菌株多重耐药率为68.8%(33/48),明显高于阴性菌株(33.3%),P<0.05。I类整合子阳性菌株和产ESBLs菌均对青霉素类、喹诺酮类、磺胺类抗生素表现出较高的耐药率。所有菌株均对亚胺培南敏感。结论I类整合子携带与产ESBLs菌株耐药有关,I类整合子阳性菌株对多种抗生素的耐药率大于整合子阴性菌株。     

整合子与多重耐药大肠埃希菌相关性研究

目的探讨整合子在多重耐药大肠埃希菌耐药性中的作用。方法对临床分离的93株多重耐药大肠埃希菌的I、Ⅱ型整合酶基因进行检测,并分析药敏结果。结果多重耐药临床分离株中Ⅰ型整合子阳性率为60.2%。所检出整合子共有3种长度即1000、1600和2000 bp;主要携带aadA和dfrA类基因盒;未检出Ⅱ型整合子。结论整合子形成是细菌产生多重耐药的重要原因。     

多重PCR方法快速检测4种主要致腹泻性大肠埃希菌

肠毒素性大肠埃希菌(ETEC)、肠致病性大肠埃希菌(EPEC)、肠出血性大肠埃希菌(EHEC)和侵袭性大肠埃希菌(EIEC)是引起腹泻的主要大肠埃希菌,威胁着食品安全和人类健康,建立同时检测4种致腹泻性大肠埃希菌的方法具有重要意义。基于ETEC LT肠毒素基因、EPEC bfpA基因、EHECO抗原基因和EIEC侵袭性质粒特异性基因,设计了4对特异性引物,通过对单一PCR反应条件的优化建立了快速检测4种主要致腹泻性大肠埃希菌的多重PCR方法,彼此之间无交叉反应。该多重PCR方法具有良好的特异性和灵敏性,对24株致病菌进行检测,所试4株致腹泻性大肠埃希菌均为PCR阳性,其他菌株则为阴性。实践证明,利用所建立的多重PCR方法对124份肉类、奶类制品及人工污染样品等进行检测,检出15份阳性,与国标(GB4789.6-1994)检测结果相同。结果表明本文建立的多重PCR方法可用于ETEC、EPEC、EHEC和EIEC的单一或混合感染的鉴别诊断及食品安全风险评估,具有良好的实用性。     

ICU病区大肠埃希菌中qnr基因的分布与耐药性的研究

目的调查温州医科大学附属第一医院ICU病区分离的大肠埃希菌基因的分布以及与耐药谱的关系,并初步探讨其在分子流行病学中的作用。方法收集2012年1-9月ICU病区分离的大肠埃希菌76株进行qnr基因检测,并通过DNA直接测序确定;分析qnr基因在ICU病区分离的大肠埃希菌的分布及其与耐药性的关系。结果根据PCR产物片段大小及测序分析,76株大肠埃希菌中共有qm基因阳性菌株46株,阳性率为60. 5% ;对阳性菌株进行DNA测序、BLAST比对,其中25株为qnrB基因,17株为qnrS基因阳性,12株基因阳性,未检测到qwC和qnrD基因。在46株qnr基因阳性菌株中有38株为产ESBL菌株,而在qnr阴性菌株中仅有5株ESBL阳性。结论该院ICU分离大肠埃希菌qnr基因携带严重,呈现出多重耐药性,多伴随呈现为产ESBL菌株。     

产ESBLs大肠埃希菌整合子及其相关基因盒的研究

目的检测产超广谱β-内酰胺酶（ESBLs）大肠埃希菌中整合子的整合酶及插入的相关基因盒情况,分析整合子对细菌耐药性的影响。方法采用K-B琼脂扩散法对45株临床分离的产ESBLs大肠埃希菌进行药敏试验;应用PCR法检测45株产ESBLs大肠埃希菌Ⅰ类、Ⅱ类和Ⅲ类整合子;对Ⅰ类整合子阳性菌进行整合子相关基因盒检测。结果45株菌中有27株（60．0％）含有Ⅰ类整合子,没有检测到Ⅱ类和Ⅲ类整合子阳性菌。在Ⅰ类整合子阳性菌中,有23株携带Ⅰ类整合子相关基因盒（85．2％）,5种不同的基因盒图谱,片段大小在600～2322bp,分离自同一科室的部分菌株携带大小相同的基因盒;Ⅰ类整合子阳性菌株的耐药率高于整合子阴性的菌株。结论Ⅰ类整合子及整合子相关基因盒在产ESBLs大肠埃希菌株中分布广泛,整合子在细菌耐药中发挥作用。     

厄他培南不敏感大肠埃希菌耐药性及同源性分析

目的探讨临床分离大肠埃希菌对碳青霉烯类抗生素的耐药机制及流行情况,为临床用药和院内感染监控提供依据。方法收集2012年8月至2013年7月温州医科大学附属第二医院厄他培南不敏感的大肠埃希菌10株,采用VITEK Compact2全自动微生物分析仪检测18种常用抗菌药物的MIC值;改良Hodge试验检测碳青霉烯酶,PCR扩增包括碳青霉烯酶基因在内的多种B．内酰胺酶基因;应用脉冲场凝胶电泳（PFGE）分析菌株同源性。结果分离菌株来自不同病区无聚集现象,标本来源以尿液为主;菌株对广谱青霉素、三代、四代头孢菌素、氟喹诺酮类和酶抑制剂复合物耐药严重,对氨基糖苷类抗生素较为敏感;PCR扩增几乎所有菌株携带ESBL基因,只有一株除外,其中主要是blarsm和、blactx-mo3株blaNOM-1基因阳性,未检出其他碳青霉烯酶基因;脉冲场凝胶电泳分析表明,菌株之间没有克隆关系。结论该院分离10株大肠埃希菌对碳青霉烯类耐药主要是存在NDM-1金属酶联合ESBL,菌株间未发现克隆传播。     

细菌染色体第1类整合子捕获耐药性基因盒反应模型的构建

【背景】整合子在细菌耐药性的获得及传播中占据重要地位,对于整合反应检测方法的改良及反应机制的研究,可以加深我们对细菌耐药性产生和播散的理解,为遏制耐药菌株的产生和播散提供新的途径。【目的】在细菌染色体上构建第1类整合子反应模型,用于评价整合酶介导的基因盒位点特异性重组。【方法】 PCR分别扩增含氯霉素耐药基因cat的CM片段、含基因盒aadA5的LacA5片段、含整合子重组位点attI1及强可变区启动子的PcS片段和插入位点两侧的同源臂,重叠延伸聚合酶链反应连接上述5个片段制备整合子模型插入片段,通过同源重组将构建好的整合子模型片段插入大肠埃希菌JM109染色体中。转入高表达第1类整合酶的质粒pHSint,在链霉素平板上筛选发生整合的菌株,并经聚合酶链反应和测序验证。【结果】构建的整合子模型片段经测序与预期一致,整合子模型片段成功插入大肠埃希菌JM109染色体中。转入高表达整合酶的质粒pHSint后,在链霉素平板上成功筛选出基因盒aadA5发生整合的菌株,经聚合酶链反应扩增并测序与预期一致。【结论】在大肠埃希菌染色体上成功构建第1类整合酶介导基因盒位点特异性重组反应模型,为进一步揭示整合子捕获耐药性基因盒的反应机制奠定基础。     

产ESBL克雷伯菌与大肠埃希菌质粒分布的初步研究

比较产超广谱 β 内酰胺酶克雷伯菌与大肠埃希菌质粒的表型与分布。用BlaTEM引物经PCR技术将 5 1株克雷伯菌与 2 9株大肠埃希菌编码产超广谱 β 内酰胺酶的质粒扩增 ,琼脂糖凝胶电泳分离目的片段后得到完整的质粒电泳图谱。克雷伯菌与大肠埃希菌存在有相同大小的产超广谱 β 内酰胺酶的质粒。 2种细菌有产ESBL相同质粒 ,PCR技术可用于产超广谱 β 内酰胺酶细菌质粒的分析。     

温度与pH值对长江水系产超广谱β-内酰胺酶大肠埃希菌耐药基因转移影响分析

研究温度和pH值对长江水系中产超广谱β-内酰胺酶(Extended-Spectrum β-Lactamases,ESBL)大肠埃希菌(Escherichia coli)耐药基因转移影响规律,为今后介水疾病的预防与控制提供理论依据。采用滤膜法分离、梅里埃微生物分析系统鉴定菌株;将由长江水系分离出的产ESBL大肠埃希菌与大肠埃希菌NK5449进行接合,观察不同温度和pH值条件下接合频率变化情况;用纸片扩散法测定耐药谱;用PCR方法分析产ESBL供体菌与转移接合子β-内酰胺酶编码基因(bla),并对供、受体菌及转移接合子进行随机扩增多态性分析,判别转移接合子与供、受体菌的同源性。温度和pH值对产ESBL大肠埃希菌耐药基因水平转移影响明显,发生接合最适宜的pH值为7.1。温度对接合频率的影响具有双重性,相同条件下,某些大肠埃希菌接合频率随环境温度的降低率急剧下降,但某些大肠埃希菌的接合频率随环境温度下降有所上升。温度和pH值对产ESBL大肠埃希菌接合频率有重要影响。     

Distribution of class 1 integrons among enteropathogenic Escherichia coli

The aim of this study was to investigate the incidence of and resistance gene content of class 1 integrons among enteropathogenic Escherichia coli (EPEC) and non-EPEC and to investigate intraspecies genetic diversity of EPEC strains isolated from children with diarrhea in Iran. Twenty-eight EPEC and 16 non-EPEC strains isolated from children with diarrhea were tested for the presence of a class 1 integron associated integrase gene (int1). Sequence analysis was performed to identify the resistance gene content of integrons. Genetic diversity and cluster analysis of EPEC isolates were also investigated using enterobacterial repetitive intergenic concensus-polymerase chain reaction (ERIC-PCR) fingerprinting. Twenty-three (82%) EPEC isolates and 11 (68.7%) non-EPEC isolates harbored the int1 gene specific to the conserved integrase region of class 1 integrons. Sequence analysis revealed the dominance of dfrA and aadA gene cassettes among the isolates of both groups. ERIC-PCR fingerprinting of EPEC isolates revealed a high diversity among these isolates. The widespread distribution of 2 resistance gene families (dfrA and aadA) among both groups of EPEC and non-EPEC isolates indicates the significance of integrons in antibiotic resistance transfer among these bacteria. Furthermore, clonal diversity of EPEC isolates harbouring a class 1 integron also suggests the circulation of these mobile elements among a diverse population of EPEC in this country.     

Serotypes and virutypes of enteropathogenic and enterohaemorrhagic Escherichia coli strains from stool samples of children with diarrhoea in Germany

Aims: To investigate the prevalence of traditional and emerging types of enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) strains in stool samples from children with diarrhoea and to characterize their virulence genes involved in the attaching and effacing (A/E) phenotype. Methods and Results: Serological and PCR‐based methods were used for detection and isolation of EPEC and EHEC strains from 861 stool samples from diarrhoeic children. Agglutination with traditional EPEC and EHEC O‐group‐specific antisera resulted in detection of 38 strains; 26 of these carried virulence factors of EPEC or EHEC. PCR screening for the eae gene resulted in isolation of 97 strains, five carried genes encoding Shiga toxins (stx), one carried the bfpA gene and 91 were atypical EPEC. The 97 EPEC and EHEC strains were divided into 36 O‐serogroups and 21 H‐types, only nine strains belonged to the traditional EPEC O‐groups O26, O55, O86 and O128. In contrast, EPEC serotypes O28:H28, O51:H49, O115:H38 and O127:H40 were found in multiple cases. Subtyping the virulence factors intimin, Tir and Tir‐cytoskeleton coupling effector protein (TccP)/TccP2 resulted in further classification of 93·8% of the 97 strains. Conclusions: Our findings show a clear advantage of the eae‐PCR over the serological detection method for identification of EPEC and EHEC strains from human patients. Significance and Impact of the Study: Molecular detection by the eae‐PCR followed by serotyping and virutyping is useful for monitoring trends in EPEC and EHEC infections and to discover their possible reservoirs.     

Phage phiC31 integrase-mediated genomic integration and long-term gene expression in the lung after nonviral gene delivery

BACKGROUND: Phage phiC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study investigated the activity of phiC31 integrase in murine lungs. METHODS: Transfections in murine alveolar epithelial (MLE12) cells were performed with Lipofectamine 2000. For in vivo gene delivery, DNA was complexed with polyethylenimine (PEI) and PEI-DNA complexes were injected intravenously into mice. Expression of luciferase in mice was monitored by in vivo bioluminsecence imaging. Genomic integration and integration into a previously described 'hotspot' were confirmed by polymerase chain reaction (PCR). RESULTS: phiC31 integrase mediated intramolecular recombination between wild-type attB and attP sites in MLE12 cells. Long-term gene expression could be observed in MLE12 cells in the presence of integrase without any selection pressure. Long-term expression of luciferase after intravenous injection of PEI-DNA complexes could be observed only in the lungs of mice which were co-injected with the integrase-encoding plasmid. Increased amounts of integrase plasmid and administration of a second dose had no effect on the level of luciferase expression achieved with a single dose, which was three orders of magnitude lower than the values observed on 'day 1' post application. Genomic integration of the transgene in the mouse lungs was confirmed by PCR. Seven out of the fifteen treated mice showed integration at the mpsL1 site, a previously described 'hot spot' from liver. CONCLUSIONS: These results provide evidence for the activity of phiC31 integrase in lungs but also emphasize the need for optimization of the system to maintain long-term gene expression at high levels.     

Investigation of diarrhoeic faecal samples for enterotoxigenic, Shiga toxin-producing and typical or atypical enteropathogenic Escherichia coli in Kashmir, India

Three hundred and twenty-six Escherichia coli isolates recovered from 326 human faecal specimens from sporadic cases of diarrhoea in Kashmir valley, India, were investigated for the presence of stx(1), stx(2), eaeA, hlyA and lt virulence genes. None of the samples was positive for stx genes or Shiga toxins by PCR or enzyme-linked immunosorbent assay. Twenty-three E. coli isolates showed the presence of the eaeA gene, whereas three isolates harboured the lt gene. Enteropathogenic E. coli (EPEC) belonged to 10 different serogroups. Out of 23 EPEC isolates, the majority (78.26%) were atypical while five (21.73%) were typical. Only one of the typical EPEC harboured the EAF plasmid. Subtyping of the eaeA gene showed the presence of eaeA-alpha(1), eaeA-beta, eaeA-xi and eaeA-eta in one, two, four and two isolates, respectively. None of the E. coli isolates possessed eaeA-delta, eaeA-epsilon and eaeA-zeta. This study further upholds the opinion that Shiga toxin-producing E. coli do not pose a major threat to human health in India and eaeA-alpha(1), eaeA-beta, eaeA-xi and eaeA-eta could be common EPEC subtypes prevalent in humans with diarrhoea in India. The present study appears to be the first report of subtype analysis of the eaeA gene from India and also records the isolation of EPEC with the eaeA-xi gene from humans.     

Metagenomic analysis of lysogeny in Tampa Bay: implications for prophage gene expression

Phage integrase genes often play a role in the establishment of lysogeny in temperate phage by catalyzing the integration of the phage into one of the host's replicons. To investigate temperate phage gene expression, an induced viral metagenome from Tampa Bay was sequenced by 454/Pyrosequencing. The sequencing yielded 294,068 reads with 6.6% identifiable. One hundred-three sequences had significant similarity to integrases by BLASTX analysis (e < or =0.001). Four sequences with strongest amino-acid level similarity to integrases were selected and real-time PCR primers and probes were designed. Initial testing with microbial fraction DNA from Tampa Bay revealed 1.9 x 10(7), and 1300 gene copies of Vibrio-like integrase and Oceanicola-like integrase L(-1) respectively. The other two integrases were not detected. The integrase assay was then tested on microbial fraction RNA extracted from 200 ml of Tampa Bay water sampled biweekly over a 12 month time series. Vibrio-like integrase gene expression was detected in three samples, with estimated copy numbers of 2.4-1280 L(-1). Clostridium-like integrase gene expression was detected in 6 samples, with estimated copy numbers of 37 to 265 L(-1). In all cases, detection of integrase gene expression corresponded to the occurrence of lysogeny as detected by prophage induction. Investigation of the environmental distribution of the two expressed integrases in the Global Ocean Survey Database found the Vibrio-like integrase was present in genome equivalents of 3.14% of microbial libraries and all four viral metagenomes. There were two similar genes in the library from British Columbia and one similar gene was detected in both the Gulf of Mexico and Sargasso Sea libraries. In contrast, in the Arctic library eleven similar genes were observed. The Clostridium-like integrase was less prevalent, being found in 0.58% of the microbial and none of the viral libraries. These results underscore the value of metagenomic data in discovering signature genes that play important roles in the environment through their expression, as demonstrated by integrases in lysogeny.     

溶原性噬菌体在猪链球菌2型分离株中的检出和鉴定

[目的]为了研究噬菌体整合酶基因在猪链球菌2型(Streptococcus suis type 2,SS2)中的分布情况.[方法]根据噬菌体整合酶基因设计引物,建立了PCR方法,并对扩增产物进行测序.[结果]结果显示,25株SS2致病菌株均扩增出目的片段,非毒力株T15、5株其它血清型猪链球菌及兰氏C群猪源链球菌未扩增出目的片段.经丝裂霉素C诱导后,SS2致病菌株出现完全的细胞溶解,而非毒力株T15未出现溶解.SS2致病株HA9801和ZY05719诱导均产生溶原性噬菌体,分别命名为SS2-HA和SS2-ZY,电镜观察,二者均头部呈正六边形,无尾部,其核酸类型为dsDNA,可鉴定为复层噬菌体科(Tectiviridae)的成员.噬菌体SS2-HA和SS2-ZY整合酶基因序列与已报道的SS2噬菌体整合酶基因序列高度同源,显示SS2噬菌体整合酶具有较高的特异性.[结论]从SS2致病株中检出溶原性噬菌体和噬菌体整合酶基因,且噬菌体整合酶基因与SS2溶菌酶释放蛋白(mrp)等7种毒力相关基因有相关性,表明SS2的溶原性噬菌体可能与其致病性有关.     

A novel and rapid method for determining integration frequency catalyzed by integron integrase intI1

We developed a faster and more convenient method to determine integration frequency mediated by integron integrase intI1. This method based on real-time fluorescent quantitative PCR. By using this method, we revealed that the integration frequency of aadA2 gene cassette was 1.87x10(-4) when integrase intI1 was present, and the background frequency was less than 5.23x10(-8) without integrase intI1.     

PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints

BACKGROUND: Gene transfer to synovium in joints has been shown to be an effective approach for treating pathologies associated with rheumatoid arthritis (RA) and related joint disorders. However, the efficiency and duration of gene delivery has been limiting for successful gene therapy for arthritis. The transient gene expression that often accompanies non-viral gene delivery can be prolonged by integration of vector DNA into the host genome. We report a novel approach for non-viral gene therapy to joints that utilizes phage phiC31 integrase to bring about unidirectional genomic integration. METHODS: Rabbit and human synovial cells were co-transfected with a plasmid expressing phiC31 integrase and a plasmid containing the transgene and an attB site. Cells were cultured with or without G418 selection and the number of neo-resistant colonies or eGFP cells determined, respectively. Plasmid rescue, PCR query, and DNA sequence analysis were performed to reveal integration sites in the rabbit and human genomes. For in vivo studies, attB-reporter gene plasmids and a plasmid expressing phiC31 integrase were intra-articularly injected into rabbit knees. Joint sections were used for histological analysis of beta-gal expression, and synovial cells were isolated to measure luciferase expression. RESULTS: We demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the transgene and attB increased the frequency of transgene expression in rabbit synovial fibroblasts and primary human RA synoviocytes. Plasmid rescue and DNA sequence analysis of plasmid-chromosome junctions revealed integration at endogenous pseudo attP sequences in the rabbit genome, and PCR query detected integration at previously characterized integration sites in the human genome. Significantly higher levels of transgene expression were detected in vivo in rabbit knees after intra-articular injection of attB-reporter gene plasmids and a plasmid expressing phiC31 integrase. CONCLUSION: The ability of phiC31 integrase to facilitate genomic integration in synovial cells and increase transgene expression in the rabbit synovium suggests that, in combination with more efficient DNA delivery methods, this integrase system could be beneficial for treatment of rheumatoid arthritis and other joint disorders.     

Bacteriophages: A possible solution to combat enteropathogenic Escherichia coli infections in neonatal goats

Due to awareness and benefits of goat rearing in developing economies, goats' significance is increasing. Unfortunately, these ruminants are threatened via multiple bacterial pathogens such as enteropathogenic Escherichia coli (EPEC). In goat kids and lambs, EPEC causes gastrointestinal disease leading to substantial economic losses for farmers and may also pose a threat to public health via the spread of zoonotic diseases. Management of infection is primarily based on antibiotics, but the need for new therapeutic measures as an alternative to antibiotics is becoming vital because of the advent of antimicrobial resistance (AMR). The prevalence of EPEC was established using bfpA gene, uspA gene and Stx1 gene, followed by phylogenetic analysis using Stx1 gene. The lytic activity of the isolated putative coliphages was tested on multi-drug resistant strains of EPEC. It was observed that a PCR based approach is more effective and rapid as compared to phenotypic tests of Escherichia coli virulence. It was also established that the isolated bacteriophages exhibited potent antibacterial efficacy in vitro, with some of the isolates (16%) detected as T4 and T4-like phages based on gp23 gene. Hence, bacteriophages as therapeutic agents may be explored as an alternative to antibiotics in managing public, livestock and environmental health in this era of AMR.     

Molecular epidemiological study of a mass outbreak caused by enteropathogenic Escherichia coli O157:H45.

We made a molecular analysis of O157:H45 Escherichia coli isolated from a mass outbreak that occurred in Obihiro City. Using DNA analysis, we confirmed this infection case as a mass outbreak. Although the isolates expressed O157 antigen, they did not produce Vero toxin. We concluded they were enteropathogenic E. coli (EPEC) because they had a bfp gene and an EAF plasmid, and further they exhibited local adherence to HEp-2 cells. We believe this is the first report of a mass outbreak by O157 EPEC, and we suggest that PCR using eae- and bfp-specific primers and HEp-2 adherence assay are useful to identify EPEC.