杭州市O139群霍乱弧菌耐药变迁及毒力基因携带

目的 研究杭州市1994～2003年分离的O139群霍乱弧菌耐药性变迁以及携带ctxA、tcpA毒力基因的情况。方法 运用K—B法和PCR，检测90株O139群霍乱弧菌对抗生索的敏感性以及是否携带ctxA、tcpA毒力基因。结果 90株O139群霍乱弧菌中无丁胺卡那耐药的菌株，13株对诺氟沙星和环丙沙星耐药；对氨苄青霉索、复方新诺明分别有5年和6年的耐药率为100％；2002、2003年耐药谱增加到7种。87．87％的O139群霍乱弧菌同时携带ctxA、tepA毒力基因。结论10年来分离自杭州的O139群霍乱弧菌的耐药情况日益严重；O139群霍乱弧菌是否携带ctxA、tcpA毒力基因在对抗生素敏感性方面差异有统计学意义。     

龙泉市首例O139群霍乱弧菌耐药性及毒力基因携带情况研究

目的：研究龙泉市首例O139群霍乱弧菌耐药性及毒力基因携带情况。方法：运用药敏试验及PCR方法．分要该霍乱弧菌的耐药性以及携带ctxA、tcpA毒力基因的情况。结果：该O139群霍乱弧菌对庆大霉素等9种抗生素多重耐药：仅对丁胺卡那、诺氟沙星和环丙沙星敏感；该O139群霍乱弧菌携带ctxA、tcpA毒力基因。结论：在对O139群霍乱弧菌引起霍乱的防治工作中应密切监测其耐药性以及携带毒力基因的情况。     

烟台地区非O1/O139群霍乱弧菌致病性分析

目的了解烟台地区非O1/O139群霍乱弧菌致病性现状。方法分别于2020年7月和11月、2021年的1月和4月对烟台市某两条河入海口采集样本32份, 获得22株非O1/O139群霍乱弧菌分离菌株, 采用脉冲场凝胶电泳(PFGE)对分离菌株进行分子分型, 采用聚合酶链式反应(PCR)方法对该22株分离菌株的毒力基因ctxA、ctxB、ace、cep、zot、rtxA、hlyA、toxR、tcpA和cri进行检测分析。结果 22株非O1/O139群霍乱弧菌菌株间的条带相似度为23.0%～100.0%, 分为17个PFGE型, 呈高度的多态性带型分布。22株非O1/O139群霍乱弧菌分离株主要携带3种毒力基因(rtxA、tcpA和zot), 其中携带rtxA和tcpA最多, 均为17株, 其次是zot, 共14株。14株菌株同时携带rtxA、tcpA和zot, 5株不携带上述毒力基因。结论烟台地区入海口流行的非O1/O139群霍乱弧菌株污染情况多样化, 主要携带具有黏附、毒素以及损伤小肠表皮细胞等方面的致病基因。     

广东省外环境O1/O139群霍乱弧菌毒力基因分型

目的 分析广东省外环境来源O1/O139群霍乱弧菌毒力基因的携带及基因分型特征,为霍乱防控提供依据.方法 选取2008 -2009年广东省O1/O139群霍乱弧菌水体分离株69株,水产品分离株16株和同期病例分离株5株,应用多重聚合酶链反应对ctxA、ace、zot、tcpA、tcpl、hlyA、ompU、toxR等8种毒力基因进行检测和分型分析.结果 90株O1/O139群霍乱弧菌均携带hlyA和toxR基因;5株病例菌株中有3株携带8种毒力基因,另外2株小川型菌株为非产毒株,基因型为hlyA+ toxR+ ompU+ zot+ tcpA+ tcpl+型和hlyA+ toxR+ tcpA+型;水体菌株中,稻叶型菌株以hlyA+ toxR+ ompU+ ace+ zot+ tcpl+型(34.15％)为主,小川型(66.67％)和O139群(70％)以hlyA+ toxR+型为主;水产品菌株中,稻叶型菌株以hlyA+ toxR+ ompU+ tcpl+型(75.00％)为主,小川型菌株各种基因型别均有分布,无明显优势基因型别.结论 广东省外环境来源O1/O139群霍乱弧菌以非产毒株广泛存在,毒力基因型别多样.     

一株非典型O1群霍乱弧菌实验室诊断与分析

目的:一株从腹泻病人分离到的O1群霍乱弧菌的实验室鉴定和毒力基因的检测与分析。方法:细菌的分离和鉴定按照卫生部疾病控制司第五版《霍乱防治手册》进行;用PCR方法对该菌株的ctxA、rtxA、Ace、TcpA、Cri、Zot 6种毒力基因进行检测。结果:经分离培养、生化反应、噬菌体-生物分型,确定该菌株是埃尔托霍乱弧菌5 e型流行株,菌落形态不典型,毒力基因TcpA、rtxA阳性,其它毒力基因均为阴性。结论:尽管该菌株从腹泻病人分离且确定为埃尔托流行株,但其霍乱肠毒素主要控制基因ctxA阴性,且菌落形态不典型,该菌是一株非典型的O1群霍乱弧菌。     

珠江河口水体霍乱弧菌监测及菌株毒力基因分布特征

目的 了解珠江河口水体中O1群和O139群霍乱弧菌的分布状况,分析菌株的分子特征和毒力基因特征.方法 对2009年1月至2010年12月从珠江河口水体中分离的59株O1群和10株O139群霍乱弧菌,采用聚合酶链反应(PCR)方法体外检测ctxA、tcpA、ace、zot、tcpl,hlyA、toxR和ompU等毒力相关基因,并进行毒力相关基因分型分析,对限制性内切酶Not Ⅰ消化后的基因组DNA进行脉冲场凝胶电泳(PFGE)分析,采用BioNumerics软件分析图谱,得到菌株带型相似性的聚类分析树状图.结果 2009-2010年共采集1 152份水体标本,分离得到O1/O139群霍乱弧菌69株,其中O1群埃尔托霍乱弧菌59株(小川型18株,稻叶型41株),O139群霍乱弧菌10株.PCR检测69株菌ctxA全部阴性,hlyA和toxR全部阳性,基因分型可分成9个型.稻叶型菌株中,34.15％(14/41)为hlyA+ toxR+ ompU+ ace+ zot+ tcpⅠ+型;小川型菌株中,66.67％(12/18)为hlyA+toxR+型;O139群菌株中,70％(7/10)为hlyA+ toxR+型.PFGE分型发现,O139群菌株PFGE相似度为69.9％～ 95.5％;O1群菌株相似度为72.8％～ 100.0％,可分成3个聚类.结论 在霍乱流行间歇期,该地区外环境水体中O1群和O139群霍乱弧菌非产毒株广泛存在,基因型别多样.     

嘉兴市霍乱弧菌分离株检测分析

目的比较分析嘉兴市霍乱弧菌菌株的血清型、毒力基因和分子分型特征。方法采用血清学和分子生物学方法对近年来分离到13株霍乱弧菌菌株进行血清型分布、毒力基因(ctxA、ace、zot、tcpA、cri和rtxA)携带和ERIC-PCR分型研究。结果 13株霍乱弧菌菌株,2株为O139群霍乱弧菌,11株为O1群霍乱弧菌,其中小川型9株,稻叶型2株。2株O139群霍乱弧菌菌株携带3种毒力基因;11株O1群霍乱弧菌菌株,除1株携带4种毒力基因外,其余10株携带全部6种毒力基因。ERIC-PCR分型分析显示,不同的霍乱弧菌菌株之间ERIC-PCR型别表现出一定的遗传多样性。结论嘉兴市霍乱弧菌菌株大多携带多种毒力基因,而且来源多样,防控工作任重而道远。     

武汉市霍乱弧菌中毒力相关基因及分类鉴定

目的为了解武汉地区历年收集的霍乱弧菌毒力相关基因分布情况,并对这些菌株进行分类鉴定。方法分别对霍乱弧菌中的ctxA、zot、ace、ompU、tcpA、toxR、tcpI与hlyA等基因进行PCR检测,并对所有霍乱弧菌进行PCR分类鉴定。结果分离的大部分霍乱弧菌菌株携带有所有的毒力基因,有3株细菌仅有个别非核心毒力基因扩增阳性;该PCR方法分类对O1型所有菌株都能很好地进行分类鉴别,对O139型则与血清型鉴定存在不一致情况。结论PCR方法对产毒型和非产毒型O1/O139霍乱弧菌以及O1/O139和非O1/O139霍乱弧菌鉴定效率非常好。     

广东省2011—2013年腹泻病例非O1/O139群霍乱弧菌耐药性及分子特征

目的分析2011—2013年广东省腹泻病例非O1/O139群霍乱弧菌的耐药性及分子特征,为霍乱防控提供依据。方法选取2011—2013年广东省腹泻病例非O1/O139群霍乱弧菌分离株20株,以及同时期海水产品分离株4株,采用抗菌药物敏感试验、毒力基因PCR检测和脉冲场凝胶电泳(PFGE)分子分型方法,分析其耐药性及分子特征。结果抗菌药物敏感性试验结果显示,分离株对复方新诺明、萘啶酸和四环素的耐药率分别为70.8%(17/24),37.5%(9/24)和20.8%(5/24);对头孢曲松和阿米卡星则完全敏感。毒力基因PCR检测结果显示,所有的菌株均携带hly A和toxR基因,而所有菌株均未检出ctxA、tcp A、ace、zot、st等5种毒力基因。菌株经NotⅠ酶切后的PFGE型别表现出明显的多样性,病例菌株与海水产品分离株的带型差别较大。结论广东省非O1/O139群霍乱弧菌毒力基因和遗传特征复杂多样,菌株对多种抗菌药物高度耐药,耐药率较高,需加强腹泻病例中非O1/O139群霍乱弧菌的菌株型别变异及耐药监测。     

112株霍乱弧菌的生物学特性、毒力基因和耐药变迁的研究

目的：了解浙江温岭霍乱弧菌的生物学特性、毒力基因和耐药情况,为霍乱防治提供科学依据.方法：以霍乱弧菌主要毒力因子基因ctx为引物,对112株霍乱弧菌（O1群92株、O139群20株）进行了PCR扩增.采用WHO推荐的改良K-B纸片法,对所有菌株进行了18种抗菌药物的药敏试验.结果：除外环境检出的4株O1群霍乱弧菌菌株外,其余的霍乱菌株均扩增出与阳性对照一致的ctx毒力基因条带.药敏试验结果显示O139群霍乱弧菌的耐药性明显高于O1群霍乱弧菌,不同年份的菌株耐药的程度不一致.结论：从所有病人中分离到的菌株均为产毒株,与致病性和流行强度有关,治疗霍乱病人应结合本地区情况,根据不同菌型选择相应的抗生素药物.     

Antimicrobial resistance of Escherichia coli O26, O103, O111, O128, and O145 from animals and humans

Susceptibilities to fourteen antimicrobial agents important in clinical medicine and agriculture were determined for 752 Escherichia coli isolates of serotypes O26, O103, O111, O128, and O145. Strains of these serotypes may cause urinary tract and enteric infections in humans and have been implicated in infections with Shiga toxin-producing E. coli (STEC). Approximately 50% of the 137 isolates from humans were resistant to ampicillin, sulfamethoxazole, cephalothin, tetracycline, or streptomycin, and approximately 25% were resistant to chloramphenicol, trimethoprim-sulfamethoxazole, or amoxicillin-clavulanic acid. Approximately 50% of the 534 isolates from food animals were resistant to sulfamethoxazole, tetracycline, or streptomycin. Of 195 isolates with STEC-related virulence genes, approximately 40% were resistant to sulfamethoxazole, tetracycline, or streptomycin. Findings from this study suggest antimicrobial resistance is widespread among E. coli O26, O103, O111, O128, and O145 inhabiting humans and food animals.     

Detection of Shiga toxin-producing Escherichia coli O26, O45, O103, O111, O113, O121, O145, and O157 serogroups by multiplex polymerase chain reaction of the wzx gene of the O-antigen gene cluster

O-antigens on the surface of Escherichia coli are important virulence factors that are targets of both the innate and adaptive immune system and play a major role in pathogenicity. O-antigens that are responsible for antigenic specificity of the strain determine the O-serogroup. E. coli O26, O45, O103, O111, O113, O121, O145, and O157 have been the most commonly identified O-serogroups associated with Shiga toxin-producing E. coli (STEC) implicated in outbreaks of human illness all over the world. A multiplex polymerase chain reaction assay was developed to simultaneously detect the eight STEC O-serogroups targeting the wzx (O-antigen-flippase) genes of all O-antigen gene clusters. The sensitivity of the multiplex polymerase chain reaction was found to be 10 colony forming units for each O-group when enriched in broth and 100 colony forming units when enriched in artificially inoculated apple juice diluted with tryptic soy broth for 16?h at 37°C. The method can be used for detecting STEC O-groups simultaneously and may be exploited for improving the safety of food products.     

Detection by multiplex real-time polymerase chain reaction assays and isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 in ground beef

Six Shiga toxin-producing Escherichia coli (STEC) serogroups, which include O26, O45, O103, O111, O121, and O145, are responsible for the majority of non-O157 STEC infections in the United States, representing a growing public health concern. Cattle and other ruminants are reservoirs for these pathogens; thus, food of bovine origin may be a vehicle for infection with non-O157 STEC. Methods for detection of these pathogens in animal reservoirs and in food are needed to determine their prevalence and to develop intervention strategies. This study describes a method for detection of non-O157 STEC in ground beef, consisting of enrichment in modified tryptic soy broth at 42°C, followed by real-time multiplex polymerase chain reaction (PCR) assays targeting stx(1), stx(2), and genes in the O-antigen gene clusters of the six serogroups, [corrected] and then immunomagnetic separation (IMS) followed by plating onto Rainbow? Agar O157 and PCR assays for confirmation of isolates. All ground beef samples artificially inoculated with 1-2 and 10-20 CFU/25?g of ground beef consistently gave positive results for all of the target genes, including the internal amplification control using the multiplex real-time PCR assays after enrichment in modified tryptic soy broth for a total of 24?h (6?h at 37°C and 18?h at 42°C). The detection limit of the real-time multiplex PCR assays was ～50 CFU per PCR. IMS for O26, O103, O111, and O145 was performed with commercially available magnetic beads, and the IMS beads for O45 and O121 were prepared using polyclonal antiserum against these serogroups. A large percentage of the presumptive colonies of each serogroup picked from Rainbow Agar O157 were confirmed as the respective serogroups; however, the percent recovery of STEC O111 was somewhat lower than that of the other serogroups. This work provides a method for detection and isolation in ground beef and potentially other foods of non-O157 STEC of major public health concern.     

DNA sequencing and identification of serogroup-specific genes in the Escherichia coli O118 O antigen gene cluster and demonstration of antigenic diversity but only minor variation in DNA sequence of the O antigen clusters of E. coli O118 and O151

The DNA sequence of the O antigen gene cluster of an Escherichia coli serogroup O118 strain was determined, and 13 open reading frames (ORFs) were identified, encoding genes required for O antigen sugar biosynthesis, transfer, and processing. Polymerase chain reaction (PCR) assays targeting the wzx (O antigen flippase) and wzy (O antigen polymerase) genes in the O antigen gene cluster of E. coli O118 were designed for identification of these serogroups. Specificity testing using strains belonging to E. coli O118 isolated from various sources, representative strains of 167 other E. coli O serogroups, and 20 non-E. coli bacteria revealed that the PCR assays were specific for E. coli O118. Thus, the PCR assays can be used for rapid identification of E. coli O118 as an alterative to typing using antisera. However, the PCR assays targeting the E. coli O118 wzx and wzy genes were also positive using E. coli serogroup O151 DNA. Therefore, the sequence of the O antigen gene cluster of E. coli O151 was determined, and it was very similar to that of E. coli O118, with only three nucleotide differences. Although the lipopolysaccharide profiles of O118 and O151 showed differences, multilocus sequence typing of E. coli O118 and O151 strains only revealed minor variation at the nucleotide level. Since E. coli O118 strains are more frequently isolated from humans, animals, and the environment than E. coli O151, serogroup O151 may likely be a minor variant of E. coli O118. Further studies are needed to elucidate this possibility.     

Development and assessment of a rapid method to detect Escherichia coli O26, O111 and O157 in retail minced beef

A molecular-based detection method was developed to detect Escherichia coli O26, O111 and O157 in minced (ground) beef samples. This method consists of an initial overnight enrichment in modified tryptone soya broth (mTSB) and novobiocin prior to DNA extraction and subsequent serogrouping using a triplex PCR. This method has a low limit of detection and results are available within 24 hours of receipt of samples. Once optimized, this rapid method was utilized to determine the prevalence of these E. coli serogroups in six hundred minced beef samples all of which were previously examined by immunomagnetic separation (IMS) and selective plating for E. coli O26 and O111. Using IMS, two E. coli O26 isolates were detected. No E. coli O111 were recovered. The multiplex PCR technique described here did not detect E. coli O111 nor O157 in any of the samples, however six minced beef samples were positive for E. coli O26 using our method, only two of these were previously detected by IMS and culture. Application of molecular methods are useful to support culture-based approaches thereby further contributing to risk reduction along the food chain.     

Taq-Man实时荧光定量PCR同时检测O1和O139群霍乱弧菌

目的针对霍乱弧菌血清群各自编码O抗原的特异性基因建立能够快速、准确地同时检测O1和O139群霍乱弧菌的荧光PCR检测方法,并克服常用PCR检测方法中经常出现的假阳性现象。方法分别以rfbM和wbfR基因作为O1和O139群霍乱弧菌的模板设计引物和探针,通过优化反应条件建立了能够同时检测O1群和O139群霍乱弧菌的二联实时荧光定量PCR方法(FQ-PCR)。对该方法的特异性、灵敏度进行评估,并对10份模拟食品样品和133份临床样品进行了检测。结果特异性试验表明,该方法能选择性检测O1和O139群霍乱弧菌,能有效地避免O141群霍乱弧菌和拟态弧菌的假阳性,特异性为100%;灵敏度试验表明,O1群霍乱弧菌和O139群霍乱弧菌检出限分别为92cfu/ml和116cfu/ml;另外,试验建立的检测方法对模拟样品和临床样品检测结果与国标法的检测结果完全一致,符合率为100%。结论该实时荧光PCR检测方法能够同时检测O1和O139群霍乱弧菌,而且特异性好、灵敏度高,能有效克服假阳性,适用于基层疾病控制、口岸检疫单位日常疫情监测和临床诊断。     

Virulence factors of Escherichia coli strains belonging to serogroups O127 and O142

A total of 102 Escherichia coli strains belonging to serogroups O127 and O142 were examined for genotypic and phenotypic characteristics. The most frequent serotypes found were O127:H21, O127:H40 and O142:H34. The virulence properties were evaluated by adhesion to HeLa cells and hybridization with gene probes for diarrhoeagenic E. coli. Most strains in the two serogroups were categorized as enteropathogenic E. coli, but enteroaggregative E. coli was also detected in both serogroups. All strains that carried the eae sequence presented the LEE region inserted in selC. Five ribotypes were detected in serogroup O127 and four in serogroup O142 and a correlation between serotypes and ribotypes was observed mainly in serogroup O142.     

霍乱弧菌O1和O139血清型I类整合子的扩增与分布研究

目的扩增霍乱弧菌不同血清型I类整合子基因片段,并分析I类整合子在霍乱弧菌O1和O139血清型中的分布. 方法收集霍乱弧菌O1和O139血清型40株,根据GenBank资料设计PCR引物,扩增霍乱弧菌I类整合子基因片段,并用限制性酶切分析法进行鉴定,分析I类整合子在不同血清型的分布情况. 结果从霍乱弧菌O1和O139血清型中均能扩增约800 bp的基因片段,扩增片段经Pst I酶切后可获得671和127 bp的片段,经HindIII酶切后可获得580、160和58 bp的片段,经HindIII 和Pst I酶切后可获得453、160、127和58 bp的片段,I类整合子在O139血清型霍乱弧菌中分布多于O1血清型. 结论 I类整合子与霍乱散发菌株耐药性的传递、血清型的分布有密切的联系.     

Prevalence and characterization of Escherichia coli O26 and O111 in retail minced beef in Ireland

Verocytotoxigenic Escherichia coli (VTEC) O157 are recognized as bacterial pathogens with significant public health impact. However, other serogroups, including O26, O111, O103, and O145, have the potential to cause the same spectrum of illness. In this study, 800 minced (ground) beef samples covering a large geographical region in Ireland were collected and tested for Escherichia coli (E. coli) O26 and E. coli O111 by conventional microbiological protocols. Two minced beef samples (0.25%) tested positive for E. coli O26, indicating fecal contamination. None of these isolates possessed verocytotoxin-encoding genes, (vt1/vt2 also known as stx1/stx2), the hemolysinencoding gene (hlyA), or the E. coli attachment-effacement (eae) gene, as determined by polymerase chain reaction (PCR). None of the beef samples analyzed contained E. coli O111. Although the E. coli O26 isolates were nonvirulent, the presence of these isolates in raw minced beef is an indication of fecal contamination and therefore potentially of public health significance.     

Validation of a method for simultaneous isolation of Shiga toxin-producing Escherichia coli O26, O103, O111, and O145 from minced beef by an international ring-trial

An isolation method described by Possé et al. (FEMS Microbiol Lett 2008;282:124-131) was satisfactorily validated in an international ring-trial using artificially contaminated minced beef samples. Until now, no validated method existed for the simultaneous isolation of Shiga toxin-producing Escherichia coli serogroups O26, O103, O111, and O145 in food. Twelve laboratories from five European countries participated and received 16 inoculated beef samples contaminated with cold-stressed cells of the four serogroups O26, O103, O111, and O145 in two levels (approximately 30 and 300 CFU 25?g?1) in duplicate. In addition, they received four non-inoculated samples. The isolation protocol comprised a selective enrichment step, a selective isolation step on a non-O157 agar plate differentiating the serogroups by color, followed by confirmation by plating on confirmation agar media and agglutination. All laboratories were able to isolate the inoculated serogroups from the samples, both for the high and the low inoculation level. Results did not differ whether in-house-prepared or ready-to-use non-O157 agar plates were used, demonstrating that by following the instructions laboratories managed to perform the complete protocol with success.