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。     

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、tcpI、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⁺tcpI⁺型和hlyA⁺toxR⁺tcpA⁺型;水体菌株中,稻叶型菌株以hlyA⁺toxR⁺ompU⁺ace+zot⁺tcpI⁺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⁺tcpI⁺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.     

饮食业从业人员健康体检中检出3株O139型非O1群霍乱弧菌

1992年10月以来,在印度和孟加拉国先后发生霍乱样疾病的严重流行,临床表现与即往和当今流行的、由O1群霍乱弧菌引起的古典型或埃尔托型霍乱无异,且临床症状严重者居多.但病原体即不与O1群霍乱弧菌多价血清发生凝集,也不与O1群霍乱单克隆抗体A、B、C发生凝集,该病原体后被命名为O139型非O1群霍乱弧菌.我国自1993年以后该菌在新疆等地相继检出.我们在1998年4月初饮食业从业人员健康体检者粪便中检出3株,该菌在我市尚属首次检出,在健康体检者粪便中检出更属罕见.     

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hlyA和toxR基因,而所有菌株均未检出ctxA、tcpA、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。     

1株O139群霍乱弧菌的分离鉴定

霍乱是由O1群和O139群霍乱弧菌引起的甲类传染病,是当今3种国际检疫传染病中最严重的一种。准确快速检测霍乱弧菌并检测毒力基因携带情况,对判断疫情的性质,进而采取适当的控制措施至关重要。2010年9月,桐乡市肠道门诊监测中检出1株0139群霍乱弧菌并作了毒力基因检测和耐药性测定,现报道如下。     

O139群霍乱弧菌耐药谱和相关耐药基因研究

目的:通过检测分析2004年-2007年31株O139群霍乱弧菌临床分离株对13种抗菌药物耐药情况和相关耐药基因携带情况,为O139群霍乱弧菌预防和治疗提供理论依据。方法:K-B纸片扩散法药物敏感实验检测细菌13种抗菌药物耐药情况,用常规PCR方法扩增TEM、aadA2、strA、strB、tetA、floR和sul1等7种耐药基因和1型耐药整合子。结果:药物敏感实验表明31株O139群霍乱弧菌临床分离株只存在一种耐药谱型,对头孢噻肟、头孢吡肟、环丙沙星和氟哌酸敏感,对氨苄西林、羧苄西林、氯霉素、复方新诺明、四环素、强力霉素、庆大霉素、卡那霉素和链霉素耐药,均扩增出TEM、aadA2、strA、strB、floR和sul1耐药基因和1型耐药整合子结构。结论:O139群霍乱弧菌已对多种抗生素耐药并存在相关耐药基因,应引起临床重视。     

无锡市锡山区首次检出1株非O1和非O139型霍乱弧菌的报告

目的对无锡市锡山区河水中检出的1株可疑霍乱弧菌进行种属鉴定。方法采集外环境标本进行霍乱弧菌分离培养,参照1999年《霍乱防治手册》第5版、2008年《霍乱诊断标准》及相关资料进行检测。结果实验中使用3家单位的血清进行血清凝集反应,结果截然相反。2家国产血清结果为阳性,泰国产血清结果为阴性。经荧光定量PCR基因核酸检测表明该菌株为非O1、非O139型霍乱弧菌。结论提示在今后工作中,对于可疑的霍乱弧菌,不能单纯靠传统的血清学诊断和生化鉴定,需要分子诊断方法,这样可以从分子水平为弧菌科细菌的种属鉴定提供更为直接的证据。     

外环境样品中检出2株带毒力基因的O139霍乱弧菌

霍乱是由霍乱弧菌所引起的急性肠道传染病，是发病急、传播快、波及范围广、易引起流行的国际检疫传染病之一。浙江省杭州市余杭区每年均有散发病例或带菌者检出，作为我省霍乱防治监测点之一，我中心每年5～10月均开展外环境样品监测，在2007年7月的监测中分离出2株带毒力基因的O139群霍乱弧菌，该菌为我中心成立以来在外环境样品中首次检出。我们对该菌的形态及生化等生物学特性进行了观察，现将检测结果报告如下。     

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。     

珠江河口水体O1群和O139群霍乱弧菌监测

目的 分析珠江河口水体O1群和O139群霍乱弧菌菌型特征,探讨环境水体监测的方法和疫情监测中的作用.方法 2006年3月至2007年2月,在珠江河口选择24个水样采集点,每月采集一次,进行O1群和O139群霍乱弧菌的分离培养,并利用实时PCR监测样品增菌液中的O1群和O139群霍乱弧菌.采样同时测定气温、水温等气象资料.用脉冲场凝胶电泳(PFGE)对分离菌株进行分型分析.结果 监测期间共采样862份,霍乱弧菌分离阳性率7.77%,实时PCR阳性率为26.33%.按月的水样检测阳性率与水温变化趋势相似;城区监测点阳性率高于其他区域,在一家海产品批发市场排水口下游检测到产毒O139群菌株;菌株的菌型构成中,分离菌株主要为非产毒株;O1群E1 Tor小川和稻叶型以及O139群菌株的分离无季节变化趋势;PFGE分析75株分离株被分为49种带型,相似性为57.4%～100%,表现出明显的多样性.结论 霍乱弧菌在珠江河口水体中广泛存在,并呈现多样性.水体监测提供产毒菌株的指示,可作为环境危险评价的指标,且能在霍乱弧菌的监测和霍乱疫情预警中发挥作用.     

Association of a disease approximating cholera caused by Vibrio cholerae of serogroups other than O1 and O139.

One hundred and six patients suffering from severe dehydrating diarrhoea were studied of whom 36 patients were positive for Vibrio cholerae. Out of 36, 15 were positive for V. cholerae O1, 10 for V. cholerae O139 and 11 for V. cholerae non-O1 non-O139. O1 and O139 were positive for the 301-bp ctxA amplicon and 471-bp tcpA amplicon indicating that the strains possessed toxigenic capability whereas no non-O1 non-O139 strain possessed ctxA or tcpA genes. Post-admission severity of purging and amount of ORS required were less in the V. cholerae non-O1 non-O139 group (P < 0.05) compared to the V. cholerae O1 and O139 groups. It appears from this study that a cholera-like clinical condition can be caused in the absence of CT as exemplified by strains of non-O1 non-O139.     

Comparison of amplified fragment length polymorphism and pulsed-field gel electrophoresis for subtyping of Vibrio cholerae serogroups O1 and O139

Molecular typing of Vibrio cholerae strains is a powerful tool for the surveillance of cholera. Amplified fragment length polymorphism (AFLP) is considered to be a powerful subtyping technique to distinguish bacterial strains at the genetic level. Optimization and standardization of AFLP protocol is required to allow data comparisons across different laboratories in a surveillance network. Here, we performed AFLP using different restriction enzymes and primer pairs for subtyping of V. cholerae serogroups O1 and O139 and compared the optimized AFLP protocol with pulsed-field gel electrophoresis (PFGE) to evaluate the applicability of AFLP for conducting epidemiological surveillance of cholera. The discriminatory index (D-value) of PFGE for serogroup O1 strains was similar when digested with NotI and SfiI, whereas that for O139 strains was higher for NotI digestion than for SfiI. EcoRI-G/MseI-T was the restriction enzyme and primer combination with highest discriminatory index used in the AFLP analysis. Capillary electrophoresis-based AFLP showed higher discriminatory power than that of polyacrylamide gel electrophoresis-based AFLP. When the two methods were compared using 72 epidemiologically unrelated serogroup O1 El Tor isolates, AFLP had a lower D-value than PFGE with NotI and SfiI digestions, respectively. For 54 epidemiologically unrelated serogroup O139 isolates, NotI PFGE had the highest discriminatory power, and SfiI PFGE and AFLP yielded almost the same but lower discriminatory power. We conclude that NotI and SfiI are both suitable for the PFGE of V. cholerae serogroup O1, whereas NotI should be defined as the primary enzyme for serogroup O139. The applicability of AFLP in V. cholerae subtyping and outbreak investigations is limited.     

Distribution and virulence of Vibrio cholerae belonging to serogroups other than O1 and O139: a nationwide survey.

The distribution and virulence of Vibrio cholerae serogroups other than O1 and O139 in India before, during and after the advent of O139 serogroup was investigated. A total of 68 strains belonging to 31 different ''O'' serogroups were identified during the study period. With the exception of O53, there was no spatial or temporal clustering of any particular non-O1 non-O139 serogroup at any given place. Two of the 68 strains examined produced cholera toxin (CT) which could only be partially absorbed with anti-CT immunoglobulin G. Tissue culture assay revealed that some of the non-O1 non-O139 strains produced factors which evoked either a cell rounding or cell elongation response depending upon the medium used. This study indicates that serogroups other than O1 and O139 should also be continuously monitored.     

Diarrheagenicity evaluation of attenuated Vibrio cholerae O1 and O139 strains in the human intestine ex vivo

The recent spread of El Tor cholera in Latin America highlights the need for a safe and economical vaccine. The main approach for developing live recombinant vaccines has been to disarm known pathogenic strains of cholera toxin leaving intact antigens involved in protection. These recombinant vaccine candidates do not cause severe diarrhea, but they are too reactogenic for wide scale usage. We describe here a test capable of determining the diarrheagenic potential of attenuated V. cholerae strains. The functional test consists in the simultaneous recording of net water movement, electrical potential difference and short-circuit current across the human intestine ex vivo. We found that human tissues incubated with supernatants from the attenuated 638, 413 and 251a V. cholerae strains caused no changes in the ion conductances and water absorption in ileal and colon tissues allowing them to be assayed in volunteers.     

O139群霍乱弧菌毒力岛区域分子特征分析

目的 比较O139群霍乱弧菌国际标准株M045和O1群E1 Tor型霍乱弧菌国际标准株N16961基因组中毒力岛区域分子特征.方法 从MO45基因组中筛选含有毒力岛上游的VC0815基因及其上游基因片段并克隆到pNEB193中命名为pNEBVC081545,绘制pNEBVC081545的限制性酶切图谱并与N16961同段序列限制性酶切图谱进行比较.在pNEBVC081545中选择VC0815基因上游的一段侧序列进行亚克隆,测序后与N16961同段序列比较.结果 pNEBVC081545限制性酶切图谱中VC0815基因上游序列图谱中第1个碱基位置是限制性内切酶位点为KpnⅠ,第732位为Sal Ⅰ,第819位为Hpa Ⅰ,第1578位为Bgl Ⅱ,第1600位为Hind Ⅱ,第1943位为Xba Ⅰ,第3247位为EcoR Ⅰ,此段序列限制性内切酶图谱与N16961同段序列限制性酶切图谱完全相同.对Sal Ⅰ和EcoR Ⅰ之间的序列进行亚克隆,并对该段序列测序,测序结果表明该段核酸序列与N16961中同段序列完全相同.结论 O139霍乱弧菌可能是E1 Tor霍乱弧菌O抗原突变而产生的菌群.     

Phenotypic and genetic analyses of 111 clinical and environmental O1, O139, and non-O1/O139 Vibrio cholerae strains from different geographical areas

A total of 111 clinical and environmental O1, O139 and non-O1/O139 Vibrio cholerae strains isolated between 1978 and 2008 from different geographical areas were typed using a combination of methods: antibiotic susceptibility, biochemical test, serogroup, serotype, biotype, sequences containing variable numbers of tandem repeats (VNTRs) and virulence genes ctxA and tcpA amplification. As a result of the performed typing work, the strains were organized into four clusters: cluster A1 included clinical O1 Ogawa and O139 serogroup strains (ctxA(+) and tcpA(+)); cluster A2 included clinical non-O1/O139 strains (ctxA(-) and tcpA(-)), as well as environmental O1 Inaba and non-O1/O139 strains (ctxA(-) and tcpA(-)/tcpA(+)); cluster B1 contained two clinical O1 strains and environmental non-O1/O139 strains (ctxA(-) and tcpA(+)/tcpA(-)); cluster B2 contained clinical O1 Inaba and Ogawa strains (ctxA(+) and tcpA(+)). The results of this work illustrate the advantage of combining several typing methods to discriminate between clinical and environmental V. cholerae strains.     

一起O139群霍乱弧菌引起的食物中毒菌株的耐药性分析

目的：探讨多重耐药0139群霍乱弧菌形成的可能分子机制。方法：对杭州一起暴发的65株0139群霍乱菌株进行药敏试验，取其中20株多重耐药菌株进行结合性转座子样SXT元件标志基因int SXT和Ⅰ类整合子的PCR检测，并对Ⅰ类整合子基因盒区扩增产物应用限制性酶切和DNA测序分析携带的基因盒。结果：65株0139群霍乱菌株中，56株(86．2％)均呈同一耐药谱，对11种抗生素多重耐药，仅对诺氟沙星、环丙沙星及丁胺卡那敏感。20株多重耐药菌株中，SXT元件标志基因int SXT均阳性，Ⅰ类整合子5’保守区、基因盒区和3’保守区也均阳性。其携带基因盒为氨基糖苷腺苷酰基转移酶基因nndA2。结论：此次暴发的多重耐药的0139群菌株均携带有SXT元件，Ⅰ类整合子在多重耐药菌株的形成中起了一定的作用。     

广西首次分离 O139群霍乱弧菌的病原学特征

目的 了解O139群霍乱弧菌的病原学特征。方法 采用经典的微生物学、噬菌体-生物分型方法进行生化、药敏和流行菌株的分型,并用家兔肠段结扎方法进行霍乱肠毒素检测。结果 4株菌与O139群霍乱诊断血清发生强凝集反应。与O1群多价血清不凝集;在庆大琼脂平板上菌落较O1群霍乱弧菌混浊、隆起。霍乱肠毒素检测均为中等毒力,对氟哌酸、丁胺卡那霉素敏感,对9种抗菌药物耐药。结论 霍乱肠毒素检测为中等毒力,具备引起流行的能力,为流行株。因此加强监测,及时做好防治措施十分必要。