2005-2014年上海市O139群霍乱弧菌的分子特征和耐药性研究

目的 分析2005-2014年上海市O139群霍乱弧菌的分子特征和耐药性。方法 以聚合酶链反应(PCR)方法检测86株O139群霍乱弧菌的霍乱毒素基因(ctxA)、溶血素基因(hlyA)、毒素协调菌毛基因(tcpA)、调控蛋白基因(toxR)和编码霍乱毒素基因的CTX基因组基因(zot、ace)。采用脉冲场凝胶电泳(PFGE)和多位点序列分型(MLST)方法对O139群霍乱弧菌菌株进行分子分型。采用WHO推荐的改良K-B纸片法,对O139群霍乱弧菌菌株进行11种抗菌药物(头孢曲松、强力霉素、诺氟沙星、环丙沙星、复方新诺明、丁胺卡那霉素、四环素、氯霉素、萘啶酸、氨苄西林、庆大霉素)敏感试验。结果 患者来源O139群霍乱弧菌菌株93.2%(41/44)为产毒株,水产来源菌株58.3%(21/36)为产毒株,6株水体来源菌株均为非产毒株。PFGE分析将O139群霍乱弧菌分为35个型别。MLST分析将O139群霍乱弧菌分为9个ST型,其中流行菌型为ST 69型,非产毒株以ST 162型为主。86株O139群霍乱弧菌经药物敏感试验分析显示O139群霍乱弧菌产毒株的耐药情况比非产毒株严重。O139群霍乱弧菌产毒株对强力霉素、复方新诺明、四环素、萘啶酸、氨苄西林、庆大霉素的耐药率高。O139群霍乱弧菌非产毒株对萘啶酸耐药严重。结论 上海市患者和水产来源O139群霍乱弧菌构成以产毒株为主。PFGE分型提示水产为霍乱疫情的主要传染源。O139群霍乱弧菌产毒株高度克隆化,流行菌型为ST 69型,耐药情况严重。     

江苏省环境来源的O139群霍乱弧菌的核酸特征分析

目的 分析江苏省2003-2005年之间从环境样本中分离到的霍乱弧菌的核酸特征,及其差异的程度.方法 将11株目的菌株的表型特征(包括血清型,耐药谱,生化特征以及El Tor霍乱弧菌的噬菌体分型)和核酸特征(包括霍乱毒素基因ctx-A、zot、ace、tcp-A的分布情况和脉冲场凝胶电泳指纹图谱)结合起来进行比较和分析.结果 11株菌株中有10株O139群霍乱弧菌,1株El Tor小川;其中6株O139同时对5种以上抗生素耐受,2株O139对4种抗生素耐受,1株0139对2种抗生素耐受,另外1株O139(属于PFGE-C型)和1株小川分别只对一种抗生素耐受;11株霍乱弧菌的生化特征没有明显差异;8株O139同时携带4种毒力相关基因,1株0139只携带zot和ace,另外1株O139(05052)和1株小川未携带这4种基因;9株O139的脉冲场凝胶电泳指纹图谱聚为一类,另外1株O139和小川各自聚为一类.结论 10株O139中有1株(属于PFGE-C型)的耐药谱、毒力相关基因分布及PFGE指纹图谱与其他9株不同,而与分离地点相同的小川相似,提示自然环境中的O139群霍乱弧菌存在着差异.     

非O1群霍乱弧菌的鉴定及耐药性分析

对本院1999年6～9月在肠道门诊检出的20株非O1群霍乱弧菌进行生化鉴定、血清学分群和药敏试验.根据1998年美国NCCLS药敏法规中新增的霍乱弧菌抗生素选择规定和判别标准[2],所选用抗生素已能满足临床治疗的需要.药敏结果提示: 抗O/129的非O1群霍乱弧菌对抗生素的敏感性比O/129敏感的非O1群霍乱弧菌有明显下降.     

O1和O139群霍乱弧菌超级整合子VchintIA基因的变异

目的 研究不同血清群(型)霍乱弧菌超级整合子整合酶基因(VchintIA)的变异及其与霍乱弧菌分类的关系。 方法 使用聚合酶链反应(PCR)和序列测定的方法对VchintIA基因进行扩增和序列分析。 结果 在实验菌株中,O1El Tor和O139群霍乱弧菌产毒株以及毒素共调菌毛(toxin co-regulated pilus, tcp)基因簇阳性的O1群非产毒株的VchintIA序列完全一致,tcp基因簇阴性的O1群非产毒株的VchintIA序列在核苷酸水平上与产毒株不同,但是在氨基酸水平上相同。O139非产毒株的VchintIA序列在核苷酸和氨基酸水平上均与产毒株的VchintIA不同。 结论 O1和O139霍乱弧菌超级整合子VchintIA存在核苷酸和氨基酸水平上的变异,O139非产毒株中VchintIA的变异与其整合子内部的结构可能相关。     

非O1/O139群霍乱弧菌:流行、致病因子和耐药

非O1/O139群霍乱弧菌引起的腹泻在许多发展中国家都有发生,其流行涉及到多个血清群。在有些地方,非O1/O139群霍乱弧菌腹泻的发病率甚至超过了O1/O139群霍乱弧菌腹泻的发病率。非O1/O139群霍乱弧菌的致病因子多种多样,肠毒素、蛋白酶、溶血素和三型分泌系统等被认为是重要的致病因子,但其致病机制非常复杂,至今仍然不完全清楚。随着抗生素使用的增加,出现了许多耐药甚至多重耐药菌株。本研究从非O1/O139群霍乱弧菌腹泻的流行情况、致病因子以及耐药方面加以综述。     

Antibiotic resistance conferred by a class I integron and SXT constin in Vibrio cholerae O1 strains isolated in Laos

Changes in the drug susceptibility pattern were observed in Vibrio cholerae O1 isolated in the Lao People's Democratic Republic during 1993 to 2000. In this study, 50 V. cholerae O1 strains were selected during this period for studying the presence of class I integron and SXT constin. Twenty-four streptomycin-resistant strains out of 26 isolated before 1997 contained a class I integron harboring the aadA1 gene cassette. Twenty-four strains isolated after 1997 contained an SXT constin (a large conjugative element). Twenty of the strains were resistant to chloramphenicol, tetracycline, streptomycin, and trimethoprim-sulfamethoxazole, while four strains were susceptible to the antibiotic tested. The resistance genes included in the SXT constins were floR, tetA, strAB, and sulII, which encode resistance to chloramphenicol, tetracycline, streptomycin, and sulfamethoxazole, respectively. The antibiotic resistance gene cluster was found to be deleted in the four susceptible strains. SXT(LAOS) did not contain dfrA1 or dfr18, which confer resistance to trimethoprim in SXT(ET) and SXT(MO10), respectively. A hot spot region of SXT(LAOS) was sequenced, and we identified two novel open reading frames showing homology to sO24 (exonuclease) and sO23 (helicase) of the genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104. Analysis of SXT(LAOS) showed that there is a continuous flux of genes among V. cholerae SXT constins which should be carefully monitored.     

O139群霍乱弧菌ctxAB基因序列分析

目的探讨O130群霍乱JX株的ctxAB基因序列,分析该株基因特点.方法采用PCR扩增技术,检测含O130群霍乱标本,对ctxAB阳性者进行核苷酸序列测定,分析基因变异情况.结果发现阳性标本中,对其中一株进行了ctxAB基因序列测定,该序列与853(O139群)和N16961(Eltor)同源性为100%,与569B(古典型)同源性99.7%.结论该研究证明本株与O1群(EVC)遗传关系最近,可能属于B型.     

Occurrence of Vibrio cholerae serogroups other than O1 and O139 in Austria

From 2000 to 2005, 13 infections due to non-O1/non-O139 Vibrio cholerae were documented in Austria. Twelve patients (8 years to 65 years old; 7 male) had symptomatic infections: diarrhea x 5, otitis x 6, septicemia once. All 5 patients who acquired their infections abroad, suffered from diarrhea. The 8 persons without travel history outside of Austria had otitis media (n = 4) or otitis externa (n = 2); the lethal case of septicemia affected a fisherman with underlying malignancy. One isolate was from an asymptomatic child. Detailed data on travel history inside Austria was available for 5 of these 8 patients: all 5 had visited or lived near Austria's largest lake. The concentration of salt in this westernmost steppe lake in Europe is approximately one-twentieth of that of sea water. Why otitis and not diarrhea is the dominating manifestation of non-O1/non-O139 infection acquired in Austria remains to be elucidated. We hypothesize that diarrhea due to Vibrio cholerae serogroups other than O1 and O139 acquired in Austria may simply be unrecognized by the standard operating procedures employed in clinical microbiology laboratories. Testing for Vibrio cholerae is not considered necessary for domestically acquired diarrhea. Only in patients who acquired diarrhea abroad, do physicians sometimes consider cholera as a differential diagnosis, thereby prompting the laboratory to use thiosulfate citrate bile salt sucrose (TCBS) agar plates.     

O139群霍乱弧菌ctxAB基因序列分析

目的 探讨O130群霍乱JX株的ctxAB基因序列，分析该株基因特点。方法 采用PCR扩增技术，检测含O130群霍乱标本，对ctxAB阳性者进行核苷酸序列测定，分析基因变异情况。结果 发现阳标本中，对其中一株进行了ctxAB基因序列测定，该序列与853（O139群）和N16961（Eltor)同源性为100％，与569B（古典型）同源性99．7％。结论 该研究证明本株与O1群（EVC）遗传关系最近，可能属于B型。     

临床分离非O1/O139群霍乱弧菌毒力及耐药特征分析

目的 了解临床分离的非O1/O139群霍乱弧菌毒力及耐药特征。方法 收集2014-2015年北京友谊医院4-10月肠道门诊分离到的非O1/O139群霍乱弧菌,采用微量肉汤稀释法检测霍乱弧菌对15种抗生素的耐药性;PCR检测霍乱弧菌的毒力相关基因。结果 35株非O1/O139群霍乱弧菌对复方新诺明的耐药率(40.0%)最高,其次是氯霉素(28.5%)和磺胺异恶唑(22.6%),对阿莫西林/克拉维酸、头孢曲松、头孢西丁、头孢吡肟及亚胺培南完全敏感。毒力基因检测显示所有菌株均携带hlyA和ompU,hapA(97.1%)、rtxA(91.4%)、Ⅵ型分泌系统T6SS(94.3%~97.1%)、Ⅲ型分泌系统T3SS(80.0%~85.7%)和nanH(62.9%)阳性率较高;主要的毒力基因型为hlyA-rtxA-hapA-ompU-nanH-vasA-vasK-vasH-vcsC-vcsV-vcsN-vspD(40.0%)。结论 临床分离非O1/O139群霍乱弧菌毒力基因多样化,对抗菌药物耐药性较高,需加强腹泻病例中非O1/O139群霍乱弧菌的毒力及耐药监测。     

Identification of Vibrio cholerae serogroups O1 and O139 by monoclonal antibodies in the slide agglutination test

The agglutinating properties of MCA-O1 of the IgG class and MCA-O139 of the IgM class towards epitopes of O-antigen of Vibrio cholerae O1 and accordingly Vibrio cholerae O139 were studied. The ascitic and cultural fluids by hybridomas F8G12 and D11 deposited in the specialized Collection of Cell Cultures of Vertebrates (Saint Petersburg) under RKKK (II) 386 D and RKKK (II) 674 D were the sources of monoclonal immunoglobulins. The advantage of diagnostic monoclonal immunoglobulins is that they are distinguished for strict specificity and their use in practical health care contributes to the higher specificity of a laboratory test for cholera and to its shorter performance.     

浙江省非O1、非O139群霍乱弧菌快速检测与毒力基因研究

目的 建立一种快速检测非O1、非O139群霍乱弧菌方法,并对浙江省部分地区海水产品中非O1、非O139 群霍乱弧菌及其携带毒力基因开展快速检测.方法 采用三糖斜面、氧化酶实验、粘丝实验、无盐胨水等简单生化进行初筛,对全部结果阳性的菌株用聚合酶链反应(PCR)方法检测霍乱弧菌外膜蛋白OmpW以及毒力基因ctxA、hl...     

Vibrio cholerae O139 multiple-drug resistance mediated by Yersinia pestis pIP1202-like conjugative plasmids

A conjugative plasmid, pMRV150, which mediated multiple-drug resistance (MDR) to at least six antibiotics, including ampicillin, streptomycin, gentamicin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole, was identified in a Vibrio cholerae O139 isolate from Hangzhou, eastern China, in 2004. According to partial pMRV150 DNA sequences covering 15 backbone regions, the plasmid is most similar to pIP1202, an IncA/C plasmid in an MDR Yersinia pestis isolate from a Madagascar bubonic plague patient, at an identity of 99.99% (22,180/22,183 nucleotides). pMRV150-like plasmids were found in only 7.69% (1/13) of the O139 isolates tested during the early period of the O139 epidemic in Hangzhou (1994, 1996, and 1997); then the frequency increased gradually from 60.00% (3/5) during 1998 and 1999 to 92.16% (47/51) during 2000 to 2006. Most (42/51) of the O139 isolates bearing pMRV150-like plasmids were resistant to five to six antibiotics, whereas the plasmid-negative isolates were resistant only to one to three antibiotics. In 12 plasmid-bearing O139 isolates tested, the pMRV150-like plasmids ranged from approximately 140 kb to 170 kb and remained at approximately 1 or 2 copies per cell. High (4.50 × 10⁻² and 3.08 × 10⁻²) and low (0.88 × 10⁻⁸ to 3.29 × 10⁻⁵) plasmid transfer frequencies, as well as no plasmid transfer (under the detection limit), from these O139 isolates to the Escherichia coli recipient were observed. The emergence of pMRV150-like or pIP1202-like plasmids in many bacterial pathogens and nonpathogens occupying diverse niches with global geographical distribution indicates an increasing risk to public health worldwide. Careful tracking of these plasmids in the microbial ecosystem is warranted.     

Molecular Characterization of ISCR1-Mediated blaPER-1 in a Non-O1, Non-O139 Vibrio cholerae Strain from China

We report the detection of PER-1 extended-spectrum β-lactamase (ESBL) in a clinical non-O1, non-O139 Vibrio cholerae strain from China. ISCR1-mediated bla_PER-1 was embedded in a complex In4 family class 1 integron belonging to the lineage of Tn1696 on a conjugative IncA/C plasmid. A free 8.98-kb circular molecule present with the ISCR1-bla_PER-1–truncated 3′-conserved sequence (CS) structure was detected in this isolate. These findings may provide insight into the mobilization of bla_PER-1.     

产色素O139群霍乱弧菌环境分离株的亲缘关系分析

目的 筛检霍乱弧菌O139血清群产褐色色素的环境分离菌株并分析其遗传近缘关系。 方法 在LBA平板上检测了1992-2010年分离保存的全国1530株O139群霍乱弧菌的色素产生能力,对其中能够产生水溶性褐色色素的菌株的马尿酸氧化酶基因VC1345进行测序,并用脉冲场凝胶电泳(PFGE)、多基因序列分型分析色素产生菌株之间的亲缘关系。 结果 筛选到不同年份不同地区的16株产色素的非产毒O139群菌株。所有的色素产生菌株具有完全一致的VC1345基因、相似的PFGE带型,多基因序列分型显示仅有2种型别。 结论 分离到的O139群产色素菌株来源于近缘的克隆群,而这些菌株之间已存在一定程度的变异。在水体中的持续存在显示其较强的环境生存能力,这些特征提示在霍乱弧菌的环境监测中需提高对这类菌株的关注。     

O139霍乱弧菌的分子流行病学特征

利用ｃｔｘＡ，Ｚｏｔ，Ａｃｅ，ＲＳ１，ｓｔ和ｉｇａ等基因探针检测５７８株霍乱弧菌，发现其中１株埃尔托型霍乱弧菌和６株Ｏ１３９霍乱弧菌不含ｃｔｘＡ，进一步分析证明这７个菌株中，除１株含ＲＳ１序列外，其他毒力基因检测均为阴性，这为菌苗候选株的筛选提供了依据。用１６ＳｒＲＮＡ基因探针检测不同地区分离的６２株Ｏ１３９菌株和Ｏ－１群埃尔托型菌株，将其分为９个１６ＳｒＲＮＡ基因型。结合ｃｔｘＡ探针检测结果，认为我国流行的Ｏ１３９霍乱弧菌有两种类型，与印度、孟加拉国分离的菌株不尽相同。本文首次报导，通过１６ＳｒＲＮＡ基因检测，可区分Ｏ－１群埃尔托型霍乱弧菌与Ｏ１３９霍乱弧菌，并建议将ｃｔｘＡ、１６ＳｒＲＮＡ两个基因作为确定Ｏ１３９霍乱弧菌分子流行病学特征的指标。     

2株与霍乱弧菌O139诊断血清发生凝集的麦氏弧菌鉴定

目的对2株疑似O139群霍乱弧菌进一步鉴定,以排除或确诊霍乱。方法以试管凝集和交叉吸收实验复核玻片凝集结果,用生化实验和BIOFOSUN 微生物鉴定系统进行生化鉴定,PCR技术检测O139霍乱弧菌特异性脂多糖基因（LPS）。结果玻片凝集阳性,试管凝集1株菌可达原诊断血清效价的1/4,1株菌达1/2,交叉吸收后均证实为非特异性凝集;未检出LPS基因;系统生化鉴定判定为麦氏弧菌。结论2株菌不是O139霍乱弧菌,而是麦氏弧菌。