2018-2021年湖南省人源非O1/非O139群霍乱弧菌药物敏感性及基因组特征

目的 了解2018—2021年间湖南省从病例分离到的非O1/非O139群霍乱弧菌的药物敏感性及基因组特征。方法 选取2018—2021年间湖南省腹泻病例肠道标本分离的非O1/非O139群霍乱弧菌分离株4株和非腹泻病例血液标本分离的非O1/非O139群霍乱弧菌分离株3株,进行药物敏感性测试以及基因组序列测定,分析其药物敏感性及基因组特征。结果 3株血液样品分离株对氨苄西林、诺氟沙星、头孢曲松、美罗培南、米诺环素、复方磺胺甲恶唑、庆大霉素、头孢噻肟、萘啶酸、环丙沙星全部敏感,1株对四环素中介,1株对氯霉素耐药;4株粪便样品分离株对诺氟沙星、头孢曲松、美罗培南、米诺环素、复方磺胺甲恶唑、庆大霉素、头孢噻肟全部敏感,1株对四环素、氯霉素中介,1株对氯霉素耐药,2株对氨苄西林、环丙沙星、萘啶酸耐药;其中1株对氨苄西林、萘啶酸、环丙沙星二重耐药;1株对氨苄西林、萘啶酸、环丙沙星、氯霉素多重耐药。7株非O1/非O139群霍乱弧菌的基因组序列表现出明显的多样性,但是部分菌株在核心基因组和泛基因组都高度相似。基因组序列中均未检出ctxAB基因、VPI-1和VSPII基因组岛。分离自粪便的菌株携带耐药基因...     

一起引起食物中毒的非O1群霍乱弧菌实验室鉴定

目的 查明引发浙江省宁波市某服饰公司食物中毒事件的病原菌.方法 对采集到可疑食品和肛拭等标本参照GB/T4789-2003标准进行细菌的分离、鉴定及血清学分型;参照霍乱防治手册检测菌株的CT毒力基因.结果 6份患者大便标本与14份轻微腹泻患者或腹部不适者大便标本中检出非O1群霍乱弧菌O29血清型9株,其中腹泻患者标本检出6株,轻微腹泻患者中检出3株,检出率为45.0%.未检出志贺菌、副溶血性弧菌、致病性大肠杆菌、O1群和O139霍乱弧菌、金黄色葡萄球菌等致病菌,剩余食物未检出志贺菌、副溶血性弧菌、致病性大肠杆菌、O1群和O139霍乱弧菌、金黄色葡萄球菌等致病菌.结论 此次食物中毒为非O1群霍乱弧菌O29血清型所致.     

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。     

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。     

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...     

霍乱弧菌中VSP-Ⅰ岛的分布和变异特征研究

目的 探讨霍乱第7次大流行基因岛1(VSP-Ⅰ)在霍乱弧菌中的分布和变异特征. 方法 从美国国立生物技术信息中心的GenBank数据库中下载所有霍乱弧菌基因组数据,以霍乱第7次大流行代表菌株N16961的VSP-Ⅰ作为参考序列,筛查所下载霍乱弧菌基因组中的VSP-Ⅰ.使用MUMmer软件进行单核苷酸多态性(SNP)分析,OrthMCL软件进行同源基因分析,BLAST软件进行序列结构变异分析和功能注释. 结果 VSP-Ⅰ广泛存在于霍乱弧菌中,其中霍乱第7次大流行的105株菌株中均携带VSP-Ⅰ,且其序列高度保守;其中94株菌的VSP-Ⅰ完全一致,仅在11株菌中分别存在1~14个SNP位点.而2株非O1非O139群菌株携带VSP-Ⅰ,但和大流行菌株相比,存在插入缺失等序列变异.同时,VSP-Ⅰ有9个共有保守基因, VC0183 虽在所有菌株中均存在,但在2株非O1非O139菌株中长度发生改变.此外,VSP-Ⅰ携带5个与该岛的转移和整合相关的基因(VC0181~VC0185), VC0178 编码马铃薯糖蛋白类似物, VC0180 编码蛋白水解酶,二者可能与毒力相关. 结论 作为霍乱第7次大流行菌株中的特异性基因簇,VSP-Ⅰ已转移至非O1非O139群菌株中,并发生序列的变异.     

我国霍乱弧菌毒力协同调节菌毛基因序列分析

目的对我国霍乱弧菌（VC）毒力协同调节菌毛（tcpA）基因进行序列分析.方法 38株VC的tcpA基因经聚合酶链反应扩增、测序和序列分析.结果 13株O139群VC产毒株和15株EVC产毒株（均为流行株）的tcpA类型与EVC标准株N16961为同一类型,共有4个位点碱基发生变异,分为5个亚型.EVC流行株中2 株非产毒株的tcpA类型,1株同N16961,1株为新类型.6株EVC非流行株均为非产毒株,分为2种新类型.2株非O1非O139群VC均为非产毒株,为2种新类型.4种新类型与国外4种类型基因和氨基酸序列比较,同源性为63.8%～84.6%和60.6%～91.7%,趋异性为17.8%～44.6%和8.8%～45.8%,tcpA基因所在的VPI毒力岛的结构是完整的,但部分基因存在一定变异.结论 O139群VC产毒株可能起源于EVC产毒株,然后再进一步分化;在非产毒VC中发现4种新类型tcpA基因.     

Distribution and content of class 1 integrons in different Vibrio cholerae O-serotype strains isolated in Thailand

In this study, 176 clinical and environmental Vibrio cholerae strains of different O serotypes isolated in Thailand from 1982 to 1995 were selected and studied for the presence of class 1 integrons, a new group of genetic elements which carry antibiotic resistance genes. Using PCR and DNA sequencing, we found that 44 isolates contained class 1 integrons harboring the aadB, aadA2, blaP1, dfrA1, and dfrA15 gene cassettes, which encode resistance to gentamicin, kanamycin, and tobramycin; streptomycin and spectinomycin; beta-lactams; and trimethoprim, respectively. Each cassette array contained only a single antibiotic resistance gene. Although resistance genes in class 1 integrons were found in strains from the same epidemic, as well as in unrelated non-O1, non-O139 strains isolated from children with diarrhea, they were found to encode only some of the antibiotic resistance expressed by the strains. Serotype O139 strains did not contain class 1 integrons. However, the appearance and disappearance of the O139 serotype in the coastal city Samutsakorn in 1992 and 1993 were associated with the emergence of a distinct V. cholerae O1 strain which contained the aadA2 resistance gene cassette. A 150-kb self-transmissible plasmid found in three O1 strains isolated in 1982 contained the aadB gene cassette. Surprisingly, several strains harbored two integrons containing different cassettes. Thus, class 1 integrons containing various resistance gene cassettes are distributed among different V. cholerae O serotypes of mainly clinical origin in Thailand.     

广州市一起非O1/O139群霍乱弧菌食物中毒分离株的病原特征分析

目的 了解2014年广州市某工厂发生的一起非O1/O139群霍乱弧菌食物中毒分离株的耐药特性及分子特征。方法 对分离到的6株非O1/O139群分离株进行生化鉴定、血清学鉴定、药物敏感性实验、毒力相关基因检测和脉冲场凝胶电泳（PFGE）分子分型分析。结果 6株分离株经生化鉴定、血清学鉴定为非O1/O139群。药物敏感性分析显示,6株分离株均对氨苄西林、头孢克肟、头孢拉定、阿米卡星、妥布霉素、庆大霉素和多粘菌素B耐药,对诺氟沙星、环丙沙星、复方新诺明、呋喃唑酮、四环素、吡哌酸、红霉素和氯霉素敏感。毒力基因聚合酶链反应检测结果显示,所有菌株均携带毒力表达调控基因（toxR）和溶血素基因（hlyA）,未检出霍乱肠毒素基因（ctxA）、毒力协同调节菌毛基因（tcpA）和肠毒素基因（ST）;5株病例分离株和1株冷柜内壁分离株经限制性内切酶NotⅠ消化后的PFGE图谱为同一型别。结论 此次食物中毒的病原体为非O1/O139群霍乱弧菌,具有共同的遗传特征,菌株出现了多重耐药,提示应加强该类菌株的监测,防止大范围扩散或暴发流行。     

Occurrence of antibiotic resistance gene cassettes aac(6')-Ib,dfrA5, dfrA12, and ereA2 in class I integrons in non-O1, non-O139 Vibrio cholerae strains in India

Molecular mechanisms of multidrug resistance in Vibrio cholerae belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were dfrA1, dfrA15, dfrA5, and dfrA12 for trimethoprim; aac(6')-Ib for amikacin and tobramycin; aadA1 and aadA2 for streptomycin and spectinomycin; and ereA2 for erythromycin resistance. To our knowledge, this is the first report of the presence of dfrA5, dfrA12, aac(6')-Ib, and ereA2 cassettes in class I integrons of V. cholerae. Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance.     

Territorial waters of the Baltic Sea as a source of infections caused by Vibrio cholerae non-O1, non-O139: report of 3 hospitalized cases

A fatal infection with temporal relation to 2 other febrile infections caused by Vibrio cholerae non-O1, non-O139 (NCV) occurred in Finland in 2003. All infections were associated with contact with seawater. The patient who died had also eaten home-salted whitefish, tested positive for NCV, preceding his symptoms. All patients had compromising factors, and all strains were distinguishable by pulsed-field gel electrophoresis and negative for the ctx gene. These 3 cases illustrate that, despite being uncommon in Finland, NCVs can cause clinically significant and even fatal infections.     

Molecular epidemiological studies of Vibrio cholerae in Bengal region

Vibrio cholerae isolates from environmental and clinical origins in the Bengal region in which epidemics of cholera break out periodically were analyzed with particular emphasis on the molecular epidemiological features. The presence of the virulence genes (ctxA, tcpA and toxR) in the isolates was analyzed by the PCR (polymerase chain reaction) method. PFGE (pulsed-field gel electrophoresis) was performed to determine the clonal relationships between the clinical and environmental strains. Antibiograms and O serovars of the isolates were also examined. O1 and O139 strains from both clinical and environmental sources were all positive for the three virulence genes while non-O1/non-O139 strains from both sources were all negative for ctxA and tcpA but positive for toxR. PFGE patterns of recent isolates of O1 and O139 were similar in each serovar regardless of origin, suggesting a clonal relationship between the clinical and environmental strains, although comparison with past isolates or isolates from different geographical area showed some differences.     

1例白血病患者血液中检出非O1非O139群霍乱弧菌

2012年8月,浦东新区儿童医学中心从1名6岁急性淋巴细胞性白血病复发患儿血液中分离到霍乱弧菌,随后浦东新区疾病预防控制中心对该分离株进行了系统生化鉴定、毒力基因及药物敏感性检测,结果为不产霍乱毒素的非O1非O139霍乱弧菌。     

2015－2017年北京市顺义区食源性疾病监测腹泻病例霍乱弧菌感染及病原学特征分析

目的研究食源性疾病监测腹泻患者霍乱弧菌感染特点及病原学特征，为科学防控和临床治疗提供依据。方法从2015－2017年北京市顺义区腹泻监测病例的粪便标本中分离培养霍乱弧菌，针对分离菌株进行毒力基因聚合酶链式反应（PCR）检测、脉冲场凝胶电泳（PFGE）、基质辅助激光解析电离飞行时间质谱（MALDI-TOF MS）图谱分析以及抗生素敏感性分析。结果2015－2017年，共收集1 105例腹泻患者，霍乱弧菌的检出率为0.54%（6/1 105），霍乱毒素基因ctxAB均为阴性，血清学分型为非O1/O139型，其中5株霍乱弧菌携带Ⅲ型分泌系统毒力基因。 菌株PFGE聚类分析结果和MALDI-TOF MS图谱聚类结果具有较好的一致性。结论霍乱弧菌流行强度不高，但其感染和病原学监测工作应在食源性疾病监测体系中得到重视。     

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.     

抗O／129的非O1群霍乱弧菌分子生物学特征研究

为了从遗传学基础上，探索抗Ｏ／１２９的非Ｏ１群霍乱弧菌的基因结构的特点，即对１９９２～１９９４年从肠道门诊急性腹泻患者粪便中分离到的２１株抗Ｏ／１２９的非Ｏ１群霍乱弧菌的分子生物学特性进行了研究。结果显示：染色体ＤＮＡ经核酸限制性内切酶ＰｓｔＩ消化后，用琼脂糖凝胶电泳分离ＤＮＡ，全部菌株表现为多样化特征的限制性酶谱；用Ｄｉｇ标记的基因探针做ｓｏｕｔｈｅｒｎｂｌｏｔ杂交，无一株菌具有Ｏ１群霍乱弧菌流行株所具有的ＣＴ毒素基因；质粒图谱分析，仅有６株菌各含有一条质粒带，而且这６株菌同时对３～８种临床常用抗生素耐药，表明该菌株对Ｏ／１２９的抗性是非质粒性的。结果表明，抗Ｏ／１２９的非Ｏ１群霍乱弧菌与Ｏ１群霍乱弧菌非流行株具相似的遗传学特性和毒力特征，因而，它不是引起霍乱暴发的病原体。     

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。     

广东省霍乱病例与环境来源霍乱弧菌的

目的比较分析广东省霍乱病例及环境来源O1/O139群霍乱弧菌抗生素敏感性和分子分型特征。方法选取2006-2007年广东省霍乱病例及相关来源、环境（珠江水体和海水产品）来源的O1/O139群霍乱弧菌。采用血清学、药物敏感性试验和分子生物学方法,研究不同来源的霍乱弧菌在菌型分布、药物敏感性、毒素基因携带以及分子分型方面的异同。结果2006-2007年,广东省共分离各类来源O1/O139群霍乱弧菌170株。其中,病例及相关来源菌株37株,环境来源菌株133株（海水产品来源37株、珠江水体96株）。两种来源菌株的菌型构成均以O1群El Tor稻叶型为主;病例及相关来源菌株以产毒株为主,ctxA毒素基因携带率（83.8%）显著高于环境菌株（4.5%）;药物敏感性试验显示,以产毒株为主的病例及相关来源菌株对萘啶酸和复方新诺明的耐药率（78.8%,78.8%）高于以非产毒株为主的环境来源菌株（50.6%,13.9%,P0.05）。环境来源菌株对多西环素的耐药率（17.7%）高于病例及相关来源菌株（0%,P0.05）。O139群菌株对氨苄西林的耐药率（70%）高于稻叶型和小川型菌株（8.9%,0%,P0.01）。脉冲场凝胶电泳（PFGE）聚类分析显示,O139群霍乱弧菌产毒株之间,O1群霍乱弧菌流行株之间以及其他的O1/O139群霍乱弧菌之间,PFGE型别表现为明显的遗传多样性。结论广东省O1/O139群霍乱弧菌来源复杂多样,霍乱防控形势严峻,需要密切注意菌株型别变异情况及菌株变异趋势。     

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。     

1株致放化疗患者口腔感染霍乱弧菌的鉴定和病原特征分析

摘要：目的?对1例食管癌术后放化疗患者口腔感染的霍乱弧菌进行菌株鉴定和病原特征分析。方法?取患者口腔分泌物进行分离培养并观察细菌形态特征，用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行蛋白质水平鉴定，并进行血清群检测；用K-B纸片扩散法进行药敏试验；提取细菌基因组DNA构建文库并进行细菌基因组重测序，通过核糖体多位点序列分型(rMLST)、平均核苷酸相似度(ANI)分析和非冗余蛋白质库(NR)比对，对菌株进行分子生物学鉴定，并进一步对其MLST分子分型和毒力基因进行分析。结果?MALDI-TOF MS鉴定分值为2.151，rMLST分析显示该菌株与霍乱弧菌吻合度为100%，ANI分析显示ANIb值为96.05%，ANIm值为96.56%，NR数据库基因注释得到霍乱弧菌相关的基因2 981个，血清群检测为非O1/O139群霍乱弧菌。药敏试验结果显示该菌对哌拉西林/他唑巴坦、氨曲南、头孢哌酮/舒巴坦、头孢他啶、头孢吡肟、庆大霉素、阿米卡星、左氧氟沙星、环丙沙星、亚胺培南、美罗培南均敏感。MLST显示该菌株为新的ST型(ST-986)，且携带toxR、hlyA、nanH、hapA、mshA、ompU、rtxC以及T6SS等毒力基因，而未检出ctxAB、tcpA、zot、ace及st等5种毒力基因。结论?检出ST-986新型非O1/O139群霍乱弧菌。临床医生应警惕免疫力低下人群中口腔感染这种病原体的可能性。