Multidrug resistant Vibrio cholerae O1 El Tor carrying classical ctxB allele involved in a cholera outbreak in South Western India

Cholera is a fatal diarrheal disease characterized with enormous fluid loss through stools. A total of 41 Vibrio cholerae isolates collected from a recent cholera outbreak in Solapur, South Western India were found to belong to serogroup O1, biotype El Tor and serotype Ogawa. Molecular analysis revealed the prevalence of different toxigenic and pathogenic genes in the isolates. All the isolates harboured rstR^ElTor allele indicating the presence of CTXΦ^ElTor. However, cholera toxin (ctxB) gene sequencing and a ctxB allele specific PCR of the isolates confirmed the presence of ctxB of classical biotype. The antibiogram profile revealed the resistance for several antibiotics including nalidixic acid, polymyxin B, streptomycin, sulfamethoxazole, trimethoprim, rifampicin and vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129). All the isolates were PCR positive for class 1 integron and SXT elements also. Fingerprinting analysis revealed the clonal relationship among the outbreak isolates. The results suggested the involvement of multidrug resistant V. cholerae El Tor biotype isolates having ctxB gene of classical biotype in the cholera outbreak.     

Plasmid mediated antibiotic resistance of Vibrio cholerae O1 biotype El Tor serotype Ogawa associated with an outbreak in Kolkata,India

ObjectiveTo determine the antibiotic resistance of Vibrio cholerae (V. cholerae) O1 biotype El Tor serotype Ogawa isolates involved in an outbreak of watery diarrhea in Kolkata, and to explore the role of plasmid in mediating antibiotic resistance.MethodsAntibiotic susceptibility and minimum inhibitory concentration (MIC) values of antibiotics for the isolated V. cholerae O1 Ogawa (n=12) were determined by disk diffusion and agar dilution methods, respectively, using ampicillin (Am), chloramphenicol (C), trimethoprim (Tm), tetracycline (T), erythromycine (Er), nalidixic acid (Nx), ciprofloxacin (Cp), amikacin (Ak) and cefotaxime (Cf). Plasmid curing of multidrug resistant (MDR) V. cholerae O1 Ogawa strains was done following ethidium bromide treatment. Following electrophoresis, the plasmid DNAs, extracted from the isolated MDR V. cholerae O1 Ogawa strains and their cured derivatives, were visualized and documented in ‘gel doc’ system.ResultsThe outbreak causing V. cholerae O1 Ogawa isolates were MDR as determined by disk diffusion susceptibility test, and MIC determination. The isolates showed three different drug resistance patterns: AmTmTErNx (for 6 isolates), TmTErCp (for 5 isolates), and AmTmNx (for one isolate), and showed uniform sensitivity to C, Ak and Cf. The loss of plasmids with the concomitant loss of resistance to Am, Tm, T and Er of the isolates occurred following ethidium bromide treatment.ConclusionsThe current findings suggest that the V. cholerae O1 Ogawa associated with the cholera outbreak were MDR, and resistance to Am, Tm, T and Er among the isolates were plasmid mediated.     

Emergence of Vibrio cholerae O1 biotype El Tor serotype Inaba causing outbreaks of cholera in Orissa, India

A total of 431 rectal swabs, collected from acute diarrheal cases at a surveillance site and at different diarrheal outbreak areas of Orissa from May to October 2005, were bacteriologically analyzed. Out of 265 culture-positive samples, Vibrio cholerae O1 was isolated in 56 samples (20.8%), of which 37 were the Inaba serotype and 19 were the Ogawa. The antibiogram profile revealed that all the V. cholerae O1 Ogawa and Inaba serotypes were uniformly sensitive to ampicillin, chloramphenicol, gentamicin, ciprofloxacin, norfloxacin and tetracycline. The V. cholerae O1 Inaba serotypes were resistant to furazolidone and nalidixic acid, while the Ogawa strains were resistant to furazolidone, nalidixic acid and neomycin. The multiplex polymerase chain reaction (PCR) assay on some selected strains of both serotypes revealed that all the strains were positive for ctxA and tcpA genes showing biotype El Tor. The present study revealed the emergence of V. cholerae O1 biotype El Tor serotype Inaba, which caused sporadic outbreaks of cholera in 2005. The outbreaks of diarrheal disorders in one geographical area of the state (in the Pattamundai area, Kendrapara district) in 2005 were due to V. cholerae O1 Ogawa, whereas the other outbreaks in other areas (Puri, Khurda and Dhenkanal districts) from August to October 2005 were due to V. cholerae O1 serotype Inaba. This is the first report that an emergence of V. cholerae O1 serotype Inaba caused sporadic outbreaks of cholera in different parts of Orissa. Switching over of V. cholerae O1 Ogawa strains to Inaba, causing diarrheal outbreaks in Orissa, needs close monitoring.     

Isolation of Vibrio cholerae O139 from the drinking water supply during an epidemic of cholera

In mid 1994, the public water supply was investigated in a medium-sized town in south India during an epidemic of cholera due to Vibrio cholerae O139. Vibrio cholerae O139 was isolated from the public water supply including one of the wells supplying the town, the central overhead tank, and domestic taps connected to the public supply. Following chlorination, the organism was no longer isolated from the water supply and the epidemic subsided. This demonstration of V. cholerae O139 in the drinking water supply of a town underlines the need for adequate treatment of the water supply.     

Vibrio cholerae O139: How great is the threat of a pandemic?

The emergence of the new strain Vibrio cholerae O139 and its rapid spread in Bangladesh and India together with its detection in several other countries, have raised the question whether this constitutes the beginning of the eighth pandemic of cholera, and if so, how large a threat it poses. In an attempt to answer this question, epidemic spread patterns of Vibrio cholerae O139 strain in Bangladesh were studied. Initially the epidemic moved quickly and affected the entire coastal and estuarine tidal plains of southern Bangladesh. In the flood plains of the northern regions it affected mostly the north‐eastern and north‐central areas, at a slower pace than in the southern areas. In the beginning the new strain totally displaced both biotypes (classic and El Tor ) of Vibrio cholerae O1. Nearly 2 years after its initial detection, striking differences in the distribution of V. cholerae O139 and O1 were observed. In most northern areas, the new strain was replaced by V. cholerae O1, whereas in the southern coastal regions, the O139 strain continues to dominate epidemics. The study suggests that the O139 strain may become endemic in the coastal ecosystem. The threat of a pandemic, therefore, may not be as large as it first seemed.     

福建省非典型O1群埃尔托型霍乱弧菌的研究

目的 探索非典型O1群埃尔托型霍乱弧菌在福建省1962-2005年霍乱菌株中的存在及其意义。方法 选择1962-2005年代表性霍乱菌株69株(稻叶21、小川48),运用PCR技术进行ctxB、tcpA、rstR、hlyA 各毒力因子的古典型(Cl)和埃尔托型(El)基因的扩增;同时对部分菌株的ctxB 和rstR 基因的序列进行测定和比对;分析福建省O1群埃尔托型霍乱弧菌(EVC)中非典型菌株出现的时间和存在意义。结果 1962年福建省分离的O1群EVC菌株中携带有ctxB-Cl、tcpA-Cl、hlyA-Cl基因;rstR-Cl基因仅在1994-2000年的霍乱菌株中检出。序列比对结果发现ctxB 基因出现新的204位碱基突变位点(T→G);同时报道两种新的ctxB 基因型别(10和11),且发现存在于同一个菌株中;ctxB基因型11的115位碱基为C,表现为古典型ctxB 位点特征,203位碱基为T,表现为埃尔托型ctxB 位点特征。结论 福建省在1962年的O1群EVC菌株中就出现杂合有ctxB-Cl、tcpA-Cl、hlyA-Cl基因的非典型EVC菌株,是迄今为止报道出现非典型EVC最早的年代;1994年以后出现整合有rstR-Cl基因的非典型EVC菌株。另外,在福建省O1群EVC中存在国内外未曾报道的新的ctxB 碱基突变位点和ctxB 基因型别;同时在同一菌株中存在不同的ctxB 基因型别杂合形式;ctxB 序列也存在不同生物型别的特异位点组合形式。福建省O1群EVC表现为独特的地方性分子特征。     

福建省不产毒O1群霍乱弧菌分子特征分析

目的 调查福建省近年从病人和外环境中分离到的不产毒O1群霍乱弧菌的毒力基因分布特征,分析菌株间的遗传相似度。方法 运用PCR扩增技术分别检测15株不产毒O1群霍乱弧菌的毒力相关基因tcpA、rstR、hlyA、zot、ace、toxR、ctxA,并通过脉冲肠凝胶电泳(PFGE)技术进行分子分型。结果15株不产毒O1群霍乱弧菌各毒力相关基因的阳检数分别为hly-ET(8/15)、tcpA-CL(6/15)、hly-Cl(4/15)、ace(3/15)、toxR(3/15),其余毒力基因均为阴性。按照100%的相似度,PFGE分为12个基因型别,无集中优势的PFGE型别;根据TENOVER原则有两个相对优势的G1、G2 PFGE群。结论 不产毒的O1群霍乱菌株不同程度地携带有相关的毒力因子,病人株和环境株毒力基因分布无明显差异,不产毒O1群霍乱菌株存在基因组多态性。     

Epidemics of severe cholera caused by El Tor Vibrio cholerae O1 Ogawa possessing the ctxB gene of the classical biotype in Orissa,India

BackgroundWe investigated the epidemic of cholera that occurred in Kashipur and Dasmantpur blocks of Orissa, reported during July–September 2007.MethodsSixty-two rectal swabs and 28 water samples collected from diarrhea patients at different hospitals and villages were bacteriologically analyzed for the identification, antibiogram, and detection of toxic genes of Vibrio cholerae.ResultsThe cholera outbreaks were caused by V. cholerae O1 Ogawa biotype El Tor in both Kashipur and Dasmantpur blocks. All the V. cholerae isolates from the clinical and environmental samples were sensitive to tetracycline, gentamicin, azithromycin, and chloramphenicol, but were resistant to ampicillin, ciprofloxacin, norfloxacin, co-trimoxazole, nalidixic acid, neomycin, and furazolidone, except the water isolates, which were sensitive to ciprofloxacin and norfloxacin. The multiplex PCR assay revealed that all the clinical and environmental V. cholerae isolates were positive for the ctxA and tcpA genes, showing biotype El Tor. Interestingly, 88% of the clinical and environmental isolates of V. cholerae were El Tor biotype with mutation at the ctxB gene of the classical strain, as confirmed by mismatch amplification of mutation (MAMA)-PCR assay.ConclusionsThis is the first report of the El Tor variant of V. cholerae O1 Ogawa having the ctxB gene of the classical strain with altered antibiogram causing epidemics of cholera in Orissa, India.     

Plasmid-borne multiple drug resistance in Vibrio cholerae serogroup O1, biotype El Tor: evidence for a point-source outbreak in Bangladesh

In 1979, an outbreak of plasmid-borne, multiply drug-resistant Vibrio cholerae serogroup O1 biotype El Tor (V. cholerae O1) occurred in the Matlab area of Bangladesh. The outbreak could have resulted from the introduction into the area of a single resistant strain or from multiple conjugations of drug-sensitive V. cholerae O1 with C plasmids in other environmental flora. Resistant strains were phage typed to determine their relatedness, and plasmid studies were conducted to determine the frequency of C plasmids in nonvibrio flora of family contacts of cholera patients. Forty-one (85%) of 48 resistant strains of V. cholerae O1 examined belonged to two closely related phage types new to the area, whereas 59 drug-sensitive strains from the same period were primarily of two different phage types. Group C plasmids were in nonvibrio strains from five of 36 family contacts of patients with drug-resistant cholera but none of 82 family contacts of patients with sensitive cholera. This outbreak most likely began from the introduction into the area of a single, multiply drug-resistant strain of V. cholerae O1. C plasmids detected in the nonvibrio flora of family contacts probably came from the resistant strain of V. cholerae O1.     

Molecular characterization of Vibrio cholerae O1 outbreak strains in Miri,Sarawak (Malaysia)

Bacterial resistance to various antimicrobial agents is common in area with high usage of antibiotics. In this study, the data on antimicrobial susceptibility patterns of Vibrio cholerae O1 from patients during an outbreak period was found to be high but variable rates of multidrug resistance. Thirty-two of 33 V. cholerae isolates harboured the tcp, ctx, zot and ace genes, suggesting their possible roles in the outbreak cases. We analyzed the molecular diversity of a total of 33 strains of V. cholerae O1 isolated from 33 patients between November 1997 and April 1998 using random amplified polymorphic DNA (RAPD) analysis. The 30 typable isolates could be separated into four major clusters containing 5, 17, 2 and 6 isolates, respectively. However, no particular RAPD pattern was predictive of a particular pattern of antibiotic susceptibility. The findings of this study showed that multiple clones seemed to be responsible for cases in the outbreaks in the study area.     

Emergence of Vibrio cholerae O1 Biotype El Tor serotype Inaba in north India

All cases of cholera that have occurred at our center in north India have been due to Vibrio cholerae O1 serotype Ogawa, including the outbreaks in 2002 and 2004. Here we report the emergence of V. cholerae O1 biotype El Tor serotype Inaba for the first time in this region since July 2004. Fifteen Inaba isolates were obtained from 32 patients suffering from cholera-like illness. The patients lived in Chandigarh and the neighboring states of Punjab, Haryana, and Himachal Pradesh. All strains were resistant to nalidixic acid and trimethoprim, and showed moderate sensitivity to amoxycillin. All were sensitive to ciprofloxacin, tetracycline, cefotaxime, amikacin, and gentamicin. All strains were found to be toxigenic when tested with a commercial reverse passive latex agglutination kit. The last reported Inaba isolate dominance in India was observed in Calcutta in 1989. There is a need to closely watch the spread of serotype Inaba, as it may cause outbreaks in other parts of India; molecular studies are warranted to understand the widespread emergence of Inaba in north India.     

Acquisition of classical CTX prophage from Vibrio cholerae O141 by El Tor strains aided by lytic phages and chitin-induced competence

The El Tor biotype of Vibrio cholerae O1, causing the current seventh pandemic of cholera, has replaced the classical biotype, which caused the sixth pandemic. The CTX prophages encoding cholera toxin in the two biotypes have distinct repressor (rstR) genes. Recently, new variants of El Tor strains that carry the classical type (CTX(class)) prophage have emerged. These "hybrid" strains apparently originate through lateral gene transfer and recombination events. To explore possible donors of the CTX(class) prophage and its mode of transfer, we tested environmental V. cholerae isolates for the presence of CTX(class) prophage and mobility of the phage genome. Of the 272 environmental V. cholerae isolates tested, 6 were found to carry the CTX(class) prophage; all of these belonged to the O141 serogroup. These O141 strains were unable to produce infectious CTX(class) phage or to transmit the prophage to recipient strains in the mouse model of infection; however, the CTX(class) prophage was acquired by El Tor strains when cultured with the O141 strains in microcosms composed of filtered environmental water, a chitin substrate, and a V. cholerae O141-specific bacteriophage. The CTX(class) prophage either coexisted with or replaced the resident CTX(ET) prophage, resulting in El Tor strains with CTX genotypes similar to those of the naturally occurring hybrid strains. Our results support a model involving phages and natural chitin substrate in the emergence of new variants of pathogenic V. cholerae. Furthermore, the O141 strains apparently represent an alternative reservoir of the CTX(class) phage genome, because the classical V. cholerae O1 strains are possibly extinct.     

霍乱弧菌O1群和O139群nhaA基因的克隆和变异性研究

目的 克隆霍乱弧菌O1群和O139群nhaA基因，并分析其变异性。方法收集我国1988—2000年散发霍乱弧菌O1群和O139群40株，用聚合酶链反应(PCR)扩增nhaA基因，克隆至pcDNA3载体，通过序列测定分析其同源性和变异性。结果 分别从霍乱弧菌Ol群和O139群扩增出约1．2kb的nhaA基因片段，基因序列分析表明，我国霍乱散发O1群和O139群的nhaA基因与Genbank中霍乱弧菌O1群野毒株参考序列同源性分别为99％和96％。编码氨基酸的突变率分别为2％和11％，nhaA基因重要的结构和功能决定簇(Aspl33、Aspl63、Aspl64、His225、Leu73和Gly338)未发生变异；第203、221位的氨基酸，O1群和O139群发生相同的变异。结论 nhaA基因编码氨基酸的变异可能是霍乱弧菌适应外环境变化的结果。     

杭州市健康人群粪便中检出O1和O139群霍乱弧菌无毒力株

目的　对杭州市饮食、服务行业人员粪便标本进行霍乱弧菌监测。方法　 2 0 0 0年 5月～ 2 0 0 1年 7月采集 2 6 2 5 2名饮食、服务行业从业人员健康体检者粪便标本 ,分别接种于碱性蛋白胨水和 4号琼脂中。通过培养特性、菌落形态、革兰染色、动力和生化试验等检查 ,对所分离的疑似霍乱弧菌菌株进行初步鉴定。采用霍乱弧菌O1群及O139群单克隆抗体的玻片凝集试验、噬菌体分型、霍乱弧菌ctxA和tcpA基因的PCR进一步鉴定各疑似霍乱菌株。 结果　上述标本中检出O1血清群和O139血清群霍乱弧菌各 2株。 2株O1血清群霍乱弧菌的噬菌体分型分别为 19k和 5k ,为埃尔托生物型非流行菌株 ;2株O139血清群霍乱弧菌的噬菌体分型分别为 2 0k和 31k ;但ctxA和tcpA基因PCR检测结果均为阴性。结论　尽管所检出的 4株霍乱弧菌均为无毒力的非流行菌株 ,但因携带者从事职业的特殊性 ,仍应引起高度重视。     

O139,O1群霍乱弧菌分子遗传特征研究

目的探讨Ｏ１３９群、Ｏ１群霍乱弧菌分子遗传特征及其相互关系。方法用聚合酶链反应（ＰＣＲ）、ＤＮＡ序列分析及随机扩增多态性ＤＮＡ（ＲＡＰＤ）,检测了３株Ｏ１３９群、３株Ｏ１群ＥｌＴｏｒ生物型和２株古典生物型霍乱弧菌。结果３株Ｏ１３９群和５株Ｏ１群霍乱弧菌均扩增出５６６ｂｐ的ＤＮＡ片段,ＤＮＡ序列源于霍乱肠毒素（ｃｔｘ）Ａ２Ｂ基因。ＲＡＰＤ产生的两种ＤＮＡ指纹图谱一致显示Ｏ１３９群与Ｏ１群ＥｌＴｏｒ生物型遗传特征极其相似,与古典生物型略有区别。结论Ｏ１３９群与Ｏ１群ＥｌＴｏｒ型存在密切的种系进化关系,前者可能由Ｏ１群ＥｌＴｏｒ生物型进化而来。     

我国主要沿海地区O1群霍乱弧菌分子分型特征分析

目的 采用MLST方法及基质辅助激光解析电离飞行时间质谱(Matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF MS)技术研究我国主要沿海地区192株O1群霍乱弧菌的种群结构、进化趋势及流行特性.方法...     

An outbreak of El Tor cholera associated with a tribal funeral in Irian Jaya, Indonesia.

An outbreak of El Tor biotype cholera occurring in a rural village in Irian Jaya, Indonesia was evaluated for risk factors associated with death from cholera. Among those dying in the village during the epidemic, a significant association between membership in one of the five tribal groups in the village complex was associated with an elevated risk of suffering a cholera death (odds ratio = 5.9). Interviews with members of the decedents' families revealed a very strong association (odds ratio = 11.6) between risk of cholera death and having attended the two day funeral of a woman who died of a cholera-like illness a few days prior to an outbreak of cholera-like diarrheal disease in the village complex. Recent flooding may have contributed to the creation of an environment conducive to cholera transmission.