Non-O1 Vibrio in acute diarrhea of the infant

The choleriform diarrhoea may be caused by Vibrio cholerae, but also by other Vibrionaceae exhibiting the cholera-toxin antigenic determinants. The authors report three instances of gastroenteritis in infants, caused by 3 strains of non-O1 Vibrio and they carry out bacteriological study on these strains and their pathogenicity-strength factors.     

Cloning and sequence of a region of Vibrio cholerae O139 Bengal and its use in PCR-based detection.

We isolated and characterized a Vibrio cholerae O139 Bengal-specific DNA region by arbitrary PCR. The fragment contains open reading frames encoding two potential glycosyltransferases possibly involved in capsular polysaccharide or lipopolysaccharide biosynthesis. In order to evaluate the possibility that this region could be used for the specific detection of V. cholerae O139 Bengal, a PCR system was established. The specificity and sensitivity of the PCR were investigated by analyzing 240 strains within the family Vibrionaceae and 178 stains of other gram-negative bacteria. All V. cholerae O139 Bengal strains tested were positive, and none of the 384 control strains were amplified. The sensitivity of the assay was 10(2) CFU/ml.     

Vibrionaceae from cases of acute diarrhoea and their antimicrobial sensitivity pattern - a five year prospective study

Over a five year period, stool samples were screened for Vibrionaceae from cases of acute diarrhoea, to study their isolation rate and their antimicrobial sensitivity pattern. All the isolates were identified by standard laboratory techniques. A total of 323 species belonging to Vibrionaceae were isolated from 4492 stool samples tested over five year period (1996-2000), giving a positivity rate of 7.2%. Maximum isolation was during the months of May to August (62.5%). Out of 323 isolates, Vibrio spp. comprised 252 and 93.3% of them were Vibrio cholerae O1 biotype El Tor. Aeromonas spp. were isolated from 71 samples and 64.8% of them were A. hydrophila. V. cholerae showed 86.8% sensitivity to amikacin followed by 73.8% to cefotaxime. Tetracycline sensitivity was only 39.6%. Aeromonas spp. also showed maximum sensitivity to amikacin (70.4%). Isolation of Vibrio spp. have increased over the years, whereas Aeromonas spp. have decreased. Amikacin sensitivity has remained within 70-80% over the years, cefotaxime sensitivity has increased and tetracycline sensitivity has decreased.     

Evaluation of DNA probes for specific detection of Vibrio cholerae O139 Bengal.

Two DNA probes, 2R1 and 2R3, prepared from a region in the chromosome specific for the lipopolysaccharide O side chains of Vibrio cholerae O139 (M.K. Waldor and J.J. Mekalanos, Lancet 343:1366, 1994) were examined for their specificity and sensitivity. Both probes did not hybridize with any strain of V. cholerae belonging to serogroups other than O139 and to any of the other species examined belonging to the family Vibrionaceae. Among the 126 strains of V. cholerae O139 examined, probe 2R1 hybridized with 125 strains while probe 2R3 hybridized with all 126 strains. Both probes were found to be highly specific and sensitive and can be used for the specific identification of V. cholerae O139.     

Changing patterns of Vibrio cholerae in sevagram between 1990 and 2005

PURPOSE: A retrospective analysis was done to note changes in prevalence, distribution of biotypes, serotypes, antibiotic susceptibility patterns and phage types of Vibrio cholerae isolated in Mahatma Gandhi Institute of Medical Sciences, Sevagram over a period of 16 years. METHODS: A total of 535 strains of V. cholerae were isolated from 10,406 stool samples and rectal swabs from January 1990 to December 2005. These comprised of serogroups O1 - 427 (79.89%), O139 - 86 (16.07%) and non O1, non O139 - 22 (4.11%). No classical V. cholerae was isolated. RESULTS: Vibrio cholerae serogroup O1 serotype Ogawa was the predominant isolate till 1992. During 1993, serogroup O139 became the main isolate; however, it completely disappeared during 1995-1996 only to reappear in 1997. Serotype Inaba in our area was conspicuous by its absence with only two strains being isolated till June 1999, but during July-December 1999, 11 out of 15 V. cholerae O1 isolates were El Tor Inaba. T4 was the predominant phage type till 1990, T2 during 1991-1994 and T27 (as per the new scheme) thereafter. Resistance to tetracycline varied between 2 and 17% for V. cholerae O1. CONCLUSIONS: The paper reports on the changing epidemiological markers of V. cholerae isolated from a rural hospital over a period of 16 years.     

霍乱弧菌O139某些生物学特性及毒力基因检测

Ｏ１３９霍乱弧菌是１９９２年发现的新型霍乱弧菌，其毒力强，危害严重。对中国、印度、孟加拉国分离的２３株Ｏ１３９菌株的部分生物学特征检查表明：对弧菌抑制剂Ｏ／１２９均具有抗性、溶原菌、山梨醇慢发酵、非溶血性。核酸分子杂交显示，所有被检Ｏ１３９菌株都具有主要的霍乱弧菌毒素基因：ｃｔｘ，ｚｏｔ，ａｃｅ和ＲＳ１序列。ＰＣＲ检测ｃｔｘ基因与Ｏ１群流行珠具有相同的扩增产物，ｔｃｐＡ基因扩增表现为与埃尔托型霍乱弧菌相似的扩增产物。兔肠段结扎测毒表明，Ｏ１３９霍乱弧菌为强毒株。因此Ｏ１３９菌与Ｏ１群霍乱弧菌流行株具有共同的毒力特征。     

Molecular epidemiology of Vibrio cholerae in the U.S. Gulf Coast.

Enterotoxigenic strains of Vibrio cholerae O-1, biotype El Tor, isolated from a case of cholera in Texas in 1973, an outbreak of cholera in Louisiana in 1978, and Louisiana sewage samples in 1980 and 1981 were analyzed for their genetic similarities. Chromosomal DNA was isolated from each strain, digested with restriction endonuclease, and analyzed by the Southern blot technique. A radioactive probe consisting of Escherichia coli heat-labile enterotoxin DNA detected cholera toxin gene sequences in these strains and demonstrated that the toxin gene sequence, if not the entire chromosomal DNA, is identical in these strains and distinctly different from other strains of V. cholerae isolated throughout the world. In addition, two strains of enterotoxigenic V. cholerae non-O-1 isolated from clinical cases, were analyzed and found to possess cholera toxin genes which differed in the DNA sequence from the V. cholerae O-1 strains. We concluded that a single strain of enterotoxigenic V. cholerae O-1 is resident in the U.S. Gulf Coast and that a second reservoir of cholera toxin genes exists in V. cholerae non-O-1 strains in Louisiana.     

Vibrio factors cause rapid fluid accumulation in suckling mice.

Non-O-1 and O-1 Vibrio cholerae and Vibrio fluvialis isolated from clinical and environmental sources were examined for virulence factor production in 3-day-old suckling mice and in Y-1 tissue culture. The responses of the suckling mice to intragastrically administered bacterial cultures were measured by intestinal fluid accumulation (FA), diarrhea, and mortality. Regardless of the O-serovar, source of isolation, or ability to produce cholera toxin, all strains of V. cholerae stimulated increased FA, which was measurable in the mice at 4 h post-inoculation. The factor(s) causing these symptoms was found to be distinct from cholera toxin by the kinetics of FA and serological difference from cholera toxin based on in vivo neutralization tests. In most instances, FA was followed by high rates of mortality. Y-1 mouse adrenal tumor cell assays also showed that many V. cholerae produced extracellular heat-labile cytotoxic factor(s), and many cholera toxin-negative strains also caused a cytotonic-like morphological response. The majority of V. fluvialis strains produced smaller amounts of cytotoxic factor(s) but no cytotoxic reactions. The factor which stimulates rapid FA in suckling mice could be one of several virulence-associated factors contributing to diarrheal disease by nontoxigenic vibrios, but this is not verified at present.     

Cholera due to altered El Tor strains of Vibrio cholerae O1 in Bangladesh

We determined the types of cholera toxin (CT) produced by a collection of 185 Vibrio cholerae O1 strains isolated in Bangladesh over the past 45 years. All of the El Tor strains of V. cholerae O1 isolated since 2001 produced CT of the classical biotype, while those isolated before 2001 produced CT of the El Tor biotype.     

Numerical taxonomy of Vibrio cholerae and related species isolated from areas that are endemic and nonendemic for cholera.

A total of 165 strains of vibrios isolated from clinical and environmental sources in the United States, India, and Bangladesh, 11 reference cultures, and 4 duplicated cultures were compared in a numerical taxonomic study using 83 unit characters. Similarity between strains was computed by using the simple matching coefficient and the Jaccard coefficient. Strains were clustered by unweighted average linkage and single linkage algorithms. All methods gave similar cluster compositions. The estimated probability of error in the study was obtained from a comparison of the results of duplicated strains and was within acceptable limits. A total of 174 of the 180 organisms studied were divided into eight major clusters. Two clusters were identified as Vibrio cholerae, one as Vibrio mimicus, one as Vibrio parahaemolyticus, three as Vibrio species, and one as Aeromonas hydrophila. The V. mimicus cluster could be further divided into two subclusters, and the major V. cholerae group could be split into seven minor subclusters. Phenotypic traits routinely used to identify clinical isolates of V. cholerae can be used to identify environmental V. cholerae isolates. No distinction was found between strains of V. cholerae isolated from regions endemic for cholera and strains from nonendemic regions.     

ICEVchInd5 is prevalent in epidemic Vibrio cholerae O1 El Tor strains isolated in India

Integrative conjugative elements (ICEs) of the SXT/R391 family are self-transmissible mobile elements mainly involved in antibiotic resistance spread among γ-Proteobacteria, including Vibrio cholerae. We demonstrated that the recently described ICEVchInd5 is prevailing in V. cholerae O1 clinical strains isolated in Wardha province (Maharashtra, India) from 1994 to 2005. Genetic characterization by ribotyping and multiple-locus SSR analysis proved the same clonal origin for V. cholerae O1 isolates in Wardha province over an 11-year period and was used to assess the correlation between strain and ICE content among ours and different Indian reference strains. In silico analysis showed the existence of at least 3 sibling ICEs of ICEVchInd5 in V. cholerae O1 El Tor reference strains, isolated in the Indian subcontinent after 1992.     

霍乱弧菌O139国内外菌株分子生物学特征比较

１９９２年１０月在印度，１２月开始在孟加拉国发生了一种新型霍乱弧菌——霍乱弧菌Ｏ１３９引起的霍乱样大流行。我国新疆南部柯坪县于１９９３年５～６月也发生了由该型菌引起的典型霍乱样腹泻暴发。分子生物学研究表明：细菌染色体ＤＮＡ经核酸限制性内切酶ＳａｌⅠ消化后，用核酸脉冲场凝胶电泳（ＰＦＧＥ）分离ＤＮＡ，所有Ｏ１３９菌株表现为一致的特征性的限制性酶谱。用１６ＳｒＲＮＡ基因为探针，染色体ＤＮＡ经ＢｇｌⅠ酶切ＰＦＧＥ分离后做ｓｏｕｔｈｅｒｎｂｌｏｔ杂交分析，结果印度、孟加拉国和中国分离的菌株分别属于不同的核糖体基因型（Ｒｉｂｏｔｙｐｅ，ＲＴ），但与新疆菌株相比，印度和孟加拉国菌株的关系更为密切。结果还显示：霍乱弧菌Ｏ１３９菌株具有基本一致的基因组特征。     

Molecular epidemiology of non-O1 Vibrio cholerae and Vibrio mimicus in the U.S. Gulf Coast region.

Ten toxigenic Vibrio cholerae non-O1 and V. mimicus strains isolated from clinical and environmental sources in the U.S. Gulf Coast region were examined for genetic relatedness. Restriction digest patterns of chromosomal DNA and Southern blot analysis with a cholera toxin gene probe revealed that the strains exhibited greater genetic divergence than the highly conserved V. cholerae O1 strains isolated from clinical and sewage samples in this region.     

Emergence of a new clone of toxigenic Vibrio cholerae O1 biotype El Tor displacing V. cholerae O139 Bengal in Bangladesh.

The emergence of Vibrio cholerae O139 Bengal in 1993, its rapid spread in an epidemic form, in which it replaced existing strains of V. cholerae O1 during 1992 and 1993, and the subsequent reemergence of V. cholerae O1 of the El Tor biotype in Bangladesh since 1994 have raised questions regarding the origin of the reemerged El Tor vibrios. We studied 50 El Tor vibrio strains isolated in Bangladesh and four other countries in Asia and Africa before the emergence of V. cholerae O139 and 32 strains isolated in Bangladesh during and after the epidemic caused by V. cholerae O139 and 32 strains isolated in Bangladesh during and after the epidemic caused by V. cholerae O139 to determine whether the reemerged El Tor vibrios were genetically different from the El Tor vibrios which existed before the emergence of V. cholerae O139. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA, cholera toxin (ctxA), and zonula occludens toxin (zot) or in DNA sequences flanking the genes showed that the El Tor strains isolated before the emergence of V. cholerae O139 belonged to four different ribotypes and four different ctx genotypes. Of 32 El Tor strains isolated after the emergence of O139 vibrios, 30 strains (93.7%) including all the clinical isolates belonged to a single new ribotype and a distinctly different ctx genotype. These results provide evidence that the reemerged El Tor strains represent a new clone of El Tor vibrios distinctly different from the earlier clones of El Tor vibrios which were replaced by the O139 vibrios. Further analysis showed that all the strains carried the structural and regulatory genes for toxin-coregulated pilus (tcpA, tcpI, and toxR). All strains of the new clone produced cholera toxin (CT) in vitro, as assayed by the GM1-dependent enzyme-linked immunosorbent assay, and the level of CT production was comparable to that of previous epidemic isolates of El Tor vibrios. Further studies are required to assess the epidemic potential of the newly emerged clone of V. cholerae O1 and to understand the mechanism of emergence of new clones of toxigenic V. cholerae.     

Use of polymerase chain reaction for detection of toxigenic Vibrio cholerae O1 strains from the Latin American cholera epidemic.

In January 1991, an outbreak of cholera started in Peru and spread throughout most of Latin America within 8 months. As of March 1992, over 450,000 cases and approximately 4,000 deaths have been reported to the Pan American Health Organization. The causative organism is toxigenic Vibrio cholerae O1 of the El Tor biotype and is distinct from the U.S. Gulf Coast strains. A polymerase chain reaction (PCR) that amplifies a 564-bp fragment of the cholera toxin A subunit gene (ctxA) was used to identify toxigenic V. cholerae O1 strains. A total of 150 V. cholerae O1 isolates were tested. They were of unknown toxin status, were associated with recent outbreaks, and were isolated from patients, food, and water. One hundred forty isolates were found to be toxigenic both by PCR and the routine diagnostic enzyme-linked immunosorbent assay. Thirty-eight known toxigenic strains isolated worldwide from 1921 to 1991 were also positive in the PCR. A collection of 18 nontoxigenic V. cholerae O1 strains, 35 Escherichia coli heat-labile-enterotoxin-I-producing strains, 26 Campylobacter strains, and 8 strains of Aeromonas hydrophila, previously reported to produce cholera toxin-like toxin, were all negative in the ctxA PCR. We conclude that this PCR is a diagnostic method that specifically detects toxin genes in V. cholerae O1 strains in a reference laboratory. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.     

Development and evaluation of a phage typing scheme for Vibrio cholerae O139

The scenario of cholera that existed previously changed in 1992 and 1993 with the emergence of toxigenic Vibrio cholerae O139 in India. The genesis of the new serogroup formed the impetus to search for O139 phages in and around the country. A total of five newly isolated phages lytic to V. cholerae O139 strains were used for the development of this phage typing scheme. These phages differed from each other and also differed from the existing O1 phages in their lytic patterns, morphologies, restriction endonuclease digestion profiles, and immunological criteria. With this scheme, 500 V. cholerae O139 strains were evaluated for their phage types, and almost all strains were found to be typeable. The strains clustered into 10 different phage types, of which type 1 (38.2%) was the dominant type, followed by type 2 (22.4%) and type 3 (18%). Additionally, a comparative study of phage types in 1993 and 1994 versus those from 1996 to 1998 for O139 strains showed a higher percentage of phage type 1 (40.5%), followed by type 3 (18.8%) during the period between 1993 and 1994, whereas phage type 2 (32. 1%) was the next major type during the period from 1996 to 1998. This scheme comprising five newly isolated phages would be another useful tool in the study of the epidemiology of cholera caused by V. cholerae O139.     

霍乱弧菌O139与O1及非O1菌关系的研究

霍乱弧菌O139是近年来出现的新型霍乱弧菌,其与O1和其它非O1霍乱弧菌之间的关系是急待解决的问题。本工作应用核酸脉冲场凝胶电泳、基因探针分子杂交、多位点酶电泳和聚合酶链式反应等分子生物学方法,对霍乱弧菌O139、O1和其它非O1的分子生物学特征进行研究。结果表明,霍乱弧菌O139具有O1流行株的主要毒力因子基因,其基因组特征也与O1群流行株相似,但相互之间有微小的差异。分子生物学数值分类表明,霍乱弧菌O139与霍乱弧菌古典型和埃尔托型流行株为一个克隆群,O139菌株与埃尔托型非流行株和其它非O1霍乱弧菌的遗传关系较远,因此O139菌株应列为霍乱病原菌。结果显示,作为霍乱的病原体,不论其表型如何,都具有特定的基因组特征和毒力基因特征。     

霍乱弧菌抑制子基因及间隔区（rstR—ig2）多态性研究

目的研究霍乱弧菌CTXφ基因组抑制子基因及间隔区（rstR-ig2）多态性。方法聚合酶链反应（PCR）及限制性片段长度多态性分析（PCR-RFLP）、型特异性引物PCR分型。结果霍乱弧菌（VC）01群古典型（CVC）5株均为class类型。VC01群埃尔托型（EVC）155株共分为4个类型：class、ET、class＋ET、class ＋newET，后2个类型为首次发现。不同地区分离的EVC均以ET类型为主，以class和class＋ET类型为辅。O139群VC82株共分为4个类型：ET、ET＋new O139、ET＋calc、ET＋calc＋newO139，后3个类型为首次发现；福建省分离的菌株以ET4-newO139类型为主，其它地区分离的均以ET类型为主。结论EVC和O139群VC的rstR-ig2基因均以ET类型为主．首次发现5种不同类型rstR-ig2基因共存的新类型。     

Isolation of nontoxigenic Vibrio cholerae O group 1 from a patient with severe gastrointestinal disease.

A nontoxigenic strain of Vibrio cholerae O group 1 was isolated in Florida from the stool of a patient with severe diarrhea. The strain had the same hemolytic and unique phage-sensitivity pattern as all toxigenic isolates from recent cases of cholera in Texas and Louisiana. Identical strains were transiently isolated from sewerage systems in two other Florida communities, suggesting that multiple human infections had occurred. This is the first indication that V. cholerae O1 strains which do not produce cholera toxin may be able to cause gastrointestinal disease in humans. The identification of these strains also raises questions about the relationship between toxigenic and nontoxigenic strains of V. cholerae O1 along the Gulf Coast of the United States.     

A retrospective macrorestrictive analysis of Vibrio cholerae eltor strains isolated under epidemic complications in the Russian Far East

A retrospective study of the clonal structure of 24 Vibrio cholerae eltor strains isolated during epidemic complications in the Russian Far East was carried out on the basis of a macrorestrictive analysis of Vibrio cholerae eltor genomic DNA using NotI and SfiI endonucleases. It was found that clonal (by amplification profiling data) toxigenic strains (n = 23) were characterized by variability of the NotI/SfiI-generated restriction profiles. The V. cholerae eltor isolated during an outbreak in the city of Yuzhno-Sakhalinsk were differentiated into four pulse types, with one dominating type detected in 76.5% of the strains. A separate NotI/SfiI-restriction profile was characteristic for the isolates from Primorskii krai. A nontoxigenic strain formed a separate dendrogram line from the toxigenic strains on the basis of the PFGE-pattern structure. The use of macrorestriction analysis was shown to be effective for deep characterization of the V. cholerae population structure and for ascertainment of molecular-epidemiological regularities in the territorial distribution of certain V. cholerae clones.