Characteristics of Vibrio cholerae O1 isolated from water of the River Ganga,Varanasi, India

Background: Vibrio cholerae is an autochthonous inhabitant of fresh and brackish water and estuarine system. Investigation of V. cholerae from the River Ganga seems important to find variation in CTX arrangement and genomic diversity. Objectives: To investigate V. cholerae O1 strains for the presence of virulence and regulatory genes, variation in number and organisation of the pre-CTXΦ and/or CTXΦ, and for the genomic diversity. Materials and Methods: Polymerase chain reaction (PCR) was used to detect virulence and regulatory genes, type of rstR and location of CTXΦ on the chromosome. Southern hybridisation was conducted to see the number and arrangement of pre-CTXΦ and CTXΦ. Ribotyping and pulsed-field gel electrophoresis were used to find genetic relatedness. Results: Seven strains gave positive results by PCR for the gene encoding for ctxA, zot, ace, tcpA (El Tor), ompU, and toxR, except one strain that was negative for the ctxA. Three strains were positive for the tcpA (El Tor), ompU and toxR genes. Determination of CTX organisation showed that among the ctx-positive strains, four harboured two copies of CTX^ETΦ arranged in tandem and two harboured one copy of CTX^ETΦ, and one ctx-negative strain harboured only one copy of pre-CTX^ETΦ. Pulsotype and ribotype analysis showed existence of at least three pulsotype and ribotypes indicating diversity in genomic content among them. Conclusion: This study thus indicates that multiple clones (ribotypes/pulsotypes) of V. cholerae O1 carrying pre-CTXΦ and/or CTXΦ and ctx-negative strains were present in the water of the River Ganga, Varanasi, India.     

Molecular Characterization of High-Level-Cholera-Toxin-Producing El Tor Variant Vibrio cholerae Strains in the Zanzibar Archipelago of Tanzania

Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR, rtxC, and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis.     

A molecular and phenotypic study of Vibrio cholerae in Iran

Vibrio cholerae is again the subject of attention on account of the current increase in the world-wide incidence of cholera. In this study, 200 clinical isolates of V. cholerae serotypes O1 and non-O1, non-O139, were collected from different provinces in Iran. The isolates were subjected to biochemical analysis, antibiogram, PCR of toxin genes, plasmid profile, ribotyping and pulsed-field gel electrophoresis (PFGE). The analysis of plasmid content showed that 33-96% of V. cholerae isolated from different provinces carry a large plasmid. PCR analysis of V. cholerae O1 showed that the genes encoding cholera toxin (ctx), toxin co-regulated pilus (tcp), accessory cholera enterotoxin (ace) and zonula occludens toxin (zot) were present in 55-97% of isolates in different provinces. Restriction fragment length polymorphism (RFLP) of BglI-digested DNA probed with five oligonucleotides revealed three different ribotype patterns in isolates of V. cholerae O1. The ribotype pattern B21 of V. cholerae O1 El Tor was found to be the predominant pattern in the isolates studied. V. cholerae non-O1, non-O139 isolates showed a single ribotype pattern. PFGE analysis also showed 10 different patterns amongst the isolates, 9 of which were in V. cholerae O1. Overall, the analysis of polymorphism of ribotypes and PFGE patterns of the isolates showed that the provinces in Iran were affected by a limited number of clones of V. cholerae O1 and non-O1, non-O139 strains.     

Resurgent Vibrio cholerae O139: Rearrangement of Cholera Toxin Genetic Elements and Amplification of rrn Operon

The unprecedented genesis of a novel non-O1 Vibrio cholerae strain belonging to serogroup O139, which caused an epidemic in late 1992 in the Indian subcontinent, and its subsequent displacement by El Tor O1 vibrios after 18 months initiated a renewed investigation of the aspects of the organism that are related to pathogenesis. The reappearance of V. cholerae O139 with altered antibiotic sensitivity compared to O139 Bengal (O139B) in late 1996 has complicated the epidemiological scenario of V. cholerae and has necessitated an examination of possible rearrangements in the genome underlying such rapid changes in the phenotypic traits. With a view to investigating whether the phenotypic changes that have occurred are associated with alteration in the genome, the genome of the resurgent V. cholerae O139 (O139R) strains were examined. Pulsed-field gel electrophoresis analysis of NotI- and SfiI-digested genomic DNA of O139R isolates showed restriction fragment length polymorphism including in the cholera toxin (CTX) genetic element locus and with O139B isolates. Analyses of the organization of the CTX genetic elements in O139R strains showed that in contrast to two copies of the elements connected by two direct-repeat sequences (RS) in most of the genomes of O139B isolates, the genomes of all O139R strains examined, except strain AS192, have three such elements connected by a single RS. While the RS present in the upstream of the CTX genetic elements in the genome of O139R is of O139B origin, the RS connecting the cores of the elements has several new restriction sites and has lost the BglII site which is supposed to be conserved in all O1 strains and O139B. The endonuclease I-CeuI, which has sites only in the rrn operons in the genomes of all organisms examined so far, has 10 sites in the genomes of O139R strains, compared to 9 in the genomes of O139B strains. The recent isolates of V. cholerae O139 have thus gained one rrn operon. This variation in the number of rrn operons within a serogroup has not been reported for any other organism. The results presented in this report suggest that like the pathogenic El Tor O1 strains, the genomes of O139 strains are undergoing rapid alterations.     

Molecular-genetic analysis of Vibrio cholerae El Tor strains of epidemic risk isolated in Siberian and Far East regions of Russia

PCR detection of biovar specificity and pathogenicity determinants has been performed in order to analyze the structural components of the genome of V. cholerae El Tor strains (n = 90) isolated during epidemic outbreaks in Siberia and the Far East, and the nucleotide sequence of the ctxB gene and the structure of ctxAB gene promoter region have been determined. As a result, toxicogenic strains V. cholerae El Tor were divided into two groups: the first group contained strains isolated at the initial stages of the seventh pandemia (in the 1970s), and they had the genotype ctxB3 ⁺ rstREl⁺ rstR ^Cl- rstC⁺TCL⁺ tbr ₄; all El Tor vibrios posing an epidemic risk isolated in the 1990s were characterized as atypical variants due to ctxB gene harboring of the classical genotype (ctxB) in their genome and were placed in the second group. The second group fell into three genotypes according to the set of tested genetic markers (ctxB, rstR, rstC, TLC, and tbr) and affect certain territories. The established variability of the genome structure of Vibrio El Tor atypical variants may serve as a marker for molecular and epidemiological analysis of cholera entry pathways and distribution and is useful for speculating on the most likely directions of pathogen evolution.     

Genome variability in the altered variants of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> biovar El Tor isolated in Russia

Molecular-genetic analysis of 56 clinical strains of the Vibrio cholerae biovar El Tor isolated during the period 1965–2010 in Russia and neighboring countries was carried out. Thirty-one isolates (55.3%) bearing the gene ctxB in the genome of the prophage CTXφ in cholera vibrios of the classical biovar were found. These strains are altered variants of the cholera El Tor pathogen. The nucleotide sequence of their genes ctxB and rstR included in the prophage CTXϕ, which are related with the production of the cholera toxin, was determined. It was found that altered variants bear different alleles of these genes (ctxB1 or ctxB7; rstR^El and/or rstR^Class) and belong to four gene types: ctxB1, rstR^Class; ctxB1, rstR^El; ctxB1, rstR^Class/rstR^El; and ctxB7, rstR^Class/rstR^El. It was shown that these gene variants produce 42 to 48 times more cholera toxin than typical strains. Isolates in which the pathogenicity island VSP-2 contains a long deletion were found among altered variants. The study data are evidence of the genomic variability of altered pathogen variants of the seventh cholera pandemic and require the development of new gene-diagnostic testing systems to improve the efficiency of epidemiological control of cholera distribution.     

Molecular Analyses of Vibrio cholerae O1 Clinical Strains,Including New Nontoxigenic Variants Isolated in Mexico during the Cholera Epidemic Years between 1991 and 2000

We studied the evolution of Vibrio cholerae O1 during the 1991 to 2000 cholera epidemic in Mexico by biochemical, serological, and molecular characterization of strains collected during this period. Strains were divided into toxigenic and nontoxigenic groups according to the presence or absence of genes encoding cholera toxin. As previously reported, we characterized two populations among toxigenic strains, which were present from the first year of the epidemic. BglI rRNA analysis revealed that these strains had ribotype profiles, denoted M5 and M6 in our study, that were identical to those previously designated Koblavi B5 or Popovic 5 and Popovic 6a or Tamayo B21a, respectively. Ribotype M5 was isolated between 1991 and 1993. This ribotype had a low level of genetic variation as detected by pulsed-field gel electrophoresis (PFGE). Ribotype M6 persisted from 1991 to 2000. However, PFGE profiles suggested that two epidemiologically unrelated strains coexisted within this single ribotype from 1995 until the end of the epidemic. We identified three new BglI ribotypes, Mx1, Mx2, and Mx3, from nontoxigenic V. cholerae O1 strains isolated between 1998 and 2000; one of them grouped strains positive for the toxin-coregulated pilus island. They differed from nontoxigenic clones isolated in Latin America and on the U.S. Gulf Coast and are probably autochthonous Mexican V. cholerae O1 variants. Most of these new variants were isolated from states surrounding the Gulf of Mexico, where the highest incidence of cholera in the country was recorded. Thus, the Mexican Gulf Coast, like the U.S. Gulf Coast, may act as an environmental reservoir of V. cholerae O1.The seventh cholera pandemic, characterized by Vibrio cholerae O1 biotype El Tor, is still present around the world. Moreover, the number of reported cholera cases has continuously increased since 2004. The World Health Organization (WHO) (http://www.who.int/wer) reported a 30% increase in cases of cholera worldwide between 2004 (101,383 cases) and 2005 (131,943 cases) and a further 79% increase between 2005 and 2006 (236,860 cases), whereas the number of countries reporting cases has remained constant. At the same time, the global case-fatality rate rose from 1.72% in 2005 to 2.66% in 2006. However, the actual numbers of cholera cases globally are estimated to be much higher than officially reported, due to underreporting and other limitations of surveillance systems. In 2006, the total number of cases reported in Africa accounted for 99% of the global total; Africa has been the continent with the highest number of officially reported cholera cases since 1996. However, in America the number of cases has greatly decreased since 1999, with only 10 cases reported from Canada and the United States (http://www.who.int/wer/2007/wer8231/en/index.html), 4 of which were indigenous to the United States.Two basic facts distinguish the seventh cholera pandemic from the other six, proposed by Pollitzer (26), occurring before 1961: (i) the epidemic clone shifted from the classic to the El Tor biotype, with an origin in Indonesia rather than the Indian subcontinent, and (ii) the seventh pandemic evolved in two waves, with the first one spreading throughout Asia between 1961 and 1966 and the second spreading to Asia, Africa, and Latin America around 1970 (20).Before the 1991 epidemic, cholera was mostly absent for a century in South, Central, and North America; a few sporadic cases were reported between 1973 and 1992 on the U.S. Gulf Coast (2), and one case was reported in 1983 in Cancun, Mexico, in a tourist from the United States (1). The epidemic began in 1991 in the coastal regions of Peru and spread rapidly to the eastern, northern, and southern countries of the continent, forming a branch of the seventh pandemic. The appearance of the disease in coastal areas of Peru remains a mystery; some studies suggest maritime transport as the source, whereas others suggest a local source (36).Following the first report in Peru in January 1991, cholera was reported in Mexico in June 1991. The number of reported cases in Mexico increased from 1991 until 1993, decreased substantially in 1994, but rose to its highest level ever recorded in 1995 (15,526 cases). Thereafter, cholera cases consistently decreased, with no cases of cholera reported by the Mexican Instituto Nacional de Diagnóstico y Referencia Epidemiológica (INDRE) (National Diagnostics and Epidemiological Reference Institute) since 2002 (32). Therefore, two epidemic cycles have been identified: between 1991 and 1994 and between 1995 and 2001.Strains responsible for the cholera epidemic in Latin America have been extensively characterized using various molecular methods such as restriction endonuclease digestion of plasmids or chromosomal DNA, ribotyping, multilocus enzyme electrophoresis, and pulsed-field gel electrophoresis (PFGE). These molecular approaches first associated the Latin American epidemic with a single strain, V. cholerae O1 biotype El Tor serotype Inaba, which was related to seventh-pandemic isolates from other parts of the world and described as ribotype B5 or 5 according to ribotype schemes reported by Koblavi et al. (21) and Popovic et al. (27) and as a distinct electrophoretic type, electrophoretic type 4, as defined by multilocus enzyme electrophoresis studies (37). A second and distinct epidemic strain, V. cholerae O1 biotype El Tor serotype Ogawa, was later associated with the Mexican epidemic. This strain, reported by Popovic et al. (27) to be ribotype 6a, identical to ribotype B21a described by Tamayo et al. (35), belonged to electrophoretic type 3 (37) and was initially isolated in 1991 in Latin America from cholera cases occurring in Mexico and Central America. It may have been responsible for a second epidemic wave (14). The emergence of the cholera epidemic in Latin America led to a closer surveillance of diarrhea diseases and to the consequent isolation of groups of V. cholerae O1 strains lacking the pathogenicity factor cholera toxin (CT) in several countries. These nontoxigenic V. cholerae O1 clusters, normally found among epidemic V. cholerae O1 strains, were reported as V. cholerae O1 “variants”; one, called Amazonia, was identified by Coelho et al. (9) in the northern region of Brazil, and a “Tucumán variant” was identified by Pichel et al. (25) in Argentina. An increasing number of these nontoxigenic V. cholerae O1 strains, as well as some nontoxigenic V. cholerae non-O1/non-O139 strains, have been reported to be associated with diarrhea disease in a number of different countries, causing mild to severe cholera-like diarrhea (31, 33). Such variants were not described in Mexico during the Latin American epidemic. The emergence of these new variants, also detected by V. cholerae repeat sequence PCR and repetitive sequence-based PCR systems (13), emphasizes the need for an accurate epidemiological tool to monitor their spread and evolution.Previous studies of Mexican V. cholerae O1 strains subtyped only isolates collected between 1991 and 1995. Consequently, as they do not cover the whole epidemic period, those findings may not be a complete representation. In this study, we selected clinical V. cholerae O1 strains collected by INDRE and isolated in different states of Mexico between 1991 and 2000 from patients with severe diarrhea associated with cholera on the basis of clinical symptoms; we examined the evolution of these strains over this period using two DNA-related typing methods, ribotyping and PFGE analysis, selected because of their extensive and successful use for molecular epidemiology surveillance of V. cholerae O1 clones.     

Heterogeneity in the organization of the CTX genetic element in strains ofVibrio choleraeO139 Bengal isolated from Calcutta,India and Dhaka,Bangladesh and its possible link to the dissimilar incidence of O139 cholera in the two locales

After a lapse of 33 months,Vibrio choleraeO139, the new serogroup associated with cholera, has re-emerged in Calcutta, India and has become the dominant serogroup causing cholera from September 1996. In neighbouring Bangladesh,V. choleraeO1 biotype El Tor continues to be the dominant cause of cholera with the O139 serogroup accounting for only a small proportion of cases. Comparison of the phenotypic traits of representative O139 strains from Calcutta and Dhaka isolated between December 1996 and April 1997 showed similar phenotypic traits with the exception that Dhaka O139 strains were susceptible to streptomycin whilst Calcutta O139 strains were resistant. The Dhaka and Calcutta O139 strains displayed identical ribotypes but showed remarkable differences in the structure and organization of the CTX genetic element. In the Dhaka O139 strains, two copies of the CTX element were arranged in tandem and this resembled the pattern displayed by the 1992 epidemic strains of O139. The Calcutta O139 strains, in contrast, carried three copies of the CTX genetic element arranged in tandem with the loss of a conservedBglII restriction site in the RS1 element and the appearance of a newHindIII site in the same region. While there may be other factors, it appears that the reorganization of the CTX genetic element in the Calcutta O139 strains may have contributed to the resurgence of this serogroup in Calcutta.     

Molecular Evidence of Cholera Outbreak Caused by a Toxigenic Vibrio cholerae O1 El Tor Variant Strain in Kelantan,Malaysia

A total of 20 Vibrio cholerae isolates were recovered for investigation from a cholera outbreak in Kelantan, Malaysia, that occurred between November and December 2009. All isolates were biochemically characterized as V. cholerae serogroup O1 Ogawa of the El Tor biotype. They were found to be resistant to multiple antibiotics, including tetracycline, erythromycin, sulfamethoxazole-trimethoprim, streptomycin, penicillin G, and polymyxin B, with 35% of the isolates being resistant to ampicillin. All isolates were sensitive to ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and kanamycin. Multiplex PCR analysis confirmed the biochemical identification and revealed the presence of virulence genes, viz., ace, zot, and ctxA, in all of the isolates. Interestingly, the sequencing of the ctxB gene showed that the outbreak strain harbored the classical cholera toxin gene and therefore belongs to the newly assigned El Tor variant biotype. Clonal analysis by pulsed-field gel electrophoresis demonstrated that a single clone of a V. cholerae strain was responsible for this outbreak. Thus, we present the first molecular evidence that the toxigenic V. cholerae O1 El Tor variant has invaded Malaysia, highlighting the need for continuous monitoring to facilitate early interventions against any potential epidemic by this biotype.Vibrio cholerae strains belonging to the O1 and O139 serogroups are agents of endemic and pandemic cholera, a potentially life-threatening diarrheal disease. As a water- and food-borne disease, cholera infection is linked to poverty and poor sanitation in many developing countries (13). From an epidemiological point of view, cholera tends to occur in explosive outbreaks throughout several regions simultaneously; likewise, extensive pandemics have followed a progressive pattern, affecting many countries across the continents and extending over many years (17). Cholera pandemics long have been believed to be exclusively associated with the toxigenic V. cholerae O1 serogroup, which consists of two biotypes: classical and El Tor. The classical biotype was responsible for the world''s first six pandemics, but the seventh pandemic was caused by the O1 El Tor biotype and was exceedingly more extensive in geographic spread and duration (13).The first non-O1 strain recognized as having caused an explosive cholera epidemic was discovered in 1992, but it did not belong to any of the 138 serogroups previously described. Thus, the new epidemic strain was designated serogroup O139 and subsequently has been linked to extensive outbreaks in various regions of Bangladesh and India (14). The discovery of its ability to cause large outbreaks and rapid spread to neighboring countries suggested the possibility that the new serogroup will facilitate the eighth pandemic of cholera if outbreaks continue to occur and more countries are affected (42). The strains of V. cholerae O1 El Tor in the seventh pandemic experienced an initial displacement by O139 strains in the Indian subcontinent (1) but recovered their prominence within a year and established a coexistence with O139 strains, causing subsequent cholera outbreaks throughout India and Bangladesh (12, 27).In Malaysia, cholera caused by V. cholerae O1 El Tor is endemic and often has been associated with sporadic outbreaks (24, 33, 46). El Tor is one of the most established biotypes within the V. cholerae O1 serogroup and is differentiated from the classical biotype based on a number of phenotypic traits, such as polymyxin B susceptibility, chicken cell agglutination, Voges-proskauer (VP) test positivity, and phage susceptibility (17). In addition, comparative genomic analysis has revealed the presence of genes that are unique to the El Tor biotype (11). Some of these genes, including the major toxin-coregulated pilus (TCP) tcpA (19) and rtxC of the repeat in toxin (RTX) cluster, have since been used as genetic markers for the identification of El Tor strains (8). The classical and El Tor biotypes also differ in the infection pattern of disease they elicit; the El Tor strains have a higher ratio of asymptomatic carriers (37), survive better in the environment and human host, and are more efficiently transmitted from host to host (http://www.ncbi.nlm.nih.gov/bookshelf/br.fegi?book=mmed&part=A1358).The public health significance of a V. cholerae isolate is routinely assessed by two critical properties: the production of cholera toxin (CT) and the possession of either the O1 or O139 antigen, which acts as a marker of epidemic potential (17). So far, agents of endemic and pandemic cholera have been represented exclusively by CT-producing V. cholerae strains. CT has been shown to be the key virulence factor responsible for the manifestation of massive, dehydrating diarrhea (21). Although CTs from classical and El Tor strains are structurally and functionally similar, differences in immunological and genetic attributes have enabled several methods to differentiate between the two biotypes. Specifically, the molecular techniques include the direct sequencing of the ctxB gene (30), a ganglioside GM₁ enzyme-linked immunosorbent assay (GM₁-ELISA) with specific monoclonal antibody to classical or El Tor CT (29), and a mismatch amplification mutation assay (MAMA) PCR to detect polymorphism at nucleotide position 203 in the ctxB genes (26).In the study presented here, we performed the genetic analysis and molecular typing of V. cholerae O1 isolated from patients during a cholera outbreak in Kelantan, Malaysia, that occurred between November and December 2009. The existence of an outbreak was established based on the region''s previous incidence rate of cholera, which was reported to be 0.0 per 100,000 for four consecutive years (2005 to 2008) (http://statistics.gov.my). The duration of the outbreak was only 21 days, and 33 cholera cases were confirmed. Among those 33, 21 victims were local Kelantanese and the remaining 12 were migrant Thai workers. The patient demographic data revealed that six different districts were involved, and the patients ranged widely in age. Isolates recovered from symptomatic patients were characterized for their antimicrobial susceptibility patterns, the presence of various toxigenic and pathogenic genes, the production of cholera toxin, the nucleotide sequence of cholera toxin B subunits, and the demonstration of the clonal relationships between these isolates. The ultimate findings from this research have provided important insights into the causative agent of the recent cholera outbreak in peninsular Malaysia.(This study was presented in part at the 2nd National Conference on Environment and Health, Kota Bharu, Kelantan, Malaysia, 17 to 18 March 2010.)     

Molecular Analysis of Non-O1, Non-O139 Vibrio cholerae Associated with an Unusual Upsurge in the Incidence of Cholera-Like Disease in Calcutta, India

There was an inexplicable upsurge in the incidence of non-O1, non-O139 Vibrio cholerae among hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, between February and March 1996. Of the 18 strains of V. cholerae isolated during this period, 15 belonged to the non-O1, non-O139 serogroups (4 belonged to O144, 3 belonged to O11, 1 each belonged to O6, O8, O12, O19, O39, and O58, and 2 strains could not be typed), 2 belonged to the O139 serogroup, and 1 belonged to the O1 serogroup. Cell-free culture supernatants of 13 representative non-O1, non-O139 V. cholerae strains evoked a distinct cytotoxic effect on CHO and HeLa cells, and the strains examined produced the nonmembrane-damaging cytotoxin. By several PCR assays, it was determined that none of the non-O1, non-O139 strains were positive for the ctxA, zot, ace, and tcpA genes and for the genes representing the heat-labile toxin, heat-stable toxin, and verotoxin of Escherichia coli and the various variants of these genes. Studies on the clonality of non-O1, non-O139 V. cholerae strains by restriction fragment length polymorphism (RFLP) analysis of rRNA genes and of other genes (hlyA, hlyU, hlx, toxR, and attRS1) and by pulsed-field gel electrophoresis (PFGE) collectively indicate that the upsurge which occurred in February and March 1996 was caused by strains belonging to different clones. Overall, there was an excellent correlation between the results of ribotyping, RFLP analysis of various genes, and PFGE, with strains belonging to a particular serogroup showing nearly identical restriction patterns and PFGE profiles. It is clear from this study that some serogroups of V. cholerae can cause diarrhea by a mechanism quite different from that of toxigenic V. cholerae O1 and O139, and we have proposed the nomenclature of enteropathogenic V. cholerae to include these serogroups.     

Genomic variations of CTXφ prophage of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> biovar El tor induced by Tn5-Mob transposone

One of the key pathogenicity factors of the cholera pathogen is the cholera toxin (CT), which is encoded by the operon ctxAB contained in the CTXφ prophage. Variations in the CTXφ prophage genome, the core region of which contains ctxAB, zot, ace, cep, orfU, and psh genes, underlies the changes in virulent properties of cholera vibrios. The mechanisms involved in remodeling this prophage genome are still vague. In this work, we demonstrate that the insertion of Tn5-Mob (Km^R) transposon into the chromosome of the Vibrio cholerae model strain MAK757 biovar El Tor, which carries two copies of the CTXφ prophage, induces the remodeling of the prophage genome, namely, the deletion of the zot, ace, cep, and orfU genes. The level of CT biosynthesis in the insertion mutants MAK757 chr::Tn5-Mob, which only retain the ctxAB operon, increased more than 2000-fold as compared to the original strain. It has been shown that the increased CT production is associated with the altered structure of the chromosomal DNA region containing a copy of the ctxAB operon, encoding this protein. This mutation in the genome of CTXφ prophage induced by Tn5-Mob is unstable. Among the 600 isolated colonies obtained after screening the MAK757 chr::Tn5-Mob transposant capable of CT overproduction on a full medium free of antibiotics, 5.8% yielded clones that lost the ctxAB operon, as well as the marker Km^R, thus becoming nontoxigenic. The emergence of V. cholerae mutants both that both overproduce CT and are nontoxigenic demonstrates the important role of genetic variations in the CTXφ prophage in the evolution of V. cholerae pathogenicity. The obtained plasmid-free strain of V. cholerae biovar El Tor with type-2 CToverproduction can be used as a producer of this toxin, which is used to manufacture preparations for cholera diagnostics and prevention.     

Comparative analysis of glucose metabolism in strains of <Emphasis Type="Italic">Vibrio cholera</Emphasis> biovar El Tor

Comparative analysis of glucose fermentation in typical strains of V. cholerae biovar El Tor isolated in the Russian Federation in 1970–1990 and highly virulent strains of genovariants imported in 1993–2012 was carried out. It was demonstrated that glucose metabolism of V. cholerae biovar El Tor genovariants changed as a result of acquisition of classical CTX prophage (or only its ctxB gene) and a corresponding increase in virulence. A phenotypical manifestation of these changes is a lack of ability to grow on a minimal nutrient medium supplemented with 1% carbohydrate, as well as compromised capacity for acetoin fermentation in the course of the Voges–Proskauer test. Possible causes of the degraded glucose metabolism are associated with the presence of SNP in gene alsD that encodes acetolactate decarboxylase enzyme and is incorporated into als operon, which, in turn, is involved in acetoin generation, as well as with hyperexpression of regulatory protein AphA that controls acetoin biosynthesis.     

Molecular evolution of Vibrio cholerae O1 strains isolated in Lima, Peru, from 1991 to 1995.

Following the emergence of cholera in Lima, Peru, in 1991, isolates of Vibrio cholerae O1 biotype El Tor recovered from patients in various parts of Lima were selected and characterized. Ribotyping and pulsed-field gel electrophoresis (PFGE) revealed four BglI ribotypes and eight NotI PFGE types among 50 V. cholerae O1 strains recovered from patients with cholera in Lima from 1991 to 1995, with certain genotypes appearing to cluster geographically. While differences in ribotype and PFGE type patterns suggest that genetic changes are occurring in the strain responsible for the Latin American cholera epidemic, more frequently than previously reported, 40 (80%) O1 strains showed an identical ribotype pattern and 41 (82%) strains showed closely related PFGE types, types 1, 2, or 3, that differed by less than three restriction fragments. All strains were susceptible to nine antibacterial agents studied. In 1991, more than 95% of the clinical V. cholerae O1 strains were serotype Inaba, whereas from 1992, serotype Ogawa began to predominate, with more than 90% of the isolates being of the Ogawa serotype in 1995. The small differences in genotypes of V. cholerae O1 is remarkable because cholera is highly seasonal in coastal areas of Peru and support the hypothesis that the epidemic strain reemerges from an environmental source. However, the relative high rate of genetic changes within V. cholerae O1 as shown by ribotyping and PFGE should be taken into consideration when typing patterns of V. cholerae O1 associated with cholera in Latin America are evaluated.     

Rapid Spread of Vibrio cholerae O1 El Tor Variant in Odisha,Eastern India,in 2008 and 2009

The emergence and spread of Vibrio cholerae O1 El Tor variant strains causing severe diarrhea has been witnessed worldwide in recent years. In the state of Odisha, India, the spread of the V. cholerae O1 El Tor variant strains was studied during outbreaks in 2008 and 2009. Analysis of 194 V. cholerae O1 Ogawa strains revealed that V. cholerae O1 El Tor variant strains are spreading gradually throughout the state, causing outbreaks replacing typical V. cholerae O1 El Tor biotype strains.     

Molecular characterization of Vibrio cholerae O1 strains isolated during cholera outbreaks in Guinea-Bissau.

In the present study, 19 strains of Vibrio cholerae O1 biotype El Tor isolated during outbreaks of cholera in Guinea-Bissau in 1987, 1994, and 1995 were characterized to investigate a possible epidemiological relationship among the isolates. On the basis of ribotyping with the restriction enzyme BglI, 5 strains isolated in 1987 showed two closely related ribotypes, while 14 strains isolated in 1994 and 1995 showed the same ribotype that was distinct from the ribotypes of strains isolated in 1987. Southern blot hybridization of BglI-digested genomic DNA with a cholera toxin probe demonstrated that the strains isolated in 1987 showed an identical cholera toxin genotype, whereas O1 strains isolated in 1994 and 1995 showed the same genotype that was distinct from the genotype of strains isolated in 1987. These results were supported by the results of antibiotic susceptibility testing, in which strains isolated in 1987 showed resistance to polymyxin B only, while each of the strains from 1994 and 1995 showed resistance to polymyxin B, trimethoprim-sulfamethoxazole, and the vibriostatic agent O/129. Although our results are based on a limited number of V. cholerae O1 strains, they suggest that the epidemic in Guinea-Bissau in 1994 and 1995 was due to the introduction of a new strain to the country.     

Cholera between 1991 and 1997 in Mexico Was Associated with Infection by Classical,El Tor,and El Tor Variants of Vibrio cholerae

Vibrio cholerae O1 biotype El Tor (ET), the cause of the current 7th pandemic, has recently been replaced in Asia and Africa by an altered ET biotype possessing cholera toxin (CTX) of the classical (CL) biotype that originally caused the first six pandemics before becoming extinct in the 1980s. Until recently, the ET prototype was the biotype circulating in Peru; a detailed understanding of the evolutionary trend of V. cholerae causing endemic cholera in Latin America is lacking. The present retrospective microbiological, molecular, and phylogenetic study of V. cholerae isolates recovered in Mexico (n = 91; 1983 to 1997) shows the existence of the pre-1991 CL biotype and the ET and CL biotypes together with the altered ET biotype in both epidemic and endemic cholera between 1991 and 1997. According to sero- and biotyping data, the altered ET, which has shown predominance in Mexico since 1991, emerged locally from ET and CL progenitors that were found coexisting until 1997. In Latin America, ET and CL variants shared a variable number of phenotypic markers, while the altered ET strains had genes encoding the CL CTX (CTX^CL) prophage, ctxB^CL and rstR^CL, in addition to resident rstR^ET, as the underlying regional signature. The distinct regional fingerprints for ET in Mexico and Peru and their divergence from ET in Asia and Africa, as confirmed by subclustering patterns in a pulsed-field gel electrophoresis (NotI)-based dendrogram, suggest that the Mexico epidemic in 1991 may have been a local event and not an extension of the epidemics occurring in Asia and South America. Finally, the CL biotype reservoir in Mexico is unprecedented and must have contributed to the changing epidemiology of global cholera in ways that need to be understood.In 1991, when cholera reemerged after being absent from Latin America for about a century, millions of people were affected, with nearly 9,000 dying in 1993 alone (13). Following its first appearance along the coast of Peru in January 1991, cholera rapidly spread to all countries in Latin America except Uruguay, reaching Mexico in June of the same year (13, 19, 25, 32). Since then, there has been a great interest in understanding the source and transmission of cholera in Latin America. Limited genetic analysis of Vibrio cholerae O1 biotype El Tor (ET) strains identified from the epidemic showed that they have a unique signature, distinguishing them from 7th pandemic ET strains (25). However, later studies showed the clonal nature of the bacterium, suggesting that the Latin American epidemic was an extension of the 7th pandemic caused by ET strains from the Western Hemisphere (34).V. cholerae O1 and O139 are the two serogroups known to cause cholera. V. cholerae O1 has two biotypes, classical (CL) and ET, which differ in specific phenotypic traits (hemolysis of sheep erythrocytes, agglutination of chicken erythrocytes, sensitivity to polymyxin B [PMB], phage susceptibilities, and Voges-Proskauer [VP] test results) (15) and genotypic traits (ctxB, acfB, tcpA, and rstR). In addition, the CL and ET biotype strains differ in two major genomic regions, namely, the Vibrio seventh pandemic pathogenicity island I (VSP-I) and VSP-II, that are unique to the 7th pandemic ET biotype (10). Biotype CL caused the first six of the seven cholera pandemics recorded between 1817 and 1923 (26), with five of these affecting the American continents. Ever since the first cases were detected in the Americas in the 1830s, endemic cholera continued to be prevalent until 1895 (18).Historically, cholera has been endemic in Asia for centuries (26), with Asia being at the center of each of the seven cholera pandemics (10, 26). Although the ET biotype was first reported in 1905 and the 6th pandemic, caused by the CL biotype, lasted until 1923, it was not until the early 1960s that the ET biotype displaced the CL biotype in Asia and became the causal agent of the 7th pandemic (33). The CL biotype maintained a low profile in its last recognized niche in the coastal ecosystem of the Bay of Bengal, before it disappeared as a causal agent of clinical disease in the 1980s (33).Over the past few years, the ET biotype causing Asiatic cholera has shown remarkable changes in its phenotypic and genetic characteristics (23). Recent molecular analysis of ET strains causing acute watery diarrhea in Bangladesh shows them to be hybrids because they possess phenotypic and genotypic traits of the CL biotype against an ET background (23). Subsequent retrospective studies showed that all of the O1 ET strains isolated in Bangladesh since 2001 have been hybrids of both the CL and ET biotypes, while those isolated before 2001 contained all the attributes of the 7th pandemic V. cholerae O1 ET strains (22). Such genetic changes among ET strains causing cholera in Latin America were also evident from the changing serotypes, electrophoretic types, ribotypes, and pulsed-field gel electrophoresis (PFGE) types (3, 9, 11, 27). While the ET prototype has been completely replaced by an altered ET in Asia and Africa (29), recent data show the nature of the ET prototype (7th pandemic) of V. cholerae O1 isolated in Peru between 1991 and 2003 (24). The Peruvian ET strains that seem to be closely linked clonally to the Asian and African ET prototype strains were shown to have a distinct region in VSP-II that differentiates them from the ET prototype strains isolated in other continents (24). To better understand the dynamics of the cholera epidemic in Latin America, the present study analyzed 91 V. cholerae O1 strains isolated from both clinical and environmental sources in Mexico between 1983 and 1997. V. cholerae O1 ET strains from Peru (1991 to 1999), Bangladesh (1985 to 2007), and Zambia (1996 to 2004) were also included for comparative purposes.     

Haitian variant Vibrio cholerae O1 Ogawa caused cholera outbreaks in Odisha

PurposeDiarrheal disorders particularly cholera cause a significant threat resulting in high morbidity and mortality in the coastal and tribal areas of Odisha. Two sequential diarrheal outbreaks reported in 2016 from Balasore and Rayagada districts of Odisha were investigated to find out the causative organisms, antibiogram profile and molecular analysis of the isolated pathogens.MethodBacteriological analysis and antibiogram profiles of the pathogens were carried out as per the standard procedure followed. The double mismatch amplification mutation (DMAMA) PCR for ctxB gene, sequencing and pulse-field gel electrophoresis (PFGE) were carried out on Vibrio cholerae O1 strains.ResultsThe rectal swabs and water samples from these districts were positive for V. cholerae O1 Ogawa biotype El Tor. The V. cholerae O1 strains isolated from Balasore district were multidrug resistant to many antibiotics which differed from the isolates of Rayagada district. The DMAMA PCR assay on all clinical and water isolates from these areas and some strains from other districts exhibited ctxB7 allele of V. cholerae O1 which correlates with the sequencing results having different pulsotypes. The Haitian variant of V. cholerae O1 strains which were compared with the V. cholerae O1 strains of 1999 and 2000 exhibited different pulsotypes.ConclusionThe present study reports cholera outbreaks due to multidrug resistant ctxB7 allele of V. cholerae O1 from both coastal (Balasore) and tribal (Rayagada) areas of Odisha.     

Mechanisms of Inflammasome Activation by Vibrio cholerae Secreted Toxins Vary with Strain Biotype

Activation of inflammasomes is an important aspect of innate immune responses to bacterial infection. Recent studies have linked Vibrio cholerae secreted toxins to inflammasome activation by using murine macrophages. To increase relevance to human infection, studies of inflammasome-dependent cytokine secretion were conducted with the human THP-1 monocytic cell line and corroborated in primary human peripheral blood mononuclear cells (PBMCs). Both El Tor and classical strains of V. cholerae activated ASC (apoptosis-associated speck-like protein-containing a CARD domain)-dependent release of interleukin-1β (IL-1β) when cultured with human THP-1 cells, but the pattern of induction was distinct, depending on the repertoire of toxins the strains produced. El Tor biotype strains induced release of IL-1β dependent on NOD-like receptor family pyrin domain-containing 3 (NLRP3) and ASC due to the secreted pore-forming toxin hemolysin. Unlike in studies with mouse macrophages, the MARTX toxin did not contribute to IL-1β release from human monocytic cells. Classical biotype strains, which do not produce either hemolysin or the MARTX toxin, activated low-level IL-1β release that was induced by cholera toxin (CT) and dependent on ASC but independent of NLRP3 and pyroptosis. El Tor strains likewise showed increased IL-1β production dependent on CT when the hemolysin gene was deleted. In contrast to studies with murine macrophages, this phenotype was dependent on a catalytically active CT A subunit capable of inducing production of cyclic AMP and not on the B subunit. These studies demonstrate that the induction of the inflammasome in human THP-1 monocytes and in PBMCs by V. cholerae varies with the biotype and is mediated by both NLRP3-dependent and -independent pathways.     

Promotion of Colonization and Virulence by Cholera Toxin Is Dependent on Neutrophils

The innate immune response to Vibrio cholerae infection is poorly understood, but this knowledge is critical for the design of safe, effective vaccines. Using an adult mouse intestinal infection model, this study examines the contribution of neutrophils to host immunity, as well as the effect of cholera toxin and other secreted factors on this response. Depletion of neutrophils from mice with anti-Ly6G IA8 monoclonal antibody led to similar survival rates of mice infected with low or moderate doses of toxigenic V. cholerae El Tor O1. At a high dose, neutropenic mice showed increased rates of survival compared to neutrophil-replete animals. Expression of cholera toxin was found to be protective to the neutropenic host, and this phenotype can be replicated by the administration of purified toxin. Neutrophils do not effectively clear colonizing bacteria from the small intestine, nor do they alter induction of early immune-modulating signals. In both neutropenic and neutrophil-replete animals, the local response to infection is characterized by expression of interleukin 6 (IL-6), IL-10, and macrophage inflammatory protein 2 alpha (MIP-2). Overall, these data indicate that the innate immune response to toxigenic V. cholerae infection differs dramatically from the host response to nontoxigenic infection or vaccination, where neutrophils are protective to the host. In the absence of neutrophils, cholera toxin induces immunomodulatory effects that increase host survival. In cholera toxin-producing strains, similar to nontoxigenic infection, accessory toxins are critical to virulence, indicating that cholera toxin and the other secreted toxins modulate the host response by different mechanisms, with both contributing to bacterial persistence and virulence.