Prototype El Tor Research Articles

Origin and Dissemination of Altered El Tor Vibrio cholerae O1 Causing Cholera in Odisha, India: Two and Half Decade's View.

The origin, spread and molecular epidemiology of altered El Tor Vibrio cholerae O1 strains isolated from cholera outbreaks/surveillance studies between 1995 and 2019 from different district of Odisha were analyzed. The stock cultures of V. cholerae O1 strains from 1995 to 2019 were analyzed through molecular analysis using different PCR assays and pulse field gel electrophoresis (PFGE) analysis. The spread map (month, year and place) was constructed to locate the dissemination of altered El Tor variants of V. cholerae O1 in this region. A total of 13 cholera outbreaks were caused by V. cholerae O1 Ogawa biotype El Tor carrying ctxB1 and ctxB7 genotypes. The ctxB1 alleles of V. cholerae O1 mostly confined to the coastal areas, whereas the ctxB7 genotypes, though originating in the coastal region of Odisha, concentrated more in the tribal areas. The positive correlation between virulence-associated genes (VAGs) was found through Pearson’s correlation model, indicative of a stronger association between the VAGs. The clonal relationship through PFGE between ctxB1 and ctxB7 genotypes of V. cholerae O1 strains exhibited 80% similarity indicating single- or multi-clonal evolution. It is evident from this study that the spread of multidrug-resistant V. cholerae O1-altered El Tor was dominant over the prototype El Tor strains in this region. The origin of altered El Tor variants of V. cholerae O1 occurred in the East Coast of Odisha established that the origin of cholera happened in the Gangetic belts of Bay of Bengal where all new variants of V. cholerae O1 might have originated from the Asian countries.

Recent Vibrio cholerae O1 Epidemic Strains Are Unable To Replicate CTXΦ Prophage Genome.

ABSTRACTCholera, an acute diarrheal disease, is caused by pathogenic strains of Vibrio cholerae generated by the lysogenization of the filamentous cholera toxin phage CTXΦ. Although CTXΦ phage in the classical biotype are usually integrated solitarily or with a truncated copy, those in El Tor biotypes are generally found in tandem and/or with related genetic elements. Due to this structural difference in the CTXΦ prophage array, the prophage in the classical biotype strains does not yield extrachromosomal CTXΦ DNA and does not produce virions, whereas the El Tor biotype strains can replicate the CTXΦ genome and secrete infectious CTXΦ phage particles. However, information on the CTXΦ prophage array structure of pathogenic V. cholerae is limited. Therefore, we investigated the complete genomic sequences of five clinical V. cholerae isolates obtained in Kolkata (India) during 2007 to 2011. The analysis revealed that recent isolates possessed an altered CTXΦ prophage array of the prototype El Tor strain. These strains were defective in replicating the CTXΦ genome. All recent isolates possessed identical rstA and intergenic sequence 1 (Ig-1) sequences and comparable rstA expression in the prototype El Tor strain, suggesting that the altered CTXΦ array was responsible for the defective replication of the prophage. Therefore, CTXΦ structures available in the database and literatures can be classified as replicative and nonreplicative. Furthermore, V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a significant change in the dissemination pattern of the current cholera pandemic occurred.IMPORTANCE Cholera is an acute diarrheal disease caused by pathogenic strains of V. cholerae generated by lysogenization of the filamentous cholera toxin phage CTXΦ. The analysis revealed that recent isolates possessed altered CTXΦ prophage array of prototype El Tor strain and were defective in replicating the CTXΦ genome. Classification of CTXΦ structures in isolated years suggested that V. cholerae epidemic strains became capable of producing CTXΦ phage particles since the 1970s. However, V. cholerae epidemic strains again lost the capacity for CTXΦ production around the year 2010, suggesting that a critical change had occurred in the dissemination pattern of the current cholera pandemic.

Epidemiological and pathogenic characteristics of Haitian variant V. cholerae circulating in India over a decade (2000–2018)

Vibrio cholerae, causative agent of the water-borne disease cholera still threatens a large proportion of world's population. The major biotypes of the pathogen are classical and El Tor. There have been recent reports of variant V. cholerae strains circulating around the world. In the present study, the epidemiological status of V. cholerae strains circulating in the country over a decade was assessed. Also, a comprehensive analysis of the difference in pathogenicity between the different biotypes of V. cholerae strains was evaluated both in-vitro and in-vivo. The amount of CT produced by different biotypes of V. cholerae strains were analyzed by GM1 ELISA and the probable reasons for the difference in toxin production was discussed. MLST analysis grouped the isolates into a single Sequence Type (ST 69) whereas PFGE analysis clustered the isolates into ten different pulsotypes revealing molecular diversity. The circulating strains were identified to produce cholera toxin and CT mRNA intermediate to the classical and prototype El Tor strains. Also, the circulating strains were identified to possess four ToxR binding sequences. In-vivo pathogenicity analysis by rabbit ileal loop fluid accumulation assay revealed the Haitian variant strains to be more hyperemic than the prototype strains.

Haitian-like genetic traits with creeping MIC of Azithromycin in Vibrio cholerae O1 isolates from Puducherry, India.

Introduction. The emergence of novel strains of Vibrio cholerae O1 El Tor biotype has gained attention due to causing several epidemics around the world. Variant strains have evolved as a result of the acquisition of genes that confer extended virulence and pathogenicity.Aim. This study aimed to determine the presence of the most recently emerging Haitian-like genetic traits among the isolates from Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, Southern India. We also wanted to detect the prevalence of the sulfamethoxazole and trimethoprim (SXT) element, which is an integrating conjugative element (ICE) and the antimicrobial resistance genes present in our isolates.Methodology. Identification of Haitian-specific alleles was done by mismatched amplification mutation assay PCR (MAMA-PCR). The presence of SXT elements was carried out by PCR by detecting int, eex, att-prfC and setR genes. Detection of antibiotic resistance determinant, sul(1,2,3); dfr(A1,18,5) for trimethoprim resistance, tet(A,B,C,D,E,Y,G,M), tet34 for tetracycline resistance and erm(A,B,C), mph(A,B), ere(A,B), msr(A,D) for azithromycin resistance were targeted by PCR. The MIC of tetracycline, ciprofloxacin and azithromycin was determined by the E-test method.Results. Of the 95 isolates, 60 % of the isolates were found to carry Haitian-specific alleles of ctxB, tcpA and rtxA gene, 100 % of the isolates were found to carry SXT elements. All the isolates harboured the four conserved genes of the SXT element, except one which had only eex, att-prfC, setR genes. About 99 % harboured sul2 and dfrA1 genes. No tet and macrolide genes were detected. We observed a progressive increase in the MIC of azithromycin ranging from 0.75 µg ml-1 to 2 µg ml-1.Conclusion. None of the isolates were the prototype El Tor biotype. All the isolates were a Haitian variant. The presence of SXT elements across all our isolates and their creeping MIC of azithromycin is a matter of concern. Further testing for other genetic determinants of resistance will be carried out in our future studies.

Novel Cholera Toxin Variant and ToxT Regulon in Environmental Vibrio mimicus Isolates: Potential Resources for the Evolution of Vibrio cholerae Hybrid Strains.

Atypical El Tor strains of Vibrio cholerae O1 harboring variant ctxB genes of cholera toxin (CT) have gradually become a major cause of recent cholera epidemics. Vibrio mimicus occasionally produces CT, encoded by ctxAB on CTXФ genome; toxin-coregulated pilus (TCP), a major intestinal colonization factor; and also the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon in V. mimicusctx-positive (ctx+) strains (i.e., V. mimicus strains containing ctx) isolated from the Bengal coast. Southern hybridization, PCR, and DNA sequencing of virulence-related genes revealed the presence of an El Tor type CTX prophage (CTXET) carrying a novel ctxAB, tandem copies of environmental type pre-CTX prophage (pre-CTXEnv), and RS1 elements, which were organized as an RS1-CTXET-RS1-pre-CTXEnv-pre-CTXEnv array. Additionally, novel variants of tcpA and toxT, respectively, showing phylogenetic lineage to a clade of V. cholerae non-O1 and to a clade of V. cholerae non-O139, were identified. The V. mimicus strains lacked the RTX (repeat in toxin) and TLC (toxin-linked cryptic) elements and lacked Vibrio seventh-pandemic islands of the El Tor strains but contained five heptamer (TTTTGAT) repeats in ctxAB promoter region similar to those seen with some classical strains of V. cholerae O1. Pulsed-field gel electrophoresis (PFGE) analysis showed that all the ctx+V. mimicus strains were clonally related. However, their in vitro CT production and in vivo toxigenicity characteristics were variable, which could be explainable by differential transcription of virulence genes along with the ToxR regulon. Taken together, our findings strongly suggest that environmental V. mimicus strains act as a potential reservoir of atypical virulence factors, including variant CT and ToxT regulons, and may contribute to the evolution of V. cholerae hybrid strains.IMPORTANCE Natural diversification of CTXФ and ctxAB genes certainly influences disease severity and shifting patterns in major etiological agents of cholera, e.g., the overwhelming emergence of hybrid El Tor variants, replacing the prototype El Tor strains of V. cholerae This report, showing the occurrence of CTXET comprising a novel variant of ctxAB in V. mimicus, points out a previously unnoticed evolutionary event that is independent of the evolutionary event associated with the El Tor strains of V. cholerae Identification and cluster analysis of the newly discovered alleles of tcpA and toxT suggest their horizontal transfer from an uncommon clone of V. cholerae The genomic contents of ToxT regulon and of tandemly arranged multiple pre-CTXФEnv and of a CTXФET in V. mimicus probably act as salient raw materials that induce natural recombination among the hallmark virulence genes of hybrid V. cholerae strains. This report provides valuable information to enrich our knowledge on the evolution of new variant CT and ToxT regulons.

Design and Construction of Vibrio cholerae Strains That Harbor Various CTX Prophage Arrays.

Toxigenic Vibrio cholerae strains arise upon infection and integration of the lysogenic cholera toxin phage, the CTX phage, into bacterial chromosomes. The V. cholerae serogroup O1 strains identified to date can be broadly categorized into three main groups: the classical biotype strains, which harbor CTX-cla; the prototype El Tor strains (Wave 1 strains), which harbor CTX-1; and the atypical El Tor strains, which harbor CTX-2 (Wave 2 strains) or CTX-3~6 (Wave 3 strains). The efficiencies of replication and transmission of CTX phages are similar, suggesting the possibility of existence of more diverse bacterial strains harboring various CTX phages and their arrays in nature. In this study, a set of V. cholerae strains was constructed by the chromosomal integration of CTX phages into strains that already harbored CTX phages or those that did not harbor any CTX phage or RS1 element. Strains containing repeats of the same kind of CTX phage, strains containing the same kind of CTX phage in each chromosome, strains containing alternative CTX phages in one chromosome, or containing different CTX phages in each chromosome have been constructed. Thus, strains with any CTX array can be designed and constructed. Moreover, the strains described in this study contained the toxT-139F allele, which enhances the expression of TcpA and cholera toxin. These characteristics are considered to be important for cholera vaccine development. Once their capacity to provoke immunity in human against V. cholerae infection is evaluated, some of the generated strains could be developed further to yield cholera vaccine strains.

Mismatch Amplification Mutation Assay (MAMA) PCR Reveals Altered El Tor Vibrio Cholerae O1 Circulating in Delhi, India

The acute diarrheal disease cholera is caused by Vibrio cholerae, a major public health problem in Asia, Africa and Latin America. V. cholerae has more than 200 known serotypes but not all the strains are pathogenic. In recent years, it has been seen that the emergence of new variants of V. cholerae O1 have carried characteristics of both classical and El Tor biotypes and these variants of V. cholerae O1 are called as ‘atypical El Tor’. These strains might have evolved from El Tor variants that acquired certain characteristics from classical genome. 30 V. cholerae O1 isolates (Table 1) were revived from stock cultures maintained at Laboratory Department in Maharishi Valmiki Infectious Diseases Hospital (MVIDH), Delhi during 2012-2014. Mismatch amplification mutation assay was used for classifying the strains into prototype El Tor, hybrid, or El Tor variant biotype based on their ctxB gene. All isolates were biochemically identified as V. cholerae biotype El Tor and serologically O1 Ogawa. All isolates were amplified with classical specific primers. Cholera continues to be endemic in large number of states in the eastern, western, northern and southern parts of India and there were 38 cholera outbreak reports published in India during 2007 to 2013. These Indian outbreaks have been associated with new El Tor variant. Key-wordsV. cholerae O1, El Tor, MAMA PCR, ctxB -------------------------------------------------IJLSSR----------------------------------------------INTRODUCTION The acute diarrheal disease cholera is caused by Vibrio cholerae, a major public health problem in Asia, Africa and Latin America. According to the World Health Organisation (WHO) estimates there were 3-5 million cholera cases and 1,00,0001,20,000 deaths each year throughout the world. V. cholerae has more than 200 known serogroups but not all the strains are pathogenic. Among them, O1 and O139 antigens are highly pathogenic and acknowledged to cause epidemic and pandemic disease. The symptom of the acute cholera is rice water stools. Access this article online Quick Response Code: Website: www.ijlssr.com DOI: 10.21276/ijlssr.2016.2.6.6 Clinical manifestations of the disease range from mild symptoms such as abdominal cramps, nausea and vomiting, to more severe symptoms such as dehydration, shock and death. A devastating cholera outbreak, that had occurred in Haiti since October 2010, involved 6, 98, 893 cases and 8,540 deaths. V. cholerae serogroup O1 is classified into two biotypes, which are termed ‘classical’ and ‘El Tor’. The current seventh pandemic of cholera is caused by the El Tor biotype was originated in 1961 from Celebes Islands in Indonesia. Spread of this biotype was recorded for the first time during 1964 in India and it made its first appearance in Delhi during June, 1965. In recent years, it has been seen that the emergence of new variants of V. cholerae O1 have carried characteristics of both classical and El Tor biotypes and these variants of V. cholerae O1 are called as ‘atypical El Tor’. These strains might have evolved from El Tor variants that acquired certain characteristics from classical genome. In this study, we focused on type of CT genotype existing among V. cholerae Research Article (Open access) Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 6 http://ijlssr.com Copyright © 2015-2016 International Journal of Life-Sciences Scientific Research Page 679 O1 isolated from the patients who were admitted in MVIDH, Delhi during 2012-2014. MATERIALS AND METHODS Collection and processing of samples 30 V. cholerae O1 isolates were revived from stock cultures were maintained at Laboratory Department in MVIDH, Delhi during 2012-2014. These samples were revived and transferred to alkaline peptone water (APW, pH-8.6) and incubated at 37°C for 4-6 hrs. After incubation, the enriched cultures were further inoculated to the thiosulphate-citrate-bile-salts-sucrose (TCBS) agar and bile salt agar (BSA, pH-8.6) (Hi-Media, Mumbai). Confirmation of V. cholerae Typical colonies appearing on TCBS/BSA were confirmed by standard biochemical tests. These isolates were further tested serologically with commercially available V. cholerae O1 polyvalent and monovalent and O139 antiserum (BD, USA and Denka Seiken, Ltd. Japan). Genomic DNA preparation Genomic DNA from each isolate was extracted using protocol described previously. PCR assays for detection of ctxB genotype Mismatch amplification mutation assay was used for classifying the strains into prototype El Tor, hybrid, or El Tor variant biotype based on their ctxB gene. In this PCR, the common forward primer for both classical and El Tor alleles was used. Two allele specific primers Re-Cla and Re-Elt were used respectively. The PCR conditions and cycles were described previously. V. cholerae O1 strains 569B (classical) and N16961 (El Tor) were used as control strains. The primers used were synthesized by Invitrogen, India and dNTPs, Taq polymerase, 10X Taq buffer used in this study were obtained from Genei, Bangalore. The primer sequences were used in this PCR is given in Table 2.

Phylogenetic Diversity of Vibrio cholerae Associated with Endemic Cholera in Mexico from 1991 to 2008.

ABSTRACTAn outbreak of cholera occurred in 1991 in Mexico, where it had not been reported for more than a century and is now endemic. Vibrio cholerae O1 prototype El Tor and classical strains coexist with altered El Tor strains (1991 to 1997). Nontoxigenic (CTX−) V. cholerae El Tor dominated toxigenic (CTX+) strains (2001 to 2003), but V. cholerae CTX+ variant El Tor was isolated during 2004 to 2008, outcompeting CTX− V. cholerae. Genomes of six Mexican V. cholerae O1 strains isolated during 1991 to 2008 were sequenced and compared with both contemporary and archived strains of V. cholerae. Three were CTX+ El Tor, two were CTX− El Tor, and the remaining strain was a CTX+ classical isolate. Whole-genome sequence analysis showed the six isolates belonged to five distinct phylogenetic clades. One CTX− isolate is ancestral to the 6th and 7th pandemic CTX+ V. cholerae isolates. The other CTX− isolate joined with CTX− non-O1/O139 isolates from Haiti and seroconverted O1 isolates from Brazil and Amazonia. One CTX+ isolate was phylogenetically placed with the sixth pandemic classical clade and the V. cholerae O395 classical reference strain. Two CTX+ El Tor isolates possessing intact Vibrio seventh pandemic island II (VSP-II) are related to hybrid El Tor isolates from Mozambique and Bangladesh. The third CTX+ El Tor isolate contained West African-South American (WASA) recombination in VSP-II and showed relatedness to isolates from Peru and Brazil. Except for one isolate, all Mexican isolates lack SXT/R391 integrative conjugative elements (ICEs) and sensitivity to selected antibiotics, with one isolate resistant to streptomycin. No isolates were related to contemporary isolates from Asia, Africa, or Haiti, indicating phylogenetic diversity.

Molecular Insights Into the Evolutionary Pathway of Vibrio cholerae O1 Atypical El Tor Variants

Pandemic V. cholerae strains in the O1 serogroup have 2 biotypes: classical and El Tor. The classical biotype strains of the sixth pandemic, which encode the classical type cholera toxin (CT), have been replaced by El Tor biotype strains of the seventh pandemic. The prototype El Tor strains that produce biotype-specific cholera toxin are being replaced by atypical El Tor variants that harbor classical cholera toxin. Atypical El Tor strains are categorized into 2 groups, Wave 2 and Wave 3 strains, based on genomic variations and the CTX phage that they harbor. Whole-genome analysis of V. cholerae strains in the seventh cholera pandemic has demonstrated gradual changes in the genome of prototype and atypical El Tor strains, indicating that atypical strains arose from the prototype strains by replacing the CTX phages. We examined the molecular mechanisms that effected the emergence of El Tor strains with classical cholera toxin-carrying phage. We isolated an intermediary V. cholerae strain that carried two different CTX phages that encode El Tor and classical cholera toxin, respectively. We show here that the intermediary strain can be converted into various Wave 2 strains and can act as the source of the novel mosaic CTX phages. These results imply that the Wave 2 and Wave 3 strains may have been generated from such intermediary strains in nature. Prototype El Tor strains can become Wave 3 strains by excision of CTX-1 and re-equipping with the new CTX phages. Our data suggest that inter-chromosomal recombination between 2 types of CTX phages is possible when a host bacterial cell is infected by multiple CTX phages. Our study also provides molecular insights into population changes in V. cholerae in the absence of significant changes to the genome but by replacement of the CTX prophage that they harbor.

CTX prophages in Vibrio cholerae O1 strains.

The classical biotype strains of the Vibrio cholerae O1 serogroup harbor the biotype-specific cholera-toxin encoding phage (CTX) CTX(cla), and the El Tor biotype strains contain CTX-1. Although the classical biotype strains have become extinct, a remnant of classical CTX phage is transferred to the El Tor biotype strains. The prototype El Tor strains, which produce the biotype-specific cholera toxin, are now being replaced by atypical El Tor variant strains producing classical biotype cholera toxin. The genome sequences of the CTX phages in atypical El Tor strains indicate that the CTX phages in atypical El Tor strains are a mosaic of CTXcla and CTX-1. Before the emergence of atypical El Tor stains in the early 1990s, unusual pre-seventh pandemic strains were isolated in the US Gulf Coast between 1973 and 1986. These strains have characteristics of atypical El Tor strains since they are El Tor biotype strains containing CTX(cla), yet the genome sequence of this CTX phage indicates that it is different from CTXcla and is therefore classified separately as CTX(US Gulf).

Genomic aspects of cholera and its relevance to cholera control

Toxigenic Vibrio cholerae belonging to serogroups O1 or O139 are water and food borne pathogens that cause the disease cholera. The fifth and sixth pandemics of cholera were caused by the classical biotype while the current seventh pandemic is caused by the El Tor biotype, which has now completely replaced the classical biotype. Molecular studies have further defined three new variants of El Tor V. cholerae O1 associated with cholera. The three variants are i) the Matlab variants which could not be biotyped by conventional methods since they had a combination of both classical and El Tor traits, ii) altered El Tor O1 strains which displayed typical traits of the El Tor biotype but carried the classical CTX prophage and iii) altered O1 El Tor isolates which harbored ctxB of classical origin. The altered El Tor variants produce higher quantities of cholera toxin as compared to the prototype El Tor and classical biotypes. The current pandemic caused by the El Tor biotype has shown subtle but distinct changes in the epidemiology and clinical presentation of the disease and also in the virulence and molecular traits. There has also been a change in the epidemiology of the disease with the burden of disease dominating in the under 5 year age group particularly in cholera endemic areas of south east Asia. Another unusual trend recently witnessed is the protracted nature of cholera outbreaks with high case fatality. Single nucleotide polymorphisms in 154 whole-genome sequences of globally and temporally representative V. cholerae isolates show that the seventh pandemic has spread from the Bay of Bengal in at least three independent but overlapping waves, and identified several transcontinental transmission events. From the studies on the genomes of V. cholerae, two facts have emerged. One is that variants of V. cholerae O1 emerge in cholera hyper endemic areas due to the intense transmission. The other fact is that all the three new waves of the El Tor variants originated from the Bay of Bengal area. Taken together, genomic studies point to the adoption of intense cholera control programs in the cholera endemic Bay of Bengal area as a strategy to eliminate cholera in these endemic areas and thereby abort the emergence and spread of cholera over the region and the globe.

The Seventh Pandemic Vibrio cholerae O1 El Tor Isolate in China Has Undergone Genetic Shifts

A total of 330 clinical Vibrio cholerae O1 serogroups from China dating between 1961 and 2010 were investigated. By phenotypic biotyping and genetic analysis, during the seventh pandemic of V. cholerae O1 in China, the isolates of hybrid biotype (mixed classical phenotypes) were present during the entire1961-2010 period, while El Tor genetic shifts appeared in 1992 and replaced the prototype El Tor from 2002 to 2010.

Vibrio choleraeO1 El Tor and O139 Bengal Strains CarryingctxBET, Bangladesh

<i>Vibrio cholerae</i>O1 El Tor and O139 Bengal Strains Carrying<i>ctxB</i>^ET, Bangladesh

Novel ctxB variants of Vibrio cholerae O1 isolates, China

We screened 650 isolates from historical collection of Vibrio cholerae O1 during the 7th cholera pandemic in China, by amplifying and sequencing the cholera toxin subunit gene ctxB. Ten isolates were identified as harboring three novel ctxB genotypes based on amino acid residue substitutions. Within them one isolate from a patient in 1964 was similar to the El Tor genotype, except for an 11 amino acid repeat sequence (LAGKREMAIIT) that was inserted after position 62. Six environmental isolates from different regions and years were identified as the Australia El Tor genotype, except at positions 36(T→A), 39(H→Y), and 55(K→N), while three environmental isolates were similar to genotype 5, except at position 24(Q→H). Sequencing of rstR, the marker gene for the CTXΦ allele typing, revealed that two isolates carried the rstR gene of the El Tor type, five carried the classical type rstR, while other isolates carried the rstR232 type. All 10 isolates contained the repeat in the toxin gene rtxC, an El Tor biotype-specific marker, and the El Tor toxin-coregulated pili subunit A gene tcpA, showing the El Tor traits of these isolates. Additionally, by phenotypic biotyping (susceptibility to polymyxin B, positive for chicken erythrocyte agglutination, and Voges–Proskauer test), all isolates except two were typical of the prototype El Tor isolate, while these two isolates had mixed classical phenotypes (hybrid biotype). Furthermore, pulsed-field gel electrophoresis analysis suggested that the new ctxB altered isolates possessed potential transmissibility and thatthey propagated in the local region(s). Taken together, these novel ctxB variants of V. cholerae O1 experienced complex hybrid and genetic exchange but belong to the El Tor lineage, and the pathogenic and epidemic potential of these lineages should be monitored.

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.

Genetic characterization ofVibrio choleraeO1 strains isolated in Zambia during 1996–2004 possessing the unique VSP-II region of El Tor variant

New variants of Vibrio cholerae O1 have appeared in different time-frames in various endemic regions, especially in Asia and Africa. Sixty-nine strains of V. cholerae O1 isolated in Zambia between 1996 and 2004 were investigated by various genotypic techniques to determine the lineage of virulence signatures and clonality. All strains were positive for Vibrio seventh pandemic Islands (VSP)-I and VSP-II and repeat toxin (RTX) gene clusters attesting their El Tor lineage. Interestingly, strains isolated in recent times (2003-2004) were identified as an altered variant (El Tor biotype that harbours El Tor type rstR but produce classical ctxB) that replaced completely the progenitor El Tor strains prevalent in 1996-1997. Recent altered variant strains differed from prototype El Tor strains isolated earlier in that these strains lacked two ORFs, VC0493 and VC0498, in the VSP-II region. PFGE analysis revealed two major clonal lineages in the strains; cluster A represented the strains isolated before 2003 and cluster B the altered strains isolated in 2003-2004. Cluster A was closely related to prototype El Tor reference strain isolated in Bangladesh in 1971. Cluster B was found to be matched with Bangladeshi altered strains but was different from the hybrid strains isolated from Mozambique and Bangladesh. This report provides important information on the genesis of altered strains of V. cholerae O1 isolated in Zambia and emphasizes the need for further studies to follow the trends of evolutionary changes.

Vibrio choleraeO1 Variant with Reduced Susceptibility to Ciprofloxacin, Western Africa

To the Editor: Many variants of choleragenic vibrios have emerged since the beginning of the seventh pandemic, indicating continuous evolution of this pathogenic agent. Variations occur mainly in genetic determinants of virulence and antimicrobial drug susceptibility. In September–October 2009, concurrent outbreaks of acute watery diarrhea in northeastern Nigeria (4,559 cases) and northern Cameroon (696 cases) were investigated by state ministries of health. We report reduced sensitivity to ciprofloxacin in Vibrio cholerae O1 strains and the atypical cholera toxin B (ctxB) genotype of these strains. In September–October 2009, stool specimens from patients in Nigeria were collected on filter paper, moistened with sterile physiologic saline, and sent at room temperature to the National Reference Center for Vibrios and Cholera at the Institut Pasteur (Paris, France). Ten V. cholerae O1 biotype El Tor serotype Ogawa strains were isolated and identified by using standard procedures. Concurrently in Cameroon, 9 V. cholerae O1 Ogawa strains isolated from patient stool samples by the bacteriology laboratory of the Pasteur Center (Garoua, Cameroon) were sent to the National Reference Center for Vibrios and Cholera. All strains were tested for antimicrobial susceptibility by MIC determination to tetracycline, trimethoprim/sulfamethoxazole, sulfonamides, ampicillin, chloramphenicol, nalidixic acid, and ciprofloxacin by using Etest (AB bioMerieux, Solna, Sweden) according to Clinical and Laboratory Standards Institute procedures and interpretative standards for V. cholerae (1). PCR amplification of the genes encoding DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) and subsequent sequencing of PCR products were performed (2). PCR was used to test for the presence of ctxA and ctxB genes, which encode the cholera toxin (CT), and the tcpA gene, which encodes the toxin-coregulated pilus. Genotyping of ctxB was performed by sequencing PCR products. All isolates showed susceptibility to tetracycline (MIC 1.5 mg/L), intermediate susceptibility to ampicillin (MICs 12–16 mg/L) and chloramphenicol (MICs 8–12 mg/L), and resistance to trimethoprim/sulfamethoxazole (MIC >32 mg/L), sulfonamides (MIC >1,024 mg/L), and nalidixic acid (MIC >256 mg/L). MICs of ciprofloxacin ranged from 0.25 to 0.5 mg/L. Sequencing of gyrA, gyrB, parC, and parE genes among all strains detected 1 mutation in gyrA (substitution of serine by isoleucine at position 83) and 1 mutation in parC (substitution of serine by leucine at position 85). Both point mutations have been associated with quinolone resistance in clinical isolates of V. cholerae (2). None of the strains had any mutations in gyrB or parE. The presence of ctxA and ctxB genes confirmed the toxigenicity of all isolates, and tcpA PCR product size and sequence identified El Tor biotype strains. The DNA sequence of ctxB was similar to that of the recently reported Orissa variant identified in India in 2007 (3). This sequence had 2 mutations resulting in histidine at position 39 and threonine at position 68 (this amino acid sequence is similar to the CT-B subunit of the reference classical strain) and a third mutation resulting in substitution of histidine by asparagine at position 20. We report atypical El Tor strains of V. cholerae O1 and their reduced susceptibility to ciprofloxacin in Nigeria and Cameroon. Since the 1990s, atypical El Tor strains that produce classical CT have been increasingly reported from countries in Asia, where they have gradually replaced the prototype El Tor strains, but they have only been reported in 2 countries in Africa (Mozambique and Zambia) (4,5). On the basis of the CT-B subunit sequence, these variants differ from variants isolated in southern Africa and from most variants isolated in Asia by having the same modified classical CT as a strain recently isolated in Orissa in eastern India (3), which has not been reported elsewhere. These findings indicate evolution of V. cholerae O1 El Tor hybrid strains. Their presence may indicate spread of strains from eastern India to Africa (6). The presence of CT-B variants in central or western Africa is of great concern because these strains may be more toxigenic (3). There is also concern for the strains isolated in this study because of their reduced susceptibility to ciprofloxacin. Although reduced susceptibility to fluoroquinolone is common in southern Asia (7,8), it was reported in Africa (Zimbabwe) only recently (9). Our findings, in addition to the report of Islam et al. (9), indicate that V. cholera with reduced sensitivity to a fluoroquinolone is present in southern and western Africa. These results highlight the need for continued monitoring of antimicrobial drug susceptibility and strain tracking to maintain an efficient cholera surveillance system.

Cholera Toxin Production by the El Tor Variant ofVibrio choleraeO1 Compared to Prototype El Tor and Classical Biotypes

Vibrio cholerae O1 El Tor variant strains produced much more cholera toxin than did prototype El Tor strains. The amount of cholera toxin produced by El Tor variant strains both in vitro and in vivo was more or less equivalent to that produced by classical strains.

Genetic analysis of CTX prophages with special reference toctxBand rstR alleles ofVibrio choleraeO139 strains isolated from Kolkata over a decade

Chronological analysis of 125 Vibrio cholerae O139 strains isolated during 1993-2005 in Kolkata revealed the prevalence of two new genotypes of cholera toxin (CT) and novel combinations of ctxB and rstR alleles resulting in variant CTX prophages. One of the new genotypes of ctxB, which first appeared in 1996 with the re-emerged V. cholerae O139 strains that had CTX Calcutta phage, was designated as genotype 4. In 1998, another new genotype, designated as genotype 5, was detected that prevailed mostly in CTX phages with El Tor rstR. The prototype El Tor CTX phage with genotype 3 gradually disappeared in O139, and since 2002 the predominant CTX prophages in O139 are Calcutta phages with genotype 4 and El Tor phages with genotype 5. Results showed that V. cholerae O139 strains of Kolkata, isolated over a decade, harboured CTX prophages in the large chromosome having no RS1 downstream of CTX prophage. During the course of its intermittent incidence over a decade, five types of O139 strains were detected based on CT genotypes. Such abrupt genetic changes in O139 strains might not favour its continued prevalence in human cases in Kolkata, India.

Vibrio choleraeO1 Hybrid El Tor Strains, Asia and Africa

<i>Vibrio cholerae</i>O1 Hybrid El Tor Strains, Asia and Africa