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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.Vibrio cholerae O1 has two biotypes, namely, classical and El Tor, which are believed to have evolved from separate lineages (7,8), and these biotypes have traditionally been differentiated by a number of phenotypic traits. Comparative genomic analyses have recently revealed a high degree of conservation among diverse strains of V. cholerae but have also shown genes that differentiate the classical biotype from the El Tor biotype (3). Apart from these phenotypic and genetic differences, there are also dissimilarities in the infection patterns of disease caused by the two biotypes. These include the occurrence of more asymptomatic than symptomatic carriers of El Tor strains, who outnumber active patients by a ratio of up to 50:1 (14), better survival of El Tor strains in the environment and in the human host, and more efficient host-to-host transmission of El Tor strains than of classical strains (5). There is firm evidence that the fifth and sixth pandemics of cholera were caused by the classical biotype, while the ongoing seventh pandemic is caused by the El Tor biotype, which has now globally replaced the classical biotype.Cholera toxin (CT), the principal toxin produced by V. cholerae O1 and O139, is responsible for most of the manifestations of the disease cholera. Based on the B subunit of CT, two immunologically related but not identical epitypes have been described: CT1 is the prototype elaborated by classical biotype strains and by U.S. Gulf Coast strains, while CT2 is produced by the El Tor biotype and O139 strains (4). Another classification identifies three types of ctxB genes based on three nonrandom base changes resulting in changes in the deduced amino acid sequence. Genotype 1 is found in strains of the classical biotype worldwide and in U.S. Gulf Coast strains, genotype 2 is found in El Tor biotype strains from Australia, and genotype 3 is found in El Tor biotype strains from the seventh pandemic and the Latin American epidemic (12). Thus, the V. cholerae O1 El Tor biotype of the ongoing seventh pandemic produces CT of the CT2 epitype and genotype 3, while the classical biotype CT belongs to the CT1 epitype and genotype 1. In this study, we examined a collection of clinical V. cholerae O1 strains isolated in Bangladesh during the past four and a half decades, using monoclonal antibodies (MAbs) produced to classical and El Tor CTs, and found that V. cholerae O1 El Tor strains isolated since 2001 in Bangladesh produce the CT subtype of the classical biotype.One hundred eighty-five strains of V. cholerae O1, consisting of 31 strains of the classical biotype isolated between 1960 and 1990 and 113 strains of the El Tor biotype and 41 hybrid strains of V. cholerae O1 (strains that could ...

PulseNet is a network that utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols with the purpose of conducting laboratory-based surveillance of foodborne pathogens. PulseNet standardized PFGE protocols are subject to rigorous testing during the developmental phase and careful evaluation during a validation process assessing its robustness and reproducibility in different laboratories. Here we describe the development and validation of a rapid PFGE protocol for subtyping Vibrio cholerae for use in PulseNet International activities. While the protocol was derived from the existing PulseNet protocol for Escherichia coli O157, various aspects of this protocol were optimized for use with V. cholerae, most notably a change of the primary and secondary restriction enzyme to SfiI and NotI, respectively, and the use of a two-block electrophoresis program. External validation of this protocol was undertaken through a collaboration between three PulseNet Asia Pacific laboratories (Public Health Laboratory Centre, Hong Kong, National Institute of Infectious Diseases, Japan, and International Center for Diarrhoeal Diseases Research-Bangladesh) and PulseNet USA. Comparison of PFGE patterns generated by each of the participating laboratories demonstrated that the protocol is robust and reproducible.

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Twenty-one atypical Shigella flexneri type 4 strains isolated from patients attending the Dhaka treatment center of the International Centre for Diarrhoeal Disease Research, Bangladesh, were extensively characterized and compared with S. flexneri serotypes 4a and 4b. The atypical strains agglutinated only with the type antigen factor 4 and did not agglutinate with any group factors, thereby excluding their characterization into serotype 4a or 4b. Of the 21 strains, 85.7% did not ferment mannitol but were able to ferment most of the sugars, whereas the remaining 14.3% strains fermented mannitol but were unable to ferment most of the sugars. Most of the strains were resistant to ampicillin, tetracycline, and trimethoprim-sulfomethoxazole. All of the strains harbored the 140-MDa plasmid, had the ipaH gene, had the sen gene (encoding Shigella enterotoxin 2), had the ability to bind Congo red, and were positive for keratoconjunctivitis in the guinea pig eye, attesting their invasive properties. All of the strains contained a middle-range plasmid (35 to 62 MDa) as well as a number of stable small plasmids, yielding mainly two plasmid profiles which were different from those of 4a and 4b strains. Conjugation and curing experiments suggested that the middle-range plasmids harbored a self-transferable multiple antibiotic resistance marker. Pulsed-field gel electrophoresis analysis of all of the tested strains yielded two types with numerous subtypes, whereas ribotyping yielded only two types which were completely different from those of types 4a and 4b. This study concluded that two different clones of atypical S. flexneri type 4 exist and strongly suggests that these are new subserotypes of S. flexneri that await further serological classification.

Forty-two episodes of Vibrio parahaemolyticus infections were detected in Beira, Mozambique, from January to May 2004. The majority of the isolates (81%) belonged to the pandemic serovars (O3:K6 and O4:K68) of V. parahaemolyticus. The pandemic serovars were positive by group-specific PCR (GS-PCR) and a PCR specific for open reading frame ORF8 (ORF8-PCR), which are molecular markers of the pandemic clone, and were positive for tdh but negative for trh. The remaining 19% of the strains also possessed the tdh gene but were GS-PCR and ORF8-PCR negative and did not belong to the pandemic serovars. Patients with V. parahaemolyticus infection were older (mean age, 27 years) than patients infected by other diarrheal agents (mean age, 21 years). Ten percent of diarrhea patients from whom no V. parahaemolyticus was cultured were severely dehydrated, but none of the V. parahaemolyticus cases were severely dehydrated. This is the first report of the isolation of pandemic strains of V. parahaemolyticus in sub-Saharan Africa and clearly indicates that the pandemic of V. parahaemolyticus has spread into the African continent.

Of 469 recently isolated Shigella flexneri strains, 452 agglutinated with Shigella flexneri-specific monoclonal antibodies. Of these, 396 could be assigned to 10 of the currently recognized 15 serotypes, with S. flexneri 2b dominating (23.2%). Of the 56 untypeable strains which showed invasive properties, 17 were serologically atypical and the remaining 39 belonged to a new serotype.Shigellosis is one of the major diarrheal diseases in Bangladesh and several other developing countries and is caused by any one of the four species or groups of Shigella, namely, S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. Each serogroup contains multiple serotypes, and at least 47 serotypes are currently recognized based on their biochemical and/or lipopolysaccharide characteristics. Based on the structure of the O antigen, a component of the lipopolysaccharide present on the outer membrane of the cell (16), 15 serotypes of S. flexneri are currently recognized. Since S. flexneri serotype 2a was found to be the major endemic serotype in developing countries, the currently available candidate vaccine is directed against S. flexneri 2a (12). It is important to determine the prevalence of various serotypes of S. flexneri in different communities worldwide and to monitor the changes over time.

PCR surveillance of the rstR genes of CTX phages in Vibrio cholerae O1 and O139 showed no relationship between the incidence of disease and changes in the rstR but showed variations in their presence in O1 and O139 strains and the occurrence of multiple types in a few strains.Of the 209 currently recognized serogroups of Vibrio cholerae, only strains belonging to serogroups O1 and O139 can cause cholera. Two major virulence gene clusters are now known to carry key virulence genes that are essential for the pathogenicity of V. cholerae O1 and O139. These gene clusters include the CTX prophage (14), which carries the ctxA and ctxB genes (the genes that encode cholera toxin [CT], which is responsible for severe diarrhea), and the toxin-coregulated pilus (TCP) pathogenicity island, which carries genes for the biosynthesis of the TCP, required for colonization of the small intestinal epithelium (7).The approximately 7-kb CTX genome consists of the core and the RS2 region. The core region encodes proteins needed for the assembly and secretion of viral particles (Psh, Cep, pIII CTX , Ace, and Zot) and also encodes CT, which is not necessary for phage morphogenesis (3), while the RS2 region represents a site-specific recombination system that allows lysogenic phage to integrate at a specific site on the host chromosome (14). The RS2 region of CTX prophage encodes proteins required for replication (RstA), phage integration (RstB), and regulation (RstR) of the lysogeny of CTX (14). An antirepressor, rstC, is carried by a satellite phage, RS1, often present adjacent to the CTX prophage in toxigenic V. cholerae O1 El Tor and O139 strains (1, 5).Diversity of the CTX phage repressor rstR has been described previously, and this diversity constitutes heteroimmunity among diverse CTX phages (8, 2). The difference in the rstR gene is also the only known genetic difference between any two different CTX phage types. The existence of at least four different rstR genes carried by different CTX phages, namely, CTX ET , CTX class , CTX Calc , and CTX Env , has been recognized (8, 2, 10). The epidemiological significance of the diversity of CTX phages is not clearly known, but at least two periods of explosive resurgence of cholera have been associated with strains showing changes in the rstR type of CTX phages. The first was the resurgence of V. cholerae O139 in August 1996 in Calcutta, India, which continued for a year (8, 9, 13), and the second was the resurgence of strain O139 in March to April of 2002 in Dhaka, Bangladesh (6). On the basis of their rstR genes and other phenotypic traits, genetic hybrids of classical and El Tor biotypes that cause cholera have been shown to exist, and these hybrids have been designated the Matlab variants of V. cholerae (11). To further document the distribution and temporal changes in the CTX phage contents of epidemic strains, we conducted a surveillance of CTX phage types by analyzing the types of rstR genes carried by a large collection of toxigenic V. cholerae strains.We selected every 10th con...