Diagnostic Dilemma of Frozen Shoulder in Post CABG Patient: A Case Report.

Frozen shoulder, also known as adhesive capsulitis, is a condition featured by stiffness and pain in shoulder joint. In this report, we present a case of 58 years old diabetic male patient with the history of coronary artery bypass grafting (CABG) 06 months back. He presented with persistent right shoulder pain for 05 months. Clinical examinations reveal restriction of the right shoulder joint movement in all directions and wasting of the right supraspinatus, infraspinatus and trapezius muscles. Both active and passive range of motions was restricted with painful right shoulder joint. Pain free abduction range was about 40 degrees in right shoulder. Plain X-ray of right shoulder joint and other relevant investigations show normal findings. Considering the clinical and laboratory findings decision was taken to treat the patient with exercise, pain killer and ultrasound therapy which were found to be optimistic.

Genetic and phenotypic analysis of Vibrio cholerae non-O1, non-O139 isolated from German and Austrian patients.

Vibrio cholerae belonging to the non-O1, non-O139 serogroups are present in the coastal waters of Germany and in some German and Austrian lakes. These bacteria can cause gastroenteritis and extraintestinal infections, and are transmitted through contaminated food and water. However, non-O1, non-O139 V. cholerae infections are rare in Germany. We studied 18 strains from German and Austrian patients with diarrhea or local infections for their virulence-associated genotype and phenotype to assess their potential for infectivity in anticipation of possible climatic changes that could enhance the transmission of these pathogens. The strains were examined for the presence of genes encoding cholera toxin and toxin-coregulated pilus (TCP), as well as other virulence-associated factors or markers, including hemolysins, repeats-in-toxin (RTX) toxins, Vibrio seventh pandemic islands VSP-1 and VSP-2, and the type III secretion system (TTSS). Phenotypic assays for hemolysin activity, serum resistance, and biofilm formation were also performed. A dendrogram generated by incorporating the results of these analyses revealed genetic differences of the strains correlating with their clinical origin. Non-O1, non-O139 strains from diarrheal patients possessed the TTSS and/or the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin, which were not found in the strains from ear or wound infections. Routine matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry (MS) analysis of all strains provided reliable identification of the species but failed to differentiate between strains or clusters. The results of this study indicate the need for continued surveillance of V. cholerae non-O1, non-O139 in Germany, in view of the predicted increase in the prevalence of Vibrio spp. due to the rise in surface water temperatures.

Development of simple and rapid PCR-fingerprinting methods for Vibrio cholerae on the basis of genetic diversity of the superintegron.

AIMS: To develop simple and rapid PCR-fingerprinting methods for Vibrio cholerae O1 (El Tor and classical biotypes) and O139 serogroup strains which cause major cholera epidemics, on the basis of the diversity of superintegron (SI) carried by these strains. METHODS AND RESULTS: PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed targeting region between integrase gene in the SI and its nearby ORF, followed by BglI digestion. Besides, a V. cholerae repeat-amplified fragment length polymorphism (VCR-AFLP) assay was also developed. In the PCR-RFLP, 94 El Tor, 29 classical and 54 O139 strains produced nine, three and six different DNA fingerprints, respectively. On the other hand, VCR-AFLP distinguished these El Tor, classical and O139 strains into five, nine and two DNA fingerprints, respectively. Combining both assays the El Tor, classical and O139 strains could be differentiated into 11, 10 and seven different types, respectively. In a comparative study, pulsed-field gel electrophoresis (PFGE) showed similar differentiation for El Tor (11 types), but lower discrimination for O139 (two types) and classical strains (five types). CONCLUSIONS: The PCR assays based on SI diversity can be used as a useful typing tool for epidemiological studies of V. cholerae. SIGNIFICANCE AND IMPACT OF STUDY: This newly developed method is more discriminatory, simple, rapid and cost-effective in comparison with PFGE, and thus can be widely applicable.

El Tor cholera with severe disease: a new threat to Asia and beyond.

During epidemics of cholera in two rural sites (Bakerganj and Mathbaria), a much higher proportion of patients came for treatment with severe dehydration than was seen in previous years. V. cholerae O1 isolated from these patients was found to be El Tor in its phenotype, but its cholera toxin (CT) was determined to be that of classical biotype. Whether the observed higher proportion of severe dehydration produced by the El Tor biotype was due to a shift from El Tor to classical CT or due to other factors is not clear. However, if cholera due to strains with increased severity spread to other areas where treatment facilities are limited, there are likely to be many more cholera deaths.

Molecular analysis of antibiotic resistance gene clusters in vibrio cholerae O139 and O1 SXT constins.

Many recent Asian clinical Vibrio cholerae E1 Tor O1 and O139 isolates are resistant to the antibiotics sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm), and streptomycin (Sm). The corresponding resistance genes are located on large conjugative elements (SXT constins) that are integrated into prfC on the V. cholerae chromosome. We determined the DNA sequences of the antibiotic resistance genes in the SXT constin in MO10, an O139 isolate. In SXT(MO10), these genes are clustered within a composite transposon-like structure found near the element's 5' end. The genes conferring resistance to Cm (floR), Su (sulII), and Sm (strA and strB) correspond to previously described genes, whereas the gene conferring resistance to Tm, designated dfr18, is novel. In some other O139 isolates the antibiotic resistance gene cluster was found to be deleted from the SXT-related constin. The El Tor O1 SXT constin, SXT(ET), does not contain the same resistance genes as SXT(MO10). In this constin, the Tm resistance determinant was located nearly 70 kbp away from the other resistance genes and found in a novel type of integron that constitutes a fourth class of resistance integrons. These studies indicate that there is considerable flux in the antibiotic resistance genes found in the SXT family of constins and point to a model for the evolution of these related mobile elements.

Diminished diarrheal response to Vibrio cholerae strains carrying the replicative form of the CTX(Phi) genome instead of CTX(Phi) lysogens in adult rabbits.

Toxigenic Vibrio cholerae strains are lysogens of CTX(Phi), a filamentous bacteriophage which encodes cholera toxin (CT). Following infection of recipient V. cholerae cells by CTX(Phi), the phage genome either integrates into the host chromosome at a specific attachment site (attRS) or exists as a replicative-form (RF) plasmid. We infected naturally occurring attRS-negative nontoxigenic V. cholerae or attenuated (CTX(-) attRS negative) derivatives of wild-type toxigenic strains with CTX(Phi) and examined the diarrheagenic potential of the strains carrying the RF of the CTX(Phi) genome using the adult rabbit diarrhea model. Under laboratory conditions, strains carrying the RF of CTX(Phi) produced more CT than corresponding lysogens as assayed by a G(M1)-based enzyme-linked immunosorbent assay and by fluid accumulation in ligated ileal loops of rabbits. However, when tested for diarrhea in rabbits, the attRS-negative strains (which carried the CTX(Phi) genome as the RF) were either negative or produced mild diarrhea, whereas the attRS-positive strains with integrated CTX(Phi) produced severe fatal diarrhea. Analysis of the strains after intestinal passage showed that the attRS-negative strains lost the phage genome at approximately a fivefold higher frequency than under in vitro conditions, and 75 to 90% of cells recovered from challenged rabbits after 24 h were CT negative. These results suggested that strains carrying the RF of CTX(Phi) are unable to cause severe disease due to rapid loss of the phage in vivo, and the gastrointestinal environment thus provides selection of toxigenic strains with an integrated CTX(Phi) genome. These results may have implications for the development of live V. cholerae vaccine candidates impaired in chromosomal integration of CTX(Phi). These findings may also contribute to understanding of the etiology of diarrhea occasionally associated with nontoxigenic V. cholerae strains.

Local production of anti-vibrio cholerae mucosal antibody in reproductive tract tissues after cholera.

To investigate whether intestinal presentation of an antigen by Vibrio cholerae, a noninvasive organism, could induce an anatomically distant mucosal immune response in reproductive tract tissues, the endocervical immune responses of women in Bangladesh were evaluated after cholera. Endocervical secretions were analyzed for secretory IgA (sIgA) antibody against the B subunit of cholera toxin (CtxB) in 9 women with cholera and 8 women with diarrhea caused by neither V. cholerae nor heat labile enterotoxin-producing Escherichia coli. Women infected with V. cholerae developed significant sIgA anti-CtxB responses in endocervical samples (P< or =.02). Antibody subtype analysis of endocervical IgA was consistent with local mucosal production (P< or =.001). Women with cholera did not develop sIgA anti-CtxB responses in serum. The ability to generate specific mucosal immune responses in reproductive tract tissues after intestinal presentation of antigen could facilitate development of vaccines effective against reproductive tract pathogens.

The efficacy of bismuth subsalicylate in the treatment of acute diarrhoea and the prevention of persistent diarrhoea.

UNLABELLED: A controlled, randomized, double-blind study in Bangladeshi children (ages 4-36 mo) with acute diarrhoea was undertaken to determine whether bismuth subsalicylate (BSS) would prevent the development of persistent diarrhoea (PD) in young children. The children were randomized to two groups: 226 were given liquid oral BSS, (as Pepto-Bismol), 100 mg/kg/d for 5 d; 225 were given placebo of identical appearance. On admission to the study, the two groups were comparable both clinically and microbiologically. Rotavirus was found in 56% of all the children, and enterotoxigenic E. coli in 31% of a subsample studied. Children treated with BSS had less severe and less prolonged illness than those treated with placebo (p = 0.057). There was, however, no difference in the development of PD between the two groups (8% and 11%). Unexpectedly, patients treated with BSS gained significantly more weight (2.3%) than those treated with placebo (0.5%; p < 0.001) during the course of the study. No toxicity of BSS was detected. CONCLUSION: Treatment with BSS had a modest therapeutic effect on acute diarrhoea, as has been previously demonstrated, but with no suggestion of a therapeutic effect on the prevention of persistent diarrhoea in this group of patients.

Molecular comparison of toxigenic clinical & non-toxigenic environmental strains of Vibrio cholerae O1 Ogawa isolated during an outbreak of cholera in south India.

BACKGROUND & OBJECTIVES: While investigating a cholera outbreak in south India, toxigenic and nontoxigenic strains of Vibrio cholerae O1 were isolated from patients and from the environment, respectively. This study was performed to compare the genetic relatedness of the patient and environmental strains to determine clonal relationships among these strains and thereby determine the source of the cholera outbreak. METHODS: The 16 strains of V. cholerae isolated from hospitalized patients and 8 environmental V. cholerae strains isolated from the environment were phenotypically and genotypically characterized using a variety of standard techniques. RESULTS: Sixteen toxigenic clinical strains and 2 nontoxigenic environmental strains belonged to O1 serogroup, Ogawa serotype and El Tor biotype. The remaining 6 nontoxigenic environmental strains were classified as non-O1, non-O139 V. cholerae. The drug resistance pattern of the clinical and environmental strains of V. cholerae showed marked differences with the patient strains being resistant to more number of drugs as compared to the environmental strains. DNA fingerprinting of the strains showed considerable diversity between toxigenic clinical and nontoxigenic environmental O1 Ogawa isolates and between the O1 and non-O1, non-O139 isolates. INTERPRETATION & CONCLUSION: In this outbreak of cholera, the O1 strains of V. cholerae from clinical and environmental sources belonged to two different clones and the environmental strains could perhaps be the future cholera outbreak causing clones.

Treatment of enterotoxigenic and enteropathogenic Escherichia coli-induced diarrhoea in children with bovine immunoglobulin milk concentrate from hyperimmunized cows: a double-blind, placebo-controlled, clinical trial.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) and enteropathogenic Escherichia coli (EPEC) are important causes of diarrhoea in young children and are associated with significant mortality rates. Passive immunization with antibodies from immunized cows has previously been shown to be effective as prophylaxis against E. coli-induced diarrhoea and therapeutically against rotavirus and cryptosporidia-induced diarrhoea. METHODS: We tested the therapeutic efficacy of an oral bovine immunoglobulin milk concentrate (BIC) from cows hyperimmunized with ETEC and EPEC strains, in a randomized, placebo-controlled study in children with E. coli-induced diarrhoea. Eighty-six children between 4-24 months of age attending the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) with E. coli-induced diarrhoea (63 EPEC/ETEC and 23 with other diarrhoeagenic E. coli) were randomly assigned to receive orally administered BIC (20 g) containing anti-ETEC/EPEC antibodies or a placebo preparation daily for 4 consecutive days. Daily stool output, intake of oral rehydration solution (ORS), stool frequency, and presence of diarrhoeagenic E. coli strains in the stool were monitored for 4 days. RESULTS: Children in the treatment group tolerated the BIC with no side effects. There were no significant differences between the two groups with regard to ORS intake, stool output, frequency of diarrhoea, or clearance of pathogen. Nor was there any significant alteration in the duration of diarrhoea. CONCLUSIONS: In contrast to the prophylactic efficacy of anti-E. coli BIC and the therapeutic efficacy of a similarly prepared anti-rotavirus BIC, antibodies from hyperimmunized cows appear to have no significant therapeutic benefit in the treatment of acute diarrhoea due to EPEC/ETEC.

The O139 serogroup of Vibrio cholerae comprises diverse clones of epidemic and nonepidemic strains derived from multiple V. cholerae O1 or non-O1 progenitors.

Sixty-four representative strains of Vibrio cholerae O139 were analyzed, to re-examine the origin of this serogroup. Ribotyping differentiated the strains into 3 HindIII and 7 BglI ribotypes. One HindIII and 5 BglI ribotypes were shared by all toxigenic O139 strains. Of 6 nontoxigenic O139 strains, 3 shared ribotypes with the toxigenic strains, carried genes encoding toxin coregulated pilus, and were susceptible to the cholera toxin-converting bacteriophage CTXPhi. The remaining 3 strains belonged to 2 different ribotypes distinct from toxigenic O139 strains and were resistant to CTXPhi and JA-1, an O139-specific lytic bacteriophage. Polymerase chain reaction amplicons corresponding to the gmhD gene carried by these 3 strains also differed from those of the toxigenic O139 strains but were identical to those of 15 environmental non-O1-non-O139 strains. Thus, the O139 antigen is present in different lineages, and this serogroup appears to comprise epidemic and nonepidemic strains derived separately from different progenitors.

Virulence genes in environmental strains of Vibrio cholerae.

The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulated expression. The ctxAB and tcpA genes are known to play a cardinal role in maintaining virulence in Vibrio cholerae, and these genes are believed to be exclusively associated with clinical strains of O1 and O139 serogroups. In this study, we examined the presence of five virulence genes, including ctxAB and tcpA, as well as toxR and toxT, which are involved in the regulation of virulence, in environmental strains of V. cholerae cultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India. PCR analysis revealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes of environmental V. cholerae strains. Sequencing of a part of the tcpA gene carried by an environmental strain showed 97.7% homology to the tcpA gene of the classical biotype of V. cholerae O1. Strains carrying the tcpA gene expressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electron microscopy of the TCP pili. Strains carrying ctxAB genes also produced cholera toxin, determined by monosialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits. Thus, this study demonstrates the presence and expression of critical virulence genes or their homologues in diverse environmental strains of V. cholerae, which appear to constitute an environmental reservoir of virulence genes, thereby providing new insights into the ecology of V. cholerae.

Sunlight-induced propagation of the lysogenic phage encoding cholera toxin.

In toxigenic Vibrio cholerae, the cholera enterotoxin (CT) is encoded by CTXPhi, a lysogenic bacteriophage. The propagation of this filamentous phage can result in the origination of new toxigenic strains. To understand the nature of possible environmental factors associated with the propagation of CTXPhi, we examined the effects of temperature, pH, salinity, and exposure to direct sunlight on the induction of the CTX prophage and studied the transmission of the phage to potential recipient strains. Exposure of cultures of CTXPhi lysogens to direct sunlight resulted in approximately 10,000-fold increases in phage titers. Variation in temperature, pH, or salinity of the culture did not have a substantial effect on the induction of the prophage, but these factors influenced the stability of CTXPhi particles. Exposure of mixed cultures of CTXPhi lysogens and potential recipient strains to sunlight significantly increased both the in vitro and in vivo (in rabbit ileal loops) transduction of the recipient strains by CTXPhi. Included in these transduction experiments were two environmental nontoxigenic (CTXPhi(-)) strains of V. cholerae O139. These two O139 strains were transduced at high efficiency by CTXPhi, and the phage genome integrated into the O139 host chromosome. The resulting CTXPhi lysogens produced biologically active CT both in vitro and in rabbit ileal loops. This finding suggests a possible mechanism explaining the origination of toxigenic V. cholerae O139 strains from nontoxigenic progenitors. This study indicates that sunlight is a significant inducer of the CTX prophage and suggests that sunlight-induced transmission of CTXPhi may constitute part of a natural mechanism for the origination of new toxigenic strains of V. cholerae.

Genomic diversity among Vibrio cholerae O139 strains isolated in Bangladesh and India between 1992 and 1998.

In order to assess the extent of genomic diversity among Vibrio cholerae O139 strains, restriction fragment length polymorphisms in two genetic loci, rrn and ctx, were studied. Analysis of 144 strains isolated from different regions of Bangladesh and India between 1992 and 1998 revealed the presence of at least six distinct ribotypes (B-I through B-VI) of which three were new ribotypes, and one of these was represented by a nontoxigenic O139 strain. Strains of ribotypes B-I through B-V shared 11 different CTX genotypes (A through K). Antimicrobial resistance patterns of the strains varied independently of their ribotypes and CTX genotypes. Results of this study suggest that V. cholerae O139 is undergoing rapid genetic changes leading to the origination of new variants, and temporal changes in antimicrobial resistance patterns may be contributing to the selection of different variants.

Immune response of Bangladeshi children with acute diarrhea who subsequently have persistent diarrhea.

BACKGROUND: Because altered immune responses may be a risk factor for persistent diarrhea, various aspects of the immune response were examined to elucidate the underlying immune mechanisms that may be involved in the development of persistent diarrhea. METHODS: Children (7-12 months of age) with watery diarrhea for 6 to 8 days from the Dhaka Hospital of the International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), were enrolled. Children were classified as having acute diarrhea (AD) or persistent diarrhea (PD) if diarrhea resolved within 14 days or persisted for more than 14 days, respectively. Uninfected control children (n = 13), from the Nutrition Follow-Up Unit of ICDDR,B were also enrolled. Of the 123 children with diarrhea who were enrolled, 85 had AD and 38 had PD. Comparisons were performed for clinical features, nutritional status (weight for age, plasma transferrin, and serum albumin levels), and immune responses: neutrophil function; peripheral blood mononuclear cell function, delayed-type hypersensitivity (DTH) responses, plasma levels of immunoglobulins, tumor necrosis factor-alpha, and interferon-gamma. Univariate analyses were conducted to assess differences among the three groups of children and between children with AD and PD. Logistic regression was performed to determine risk factors for PD. RESULTS: There were no differences in clinical features and nutritional status among the groups of children studied. More children in whom PD developed had a negative DTH response to tuberculin than those with AD (P = 0.021). Also, a negative DTH response to tuberculin was a significant risk factor for PD (odds ratio [OR] = 3.8, 95% confidence interval [CI] = 1.5-9.9). CONCLUSIONS: Children with acute diarrhea with a negative DTH response to tuberculin are more likely to have development of persistent diarrhea.

Case-control study of enteropathogens associated with childhood diarrhea in Dhaka, Bangladesh.

The International Centre for Diarrhoeal Disease Research, Bangladesh, is a major center for research into diarrheal diseases. The center treats more than 100,000 patients a year. To obtain useful information representative of all patients, a surveillance system in which a 4% systematic sample of all patients is studied in detail, including etiological agents of diarrhea, was installed in October 1979. The first paper on etiology for the surveillance patients was published in 1982, which identified a potential enteric pathogen in 66% of patients. In subsequent years, several new agents of diarrhea have been identified. To assess the importance of a broader spectrum of diarrheal agents including the ones identified relatively recently, we studied 814 children with diarrhea. The children were up to 5 years of age and were part of the surveillance system. They were matched with an equal number of community controls without diarrhea. The study was conducted from February 1993 to June 1994. A potential enteric pathogen was isolated from 74.8% of diarrheal children and 43.9% of control children (P = 0.0001). Even though the first study was not a case-control study, it identified rotavirus, Campylobacter jejuni, enterotoxigenic Escherichia coli, Shigella spp. , and Vibrio cholerae O1 as major pathogens. The present study identified these pathogens as being significantly associated with diarrhea. In addition, the study also identified six additional agents, including enteropathogenic E. coli, Aeromonas spp., V. cholerae O139, enterotoxigenic Bacteroides fragilis, Clostridium difficile, and Cryptosporidium parvum, as being significantly associated with diarrhea. Plesiomonas shigelloides, Salmonella spp., diffusely adherent E. coli, enteroaggregative E. coli, Entamoeba histolytica, and Giardia lamblia were not significantly associated with diarrhea. Enteroinvasive E. coli, enterohemorrhagic E. coli, and Cyclospora cayetanensis were not detected in any of the children. The major burden of diseases due to most pathogens occurred in the first year of life. As in the previous study, seasonal patterns were seen for diarrhea associated with rotavirus, V. cholerae, and enterotoxigenic E. coli, and infections with multiple pathogens were common. With a few exceptions, these findings are in agreement with those from other developing countries. This knowledge of a broader spectrum of etiological agents of diarrhea in the surveillance patients will help us plan studies into various aspects of diarrheal diseases in this population.

Lysogenic conversion of environmental Vibrio mimicus strains by CTXPhi.

The filamentous bacteriophage CTXPhi, which encodes cholera toxin (CT) in toxigenic Vibrio cholerae, is known to propagate by infecting susceptible strains of V. cholerae by using the toxin coregulated pilus (TCP) as its receptor and thereby causing the origination of new strains of toxigenic V. cholerae from nontoxigenic progenitors. Besides V. cholerae, Vibrio mimicus strains which are normally TCP negative have also been shown to occasionally produce CT and cause diarrhea in humans. We analyzed nontoxigenic V. mimicus strains isolated from surface waters in Bangladesh for susceptibility and lysogenic conversion by CTXPhi and studied the expression of CT in the lysogens by using genetically marked derivatives of the phage. Of 27 V. mimicus strains analyzed, which were all negative for genes encoding TCP but positive for the regulatory gene toxR, 2 strains (7.4%) were infected by CTX-KmPhi, derived from strain SM44(P27459 ctx::km), and the phage genome integrated into the host chromosome, forming stable lysogens. The lysogens spontaneously produced infectious phage particles in the supernatant fluids of the culture, and high titers of the phage could be achieved when the lysogens were induced with mitomycin C. This is the first demonstration of lysogenic conversion of V. mimicus strains by CTXPhi. When a genetically marked derivative of the replicative form of the CTXPhi genome carrying a functional ctxAB operon, pMSF9.2, was introduced into nontoxigenic V. mimicus strains, the plasmid integrated into the host genome and the strains produced CT both in vitro and inside the intestines of adult rabbits and caused mild-to-severe diarrhea in rabbits. This suggested that in the natural habitat infection of nontoxigenic V. mimicus strains by wild-type CTXPhi may lead to the origination of toxigenic V. mimicus strains which are capable of producing biologically active CT. The results of this study also supported the existence of a TCP-independent mechanism for infection by CTXPhi and showed that at least one species of Vibrio other than V. cholerae may contribute to the propagation of the phage.

Molecular characterization of a new ribotype of Vibrio cholerae O139 Bengal associated with an outbreak of cholera in Bangladesh.

Vibrio cholerae O139 Bengal initially appeared in the southern coastal region of Bangladesh and spread northward, causing explosive epidemics during 1992 and 1993. The resurgence of V. cholerae O139 during 1995 after its transient displacement by a new clone of El Tor vibrios demonstrated rapid changes in the epidemiology of cholera in Bangladesh. A recent outbreak of cholera in two north-central districts of Bangladesh caused by V. cholerae O139 led us to analyze strains collected from the outbreak and compare them with V. cholerae O139 strains isolated from other regions of Bangladesh and neighboring India to investigate their origins. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA (ribotype) revealed that the recently isolated V. cholerae O139 strains belonged to a new ribotype which was distinct from previously described ribotypes of toxigenic V. cholerae O139. All strains carried the genes for toxin-coregulated pili (tcpA and tcpI) and accessory colonization factor (acfB), the regulatory gene toxR, and multiple copies of the lysogenic phage genome encoding cholera toxin (CTXPhi) and belonged to a previously described ctxA genotype. Comparative analysis of the rfb gene cluster by PCR revealed the absence of a large region of the O1-specific rfb operon downstream of the rfaD gene and the presence of an O139-specific genomic region in all O139 strains. Southern hybridization analysis of the O139-specific genomic region also produced identical restriction patterns in strains belonging to the new ribotype and those of previously described ribotypes. These results suggested that the new ribotype of Bengal vibrios possibly originated from an existing strain of V. cholerae O139 by genetic changes in the rRNA operons. In contrast to previously isolated O139 strains which mostly had resistance to trimethoprim, sulfamethoxazole, and streptomycin encoded by a transposon (SXT element), 68.6% of the toxigenic strains analyzed in the present study, including all strains belonging to the new ribotype, were susceptible to these antibiotics. Molecular analysis of the SXT element revealed possible deletion of a 3.6-kb region of the SXT element in strains which were susceptible to the antibiotics. Thus, V. cholerae O139 strains in Bangladesh are also undergoing considerable reassortments in genetic elements encoding antimicrobial resistance.

Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae.

Cholera caused by toxigenic Vibrio cholerae is a major public health problem confronting developing countries, where outbreaks occur in a regular seasonal pattern and are particularly associated with poverty and poor sanitation. The disease is characterized by a devastating watery diarrhea which leads to rapid dehydration, and death occurs in 50 to 70% of untreated patients. Cholera is a waterborne disease, and the importance of water ecology is suggested by the close association of V. cholerae with surface water and the population interacting with the water. Cholera toxin (CT), which is responsible for the profuse diarrhea, is encoded by a lysogenic bacteriophage designated CTXPhi. Although the mechanism by which CT causes diarrhea is known, it is not clear why V. cholerae should infect and elaborate the lethal toxin in the host. Molecular epidemiological surveillance has revealed clonal diversity among toxigenic V. cholerae strains and a continual emergence of new epidemic clones. In view of lysogenic conversion by CTXPhi as a possible mechanism of origination of new toxigenic clones of V. cholerae, it appears that the continual emergence of new toxigenic strains and their selective enrichment during cholera outbreaks constitute an essential component of the natural ecosystem for the evolution of epidemic V. cholerae strains and genetic elements that mediate the transfer of virulence genes. The ecosystem comprising V. cholerae, CTXPhi, the aquatic environment, and the mammalian host offers an understanding of the complex relationship between pathogenesis and the natural selection of a pathogen.