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.

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.

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.

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.

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.

A monoclonal antibody to Shigella dysenteriae serotype 13 cross-reacting with Shiga toxin.

A monoclonal antibody (mAb ICT6) was produced against the newly described Shigella dysenteriae serotype type 13. The mAb was of IgM isotype and recognized purified Shiga toxin in ELISA and immunoblot. It also recognized periplasmic extract S. dysenteriae type 13 in immunoblot as did an affinity-purified polyclonal rabbit antiserum and a previously described monoclonal antibody to the B subunit of Shiga toxin. The mAb ICT6 did not neutralize the cytotoxic effects or S. dysenteriae type 13, Shiga toxin or periplasmic extracts of S. dysenteriae type 1 for HeLa cells.

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.

Sharing of virulence-associated properties at the phenotypic and genetic levels between enteropathogenic Escherichia coli and Hafnia alvei.

Seven strains of Hafnia alvei isolated from diarrhoeal stools of children resembled enteropathogenic Escherichia coli (EPEC) in that they produced attaching-effacing (AE) lesions in rabbit ileal loops and fluorescent actin staining in infected HEp-2 cells. In addition, a DNA probe from a chromosomal gene required by EPEC to produce AE lesions, hybridised to chromosomal DNA from all seven H. alvei strains. These findings indicate that there is a sharing of virulence-associated properties at the phenotypic and genetic levels by H. alvei and EPEC. H. alvei strains with these properties should be considered diarrhoeagenic.

A comparative study of specific gene probes and standard bioassays to identify diarrhoeagenic Escherichia coli in paediatric patients with diarrhoea in Bangladesh.

We compared the usefulness of gene probes with standard bioassays to identify diarrhoeagenic Escherichia coli amongst isolates from Bangladeshi children under 1 year of age with diarrhoea. E. coli isolates were analysed with specific gene probes for localised adhesiveness (LA), diffuse adhesiveness (DA), heat-labile toxin (LT), heat-stable toxin (ST), Shiga-like toxins (SLT I and SLT II), and enteroinvasiveness, and in bioassays for production of enterotoxins and cytotoxins, and for cell adherence. With 1136 isolates from 387 patients, there was general agreement between the two assay methods. When there was disparity, gene-probe-positive isolates gave negative results in the corresponding bioassay. In the HeLa cell adherence assay, 94% of the LA probe-positive isolates and 91.6% of the DA probe-positive isolates gave positive bioassay results for LA and DA respectively. Thirty-six of 39 LT probe-positive isolates and 73 of 86 ST probe-positive isolates gave positive results in the bioassays. Of 28 isolates that gave negative results in the suckling mouse assay but were initially positive with the probe for ST, 15 were later found to hybridize with the cloning vector for the ST probe. Addition of denatured vector DNA at a concentration of 10 micrograms/ml in the hybridisation solution eliminated these false positive results. None of the other probe-positive isolates hybridised with any of the cloning vectors used. The DNA hybridisation assay appeared to be a convenient alternative to bioassays for screening large numbers of isolates in epidemiological investigation.

Characterisation of Plesiomonas shigelloides strains that share type-specific antigen with Shigella flexneri 6 and common group 1 antigen with Shigella flexneri spp. and Shigella dysenteriae 1.

Three strains of Plesiomonas shigelloides isolated from patients with diarrhoea were agglutinated with Shigella flexneri 6 antiserum in slide and tube tests. All the strains were also agglutinated with a monoclonal antibody to the common group 1 antigen shared between S. flexneri serotypes and S. dysenteriae type 1. Further studies with one strain also showed sharing of antigenicity in an enzyme-linked immunosorbent assay. The results suggest that the strains share type-specific antigen with S. flexneri 6 and the common group 1 antigen with S. flexneri serotypes and S. dysenteriae 1. The sharing of antigens may have implications for cross-protection. One strain adhered to HEp-2 cell monolayers. None of the strains contained high mol. wt plasmids and there was no sequence homology with the invasiveness plasmid of Shigella spp. in DNA probe hybridisation. They were susceptible to the commonly used antibiotics. However, they possessed four other virulence-associated properties of Shigella spp. that included Congo-red binding, hydrophobicity, toxicity to HeLa cells and HEp-2 cell invasiveness (although they gave negative results in the Sereny test for invasiveness). These data suggest that the three unique strains might be considered pathogenic. Studies in animal models and human volunteers would be necessary to establish their pathogenic potential.

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.

Effect of vitamin-A deficiency on plaque forming response of antibody producing spleen cells against Salmonella typhimurium in rats.

Plaque forming response of antibody producing spleen cells against Salmonella typhimurium was studied in vitamin-A deficient and normal rats after 3, 6, 9 and 12 days of injecting the antigen. Vitamin-A deficient rats were found to have significantly decreased (P less than 0.001) number of antibody plaque forming cells in the spleen as compared to normal rats in all cases. Serum total protein and serum Vitamin-A levels were significantly (P less than 0.001) lower in the vitamin-A deficient rats as compared to the controls and immunization caused no significant change in these parameters. The average spleen weights were increased in both the groups on immunization but this increase was comparatively more in case of the control rats.

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.

Hypotonic oral rehydration solution in acute diarrhoea: a controlled clinical trial.

In a controlled trial, a hypotonic oral rehydration solution (ORS) (Na+67, K+20, Cl-66, citrate 7, glucose 89 mmol/l osmolality 249 mosmol/kg) was compared with a standard WHO-ORS (Na+90, K+20, Cl-80, citrate 10, glucose 111 mmol/l, osmolality 311 mosmol/kg) in 60 children aged 5-24 months with acute watery diarrhoea. In the hypotonic ORS group, stool frequency, proportion of children who vomited, ORS requirements and purging rate over 24-48 h were reduced by 33% (p = 0.01), 30% (p = 0.02), 21% (p = 0.067) and 21% (p = 0.03), respectively. The proportion of children who vomited and the purging rate over 48 h were reduced by 23% (p = 0.03) and 10% (p = 0.097), respectively. Serum electrolytes after 48 h were comparable. The beneficial effect of hypotonic ORS was most marked in, and largely contributed by, the subgroup negative for rotavirus.

Aetiological, clinical and epidemiological characteristics of a seasonal peak of diarrhoea in Dhaka, Bangladesh.

During the spring peak of diarrhoea in Bangladesh, 113 consecutive patients who represented a systematic 4% sample of all patients attending an urban diarrhoea treatment facility between 18 and 23 April 1995 were studied. The study was conducted to characterize enteric pathogens associated with the spring peak of the diarrhoea outbreak in Bangladesh and to describe clinical and epidemiological features of the patients. The spring peak is traditionally thought to be mostly due to V. cholerae O1. However, the most common cause of diarrhoea among the study patients was enterotoxigenic Escherichia coli (36%) followed by Vibrio cholerae O1 (23%). The V. cholerae O1 patients attended significantly (p < 0.01) sooner after onset of diarrhoea than enterotoxigenic E. coli (ETEC) patients. Studies of behavioural and environmental characteristics are important to determine risk factors for observed higher proportion of ETEC infection during seasonal diarrhoea peaks.

Distribution of Zonula occludens toxin (zot) gene among clinical isolates of Vibrio cholerae O1 from Bangladesh and Africa.

Seventy-two clinical isolates of Vibrio cholerae O1 from Bangladesh, and 12 and 9 isolates respectively from Tanzania and Nigeria were screened for sequences homologous to zonula occludens toxin (zot) and cholera toxin (ctx) genes. As observed previously, all isolates in the present study also possessed sequences for both toxins which suggested that zot does not occur independent of ctx. It appears that along with the virulence genes located in the "virulence cassette" region of the bacterial chromosome, zot may play a role in the pathogenesis of cholera.

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.

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.