Vibrio cholerae O139 bacteriophages

Cholera bacteriophages have been isolated from 27 lysogenic cultures of V. cholerae O139. As shown the pages under study belong to two morphological groups A1 and F1 and serological types II and XII. The use of prophage typing and the sensitivity test to specific phage made it possible to differentiate V. cholerae strains, serogroup O139.     

Pili of a Vibrio cholerae O139

The pili of a strain of Vibrio cholerae O139 were purified and characterized. They were morphologically, electrophoretically and immunologically indistinguishable from the pili with 16 kDa subunit protein of V. cholerae O1. All 22 strains of V. cholerae O139 examined possessed the pili. The pili were different in hemagglutination inhibition pattern from V. cholerae O1 16K pili.     

Multiple antibiotic resistance of Vibrio cholerae serogroup O139 in China from 1993 to 2009

Regarded as an emerging diarrheal micropathogen, Vibrio cholerae serogroup O139 was first identified in 1992 and has become an important cause of cholera epidemics over the last two decades. O139 strains have been continually isolated since O139 cholera appeared in China in 1993, from sporadic cases and dispersed foodborne outbreaks, which are the common epidemic types of O139 cholera in China. Antibiotic resistance profiles of these epidemic strains are required for development of clinical treatments, epidemiological studies and disease control. In this study, a comprehensive investigation of the antibiotic resistance of V. cholerae O139 strains isolated in China from 1993 to 2009 was conducted. The initial O139 isolates were resistant to streptomycin, trimethoprim-sulfamethoxazole and polymyxin B only, while multidrug resistance increased suddenly and became common in strains isolated after 1998. Different resistance profiles were observed in the isolates from different years. In contrast, most V. cholerae O1 strains isolated in the same period were much less resistant to these antibiotics and no obvious multidrug resistance patterns were detected. Most of the non-toxigenic strains isolated from the environment and seafood were resistant to four antibiotics or fewer, although a few multidrug resistant strains were also identified. These toxigenic O139 strains exhibited a high prevalence of the class I integron and the SXT element, which were rare in the non-toxigenic strains. Molecular subtyping of O139 strains showed highly diverse pulsed-field gel electrophoresis patterns, which may correspond to the epidemic state of sporadic cases and small-scale outbreaks and complex resistance patterns. Severe multidrug resistance, even resistance transfers based on mobile antibiotic resistance elements, increases the probability of O139 cholera as a threat to public health. Therefore, continual epidemiological and antibiotic sensitivity surveillance should focus on the occurrence of multidrug resistance and frequent microbial population shifts in O139 strains.     

Characterization of Vibrio cholerae O139 Synonym Bengal Isolated from Patients with Cholera-Like Disease in Bangladesh

Vibrio cholerae O139 (synonym Bengal), a novel serovar of V. cholerae, is the causative agent of large outbreaks of cholera-like illness currently sweeping India and Bangladesh. Eight randomly selected V. cholerae O139 isolates were studied for their biological properties, which were compared with those of V. cholerae O1 and other V. cholerae non-O1. The V. cholerae O139 isolates were characterized by the production of large amount of cholera toxin, hemagglutination, weak hemolytic properties, resistance to polymyxin B, lysogeny with, and production of, kappa type phage (4/8 isolates only), and resistance to both classical and El Tor-specific phages. Thus, V. cholerae O139 isolates had an overall similarity with V. cholerae O1 El Tor.     

Seasonal Cholera Caused by Vibrio cholerae Serogroups O1 and O139 in the Coastal Aquatic Environment of Bangladesh

Since Vibrio cholerae O139 first appeared in 1992, both O1 El Tor and O139 have been recognized as the epidemic serogroups, although their geographic distribution, endemicity, and reservoir are not fully understood. To address this lack of information, a study of the epidemiology and ecology of V. cholerae O1 and O139 was carried out in two coastal areas, Bakerganj and Mathbaria, Bangladesh, where cholera occurs seasonally. The results of a biweekly clinical study (January 2004 to May 2005), employing culture methods, and of an ecological study (monthly in Bakerganj and biweekly in Mathbaria from March 2004 to May 2005), employing direct and enrichment culture, colony blot hybridization, and direct fluorescent-antibody methods, showed that cholera is endemic in both Bakerganj and Mathbaria and that V. cholerae O1, O139, and non-O1/non-O139 are autochthonous to the aquatic environment. Although V. cholerae O1 and O139 were isolated from both areas, most noteworthy was the isolation of V. cholerae O139 in March, July, and September 2004 in Mathbaria, where seasonal cholera was clinically linked only to V. cholerae O1. In Mathbaria, V. cholerae O139 emerged as the sole cause of a significant outbreak of cholera in March 2005. V. cholerae O1 reemerged clinically in April 2005 and established dominance over V. cholerae O139, continuing to cause cholera in Mathbaria. In conclusion, the epidemic potential and coastal aquatic reservoir for V. cholerae O139 have been demonstrated. Based on the results of this study, the coastal ecosystem of the Bay of Bengal is concluded to be a significant reservoir for the epidemic serogroups of V. cholerae.     

Seasonal cholera caused by Vibrio cholerae serogroups O1 and O139 in the coastal aquatic environment of Bangladesh

Since Vibrio cholerae O139 first appeared in 1992, both O1 El Tor and O139 have been recognized as the epidemic serogroups, although their geographic distribution, endemicity, and reservoir are not fully understood. To address this lack of information, a study of the epidemiology and ecology of V. cholerae O1 and O139 was carried out in two coastal areas, Bakerganj and Mathbaria, Bangladesh, where cholera occurs seasonally. The results of a biweekly clinical study (January 2004 to May 2005), employing culture methods, and of an ecological study (monthly in Bakerganj and biweekly in Mathbaria from March 2004 to May 2005), employing direct and enrichment culture, colony blot hybridization, and direct fluorescent-antibody methods, showed that cholera is endemic in both Bakerganj and Mathbaria and that V. cholerae O1, O139, and non-O1/non-O139 are autochthonous to the aquatic environment. Although V. cholerae O1 and O139 were isolated from both areas, most noteworthy was the isolation of V. cholerae O139 in March, July, and September 2004 in Mathbaria, where seasonal cholera was clinically linked only to V. cholerae O1. In Mathbaria, V. cholerae O139 emerged as the sole cause of a significant outbreak of cholera in March 2005. V. cholerae O1 reemerged clinically in April 2005 and established dominance over V. cholerae O139, continuing to cause cholera in Mathbaria. In conclusion, the epidemic potential and coastal aquatic reservoir for V. cholerae O139 have been demonstrated. Based on the results of this study, the coastal ecosystem of the Bay of Bengal is concluded to be a significant reservoir for the epidemic serogroups of V. cholerae.     

Antigenic specificity of Vibrio cholerae O139 nitrosoguanidine mutants

The action of nitrosoguanidine (NG) on the culture of V. cholerae O139 P-16064 resulted in the appearance of mutant 16064 NG6, not agglutinating with commercial diagnostic serum O139. Its incapacity of agglutination was due to the sorption of the specific serum with strains V. cholerae O22 and R-variant RCA-385, which caused the loss of antibodies to common determinants. Experiments with the sorption of immune sera made it possible to suggest that one of the determinants of LPS O139, phosphate-galactose, was absent in NG mutant.     

Filamentous Phage fs1 of Vibrio cholerae O139

Filamentous phage, fs1, was obtained from Vibrio cholerae O139. The lysogenized strains produced a large amount of fs1 phage in the culture supernatant. This phage was previously reported as novel fimbriae of that organism. The genome of the phage was a 6.5 kb single-stranded DNA. The capsid of fs1 consists of a small molecule peptide (about 2.5 kDa).     

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.     

Purification and characterization of Vibrio cholerae O139 fimbriae

Abstract A Vibrio cholerae O139 (strain Al-1841) isolated from a patient with a cholera-like disease in Bangladesh predominantly produced new curved, wavy fimbriae (Al-1841 fimbriae) and small numbers of previously reported V. cholerae non-O1 S7-like pili. The former was purified and characterized. The molecular mass of the Al-1841 fimbrial subunit was less than 2.5 kDa, and it was immunologically different from that of V. cholerae non-O1 S7 pili. This novel fimbrial antigen was detected in all 182 Gram-negative strains from five genera tested but was absent from the Gram-positive bacteria tested. The purified Al-1841 fimbriae did not agglutinate human or rabbit erythrocytes.     

Viability of the nonculturable Vibrio cholerae O1 and O139

Vibrio cholerae is capable of transforming into a viable but nonculturable (VBNC) state, and, in doing so, undergoes alteration in cell morphology. In the study reported here, Vibrio cholerae O1 and O139 cells were maintained in laboratory microcosms prepared with 1% Instant Ocean and incubated at 4 degrees C, i.e., conditions which induce the VBNC state. Cells were fixed at different stages during entry into the VBNC state and, when no growth was detectable on solid or in liquid media, the ultrastructure of these cells was examined, using both transmission and scanning electron microscopy. As shown in earlier studies, the cells became smaller in size and changed from rod to ovoid or coccoid morphology, with the central region of the cells becoming compressed and surrounded by denser cytoplasm. Because the coccoid morphology, indicative of the VBNC state is common for Vibrio cholerae in the natural environment, as well as in starved cells (Baker et al., 1983; Hood et al., 1986) viability of the coccoid, viable but nonculturable cell was investigated. The percentage of coccoid (VBNC) cells showing metabolic activity and retention of membrane integrity was monitored using direct fluorescence staining (LIVE/DEAD BacLight Bacterial Viability kit), with 75 to 90% of the viable but nonculturable coccoid cells found to be metabolically active by this test. Furthermore, the proportion of actively respiring cells, using the redox dye, 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC), relative to total cells, the latter determined by DAPI staining, ranged from 10 to 50%. VBNC coccoid cells retained the antigenic determinants of Vibrio cholerae O1 and O139, respectively, evidenced by positive reaction with monoclonal fluorescent antibody. Viability was further established by susceptibility of the VBNC cells to chlorine, copper sulfate, zinc sulfate, and formaldehyde. Since retention of cell membrane integrity is a determining characteristic of viable cells, DNA was extracted from VBNC cells in microcosms maintained for two months and for one year. Conservation of cholera toxin and toxin-associated genes, ctxA, toxR, tcpA, and zot in chromosomal DNA of VBNC cells was demonstrated using PCR and employing specific primers. It is concluded that not only do VBNC V cholerae O1 and O139 retain viability up to one year, but genes associated with pathogenicity are retained, along with chromosomal integrity.     

Impact of rapid urbanization on the rates of infection by Vibrio cholerae O1 and enterotoxigenic Escherichia coli in Dhaka, Bangladesh

Background

In Bangladesh, increases in cholera epidemics are being documented with a greater incidence and severity. The aim of this prospective study was to identify the prevalence and importance of V. cholerae O1 and enterotoxigenic Escherichia coli (ETEC) as causal agents of severe diarrhea in a high diarrhea prone urban area in Dhaka city.

Methodology

Systematic surveillance was carried out on all diarrheal patients admitted from Mirpur between March 2008 to February 2010 at the ICDDR, B hospital. Stool or rectal swabs were collected from every third diarrheal patient for microbiological evaluation.

Principal Findings

Of diarrheal patients attending the hospital from Mirpur, 41% suffered from severe dehydration with 39% requiring intravenous rehydration therapy. More diarrheal patients were above five years of age (64%) than those below five years of age (36%). About 60% of the patients above five years of age had severe dehydration compared with only 9% of patients under five years of age. The most prevalent pathogen isolated was Vibrio cholerae O1 (23%) followed by ETEC (11%). About 8% of cholera infection was seen in infants with the youngest children being one month of age while in the case of ETEC the rate was 11%. Of the isolated ETEC strains, the enterotoxin type were almost equally distributed; ST accounted for 31% of strains; LT/ST for 38% and LT for 31%.

Conclusion

V. cholerae O1 is the major bacterial pathogen and a cause of severe cholera disease in 23% of patients from Mirpur. This represents a socioeconomic group that best reflects the major areas of high cholera burden in the country. Vaccines that can target such high risk groups in the country and the region will hopefully be able to reduce the disease morbidity and the transmission of pathogens that impact the life and health of people.     

Characterization of an Enterotoxin Produced by Vibrio cholerae O139

A cholera-like enterotoxin was purified from Vibrio cholerae O139 strain AI-1841 isolated from a diarrheal patient in Bangladesh. Its characteristics were compared with that of cholera toxins (CTs) of classical strain 569B and El Tor strain KT25. Al-1841 produced as much toxin as O1 strains. The toxins were indistinguishable in terms of their migration profiles in conventional polyacrylamide gel disc electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectrofocusing as well as their affinity for hydroxyapatite. The skin permeability factor activity and the fluid accumulation induced in rabbit ileal loops of the toxin of AI-1841 were identical to those of the CTs. Three toxins equally reacted against anti-569B CT antiserum in Western blotting, and their B subunits formed a precipitin line against any anti-B subunit antiserum by double gel immunodiffusion. Anti-569B CTB antibody neutralized the three toxins in their PF activities and enterotoxicities. The amino acid sequence of 1841 toxin B subunit was identical with that of KT25 CTB, corresponding to the DNA sequence of ctxB from El Tor strains of the seventh pandemic. We concluded 1841 toxin was identical to CT of the seventh pandemic El Tor vibrios.     

Novel Vibrio cholerae O139 genes involved in lipopolysaccharide biosynthesis.

The sequence of part of the rfb region of Vibrio cholerae serogroup O139 and the physical map of a 35-kb region of the O139 chromosome have been determined. The O139 rfb region presented contains a number of open reading frames which show similarities to other rfb and capsular biosynthesis genes found in members of the Enterobacteriaceae family and in V. cholerae O1. The cloned and sequenced region can complement the defects in O139 antigen biosynthesis in transposon insertions within the O139 rfb cluster. Linkage is demonstrated among IS1358 of V. cholerae O139, the rfb region, and the recently reported otnA and otnB genes (E. M. Bik, A. E. Bunschoten, R. D. Gouw, and F. R. Mooi, EMBO J. 14:209-216, 1995). In addition, the whole of this region has been linked to the rfaD gene. Furthermore, determination of the sequence flanking IS1358 has revealed homology to other rfb-like genes. The exact site of insertion with respect to rfaD is defined for the novel DNAs of both the Bengal and the Argentinian O139 isolates.     

Distribution of Vibrio cholerae O1 antigen biosynthesis genes among O139 and other non-O1 serogroups of Vibrio cholerae

The organization and distribution of the genes responsible for O antigen biosynthesis in various serogroups of Vibrio cholerae were investigated using several DNA probes derived from various regions of the genes responsible for O1 antigen biosynthesis. Based on the reactivity pattern of the probes against the various serogroups, the cluster of genes responsible for the O1 antigen biosynthesis could be broadly divided into six groups, designated as class 1-6. The class 3 cluster of genes corresponding to gmd to wbeO, wbeT and a part of wbeU was specific for only the O1 serogroup. The other cluster of genes (class 1, 2, 4-6) reacted with other serogroups of V. cholerae. These data indicate that serotype conversion in V. cholerae does not depend on a simple mutational event but may involve horizontal gene transfer not only between V. cholerae strains but also between V. cholerae and species other than V. cholerae.     

Characterization of a clinical Vibrio cholerae O139 isolate from Mexico

Pathogenic strains of Vibrio cholerae O139 possess the cholera toxin A subunit (ctxA) gene as well as the gene for toxin co-regulated pili (tcpA). We report the isolation of a ctxA-negative, tcpA-negative V. cholerae O139 strain (INDREI) from a patient in Mexico diagnosed with gastrointestinal illness. Certain phenotypic characteristics of this strain were identical to those of V. cholerae O1 biotype El Tor. Unlike ctxA-positive V. cholerae O139 strains, this strain was sensitive to a wide panel of antibiotics, including ampicillin, chloramphenicol, ciprofloxacin, gentamicin, furazolidone, nalidixic acid, nitrofurantoin, tetracycline, trimethoprim-sulfamethoxazole, and streptomycin, but was resistant to polymyxin B. Ribotype and pulsed-field gel electrophoresis profiles of INDRE1 differed from those of ctxA-positive V. cholerae O139 and other V. cholerae strains. Phenotypic characteristics of the Mexico strain were similar to those reported for V. cholerae O139 isolates from Argentina and Sri Lanka.     

Adherence to human small intestines of capsulated Vibrio cholerae O139

Abstract Capsulated cells of V. cholerae O139 adhered to formalis-fixed or native mucosa of the small intestines from an adult and a child. The primary adherence target was mucus. Capsulated O139 cells adhered better to the antigen sampling cells (M cells) of ileal Peyer's patch than to the absorptive cells. O139 cells on the mucosa appeared as small aggregates. Similar organisms were found on the mucosa of duodenal biopsy samples from patients infected with V. cholerae O139. The findings indicated that capsulated cells of V. cholerae O139 tend to autoagglutinate and contribute to the effective adherence to the intestinal mucosa.     

Isolation of Vibrio cholerae O139 synonym Bengal from the aquatic environment in Bangladesh: implications for disease transmission.

Currently, Bangladesh is experiencing an epidemic of acute watery diarrhea caused by Vibrio cholerae O139. Surface waters were collected and cultured for vibrious following enrichment. Twelve percent (11 of 92) of samples yielded V. cholerae O139, and all of them were positive for cholera toxin. The data suggest that V. cholerae O139 is easily culturable from surface water samples.