Structural studies of a polysaccharide from Vibrio parahaemolyticus strain AN-16000.

The structure of a polysaccharide from Vibrio parahaemolyticus strain AN-16000 has been investigated. The sugar and absolute configuration analysis revealed d-Glc, d-GalN, d-QuiN and l-FucN as major components. The PS was subjected to dephosphorylation with aqueous 40% HF to obtain an oligosaccharide that was analyzed by (1)H and (13)C NMR spectroscopy. The HR-MS spectrum of the oligosaccharide revealed a pentasaccharide composed of two Glc residues, one QuiNAc and one GalNAc, one FucNAc, as well as a glycerol moiety. The structure of the PS was determined using (1)H, (13)C, (15)N and (31)P NMR spectroscopy; inter-residue correlations were identified by (1)H,(13)C-heteronuclear multiple-bond correlation, (1)H,(1)H-NOESY and (1)H,(31)P-hetero-TOCSY experiments. The PS backbone has the following teichoic acid-like structure: →3)-d-Gro-(1-P-6)-ß-d-Glcp-(1→4)-α-l-FucpNAc-(1→3)-ß-d-QuipNAc-(1→ with a side-chain consisting of α-d-Glcp-(1→6)-α-d-GalpNAc-(1→ linked to the O3 position of the FucNAc residue.

Structural studies of the O-antigenic polysaccharide from Plesiomonas shigelloides strain AM36565.

The structure of the repeating unit of the O-antigenic polysaccharide from Plesiomonas shigelloides strain AM36565 has been determined. Component analysis and (1)H and (13)C NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by (1)H,(13)C heteronuclear multiple-bond correlation, (1)H,(1)H-NOESY, and (1)H,(13)C-HSQC-(1)H,(1)H-NOESY experiments. The O-antigen polysaccharide is composed of repeating units with the following structure: →3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→4)[ß-D-GalpNAc-(1→3)]-α-D-GlcpNAc-(1→, in which the monosaccharide side-chain substitutes the backbone in half of the repeating units. A matrix-assisted laser desorption/ionization mass spectrometry experiment suggested that the polysaccharide consists of two regions, one with tetrasaccharide repeating units and one with trisaccharide repeating units.

Multi-drug resistant Vibrio cholerae O1 variant El Tor isolated in northern Vietnam between 2007 and 2010.

Since 2007, there has been a re-emergence of cholera outbreaks in northern Vietnam. To understand the molecular epidemiological relatedness and determine the antibiotic susceptibility profiles of responsible V. cholerae O1 outbreak strains, a representative collection of 100 V. cholerae O1 strains was characterized. V. cholerae O1 strains isolated from diarrhoeal patients in northern Vietnam between 2007 and 2010 were investigated for antibiotic susceptibility and characterized by using phenotypic and genotypic tests, including PFGE analysis. Ten clinical V. cholerae O1 isolates from Bangladesh and Zimbabwe were included for comparison. The results revealed that all isolates were resistant to co-trimoxazole and nalidixic acid, 29 % were resistant to tetracycline and 1 % were resistant to azithromycin. All strains were susceptible to ampicillin-sulbactam, doxycycline, chloramphenicol and ciprofloxacin and 95 % were susceptible to azithromycin. MIC values did show reduced susceptibility to fluoroquinolones and 63 % of the strains were intermediately resistant to tetracycline. The isolates expressed phenotypic traits of both serogroup O1 Ogawa and El Tor and harboured an rstR El Tor and ctxB classical biotype. Among the outbreak isolates, only a single PFGE pattern was observed throughout the study period. This study shows that multi-drug resistant V. cholerae altered El Tor producing classical CT strains are now predominant in northern Vietnam.

Phenotypic, genotypic, and antibiotic sensitivity patterns of strains isolated from the cholera epidemic in Zimbabwe.

This paper details the phenotypic, genotypic, and antibiotic sensitivity patterns of 88 Vibrio cholerae strains from Zimbabwe. Of the 88 strains, 83 were classified as "altered El Tor" and 5 as "hybrid El Tor" strains. All of the strains were susceptible to tetracycline, doxycycline, ciprofloxacin, and azithromycin by disc diffusion, but susceptibility to tetracycline and azithromycin diminished when observed using the MIC method.

Toxin production by Aeromonas sobria in natural environments: river water vs. seawater.

Aeromonas are water-borne pathogens. They are halotolerant, which means that they can survive in environments whose salt content corresponds to that of seawater (3.0% NaCl). However, the presence of Aeromonas in seawater is extremely rare compared with that in river water. In this study, we tested the ability of Aeromonas sobria to produce toxins in river water and seawater. First, we cultured A. sobria on skim milk agar plates supplemented with either river water (SARW) or seawater (SASW). The bacteria grew on both plates. A clear zone around the bacteria was generated in SARW. However, such a zone was not observed in SASW, suggesting that proteases were not generated in SASW. Subsequently, we cultured A. sobria in a nutrient broth supplemented with either river water (NRW) or with seawater (NSW), and examined the protease activity of their culture supernatants. The protease activity of the culture supernatant from NSW was extremely low compared to that from NRW. The immunoblotting analysis showed that serine protease (ASP) was not produced by the culture in NSW. By contrast, aerolysin-like hemolysin was produced in all conditions examined in this study. This indicates that the salinity of water is deeply involved in the production of ASP by A. sobria.

Genetic diversity of El Tor strains of Vibrio cholerae O1 with hybrid traits isolated from Bangladesh and Mozambique.

Vibrio cholerae O1 strains that are hybrids between the classical and El Tor biotypes were isolated during two consecutive years (2004-2005) from diarrheal patients in Mozambique. Similar variants isolated in Bangladesh and recently isolated El Tor strains were analyzed for genetic diversity. Pulsed-field gel electrophoresis (PFGE) analysis using the restriction enzyme NotI, resulted in 18-21 bands showed five closely related PFGE patterns that were distributed similarly in both years (2004-2005) among the 80 strains tested in Mozambique. Overall based on the PFGE patterns the hybrids indicated an El Tor lineage. The restriction patterns of whole-chromosomal DNA grouped the hybrid strains from Mozambique into a separate cluster from Bangladeshi clinical and environmental hybrid strains. A high molecular weight band of 398kb that contain rstR allele of the classical type was detected from all hybrid strains, which was absent in all conventional classical and El Tor strains. This band could be designated as a marker for the hybrid strains. This study suggests that hybrid strains from Mozambique are closely related to each other, different from Bangladeshi hybrid strains that are diverse in nature and all hybrid strains differed markedly from conventional classical and El Tor strains.

Toxin(s), other than cholera toxin, produced by environmental non O1 non O139 Vibrio cholerae.

A total of 39 Vibrio cholerae non O1 non O139 strains were isolated from surface waters of different parts of Dhaka City, Bangladesh. All these strains showed lack of ctx or zot gene, as demonstrated by the PCR analysis. Eighteen representative strains were tested for enterotoxin production using a rabbit ileal loop model, of which live cells of 8 strains and culture filtrates of 6 strains produced fluid accumulation in ileal loops. However, none of them produced heat stable toxin (ST), as detected by suckling mouse assay. On the other hand, 15% of isolates produced cytotoxin as detected by the Chinese Hamster Ovary (CHO) cell assay. Fifty times concentrated culture filtrates of the representative strains did not give any precipitin band against the anti-cholera toxin, suggesting the strains produced an enterotoxin, which is antigenically different from known cholera toxin (CT). Eighty percent of the total isolates were found to be positive for heat labile haemolysin detected by tube method, whereas, 39% were found positive by the Christie-Atkins-Munch-Petersen (CAMP) method. However, 87% of the isolates were positive for haemagglutinin/protease and all of the strains were positive for mannose-sensitive-haemagglutinin assay.

Evaluation of three rapid diagnostic tests for cholera: does the skill level of the technician matter?

OBJECTIVE: To evaluate SMART, Medicos Dip Stick and an Institut Pasteur (IP) cholera dipstick tests for accuracy and ease of use. METHOD: Every 50th patient presenting with diarrhoea at ICDDR,B between 1 April 2003 and 30 November 2003 was enrolled. The rapid diagnostic tests were performed by field and laboratory technicians, and sensitivity (Se), specificity (Sp), positive (PPV) and negative (NPV) predictive values calculated. RESULTS: We isolated Vibrio cholerae O1 from 116 (38%) of 304 patients. The Se, Sp, PPV and NPV of the SMART test were 58%, 95%, 84% and 84% for field technicians, and 83%, 88%, 83% and 88% for laboratory technicians. The Se, Sp, PPV and NPV of the IP dipstick test were 93%, 67%, 63% and 94% for field technicians, and 94%, 76%, 70% and 95% for laboratory technicians. The Se, Sp, PPV and NPV of the Medicos test were 84%, 79%, 71% and 90% for field technicians, and 88%, 80%, 72% and 92% for laboratory technicians. A high proportion of indeterminates (30%) hampered the performance of the SMART test. The IP dipstick had the highest Se, irrespective of technician skill level. CONCLUSION: The IP dipstick is the most appropriate rapid diagnostic assay for the detection of V. cholerae O1 in locations where the skill level of personnel may be low, such as remote areas or refugee camp settings. High cost may limit the utility of any diagnostic test in the developing world.

TnphoA mutants of Providencia alcalifaciens with altered invasiveness of HEp-2 cells.

Recent studies have shown that Providencia alcalifaciens is a diarrhoeal pathogen. It may cause diarrhoea by an invasive mechanism, as it invades cultured mammalian cells in vitro and intestinal epithelial cells of experimentally inoculated rabbits in vivo. To locate the gene(s) involved in invasion, TnphoA mutants of a diarrhoeal isolate of P. alcalifaciens were generated. Compared with the parent strain, these mutants exhibited negligible invasion and actin condensation in HEp-2 cells. TnphoA insertion was located in fragments of 4.9 kb and 11.1 kb of the bacterial chromosome by Southern blot. These mutants did not secrete a 28-kDa protein, which may be involved in invasion. It should be possible now to study the gene(s) involved in invasion of P. alcalifaciens with these mutants. This investigation is another example of the usefulness of TnphoA mutagenesis in the study of bacterial virulence genes.