Pathogenicity of non-O1/ O139 Vibrio cholerae and its induced immune response in Macrobrachium rosenbergii.

Outbreaks of mass mortalities occurred in Macrobrachium rosenbergii farms in Gaoyou county, Jiangsu Province of China. The bacterial isolates from M. rosenbergii exhibited the same phenotypic traits and biochemical characteristics, and were identified as non-O1/O139 Vibrio cholerae according to biochemical characteristics and molecular identification. In challenge test, M. rosenbergii infected with non-O1/O139 V. cholerae GXFL1-4 developed similar pathological signs to the naturally diseased prawns, and LD50 of the strain to M. rosenbergii was 4.5 × 106 CFU/mL at 96 h post-infection. Histopathological analysis revealed that hepatopancreas and intestines of diseased M. rosenbergii exhibited obvious inflammatory responses to non-O1/O139 V. cholerae infection. Detection virulence factors of the strain GXFL1-4 showed that the bacteria produced caseinase, lipase, amylase, lecithinase and hemolysin, and carried toxR, hlyA, ompW, ompU, hap, rtxA and rtxC virulence related genes, supporting the strong virulence to M. rosenbergii. Additionally, the immune related gene expression in M. rosenbergii evaluated by qRT-PCR analysis showed that HSP70, Crustin, Lysozyme, TRL1, ALF1, Lectin, Peroxinectin, proPO and SOD immune related genes were significantly up-regulated at 6 and 12 h after infection with GXFL1-4. The results of our study suggested that non-O1/O139 V. cholerae was an etiological element in the mass mortalities of M. rosenbergii and this study provided preliminary insights into the diversity in the immune response of M. rosenbergii to the bacterial invasion.

Environmental Vibrio cholerae non O1/ non O139 from the Gangetic delta: a diarrhoeal disease purview.

Diarrhoea still remains an unsolved enigma in developing countries, a major concern for the health planners. We targeted the abundance and toxicity of Vibrio cholerae non-O1/non-O139 (NOVC) in Gangetic riverine-estuarine ecosystem. A total of 74 V. cholerae were isolated from 120 water samples (68 NOVC, 6 V. cholerae O1) from two sampling sites off river Ganges. V. cholerae showed distinct seasonality, with steady increase from summer to monsoon, steep ascent in post-monsoon and an abrupt decline in winter. Highest number of NOVC was isolated form Howrah, attributed to low salinity and high anthropogenic influence. Environmental NOVC harboured hlyA (94.0 %), rtxA (81.0 %) and toxR (28.0 %) genes. About 23.4 % of the hlyA harbouring NOVC showed haemolytic activity. Accessory toxin genes (tlcR, toxT, RJ and LJ and aldA), among 3-5 % of the NOVC carry significant health implications. Haemolytic activity and biofilm formation in NOVC, during unfavourable conditions, facilitates gene transfer and emphasises the role of environmental NOVC in diarrhoeal incidence in South Bengal, India.

Vibriosis, not cholera: toxigenic Vibrio cholerae non-O1, non-O139 infections in the United States, 1984-2014.

Toxigenic strains of Vibrio cholerae serogroups O1 and O139 have caused cholera epidemics, but other serogroups - such as O75 or O141 - can also produce cholera toxin and cause severe watery diarrhoea similar to cholera. We describe 31 years of surveillance for toxigenic non-O1, non-O139 infections in the United States and map these infections to the state where the exposure probably originated. While serogroups O75 and O141 are closely related pathogens, they differ in how and where they infect people. Oysters were the main vehicle for O75 infection. The vehicles for O141 infection include oysters, clams, and freshwater in lakes and rivers. The patients infected with serogroup O75 who had food traceback information available ate raw oysters from Florida. Patients infected with O141 ate oysters from Florida and clams from New Jersey, and those who only reported being exposed to freshwater were exposed in Arizona, Michigan, Missouri, and Texas. Improving the safety of oysters, specifically, should help prevent future illnesses from these toxigenic strains and similar pathogenic Vibrio species. Post-harvest processing of raw oysters, such as individual quick freezing, heat-cool pasteurization, and high hydrostatic pressurization, should be considered.

Genomic and phenotypic characterization of Vibrio cholerae non-O1 isolates from a US Gulf Coast cholera outbreak.

Between November 2010, and May 2011, eleven cases of cholera, unrelated to a concurrent outbreak on the island of Hispaniola, were recorded, and the causative agent, Vibrio cholerae serogroup O75, was traced to oysters harvested from Apalachicola Bay, Florida. From the 11 diagnosed cases, eight isolates of V. cholerae were isolated and their genomes were sequenced. Genomic analysis demonstrated the presence of a suite of mobile elements previously shown to be involved in the disease process of cholera (ctxAB, VPI-1 and -2, and a VSP-II like variant) and a phylogenomic analysis showed the isolates to be sister taxa to toxigenic V. cholerae V51 serogroup O141, a clinical strain isolated 23 years earlier. Toxigenic V. cholerae O75 has been repeatedly isolated from clinical cases in the southeastern United States and toxigenic V. cholerae O141 isolates have been isolated globally from clinical cases over several decades. Comparative genomics, phenotypic analyses, and a Caenorhabditis elegans model of infection for the isolates were conducted. This analysis coupled with isolation data of V. cholerae O75 and O141 suggests these strains may represent an underappreciated clade of cholera-causing strains responsible for significant disease burden globally.

ROLE OF HLYA-POSITIVE VIBRIO CHOLERAE NON-O1/NON-O139 ON APOPTOSIS AND CYTOTOXICITY IN A CHINESE HAMSTER OVARY CELL LINE.

Vibrio cholerae non-O1/non-O139 is capable of producing sporadic outbreaks of cholera-like diarrhea; however, the pathogenic mechanisms of this bacterium remain unclear. The objectives of this study were to: 1) compare the apoptosis induction and cytotoxicity between hlyA-positive and hlyA-negative strains of V. cholerae non-O1/non-O139; 2) clarify the molecular mechanisms by which these strains induce apoptosis; and 3) compare clinical and environmental V. cholerae non-O1/non-O139 isolates with respect to cytotoxicity and ability to induce apoptosis. Using cytotoxicity and apoptosis assays, it was shown that hlyA-positive strains of V. cholerae non-O1/non-O139 had significantly higher cytotoxic activity (70.6%) and levels of apoptosis induction (59.6%) than hlyA- negative strains (37.0% and 37.5%, respectively). Western blot analyses revealed that hlyA-positive strains had significantly increased expression of Bax; active caspase-3 and -9; and significantly decreased expression of NF-κB and Bcl-2 relative to hlyA-negative strains. Expression of BID did not differ significantly between hlyA-positive and negative strains. The truncated BID was not found, indicating that V. cholerae non-O1/non-O139 induces apoptosis through a mitochondria- dependent apoptosis pathway and not an extrinsic pathway. V. cholerae non-O1/ non-O139 isolated from clinical sources exhibited significantly higher cytotoxic activity (79%) and levels of apoptosis induction (65.2%) than bacteria isolated from environmental sources (63% and 54.6%, respectively), suggesting that the clini- cal isolates may have other virulence-associated genes besides hlyA. Our results indicate that hlyA products play a role in cytotoxicity and apoptosis induction and that a mitochondria-dependent apoptosis pathway is involved.

[Role of galactose-specific receptor--lectin in bactericidal activity of hemolysin of Vibrio cholerae non O1/O139].

AIM: To study the role lectin galactose-specific receptor of Vibrio cholerae hemolysin in receptor mechanisms of bacterial cells lysis. MATERIALS AND METHODS: Five strains of V. cholerae eltor ctx- Hly+ and 5 strains V. cholerae eltorctx+ Hly(-) isolated from patients and environment as well as 6 strains from Shigella genus, 3 strains of Escherichia coli, 2 strains of V. cholerae eltor, and 1 strain of V. cholerae non O1 were used in the study. Preparation P-11702 obtained from strain of V. cholerae non O1 was used for the study of bactericidal activity of hemolysin. For neutralization of bactericidal effect antihemolytic serum prepared against P-11702 as well as 1% solutions of carbohydrates (galactose, sucrose, glucose, and N-acetyl-D-galactosamine) were used. RESULTS: It was shown that lytic activity of hemolysin against cholera vibrios and indicator cultures for detection of vibriocins was neutralized by galactose as well as hemolysis of erythrocytes was neutralized by specific antihemolytic serum. Sucrose, glucose, and N-acetyl-D-galactosamine did not have effect on bactericidal and hemolytic activity of hemolysin. CONCLUSION: Protein-carbohydrate receptor binding of galactose-specific lectin has an important role in realization of bactericidal effect of V. cholerae hemolysin.

Molecular diversification in the quorum-sensing system of Vibrio cholerae: Role of natural selection in the emergence of pandemic strains.

Two haplotypes of the Vibrio cholerae quorum-sensing system regulator hapR are described: hapR1, common among nonpandemic, non-O1, non-O139 strains, and hapR2, associated with pandemic O1 and O139 and epidemic O37 V. cholerae strains. The hapR2 has evolved under strong natural selection, implying that its fixation was influenced by conditions that led to cholera pandemics.

Association of ompU gene in Vibrio cholerae from patients and environment with bile resistance.

The objective of this study was to determine whether Vibrio cholerae, possessing ompU isolated from patients and the environment, conferred bile resistance and whether other virulence genes were also related to bile resistance. Fifty-two V cholerae O1 and non-O1 isolates were examined by PCR for the presence of the virulence-associated and regulatory genes, ctxA, tcpA, zot, ace, ompU, toxR, hlyA and stn/sto. V. cholerae possessing ompU resistant to equal or greater than 10% sodium deoxycholate were found in 93% of isolates but only in 9% of V. cholerae isolates not possessing ompU. The effects of other virulence genes on bile resistance could not be ascertained in this study. Thus V cholerae non-O1 with ompU and possibly other virulence genes isolated from the environment have the potential of affecting public health.

[Biofilm formation and response to oxidative stress in Pseudomonas aeruginosa and Vibrio cholerae non-O1 depending on culture media]. / Tvorba biofilmu a odpoved' na oxidacný stres u kmenov Pseudomonas aeruginosa a Vibrio cholerae non-O1 v závislosti na kultivacných médiách.

OBJECTIVE: To evaluate the effect of six culture media (five complex and one mineral) on biofilm formation and response to oxidative stress in Pseudomonas aeruginosa (3 strains) and Vibrio cholerae non-O1 (3 strains). METHODS: Biofilm formation was quantitatively determined by a crystal violet absorption assay. The bacterial response to oxidative stress evoked by hydrogen peroxide was visualized as a zone of clearing around the disc after 24 h incubation at 37 degrees C. RESULTS: For both of the bacterial species studied, biofilm formation was the highest after cultivation in tryptone soya broth (TSM) or in TSM supplemented with 8% glucose (TSM+GL), being the lowest in mineral medium (MM). V. cholerae non O1 strains were 1.4 to 4.3 times more responsive on average to oxidative stress depending on culture medium as compared with P. aeruginosa strains. The culture medium had no significant effect on H2O2 evoked by response to oxidative stress in vibrios in contrast to P. aeruginosa. In P. aeruginosa, the highest mean resistance to H2O2 was observed after cultivation in peptone water while the most sensitive cells were found after incubation in TSM+GL and MM. CONCLUSION: The culture medium composition influnced biofilm formation in both of the bacterial species tested and had a considerable effect on response to oxidative stress in P. aeruginosa.

[Microbiological characterization of non-O1 Vibrio cholerae isolated in Cuba]. / Caracterización microbiológica de cepas de Vibrio cholerae no-O1 aisladas en Cuba.

The study of 422 non-01 Vibrio cholerae strains from nine provinces, 9 of them isolated from a water-borne disease outbreak, was performed. All the strains exhibited antimicrobial susceptibility and virulence factors. The nine strains from the outbreak were subjected to a DNA macrorestriction study based on the pulsed field electrophoresis technique. For the first time in Cuba and the Caribbean. The circulation of atypical non-01 V cholerae strains (resistent to vibriostatic compound 0129 and trimethoprim/sulfamethoxazole). The behavior of antimicrobial susceptibility evinced for the first time the circulation of two different resistence patterns in Cuba (ampicilline, trimethoprim/ sulfamethoxazole, sulfonamide and tetracycline, trimethoprim/ sulfamethoxazole, sulfonamide). The frequency of trimethoprim/ sulfamethoxazole-resistent strains was similar during the whole period of study. However, resistance to ampicilline decreased whereas resistance to tetracycline increased. The main found virulence factors were gelatinase, hemolysine, elastase and adherence to Hep-2 cells. On the other hand, the outbreak strains showed higher percentages than the others due to the presence of heat-liable toxin and fimbriae. The results of the molecular and epidemiological studies allowed giving a speedy and accurate response that explained the etiology of the first food-borne disease outbreak.

Influence of environmental factors on the presence of Vibrio cholerae in the marine environment: a climate link.

Evidence indicates that the atmospheric and oceanic processes that occur in response to increased greenhouse gases in the broad-scale climate system may already be changing the ecology of infectious diseases. Recent studies have shown that climate also influences the abundance and ecology of pathogens, and the links between pathogens and changing ocean conditions, including human diseases such as cholera. Vibrio cholerae is well recognized as being responsible for significant mortality and economic loss in developing countries, most often centered in tropical areas of the world. Within the marine environment, V. cholerae is found attached to surfaces provided by plants, filamentous green algae, copepods, crustaceans, and insects. The specific environmental changes that amplified plankton and associated bacterial proliferation and govern the location and timing of plankton blooms have been elucidated. Several studies have demonstrated that environmental non-O1 and non-O139 V. cholerae strains and V. cholerae O1 El Tor and O139 are able to form a three-dimensional biofilm on surfaces which provides a microenvironment, facilitating environmental persistence within natural aquatic habitats during interepidemic periods. Revealing the influence of climatic/environmental factors in seasonal patterns is critical to understanding temporal variability of cholera at longer time scales to improve disease forecasting. From an applied perspective, clarifying the mechanisms that link seasonal environmental changes to diseases' dynamics will aid in developing strategies for controlling diseases across a range of human and natural systems.

[Factors influencing the capacity of cellular substrate adherence of vibrio cholerae O1 and non O1 strains]. / Studiul factorilor care influenteaza capacitatea de aderenta la substratul celular a tulpinilor de Vibrio cholerae O1 si non O1.

Bacterial adherence to eukariotic cells represents an important step of tissue colonization and is mediated by specific molecules called adhesins. Bacterial adherence to cellular substrate is a very complex process consisting in specific interactions between the surface of host cell and bacterial cell surface respectively. Adherence to cellular substrate confers selective advantages to bacterial cells, as: rapid growth rate by shorter lag period and protection against antibodies and lysozime. Adherence and colonization of small bowel represent the early steps of cholera infection (1, 2). The purposes of this study were to characterize the adherence ability of 46 Vibrio cholerae O1 and non O1 strains with different sources of isolation (acute diarrhea, water sources) to HEp-2 cell; to determine the influence of different factors (culture media, bacterial culture growth phase, proteolytic enzymes, carbohydrates and polyvalent agglutinant anti V. cholerae O1 serum) on the bacterial adherence capacity. Adherence capacity was assayed using the qualitative Cravioto's method. The adherence ability was appreciated by semi quantitative ("+", "++" and "+++") and quantitative assays. The adherence pattern of the tested strains was predominantly a diffuse one. The agar medium proved to be the most appropriate for the early and maximal expression of adhesion molecules, by comparison with nutritive broth and alkaline peptone water. Manose in different concentrations (1% and 3%) inhibited the adherence ability, demonstrating the role of manose-sensitive haemagglutinating fimbriae (MSHA) in mediating the adherence of V. cholerae strains to cellular substrate. Trypsine has no notable effect on the adherence ability, suggesting that the major V. cholerae adhesion molecules are not essentially of protein nature, so that the afimbrial adhesins could also play an important role in bacterial adhesion to eukariotic cells. The agglutinant polyvalent anti-V. cholerae O1 serum had the most significant inhibitory effect on the adherence ability, which was completely abolished in the presence of sub-agglutinant dilutions of serum titer (1/60-1/120) and partially reduced at titers ranging from 1/240 to 1/920. This inhibitory effect could be explained by bacterial agglutination, but also by the specific blocking of some surface structure implicated in the adherence process (i.e. lipopolysaccharides, as demonstrated by the inhibitory effect of sub-agglutinant serum titers). The inhibitory effect of polyvalent anti-V. cholerae O1 serum was limited to O1, but was not evident for the non O1 serogroups, demonstrating that the serum antibodies are acting on serogroup specific antigenic fractions.