Kegg Pathway: Cholera - Environment

PLoS One. 2009; 4(11): e7806
Ou G, Rompikuntal PK, Bitar A, Lindmark B, Vaitkevicius K, Wai SN, Hammarström ML

BACKGROUND: Vibrio Cholerae is the causal intestinal pathogen of the diarrheal disease Cholera. It secretes the protease PrtV, which protects the bacterium from invertebrate predators but reduces the ability of Vibrio-secreted factor(s) to induce interleukin-8 (IL-8) production by human intestinal epithelial cells. The aim was to identify the secreted component(s) of V. Cholerae that induces an epithelial inflammatory response and to define whether it is a substrate for PrtV. METHODOLOGY/PRINCIPAL FINDINGS: Culture supernatants of wild type V. Cholerae O1 strain C6706, its derivatives and pure V. Cholerae cytolysin (VCC) were analyzed for the capacity to induce changes in cytokine mRNA expression levels, IL-8 and tumor necrosis factor-alpha (TNF-alpha) secretion, permeability and cell viability when added to the apical side of polarized tight monolayer T84 cells used as an in vitro model for human intestinal epithelium. Culture supernatants were also analyzed for hemolytic activity and for the presence of PrtV and VCC by immunoblot analysis. CONCLUSIONS/SIGNIFICANCE: We suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-alpha in tight monolayers. Pure VCC at a concentration of 160 ng/ml caused an inflammatory response that reached the magnitude of that caused by Vibrio-secreted factors, while higher concentrations caused epithelial cell death. The inflammatory response was totally abolished by treatment with PrtV. The findings suggest that low doses of VCC initiate a local immune defense reaction while high doses lead to intestinal epithelial lesions. Furthermore, VCC is indeed a substrate for PrtV and PrtV seems to execute an Environment-dependent modulation of the activity of VCC that may be the cause of V. Cholerae reactogenicity.

J Environ Health. 2009 Oct; 72(3): 24-7; quiz 38
DeGolier L, Ledin K, Vargulich G, Patnaik JL, Wilson B, Vogt RL

Cases of non-Cholera Vibrio illness are typically associated with exposure to shellfish from marine coastal areas (U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, 2009), not landlocked states such as Colorado. In 2004, a 2.8-fold increase in the incidence of non-Cholera Vibrio cases in the Tri-County Health Department (TCHD) jurisdiction of Colorado prompted scrutiny of shellfish practices in local retail food establishments. Forty-three percent of establishments serving raw shellfish in the TCHD jurisdiction were in violation of one or more sections of the Colorado Retail Food Establishment Rules and Regulations (Colorado Department of Public Health and Environment, 2007a). The frequency of violations and the underutilization of safer, post-harvest processed shellfish may result in significant hazards to consumers if these practices continue.

Curr Microbiol. 2009 Oct 20;
Kumar P, Peter WA, Thomas S

Vibrio Cholerae, the causative agent of Cholera is ubiquitously distributed in aquatic Environment particularly in coastal waters, estuaries, and rivers. In the present investigation, a multiplex PCR assay was developed for the detection of virulence-associated genes (rtxA, tcpA, ctxA, hlyA, and sto) in Environmental isolates of V. Cholerae. A total of 90 strains isolated from different Environmental sources were screened for the presence of virulence-associated genes. Our results showed that this method represents a simple, cost effective, and robust tool for rapid detection of virulence-associated genes. This multiplex PCR can be used for examining prevalence of virulence-associated genes and hence will be useful for better understanding of epidemiology of Environmental V. Cholerae.

Exp Parasitol. 2009 Oct 6;
Sandström G, Saeed A, Abd H

Acanthamoeba is a genus of free-living amoebae found to be able to host many bacterial species living in the Environment. Acanthamoebae and Vibrio Cholerae are found in the aquatic Environments of Cholera endemic areas. Previously it has been shown that V. Cholerae O1 and O139 can survive and grow in Acanthamoeba castellanii. The aim of this study was to examine the ability of Acanthamoeba polyphaga to host V. Cholerae O1 and O139. The interaction between A. polyphaga and V. Cholerae strains was studied by means of viable amoeba cell counts and viable count of the bacteria in the absence and presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Electron microscopy was used to determine the localization of V. Cholerae inside A. polyphaga. The results showed that A. polyphaga enhanced growth and survival of V. Cholerae, which grew and survived inside the amoeba cells for 2weeks. The electron microscopy showed that A. polyphaga hosted intracellular V. Cholerae localized in the vacuoles of amoeba cell. Neither the presence of V. Cholerae together with A. polyphaga nor the intracellular localization of the bacteria inhibited growth and survival of A. polyphaga. The outcome of the interaction between these microorganisms may support strongly the role of A. polyphaga as host for V. Cholerae O1 and O139.

Curr Top Microbiol Immunol. 2009; 337: 37-59
Ritchie JM, Waldor MK

Vibrio Cholerae is a curved Gram-negative rod that causes the diarrheal disease Cholera. One hundred and twenty five years of study of V. Cholerae microbiology have made this lethal pathogen arguably the most well-understood non-invasive mucosal pathogen. Over the past 25 years, modern molecular techniques have permitted the identification of many genes and cellular processes that are critical for V. Cholerae colonization of the gastrointestinal tract. Review of the literature reveals that there are two classes of genes that influence V. Cholerae colonization of the suckling mouse intestine, the most commonly used animal model to study V. Cholerae pathogenesis. Inactivation of one class of genes results in profound attenuation of V. Cholerae intestinal colonization, whereas inactivation of the other class of genes results in only moderate colonization defects. The latter class of genes suggests that V. Cholerae may colonize several intestinal niches that impose distinct requirements and biological challenges, thus raising the possibility that there is physiologic heterogeneity among the infecting population. Efficient V. Cholerae intestinal colonization and subsequent dissemination to the Environment appears to require temporally ordered expression of sets of genes during the course of infection. Key challenges for future investigations of V. Cholerae pathogenicity will be to assess the degree of heterogeneity in the infecting population, whether such heterogeneity has functional significance, and if stochastic processes contribute to generation of heterogeneity in vivo.

PLoS One. 2009; 4(10): e7352
Olivier V, Queen J, Satchell KJ

Vibrio Cholerae colonizes the small intestine of adult C57BL/6 mice. In this study, the physical and genetic parameters that facilitate this colonization were investigated. Successful colonization was found to depend upon anesthesia with ketamine-xylazine and neutralization of stomach acid with sodium bicarbonate, but not streptomycin treatment. A variety of common mouse strains were colonized by O1, O139, and non-O1/non-O139 strains. All combinations of mutants in the genes for hemolysin, the multifunctional, autoprocessing RTX toxin (MARTX), and hemagglutinin/protease were assessed, and it was found that hemolysin and MARTX are each sufficient for colonization after a low dose infection. Overall, this study suggests that, after intragastric inoculation, V. Cholerae encounters barriers to infection including an acidic Environment and an immediate immune response that is circumvented by sodium bicarbonate and the anti-inflammatory effects of ketamine-xylazine. After initial adherence in the small intestine, the bacteria are subjected to additional clearance mechanisms that are evaded by the independent toxic action of hemolysin or MARTX. Once colonization is established, it is suggested that, in humans, these now persisting bacteria initiate synthesis of the major virulence factors to cause Cholera disease. This adult mouse model of intestinal V. Cholerae infection, now well-characterized and fully optimized, should serve as a valuable tool for studies of pathogenesis and testing vaccine efficacy.

J Neurosci. 2009 Sep 23; 29(38): 11954-64
Yoshida K, Li X, Cano G, Lazarus M, Saper CB

Sympathetic premotor neurons in the rostral medullary raphe (RMR) regulate heat conservation by tail artery vasoconstriction and brown adipose tissue thermogenesis. These neurons are a critical relay in the pathway that increases body temperature. However, the origins of the inputs that activate the RMR during cold exposure have not been definitively identified. We investigated the afferents to the RMR that were activated during cold by examining Fos expression in retrogradely labeled neurons after injection of Cholera toxin B subunit (CTb) in the RMR. These experiments identified a cluster of Fos-positive neurons in the dorsomedial hypothalamic nucleus and dorsal hypothalamic area (DMH/DHA) with projections to the RMR that may mediate cold-induced elevation of body temperature. Also, neurons in the median preoptic nucleus (MnPO) and dorsolateral preoptic area (DLPO) and in the A7 noradrenergic cell group were retrogradely labeled but lacked Fos expression, suggesting that they may inhibit the RMR. To investigate whether individual or common preoptic neurons project to the RMR and DMH/DHA, we injected CTb into the RMR and Fluorogold into the DMH/DHA. We found that projections from the DLPO and MnPO to the RMR and DMH/DHA emerge from largely separate neuronal populations, indicating they may be differentially regulated. Combined cell-specific lesions of MnPO and DLPO, but not lesions of either one alone, caused baseline hyperthermia. Our data suggest that the MnPO and DLPO provide parallel inhibitory pathways that tonically inhibit the DMH/DHA and the RMR at baseline, and that hyperthermia requires the release of this inhibition from both nuclei.

Trans Am Clin Climatol Assoc. 2009; 120: 119-28
Constantin de Magny G, Colwell RR

Vibrio Cholerae, the causative agent of Cholera, is naturally present in the Environment and autochthonous to coastal and estuarine ecosystems. V. Cholerae is associated with copepods for its survival and multiplication in the natural Environment. Changes in the density of its reservoir may result in modification of the bacterial population size in the Environment. In this context, climate and/or Environmental changes will influence the emergence of Cholera in human populations. Several human pathogens are naturally occurring in the aquatic Environment and can pose a threat to public health, including V. Cholerae. We present results of a project, the goal of which was to improve the understanding of Environmental factors associated with occurrence and distribution of the causative agent of Cholera in time and space. The system that was developed provides real-time as well as short-term to seasonal forecasts of the likelihood of occurrence of V. Cholerae in the Chesapeake Bay. The system, and potential future improved versions of it, may be useful to public health officials concerned with Environmental factors influencing human health.

Appl Environ Microbiol. 2009 Nov; 75(22): 6981-5
Huang J, Zhu Y, Wen H, Zhang J, Huang S, Niu J, Li Q

Vibrio Cholerae is a natural inhabitant of the aquatic Environment. However, its toxigenic strains can cause potentially life-threatening diarrhea. A quadruplex real-time PCR assay targeting four genes, the Cholera toxin gene (ctxA), the hemolysin gene (hlyA), O1-specific rfb, and O139-specific rfb, was developed for detection and differentiation of O1, O139, and non-O1, non-O139 strains and for prediction of their toxigenic potential. The specificity of the assay was 100% when tested against 70 strains of V. Cholerae and 31 strains of non-V. Cholerae organisms. The analytical sensitivity for detection of toxigenic V. Cholerae O1 and O139 was 2 CFU per reaction with cells from pure culture. When the assay was tested with inoculated water from bullfrog feeding ponds, 10 CFU/ml could reliably be detected after culture for 3 h. The assay was more sensitive than the immunochromatographic assay and culture method when tested against 89 bullfrog samples and 68 water samples from bullfrog feeding ponds. The applicability of this assay was confirmed in a case study involving 15 bullfrog samples, from which two mixtures of nontoxigenic O1 and toxigenic non-O1/non-O139 strains were detected and differentiated. These data indicate that the quadruplex real-time PCR assay can both rapidly and accurately detect/identify V. Cholerae and reliably predict the toxigenic potential of strains detected.

J Infect Dev Ctries. 2009; 3(6): 408-11
Talavera A, Pérez EM

BACKGROUND: Cholera remains a global threat and is one of the key indicators of social development. While the disease no longer poses a menace to countries with minimum standards of hygiene, it remains a serious challenge to countries where access to safe drinking water and adequate sanitation cannot be guaranteed. The objective of this work was to analyse the results obtained when contrasting the reports of the World Health Organization (WHO) about Cholera disease with those of the World Bank List of economies (countries). METHODOLOGY: Data were obtained from reports of two international organizations, the report on Cholera disease incidence of the World Health Organization and the World Bank's classification of countries attending to their income. RESULTS: We determined that low-income countries are more affected by Cholera disease than countries with middle or high income. This difference was reflected in the percent of countries, the total number of reported cases, the number of cases per 100,000 habitants, as well as in the reported mortality. These results support the phrase "Cholera disease is a disease of poverty." CONCLUSIONS: We consider that economic development is an important factor in the morbidity and mortality of Cholera, together with Environment, climate, culture, medical management, political intention, and the intrinsic factors of the system.

Am J Public Health. 2009 Nov; 99(11): 1982-7
Sasaki S, Suzuki H, Fujino Y, Kimura Y, Cheelo M

OBJECTIVES: We investigated the association between precipitation patterns and Cholera outbreaks and the preventative roles of drainage networks against outbreaks in Lusaka, Zambia. METHODS: We collected data on 6542 registered Cholera patients in the 2003-2004 outbreak season and on 6045 Cholera patients in the 2005-2006 season. Correlations between monthly Cholera incidences and amount of precipitation were examined. The distribution pattern of the disease was analyzed by a kriging spatial analysis method. We analyzed Cholera case distribution and spatiotemporal cluster by using 2590 Cholera cases traced with a global positioning system in the 2005-2006 season. The association between drainage networks and Cholera cases was analyzed with regression analysis. RESULTS: Increased precipitation was associated with the occurrence of Cholera outbreaks, and insufficient drainage networks were statistically associated with Cholera incidences. CONCLUSIONS: Insufficient coverage of drainage networks elevated the risk of Cholera outbreaks. Integrated development is required to upgrade high-risk areas with sufficient infrastructure for a long-term Cholera prevention strategy.

Nat Rev Microbiol. 2009 Oct; 7(10): 693-702
Nelson EJ, Harris JB, Morris JG, Calderwood SB, Camilli A

Zimbabwe offers the most recent example of the tragedy that befalls a country and its people when Cholera strikes. The 2008-2009 outbreak rapidly spread across every province and brought rates of mortality similar to those witnessed as a consequence of Cholera infections a hundred years ago. In this Review we highlight the advances that will help to unravel how interactions between the host, the bacterial pathogen and the lytic bacteriophage might propel and quench Cholera outbreaks in endemic settings and in emergent epidemic regions such as Zimbabwe.

J Appl Microbiol. 2009 Jul 29;
Choi S, Dunams D, Jiang SC

Abstract Aims: Vibrio Cholerae is an important bacterial pathogen that causes global Cholera epidemic. Although they are commonly found in coastal waters around the world, most Environmental isolates do not contain Cholera toxin genes. This study investigates vibriophages in southern California coastal waters and their ability to transfer Cholera toxin genes. Methods and Results: Lytic phages infecting V. Cholerae were isolated from Newport Bay, California, between May and November, while none was found in winter. Some of the phage isolates can infect multiple Environmental V. Cholerae strains and El Tor strains. All phages contained double-stranded DNA. Transduction experiments using kanamycin-resistant gene marked CTXPhi demonstrated that some Environmental vibriophages can transfer CTXPhi genes from O1 El Tor strain to Environmental non-O1/O139 V. Cholerae via generalized transduction. Conclusions: Vibriophages are important components of the natural aquatic ecosystem. They play an important role in influencing the dynamics and evolution of V. Cholerae in the Environment. Significance and Impact of the Study: This study demonstrates the significance of vibriophages in the coastal Environment and transduction as one of the mechanisms of pathogenicity evolution among Environmental V. Cholerae.

J Bacteriol. 2009 Nov; 191(21): 6632-42
Pratt JT, McDonough E, Camilli A

Signaling through the second messenger cyclic di-GMP (c-di-GMP) is central to the life cycle of Vibrio Cholerae. However, relatively little is known about the signaling mechanism, including the specific external stimuli that regulate c-di-GMP concentration. Here, we show that the phosphate responsive regulator PhoB regulates an operon, acgAB, which encodes c-di-GMP metabolic enzymes. We show that induction of acgAB by PhoB positively regulates V. Cholerae motility in vitro and that PhoB regulates expression of acgAB at late stages during V. Cholerae infection in the infant mouse small intestine. These data support a model whereby PhoB becomes activated at a late stage of infection in preparation for dissemination of V. Cholerae to the aquatic Environment and suggest that the concentration of exogenous phosphate may become limited at late stages of infection.

Proc Natl Acad Sci U S A. 2009 Sep 8; 106(36): 15442-7
Chun J, Grim CJ, Hasan NA, Lee JH, Choi SY, Haley BJ, Taviani E, Jeon YS, Kim DW, Lee JH, Brettin TS, Bruce DC, Challacombe JF, Detter JC, Han CS, Munk AC, Chertkov O, Meincke L, Saunders E, Walters RA, Huq A, Nair GB, Colwell RR

Vibrio Cholerae, the causative agent of Cholera, is a bacterium autochthonous to the aquatic Environment, and a serious public health threat. V. Cholerae serogroup O1 is responsible for the previous two Cholera pandemics, in which classical and El Tor biotypes were dominant in the sixth and the current seventh pandemics, respectively. Cholera researchers continually face newly emerging and reemerging pathogenic clones carrying diverse combinations of phenotypic and genotypic properties, which significantly hampered control of the disease. To elucidate evolutionary mechanisms governing genetic diversity of pandemic V. Cholerae, we compared the genome sequences of 23 V. Cholerae strains isolated from a variety of sources over the past 98 years. The genome-based phylogeny revealed 12 distinct V. Cholerae lineages, of which one comprises both O1 classical and El Tor biotypes. All seventh pandemic clones share nearly identical gene content. Using analogy to influenza virology, we define the transition from sixth to seventh pandemic strains as a "shift" between pathogenic clones belonging to the same O1 serogroup, but from significantly different phyletic lineages. In contrast, transition among clones during the present pandemic period is characterized as a "drift" between clones, differentiated mainly by varying composition of laterally transferred genomic islands, resulting in emergence of variants, exemplified by V. Cholerae O139 and V. Cholerae O1 El Tor hybrid clones. Based on the comparative genomics it is concluded that V. Cholerae undergoes extensive genetic recombination via lateral gene transfer, and, therefore, genome assortment, not serogroup, should be used to define pathogenic V. Cholerae clones.

FEMS Microbiol Lett. 2009 Oct; 299(2): 166-74
McGinnis MW, Parker ZM, Walter NE, Rutkovsky AC, Cartaya-Marin C, Karatan E

Vibrio Cholerae, the causative agent of the devastating diarrheal disease Cholera, can form biofilms on diverse biotic and abiotic surfaces. Biofilm formation is important for the survival of this organism both in its natural Environment and in the human host. Development of V. Cholerae biofilms are regulated by complex regulatory networks that respond to Environmental signals. One of these signals, norspermidine, is a polyamine that enhances biofilm formation via the NspS/MbaA signaling system. In this work, we have investigated the role of the polyamine spermidine in regulating biofilm formation in V. Cholerae. We show that spermidine import requires PotD1, an ortholog of the periplasmic substrate-binding protein of the spermidine transport system in Escherichia coli. We also show that deletion of the potD1 gene results in a significant increase in biofilm formation. We hypothesize that spermidine imported into the cell hinders biofilm formation. Exogenous spermidine further reduces biofilm formation in a PotD1-independent, but NspS/MbaA-dependent, manner. Our results suggest that polyamines affect biofilm formation in V. Cholerae via multiple pathways involving both transport and signaling networks.

PLoS Med. 2009 Jun 30; 6(6): e1000102

Appl Environ Microbiol. 2009 Sep; 75(17): 5439-44
Grim CJ, Zo YG, Hasan NA, Ali A, Chowdhury WB, Islam A, Rashid MH, Alam M, Morris JG, Huq A, Colwell RR

A species-specific RNA colony blot hybridization protocol was developed for enumeration of culturable Vibrio Cholerae and Vibrio mimicus bacteria in Environmental water samples. Bacterial colonies on selective or nonselective plates were lysed by sodium dodecyl sulfate, and the lysates were immobilized on nylon membranes. A fluorescently labeled oligonucleotide probe targeting a phylogenetic signature sequence of 16S rRNA of V. Cholerae and V. mimicus was hybridized to rRNA molecules immobilized on the nylon colony lift blots. The protocol produced strong positive signals for all colonies of the 15 diverse V. Cholerae-V. mimicus strains tested, indicating 100% sensitivity of the probe for the targeted species. For visible colonies of 10 nontarget species, the specificity of the probe was calculated to be 90% because of a weak positive signal produced by Grimontia (Vibrio) hollisae, a marine bacterium. When both the sensitivity and specificity of the assay were evaluated using lake water samples amended with a bioluminescent V. Cholerae strain, no false-negative or false-positive results were found, indicating 100% sensitivity and specificity for culturable bacterial populations in freshwater samples when G. hollisae was not present. When the protocol was applied to laboratory microcosms containing V. Cholerae attached to live copepods, copepods were found to carry approximately 10,000 to 50,000 CFU of V. Cholerae per copepod. The protocol was also used to analyze pond water samples collected in an area of Cholera endemicity in Bangladesh over a 9-month period. Water samples collected from six ponds demonstrated a peak in abundance of total culturable V. Cholerae bacteria 1 to 2 months prior to observed increases in pathogenic V. Cholerae and in clinical cases recorded by the area health clinic. The method provides a highly specific and sensitive tool for monitoring the dynamics of V. Cholerae in the Environment. The RNA blot hybridization protocol can also be applied to detection of other gram-negative bacteria for taxon-specific enumeration.

J Travel Med. 2009 May-Jun; 16(3): 179-85
Chongsuvivatwong V, Chariyalertsak S, McNeil E, Aiyarak S, Hutamai S, Dupont HL, Jiang ZD, Kalambaheti T, Tonyong W, Thitiphuree S, Steffen R

BACKGROUND: Current data on risk of travelers' diarrhea (TD) among visitors to Thailand largely comes from US military personnel, Peace Corps volunteers, or expatriates. We performed a 14-month systematic study of the incidence rate and characteristics of TD and a smaller study of etiology of the disease among visitors to Phuket and Chiang Mai. METHODS: One randomly selected day each week from August 2005 until October 2006, data were collected from foreign tourists departing from airports serving Phuket and Chiang Mai. A separate subgroup of subjects with TD acquired in Phuket were invited to submit a stool sample for enteropathogens. RESULTS: Based on 22,401 completed questionnaires, the attack rate for TD was highest among residents from Australia or New Zealand (16%), while those from the United States and Europe had attack rates of 7% to 8%. Independent risk factors for the development of TD were eating outside the hotel and eating meat. In contrast, a history of drinking tap water and consuming ice cream were protective. In 56 subjects studied for etiology, Aeromonas spp were found in 8 subjects (14%), enterotoxigenic Escherichia coli (ETEC) or Vibrio spp each was found in 7 (13%) with O1 V. Cholera (Cholera) seen in one, mixed pathogens were found in 3 (5%), with no pathogen being detected in 33 (59%). CONCLUSIONS: Phuket and Chiang Mai should not be considered high-risk destinations for development of TD among US and European travelers to Thailand. In the study, Aeromonas, ETEC, and Vibrio spp were the most frequent enteropathogens identified.

J Bacteriol. 2009 Aug; 191(16): 5116-22
Lainhart W, Stolfa G, Koudelka GB

Bacterially derived exotoxins kill eukaryotic cells by inactivating factors and/or pathways that are universally conserved among eukaryotic organisms. The genes that encode these exotoxins are commonly found in bacterial viruses (bacteriophages). In the context of mammals, these toxins cause diseases ranging from Cholera to diphtheria to enterohemorrhagic diarrhea. Phage-carried exotoxin genes are widespread in the Environment and are found with unexpectedly high frequency in regions lacking the presumed mammalian "targets," suggesting that mammals are not the primary targets of these exotoxins. We suggest that such exotoxins may have evolved for the purpose of bacterial antipredator defense. We show here that Tetrahymena thermophila, a bacterivorous predator, is killed when cocultured with bacteria bearing a Shiga toxin (Stx)-encoding temperate bacteriophage. In cocultures with Tetrahymena, the Stx-encoding bacteria display a growth advantage over those that do not produce Stx. Tetrahymena is also killed by purified Stx. Disruption of the gene encoding the StxB subunit or addition of an excess of the nontoxic StxB subunit substantially reduced Stx holotoxin toxicity, suggesting that this subunit mediates intake and/or trafficking of Stx by Tetrahymena. Bacterially mediated Tetrahymena killing was blocked by mutations that prevented the bacterial SOS response (recA mutations) or by enzymes that breakdown H(2)O(2) (catalase), suggesting that the production of H(2)O(2) by Tetrahymena signals its presence to the bacteria, leading to bacteriophage induction and production of Stx.