Attachment of Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters

Vibrio cholerae serogroup O1, the causative agent of cholera, is capable of surviving in aquatic environments for extended periods and is considered an autochthonous species in estuarine and brackish waters. These environments contain numerous elements that may affect its ecology. The studies reported here examined physical interactions between V. cholerae O1 and natural plankton populations of a geographical region in Bangladesh where cholera is an endemic disease. Results showed that four of five clinical V. cholerae O1 strains and endogenous bacterial flora were attached preferentially to zooplankton molts (exuviae) rather than to whole specimens. One strain attached in approximately equal numbers to both exuviae and whole specimens. V. cholerae O1 also attached to several phytoplankton species. The results show that V. cholerae O1 can bind to diverse plankton species collected from an area where cholera is an endemic disease, with potentially significant effects on its ecology.     

Attachment of Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters.

Vibrio cholerae serogroup O1, the causative agent of cholera, is capable of surviving in aquatic environments for extended periods and is considered an autochthonous species in estuarine and brackish waters. These environments contain numerous elements that may affect its ecology. The studies reported here examined physical interactions between V. cholerae O1 and natural plankton populations of a geographical region in Bangladesh where cholera is an endemic disease. Results showed that four of five clinical V. cholerae O1 strains and endogenous bacterial flora were attached preferentially to zooplankton molts (exuviae) rather than to whole specimens. One strain attached in approximately equal numbers to both exuviae and whole specimens. V. cholerae O1 also attached to several phytoplankton species. The results show that V. cholerae O1 can bind to diverse plankton species collected from an area where cholera is an endemic disease, with potentially significant effects on its ecology.     

Genetics of stress adaptation and virulence in toxigenic Vibrio cholerae

Vibrio cholerae, a Gram-negative bacterium belonging to the gamma-subdivision of the family Proteobacteriaceae is the etiologic agent of cholera, a devastating diarrheal disease which occurs frequently as epidemics. Any bacterial species encountering a broad spectrum of environments during the course of its life cycle is likely to develop complex regulatory systems and stress adaptation mechanisms to best survive in each environment encountered. Toxigenic V. cholerae, which has evolved from environmental nonpathogenic V. cholerae by acquisition of virulence genes, represents a paradigm for this process in that this organism naturally exists in an aquatic environment but infects human beings and cause cholera. The V. cholerae genome, which is comprised of two independent circular mega-replicons, carries the genetic determinants for the bacterium to survive both in an aquatic environment as well as in the human intestinal environment. Pathogenesis of V. cholerae involves coordinated expression of different sets of virulence associated genes, and the synergistic action of their gene products. Although the acquisition of major virulence genes and association between V. cholerae and its human host appears to be recent, and reflects a simple pathogenic strategy, the establishment of a productive infection involves the expression of many more genes that are crucial for survival and adaptation of the bacterium in the host, as well as for its onward transmission and epidemic spread. While a few of the virulence gene clusters involved directly with cholera pathogenesis have been characterized, the potential exists for identification of yet new genes which may influence the stress adaptation, pathogenesis, and epidemiological characteristics of V. cholerae. Coevolution of bacteria and mobile genetic elements (plasmids, transposons, pathogenicity islands, and phages) can determine environmental survival and pathogenic interactions between bacteria and their hosts. Besides horizontal gene transfer mediated by genetic elements and phages, the evolution of pathogenic V. cholerae involves a combination of selection mechanisms both in the host and in the environment. The occurrence of periodic epidemics of cholera in endemic areas appear to enhance this process.     

Gene Fitness Landscapes of Vibrio cholerae at Important Stages of Its Life Cycle

Vibrio cholerae has evolved to adeptly transition between the human small intestine and aquatic environments, leading to water-borne spread and transmission of the lethal diarrheal disease cholera. Using a host model that mimics the pathology of human cholera, we applied high density transposon mutagenesis combined with massively parallel sequencing (Tn-seq) to determine the fitness contribution of >90% of all non-essential genes of V. cholerae both during host infection and dissemination. Targeted mutagenesis and validation of 35 genes confirmed our results for the selective conditions with a total false positive rate of 4%. We identified 165 genes never before implicated for roles in dissemination that reside within pathways controlling many metabolic, catabolic and protective processes, from which a central role for glycogen metabolism was revealed. We additionally identified 76 new pathogenicity factors and 414 putatively essential genes for V. cholerae growth. Our results provide a comprehensive framework for understanding the biology of V. cholerae as it colonizes the small intestine, elicits profuse secretory diarrhea, and disseminates into the aquatic environment.     

Quorum sensing enhances the stress response in Vibrio cholerae

Vibrio cholerae lives in aquatic environments and causes cholera. Here, we show that quorum sensing enhances V. cholerae viability under certain stress conditions by upregulating the expression of RpoS, and this regulation acts through HapR, suggesting that a quorum-sensing-enhanced stress response plays a role in V. cholerae environmental survival.     

Toxigenic Vibrio cholerae in the aquatic environment of Mathbaria, Bangladesh

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.     

Method of DNA extraction and application of multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae O1 and O139 from aquatic ecosystems

Vibrio cholerae is a free-living bacterium found in water and in association with plankton. V. cholerae non-O1/non-O139 strains are frequently isolated from aquatic ecosystems worldwide. Less frequently isolated are V. cholerae O1 and V. cholerae O139, the aetiological agents of cholera. These strains have two main virulence-associated factors, cholera toxin (CT) and toxin co-regulated pilus (TCP). By extracting total DNA from aquatic samples, the presence of pathogenic strains can be determined quickly and used to improve a microbiological risk assessment for cholera in coastal areas. Some methods suggested for DNA extraction from water samples are not applicable to all water types. We describe here a method for DNA extraction from coastal water and a multiplex polymerase chain reaction (PCR) for O1 and O139 serogroups. DNA extraction was successfully accomplished from 117 sea water samples collected from coastal areas of Perú, Brazil and the USA. DNA concentration in all samples varied from 20 ng to 480 micro g micro l-1. The sensitivity of the DNA extraction method was 100 V. cholerae cells in 250 ml of water. The specificity of multiplex O1/O139 PCR was investigated by analysing 120 strains of V. cholerae, Vibrio and other Bacteria species. All V. cholerae O1 and O139 tested were positive. For cholera surveillance of aquatic environments and ballast water, total DNA extraction, followed by V. cholerae PCR, and O1/O139 serogroup and tcpA/ctxA genes by multiplex PCR offers an efficient system, permitting risk analysis for cholera in coastal areas.     

Vibrio cholerae and cholera: out of the water and into the host

The facultative human pathogen Vibrio cholerae can be isolated from estuarine and aquatic environments. V. cholerae is well recognized and extensively studied as the causative agent of the human intestinal disease cholera. In former centuries cholera was a permanent threat even to the highly developed populations of Europe, North America, and the northern part of Asia. Today, cholera still remains a burden mainly for underdeveloped countries, which cannot afford to establish or to maintain necessary hygienic and medical facilities. Especially in these environments, cholera is responsible for significant mortality and economic damage. During the last three decades, intensive research has been undertaken to unravel the virulence properties and to study the epidemiology of this significant human pathogen. More recently, researchers have been elucidating the environmental lifestyle of V. cholerae. This review provides an overview of the current knowledge of both the host- and environment-specific physiological attributes of V. cholerae.     

Colonization of the gut of the blue crab (Callinectes sapidus) by Vibrio cholerae

Attachment of Vibrio cholerae to the mucosal surface of the intestine is considered to be an important virulence characteristic. Vibrio cholerae, an autochthonous member of brackish water and estuarine bacterial communities, also attaches to crustacea, a significant factor in multiplication and survival of V. cholerae in nature. The ability of V. cholerae to attach to the gut wall of the blue crab (Callinectes sapidus) was examined, and attachment was observed only in the hindgut and not the midgut of crabs, confirming a requirement for chitin in the attachment of V. cholerae to invertebrate and zooplankton surfaces. The new finding of attachment of V. cholerae to the hindgut of crabs may be correlated with the epidemiology and transmission of cholera in the aquatic environment. The crab model may also prove useful in elucidating the mechanism(s) of ion transport in crustacea.     

Colonization of the gut of the blue crab (Callinectes sapidus) by Vibrio cholerae.

Attachment of Vibrio cholerae to the mucosal surface of the intestine is considered to be an important virulence characteristic. Vibrio cholerae, an autochthonous member of brackish water and estuarine bacterial communities, also attaches to crustacea, a significant factor in multiplication and survival of V. cholerae in nature. The ability of V. cholerae to attach to the gut wall of the blue crab (Callinectes sapidus) was examined, and attachment was observed only in the hindgut and not the midgut of crabs, confirming a requirement for chitin in the attachment of V. cholerae to invertebrate and zooplankton surfaces. The new finding of attachment of V. cholerae to the hindgut of crabs may be correlated with the epidemiology and transmission of cholera in the aquatic environment. The crab model may also prove useful in elucidating the mechanism(s) of ion transport in crustacea.     

Adult non-biting midges: possible windborne carriers of Vibrio cholerae non-O1 non-O139

Vibrio cholerae is a waterborne bacterium native to the aquatic environment. There are over 200 known serogroups yet only two cause cholera pandemics in humans. Direct contact of human sewage with drinking water, sea-born currents and marine transportation, represent modes of dissemination of the bacteria and thus the disease. The simultaneous cholera outbreaks that occur sometimes in distant localities within continental landmasses are puzzling. Here we present evidence that flying, non-biting midges (Diptera; Chironomidae), collected in the air, carry viable non-O1 non-O139 serogroups of V. cholerae. The association of V. cholerae with chironomid egg masses, which serve as a V. cholerae reservoir, was further confirmed. In simulated field experiments, we recorded the transfer of environmental V. cholerae by adult midges from the aquatic environment into bacteria-free water-pools. In laboratory experiments, flying adult midges that emerged from V. cholerae (O1 or O139) contaminated water transferred the green fluorescent protein (GFP)-tagged pathogenic bacteria from one laboratory flasks to another. Our findings show that aerial transfer by flying chironomids may play a role in the dissemination of V. cholerae in nature.     

Vibrio cholerae O1 strains are facultative intracellular bacteria, able to survive and multiply symbiotically inside the aquatic free-living amoeba Acanthamoeba castellanii

Vibrio cholerae species are extracellular, waterborne, gram-negative bacteria that are overwhelmed by predators in aquatic environments. The unencapsulated serogroup V. cholerae O1 and encapsulated V. cholerae O139 cause epidemic and pandemic outbreaks of cholera. It has recently been shown that the aquatic and free-living amoeba Acanthamoeba castellanii is not a predator to V. cholerae O139; rather, V. cholerae O139 has shown an intracellular compatibility with this host. The aim of this study was to examine the ability of V. cholerae O1 classical and El Tor strains to grow and survive in A. castellanii. The interaction between A. castellanii and V. cholerae O1 strains was studied by means of amoeba cell counts and viable counts of the bacteria in the absence or presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Confocal microscopy and electron microscopy were used to determine the intracellular localization of V. cholerae in A. castellanii. The results showed that V. cholerae O1 classical and El Tor strains grew and survived intracellularly in the cytoplasm of trophozoites, and that the bacteria were also found in the cysts of A. castellanii. The interaction showed a facultative intracellular behaviour of V. cholerae O1 classical and El Tor strains and a possible role of A. castellanii as an environmental host of V. cholerae species.     

A glimpse into the expanded genome content of Vibrio cholerae through identification of genes present in environmental strains

Vibrio cholerae has multiple survival strategies which are reflected both in its broad distribution in many aquatic environments and its high genotypic diversity. To obtain additional information regarding the content of the V. cholerae genome, suppression subtractive hybridization (SSH) was used to prepare libraries of DNA sequences from two southern California coastal isolates which are divergent or absent in the clinical strain V. cholerae O1 El Tor N16961. More than 1,400 subtracted clones were sequenced. This revealed the presence of novel sequences encoding functions related to cell surface structures, transport, metabolism, signal transduction, luminescence, mobile elements, stress resistance, and virulence. Flanking sequence information was determined for loci of interest, and the distribution of these sequences was assessed for a collection of V. cholerae strains obtained from southern California and Mexican environments. This led to the surprising observation that sequences related to the toxin genes toxA, cnf1, and exoY are widespread and more common in these strains than those of the cholera toxin genes which are a hallmark of the pandemic strains of V. cholerae. Gene transfer among these strains could be facilitated by a 4.9-kbp plasmid discovered in one isolate, which possesses similarity to plasmids from other environmental vibrios. By investigating some of the nucleotide sequence basis for V. cholerae genotypic diversity, DNA fragments have been uncovered which could promote survival in coastal environments. Furthermore, a set of genes has been described which could be involved in as yet undiscovered interactions between V. cholerae and eukaryotic organisms.     

Dependent population dynamics between chironomids (nonbiting midges) and Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, is a natural inhabitant of the aquatic ecosystem. Chironomid (nonbiting midges) egg masses were recently found to harbour V. cholerae non-O1 and non-O139, providing a natural reservoir for the cholera bacterium. Chironomid populations and the presence of V. cholerae in chironomid egg masses were monitored. All V. cholerae isolates were able to degrade chironomid egg masses. The following virulence associated genes were detected in the bacterial isolates: hapA (100%), toxR (100%), hlyA (72%) and ompU (28%). The chironomid populations and the V. cholerae in their egg masses followed the phenological succession and interaction of host-pathogen population dynamics. A peak in the chironomid population was followed by a peak in the V. cholerae population. If such a connection is further substantiated for the pathogenic serogroups of V. cholerae in endemic areas of the disease, it may lead to a better understanding of the role of chironomids as a host for the cholera bacterium.     

The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139

Throughout most of history, epidemic and pandemic cholera was caused by Vibrio cholerae of the serogroup O1. In 1992, however, a V. cholerae strain of the serogroup O139 emerged as a new agent of epidemic cholera. Interestingly, V. cholerae O139 forms biofilms on abiotic surfaces more rapidly than V. cholerae O1 biotype El Tor, perhaps because regulation of exopolysaccharide synthesis in V. cholerae O139 differs from that in O1 El Tor. Here, we show that all flagellar mutants of V. cholerae O139 have a rugose colony morphology that is dependent on the vps genes. This suggests that the absence of the flagellar structure constitutes a signal to increase exopolysaccharide synthesis. Furthermore, although exopolysaccharide production is required for the development of a three-dimensional biofilm, inappropriate exopolysaccharide production leads to inefficient colonization of the infant mouse intestinal epithelium by flagellar mutants. Thus, precise regulation of exopolysaccharide synthesis is an important factor in the survival of V. cholerae O139 in both aquatic environments and the mammalian intestine.     

Effect of mild acid pH on the functioning of bacterial membranes in Vibrio cholerae

In this paper, we initiated the first two-dimensional electrophoresis map of Vibrio cholerae, the aetiological agent of cholera disease. In this pathogen the efficient adaptation to detrimental conditions plays an important role in its survival in both the aquatic reservoir and human intestine. By proteome analysis we investigated the effect of mild acid treatment on the physiology of V. cholerae. More than 50 proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database searching. Amongst them, pH regulated proteins belong to various functional classes such as intermediary metabolism and bacterial envelope. Several proteins whose accumulation level was decreased in response to acidic pH are known to be involved in the organization and the functioning of membranes, including lipopolysaccharide. Consistent with this, we observed an increased susceptibility to hydrophobic drugs, a loss of motility and a reduction in the ability to form a biofilm in cells grown at pH 6. Our results suggest that V. cholerae is able to sense a moderate decrease in pH and to modify accordingly its structure and physiology.