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REVIEWS REVIEWS REVIEWS 80 Environmental microbe and human pathogen: the ecology and microbiology of Vibrio cholerae Kathryn L Cottingham1, Deborah A Chiavelli1,2, and Ronald K Taylor2 Vibrio cholerae, the bacterium that causes cholera, is a fascinating microorganism that occupies two distinct habitats: aquatic environments and human intestines. Although its activity in human hosts has received a great deal of attention because of its effect on human health, the microbe’s behavior in natural ecosystems has received much less consideration. In this paper we briefly review the microbiology and ecology of V. cholerae, before highlighting four important areas for further research. First, V. cholerae acts as a typical heterotrophic bacterium while in aquatic environments, mineralizing organic matter for reuse within the food web. In addition, three key processes affect the microorganism’s population dynamics and therefore its role in ecosystem processes: a viable-but-non-culturable (VBNC) state, attachment to environmental substrates, and bacterivory. Several factors link V. cholerae in its human and aquatic habitats, especially human activities that influence its growth conditions while outside the body. These activities create feedbacks between humans and the environment that are at present not well understood, but which are likely to alter the persistence and spread of the resulting disease. Front Ecol Environ 2003, 1(2), 80–86 C holera is a more persistent and global health problem now than it was a few decades ago (Colwell 1996). This infectious disease is endemic in areas such as India and Bangladesh, and is emerging or re-emerging elsewhere, particularly in developing nations with inadequate sanitation (Faruque et al. 1998). Cholera outbreaks often accompany natural disasters and destabilization due to wars, even in non-endemic areas (Faruque et al. 1998), while climate change and increased globalization will put additional areas at risk for future outbreaks (Ruiz et al. 2000; Harvell et al. 2002). Taken together, these observations suggest that an improved understanding of the factors controlling cholera outbreaks would be beneficial to human health worldwide. Cholera is one of many human diseases with a substantial environmental component. The disease begins with In a nutshell: • Vibrio cholerae, the bacterium that causes cholera, exists in both human intestines and aquatic ecosystems • Human activity and the resulting ecological changes directly affect the bacterium’s persistence and spread in aquatic environments • Research described here on the behaviour of V. cholerae outside the body will help the development of strategies to combat this worldwide health problem 1 Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 ([email protected]); 2Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH 03755 www.frontiersinecology.org the ingestion of the causal bacterium Vibrio cholerae (Figure 1) via contaminated water or food. The microbe makes its way to the small intestine, where it attaches and begins to produce cholera toxin (CT). Sufficient quantities of CT cause severe diarrhea and the shedding of many bacteria into the environment – up to 1013 per infected individual per day (Mintz et al. 1994), facilitating disease spread. Although scientists had long thought that the bacterium survived outside mammalian intestines only for brief periods, research in the past few decades shows that V. cholerae are an abundant, naturally occurring component of freshwater, estuarine, and marine ecosystems worldwide (Islam et al. 1997). As such, the bacterium inhabits two very distinct habitats – human hosts and aquatic ecosystems – and is tremendously flexible in its activities in order to survive in each habitat. We need a much better understanding of the relationship between the dynamics of V. cholerae in humans and aquatic environments. Microbiology and pathogenesis Microbiological studies of V. cholerae began with its identification as the causal agent of cholera in 1884. Since then, we have learned a great deal about its biochemistry, genetics, and behavior in the human host (Faruque et al. 1998; Reidl and Klose 2002). Although there are more than 200 known serogroups of V. cholerae, only two, O1 and O139, are known to cause epidemic disease (Faruque et al. 1998; Reidl and Klose 2002). Strains from these closely related groups have very similar antigens on their © The Ecological Society of America Ecology and microbiology of Vibrio cholerae outermost layers, suggesting that surface properties are a key component in the ability to cause disease. Historically, cholera outbreaks were caused by V. cholerae O1, but O139 became the major cause of epidemic cholera in some regions during the 1990s (Faruque et al. 1998). The O1 serogroup is further divided into two biotypes: classical and El Tor. Classical strains predominated in clinical samples until El Tor was identified in 1961. Since then, El Tor strains have gradually displaced classical strains throughout much of the world (Colwell 1996; Faruque et al. 1998). Whether these shifts between the Figure 1. Vibrio cholerae. O1 classical, O1 El Tor, and O139 strains are related to environmental fitness, altered patho- level and attachment could affect their relative vulneragenicity, increased transmissibility, or other factors is not bility to grazers. yet known. Since most environmental strains of V. cholerae do not Viable but not culturable cause epidemic disease in humans, an interesting question is how epidemic strains arise. Horizontal gene trans- As described by microbiologists, the viable-but-non-culmission through bacteriophages (viruses that infect bac- turable (VBNC) state is a response to rapid transitions in teria) and other vectors plays a major role in creating environmental conditions, including temperature and pathogenic V. cholerae by conveying genes involved in osmolarity (McDougald et al. 1998). When a bacterial the colonization of humans and the production of cholera cell enters a VBNC state, it loses its flagellum and toxin (Islam et al. 1997). The most critical genes are clus- changes to a smaller, spherical form (McDougald et al. tered together in two regions of the bacterial genome – a 1998; Huq et al. 2000). VBNC cells do not reproduce on pathogenicity island and a lysogenic bacteriophage standard microbiological media, and have decreased (Faruque et al. 1998). The vibrio pathogenicity island nucleic acid content and reduced respiration and meta(VPI) encodes a number of genes, including those bolic rates (McDougald et al. 1998). The VBNC pherequired to express the toxin coregulated pilus (TCP), nomenon has been described for a number of pathogenic which is essential for intestinal colonization. The lyso- bacteria, including V. cholerae, V. vulnificus, Escherichia genic bacteriophage CTX-, which harbors the genes for coli, and Campylobacter jejuni (McDougald et al. 1998). cholera toxin, uses the TCP to enter only those strains of Laboratory experiments indicate that V. cholerae can exist V. cholerae capable of colonizing humans. It then lysoge- in the VBNC state for long periods, presumably allowing nizes invaded cells by integrating its entire genome into them to survive unfavorable environmental conditions that of the host bacterium. Thus, a partnership between (Huq et al. 2000). The VBNC state is of critical importance to public CTX- and V. cholerae expressing TCP is required to health microbiologists, who need to be able to reliably cause a cholera epidemic. detect V. cholerae and other pathogenic bacteria in the environment, particularly since VBNC cells can cause dis Ecology of V. cholerae ease (Colwell et al. 1996). Consequently, both antibodyIn aquatic environments, V. cholerae probably acts as a and nucleic-acid-based approaches have been developed typical heterotrophic bacterium. These microorganisms, to detect pathogens in natural waters, regardless of their which cannot synthesize their own food, are responsible culturability (Theron and Cloete 2002). These methods for much of the mineralization of organic matter, and have identified V. cholerae in the VBNC state in the can be important components of aquatic food webs and majority of water and plankton samples tested (Huq et al. nutrient cycles (Cole 1999; Cotner and Biddanda 1990; Huq et al. 2000), with a higher incidence of cultur2002). Like other organisms, bacteria react to environ- able V. cholerae during outbreak periods in both mental conditions by increasing or decreasing their Bangladesh and Peru (Huq et al. unpublished). metabolic rates, and can enter dormant and/or non-culThe VBNC state is controversial, however, because turable states. They can also change the quality of their researchers do not yet agree on whether it is reversible. microhabitat by switching between free-living (unat- Can cells be resuscitated from a VBNC state after envitached) and surface-attached forms and by aggregating ronmental conditions improve, or do the few remaining into biofilms while attached. Importantly, both activity culturable cells simply regrow (McDougald et al. 1998; © The Ecological Society of America www.frontiersinecology.org 81 Courtesy of Louisa Howard, Thomas Kirn, and Niranjan Bose KL Cottingham et al. Ecology and microbiology of Vibrio cholerae 82 Kell et al. 1998)? Findings to date are equivocal, but resolving this controversy is important to how we interpret field measurements of bacterial abundance, and how we relate dynamics in the aquatic environment to human disease outbreaks. For example, if VBNC V. cholerae can be resuscitated (as demonstrated by Wai et al. 1996), then the factors that trigger the transition back to an active, culturable state are likely to be very important to both cholera epidemiology and ecosystem processes. Attachment Like other heterotrophic bacteria, V. cholerae has been found attached to a wide variety of aquatic organisms, especially plankton (Table 1; Figure 2). Although attachment to a living host increases V. cholerae’s survival and growth in the laboratory (Huq et al. 1984a; Islam et al. 1999), its effect on natural microbial populations or ecosystem processes is not known. The surfaces of planktonic organisms are resource-rich microhabitats, since V. cholerae can metabolize chitin, the surface material of crustacean zooplankton, and mucilage from the outer surfaces of some phytoplankton (Islam et al. 1994). Living plankton also supply nutrients to bacteria through excretion or exudates (Islam et al. 1994), while host movement may prevent the depletion of nutrients in the surrounding water (Threlkeld et al. 1993). Furthermore, various plankton offer very different substrate qualities for attached V. cholerae (Tamplin et al. 1990). For example, copepods offer a more stable attachment substrate than cladocerans, since copepods have a terminal molt stage while cladocerans molt throughout their lifetimes. Phytoplankton, on the other hand, provide a very nutrient-rich habitat, especially nitrogen-fixing taxa like Anabaena (Islam et al. 1994). The macroscopic detrital aggregates known as “marine snow” and “lake snow” (Figure 3) may also provide a habi- KL Cottingham et al. Table 1. Organisms to which Vibrio cholerae is known to attach Phytoplankton Cyanobacteria – Anabaena Chlorophytes – Volvox, desmids, Rhizoclonium Diatoms – Skeletonema Dinoflagellates Zooplankton Copepods – Acartia, Cyclops, Diaptomus Cladocerans – Daphnia, Bosmina, Bosminopsis, Ceriodaphnia, Diaphanosoma, rotifers Macrophytes Marine taxa – Ulva, Entermorpha, Ceramium, Polysiphonia Freshwater taxa – Eichhornia (water hyacinth), Lemna (duckweed) Benthos Prawns – Penaeus, Metapenaeus, Macrobrachium Oysters Crabs Chironomid egg masses Fish Sea mullet Tamplin et al. 1990; Huq et al. 1990; Islam et al. 1994; Colwell 1996 tat for attached V. cholerae, although this possibility is only beginning to be explored. Work with non-pathogenic taxa indicates that detrital aggregates provide resource-rich microhabitats, to the extent that their associated bacteria can account for a large fraction of microbial productivity in the open ocean (Azam et al. 1994; Grossart and Simon 1998). To date, microbial ecologists have overlooked live plankton as potential hosts for highly productive bacterial communities, while microbiologists are only beginning to consider whether V. cholerae attaches to detrital aggregates – perhaps because microbial ecology tends to focus on bacteria as decomposers, while microbiology focuses on host–parasite interactions. Once attached to either living hosts or detrital aggregates, the complexity of bacterial communities varies from unassociated cells to wellorganized, three-dimensional structures called biofilms (Davey and O’Toole 2000; Figure 4). Biofilms increase bacterial productivity and can provide protection from harmful environmental conditions, including chlorination and antibiotics (O’Toole et al. 2000; Davey and O’Toole 2000). V. cholerae biofilm activity may occur on both planktonic and detrital substrates, but it is virtually unstudied under natural conditions. However, laboratory studies suggest that biofilm activity affects V. cholerae abundance, Figure 2. V. cholerae expressing green fluorescent protein (a) attached to and (b) in the growth rate, and persistence in poor gut of the cladoceran filter-feeder Daphnia. conditions (Yildiz and Schoolnik www.frontiersinecology.org © The Ecological Society of America KL Cottingham et al. Ecology and microbiology of Vibrio cholerae 1999; Haugo and Watnick 2002) – factors that are likely to be important in aquatic environments. The effects of water quality and surface material on attachment, and later detachment, remain a key question in cholera epidemiology. Several studies have evaluated V. cholerae attachment under different physical and chemical conditions (Huq et al. 1984b; McCarthy 1996; Hood and Winter 1997; Chiavelli et al. 2001), although none address interactions between factors. Like the VBNC state, attachment appears to be an adaptive response to suboptimal conditions. At present, however, we know very little about the individual contributions of the VBNC state and attachment to the dynamics of V. cholerae in aquatic ecosystems, and even less about how these processes interact. To complicate the situation still further, both processes are likely to affect bacterivory – the consumption of bacteria by other microorganisms. Bacterivory Bacterivores, including heterotrophic nanoflagellates, protozoans, rotifers, and cladocerans, may strongly regulate bacterial populations (Cole 1999; Jürgens and Matz 2002). Bacterivory is most important in situ- Figure 3. Bacteria attached to marine snow from the German Waddensea, ations where predation rates exceed bacterial visualized using SybrGold staining (x400). population growth rates. The extent to which V. cholerae population dynamics are regulated by copepods do not generally feed on bacteria (Cole 1999). bacterivores has not yet been investigated. However, we Attachment to plankton or detritus should provide a hypothesize that activity level and attachment status refuge from grazers, regardless of activity level, since bacaffect the rates of consumption of V. cholerae, since graz- teria attached to surfaces are less vulnerable to predation ing on non-pathogenic bacteria is a function of bacterial (Cole 1999). activity, bacterial cell size, and grazer size (Cole 1999; Jürgens and Matz 2002). For example, protistan grazers Linking the environment to outbreaks selectively remove active cells, while cladocerans feed indiscriminately on both active and inactive (VBNC) In areas where cholera is endemic, disease outbreaks cells (Cole 1999; Langenheder and Jürgens 2001). begin when humans are infected with V. cholerae from the However, protists can be limited by bacterial size, so that environment, and may then be accelerated by fecal contcells larger than 2.4 m are rarely grazed (Pernthaler et al. amination (Franco et al. 1997). There are typical seasonal 1996). V. cholerae is rod-shaped and measures approxi- patterns to endemic outbreaks (Colwell 1996). In parts of mately 3 m long by 1 m wide when active, and Bangladesh, for example, there is usually one outbreak becomes a sphere of about 1 m in diameter when in the before the monsoons (March–June) and a second, larger outbreak following the monsoons (September– VBNC state. We believe that filter-feeding cladoceran zooplankton December) (Islam et al. 1993; Longini et al. 2002). In like Daphnia, but not protists, graze active V. cholerae due Peru, outbreaks occur annually, following spring warming to their large size. In contrast, V. cholerae in the VBNC (Franco et al. 1997). Outbreaks in endemic areas are state should be vulnerable to grazing by both protists and often geographically localized (Tauxe et al. 1994; Faruque cladocerans, based on size constraints, although protists et al. 1998) and appear to coincide with seasonal changes may not select inactive cells. Furthermore, we suggest that in plankton abundance (Islam et al. 1994; Colwell 1996; zooplankton communities dominated by Daphnia are bet- Lobitz et al. 2000). Despite the apparent relationship between cholera outter at reducing the abundance of free-living V. cholerae populations than copepod-dominated communities, since breaks and plankton numbers, it is unclear whether plank© The Ecological Society of America www.frontiersinecology.org Courtesy of Hans-Peter Grossart 83 Ecology and microbiology of Vibrio cholerae KL Cottingham et al. aquatic environment (Faruque et al. 1998), and then be enhanced in their infectivity by a trip through a human host (Merrell et al. 2002). 84 Courtesy of Paula Watnick Anthropogenic effects Figure 4. V. cholerae biofilm. ton blooms actually cause cholera outbreaks, since a large number of physical, chemical, and biological factors differ during outbreak periods. For example, seasonal cholera outbreaks in Bangladesh coincide with times of both high nutrient availability and high plankton populations (Colwell 1996), either of which could increase V. cholerae abundance. However, it is not clear whether V. cholerae is responding to improved water conditions, more plankton surfaces, more of a particular kind of plankton, or other factors. Careful monitoring and statistical analyses of all relevant physical, chemical, biological, and epidemiological variates, followed by experimental manipulation of the putative causal factors, is needed to untangle the triggers of cholera outbreaks. The potential for an environmental reservoir of pathogenicity genes is a second important link between V. cholerae in aquatic ecosystems and those attacking human hosts (Faruque et al. 1998; Reidl and Klose 2002; Faruque and Nair 2002). Interestingly, many V. cholerae isolated from the environment, including strains other than the epidemic-associated O1 or O139 serogroups, contain the VPI and CTX- ( Faruque and Nair 2002). Whether these strains serve as precursors to epidemic strains or as a source for pathogenesis genes that can be transferred to O1 or O139 strains is not yet known, but at least some of the environmental strains that harbor the pathogenicity genes are capable of infection in an infant mouse cholera model (Boyd and Waldor 2002). Thus, new clones capable of causing disease may emerge from the www.frontiersinecology.org Humans have the potential to affect V. cholerae abundance and transmission at multiple spatial scales. At a local scale, any human activities that alter water characteristics, such as temperature and nutrient concentrations, may affect V. cholerae directly, by altering conditions for growth, or indirectly, by altering the distribution, abundance, and composition of the plankton (and therefore the potential for attachment or bacterivory). Such activities include land-use change, pollution (including sewage), aquaculture and fisheries management, the introduction of exotic species, and the reduction of biodiversity. On a broader spatial scale, human-induced climate change could alter V. cholerae dynamics by changing the microbe’s seasonal regimes or facilitating its spread to new areas. Effects of climate variability on cholera are already documented (Pascual et al. 2002); outbreaks occur during periods of higher lake and river temperatures (Franco et al. 1997) and higher sea surface temperature and height (Lobitz et al. 2000), and have been linked to the El Niño Southern Oscillation (Pascual et al. 2000). Cholera dynamics could be further altered by changes in drought–monsoon cycles and rising sea levels, both of which are expected under current climate change scenarios (Patz and Khaliq 2002). In addition, increased global temperatures could promote colonization or establishment of epidemic strains in new areas, as has happened for other diseases (Harvell et al. 2002). Finally, long-distance transmission through a globalized economy appears increasingly likely. Past cholera epidemics have been linked to ballast water transmission, international food transport, and travelers moving from endemic to non-endemic areas of infection (Tauxe et al. 1994). As each of these activities continues to increase, we need to better understand the feedbacks between human activities, V. cholerae in the environment, and cholera outbreaks. Perspectives The persistence of V. cholerae in aquatic environments, the lack of an effective vaccine, and increasing antibiotic resistance among strains isolated from cholera patients all suggest that cholera will not be eradicated in the foreseeable future. As with other tropical diseases, there is growing concern that the combination of climate change, anthropogenic disturbance of local environments, and unintentional transport due to travel and trade will expand the ranges of endemic strains, and consequently create more focal points for cholera outbreaks. A more complete understanding of the ecology of V. cholerae is © The Ecological Society of America KL Cottingham et al. critical to the prediction and management of the associated disease. Of the suggestions for further research given above, four topics are worthy of particular attention. (1) V. cholerae provides an excellent model organism for probing the function of heterotrophic bacteria in natural ecosystems. Traditionally, microbial ecology has treated bacteria as a “black box”, by necessity ignoring the identity and behavior of individual taxa (Leff and Lemke 1998). However, recent developments in molecular, genetic, and genomic tools make it possible to follow particular species and to evaluate their role from the population, community, and ecosystem perspectives (Jackson et al. 2002). V. cholerae is a well-studied pathogen that has been extensively characterized (Faruque et al. 1998; Reidl and Klose 2002), providing a wealth of information that can be exploited for ecological studies. A number of interesting questions remain unanswered. How does the productivity of this model organism vary in free-living versus attached states? How do productivity and population dynamics, including biofilm development, change over time once V. cholerae is attached to a host? Do these dynamics vary on different hosts? How does bacterivory affect population dynamics and ecosystem functioning? What environmental signals cue entry into and out of a VBNC state? (2) The dual habitats and complex life history of V. cholerae, when combined with the availability of a fully sequenced genome (Heidelberg et al. 2000), make it an excellent model for exploring the genetic basis of bacterial behavior. Microarray technology, which allows the simultaneous quantification of the expression of each gene (Gibson 2002), should be a particularly valuable tool. How does gene expression differ in aquatic environments as opposed to human hosts? What genes are involved in attachment and the VBNC state? Are the same genes involved in attachment to human hosts as to plankton? What, if anything, do genes on the VPI and CTX- do in aquatic environments? What are the correlations between infectivity to humans and how the bacterium behaves in aquatic environments? Recent work with other pathogenic bacteria indicates that virulence is linked to environmental performance (Hogan and Kolter 2002). (3) Many gaps remain in our understanding of V. cholerae’s behavior in aquatic ecosystems. For example, how do the VBNC state, attachment, and vulnerability to bacterivores vary across the salinity gradient from freshwater to estuarine to marine systems? How do population dynamics differ when V. cholerae is free-living and when it is attached? Does V. cholerae form biofilms on living hosts or detrital aggregates? Does biofilm formation change the tendency to go into a VBNC state or to be pathogenic to humans? Better natural history information, in conjunction with experimental ecology and genetic research, should improve the management of V. cholerae populations in nature. (4) The interactions between bacterial genetics and the ecology and evolutionary biology of V. cholerae are also © The Ecological Society of America Ecology and microbiology of Vibrio cholerae worthy of attention, particularly as regards the emergence of new pathogenic strains. We currently know little about the ecological and evolutionary success of different strains of V. cholerae, although there is some evidence for important ecological and clinical differences (Chiavelli et al. 2001; Longini et al. 2002). For example, what are the adaptive advantages of attachment and the VBNC state? Do V. cholerae O1 classical, O1 El Tor, and O139 show the same tendencies to attach to hosts? Do all strains have the ability to enter the VBNC state? Are all strains still infective while in the VBNC state? Are there differences in grazing susceptibility? Does the presence of pathogenicity genes, including the VPI and CTX-, alter V. cholerae’s ability to survive and reproduce in aquatic environments? How does V. cholerae evolve when introduced into new habitats? Answers to these questions will improve our understanding of how the human and aquatic habitats of V. cholerae are linked, and will increase the likelihood of developing effective control strategies. 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