Diversity, sources, and detection of human bacterial




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DRAFT – NOT FOR DISTRIBUTION

Diversity, sources, and detection of human bacterial

pathogens in the marine environment


Janelle R. Thompson, Luisa A. Marcelino, Martin F. Polz

Massachusetts Institute of Technology

  1. Introduction

  2. Diversity and Ecology

Pathogenic Species

Environmental Associations

Abiotic Factors

  1. Routes of Transmission

Seafood Consumption

Seawater Exposure

Aerosol Exposure

Marine Zoonoses

  1. Indicators for Marine Risk Assessment

Indicators for Sewage Pollution

Indicators for Non-Sewage Related Risk

  1. Detection and Quantification

Culture-based Methods

Immunological Methods

Nucleic Acid-Based Methods

  1. Outlook


1. INTRODUCTION

Disease outbreaks in marine organisms appear to be escalating worldwide (Harvell et al., 1999; Harvell et al., 2002) and a growing number of human bacterial infections have been associated with recreational and commercial uses of marine resources (Tamplin, 2001). Whether these increases reflect better reporting or global trends is a subject of active research [reviewed in (Harvell et al., 1999; Rose et al., 2001; Harvell et al., 2002; Lipp et al., 2002)]; however, in light of heightened human dependence on marine environments for fisheries, aquaculture, waste disposal and recreation, the potential for pathogen emergence from ocean ecosystems requires investigation.

A surprising number of pathogens have been reported from marine environments and the probability of their transmission to humans is correlated to factors that affect their distribution. Both indigenous and introduced pathogens can be the cause of illness acquired from marine environments and their occurrence depends on their ecology, source and survival. To judge the risk from introduced pathogens, levels of indicator organisms are routinely monitored at coastal sites. However, methods targeting specific pathogens are increasingly used and are the only way to judge or predict risk associated with the occurrence of indigenous pathogen populations.

In this chapter, we review the recognized human pathogens that have been found in associations with marine environments (section 2), the potential routes of transmission of marine pathogens to humans, including seafood consumption, seawater exposure (including marine aerosols), and marine zoonoses (section 3), and we discuss the methods available to assess the public-health risks associated with marine pathogens (sections 4 and 5).


2. DIVERSITY AND ECOLOGY

Our current knowledge of the diversity and ecology of bacterial pathogens associated with marine environments stems from (i) clinical accounts of marine-acquired illnesses, (ii) disease outbreaks of known etiology in marine animals, and (iii) testing of marine environments for the presence of pathogen populations. In particular, surveys of environmental microbial communities based on 16S ribosomal RNA gene sequence diversity have revealed a large number of organisms closely related to human pathogens; however, the public health risk of many of these pathogen-like populations remains unknown. This is largely due to a poorly defined relationship between clinical isolates and pathogen-like populations detected in the environmental because many methods used to detect environment populations do not possess high enough resolution to discriminate virulent from harmless strains.

The genetic elements encoding virulence properties are not uniformly distributed among strains within a potentially pathogenic species. For marine pathogens, this has been explored in some detail in Vibrio species. Environmental populations of V. cholera are characterized by heterogeneous distributions of multiple virulence factors, combinations of which regulate the epidemic potential [e.g. (Faruque et al., 1998; Karaolis et al., 1998; Chakraborty et al., 2000)]. Similarly, comparisons of the genomic diversity of clinical and environmental V. vulnificus isolates suggests that seafood-borne human-infections are established by a single highly-virulent strain among coexisting genetically heterogenous populations (Jackson et al., 1997). However, what leads to the occurrence of one strain over another remains poorly understood.

To what extent environmental conditions select for strains possessing human virulence factors is an area of increased research [e.g. (Tamplin et al., 1996; Jackson et al., 1997; Faruque et al., 1998; Chakraborty et al., 2000)]. Such factors may include attachment mechanisms to organic matter, motility, secretion of lytic compounds, and the ability to grow rapidly under nutrient-replete conditions. Transfer of virulence properties between different species has been observed (Faruque et al., 1999; Boyd et al., 2000), and specific virulence factors (e.g., haemolysins, toxins, attachment pilli) may be borne on mobile genetic elements. Thus, environmental interaction may confer enhanced pathogenicity on a subset of an environmental population. In general, the marine environment may be a powerful incubator for new combinations of virulence properties due to the extremely large overall population size of bacterial populations and efficient mixing time-scales. These natural phenomena may be further enhanced by human activity such as increased sewage input and ballast water transport (Ruiz et al., 2000) both of which introduce microbial species across geographical barriers.


Pathogenic Species

The known diversity of human pathogens in the Ocean continues to expand as the virulence of emerging pathogens is recognized. Pathogens associated with marine environments and their observed routes of transmission to humans are presented in Table 1. Of the 23 lineages currently characterized within the domain Bacteria by 16S rRNA phylogeny (Cole et al., 2003), six harbor human pathogens, and of these six, all lineages contain strains found as human and/or animal pathogens in marine environments [i.e., the Bacteroides-Flavobacterium group (Bernardet, 1998), the Spirochetes, the Gram-positive Bacteria, the Chlamydia (Johnson and Lepennec, 1995; Kent et al., 1998), the Cyanobacteria (Carmichael, 2001) and the Proteobacteria] (see also references in Table 1).

A large majority of known marine pathogens belong to the gamma-Proteobacteria. Within these, the genus Vibrio alone contains 11 recognized human pathogens including V. cholera, the etiological agent of epidemic cholera, and the hazardous seafood poisoning agents V. vulnificus and V. parahaemolyticus. Many more vibrios are associated with diseases in marine animals, and only a handful of the 40 or more species currently described within the genus appear to be benign. Other notable gamma-proteobacterial pathogens are members of the Aeromonas and Shewanella genera, which are also widely distributed throughout marine environment. The proportion of marine human pathogenic species within the gamma-Proteobacteria is in contrast to terrestrial environments where groups such as the alpha-Proteobacteria and the spirochetes also contain many pathogenic members. Such discrepancy could reflect differing evolutionary trajectories of marine and terrestrial communities, or could reflect preferential culturability of gamma-proteobacterial pathogens as has generally been observed for heterotrophic gamma Proteobacteria from the marine environment [e.g. (Eilers et al., 2000b)].

The deeply branching lineages of the Gram-positive bacteria also contain a high diversity of recognized marine pathogens. The Mycobacterium group is represented with several notable human pathogens including agents of tuberculosis, skin disease, and an expanding diversity of fish and marine mammal pathogens (Saubolle et al., 1996; Kusuda and Kawai, 1998; Dobos et al., 1999; Rhodes et al., 2001). Other Gram-positive human pathogens found in associations with marine environments include members of the Clostridia, Listeria, Rhodococcus, Streptococcus and Mycoplasma group (Table 1).


Environmental associations

Marine pathogens are often found in association with the guts and skin of marine animals, phytoplankton, sediments and suspended detritus. The association of pathogens with marine biota has been compared to vector-borne disease in terrestrial environments as variability in environmental conditions can affect both the vector distribution and the amplification of the pathogen within the host (Lipp et al., 2002). For example, algal and zooplankton blooms can promote proliferation of associated bacterial communities by providing microenvironments favoring growth and by exuding nutrients into the water (Lipp et al., 2002). Associations between zooplankton and pathogenic Vibrio and Aeromonas species have been observed (Kaneko and Colwell, 1978; Colwell, 1996; Dumontet et al., 2000; Heidelberg et al., 2002a). Correlations of the dynamics of attached pathogenic Vibrio species to seasonal algal and zooplankton blooms have been established (Kaneko and Colwell, 1978; Colwell, 1996; Heidelberg et al., 2002a) and may exist for additional algal and zooplankton associated pathogens. Incidence of epidemic cholera in Bangladesh has also been correlated to seasonal algal and zooplankton blooms, suggesting a link between the abundance of marine indigenous pathogens and outbreaks of human disease (Colwell, 1996).

Animal activity can temporarily raise levels of pathogens in local environments. For example, C. botulinum spores were found enriched in marine sediments with high overlying fish abundance (Huss, 1980). In particular, filter-feeding shellfish are effective concentrators of small particles and have been shown to contain diverse bacteria in their tissues including indigenous pathogens (Olafsen et al., 1993; Lipp and Rose, 1997) and marine contaminants. Enteric (Burkhardt et al., 1992), Campylobacter (Abeyta et al., 1993; Endtz et al., 1997) and Listeria species (Colburn et al., 1990) were all detected in shellfish samples. Thus, it is not surprising that shellfish has long been recognized as a potential source for marine-acquired illness.

Active growth of certain marine pathogens may depend on association with nutrient-rich environments such as animal guts or organic-rich sediments. Gastrointestinal tracts of marine animals have been shown to harbor a wide diversity of organisms closely related to bacterial pathogens (MacFarlane et al., 1986; Oxley et al., 2002). Similarly, organisms commonly associated with sediment environments include enteric pathogens (Grimes et al., 1986), and members of the genera Vibrio (Watkins, 1985; Hoi et al., 1998; Dumontet et al., 2000), Aeromonas (Dumontet et al., 2000), Shewanella (Myers and Nealson, 1990), Clostridia (Huss, 1980) and Listeria (Colburn et al., 1990). Populations dislodged from guts or sediments may occur as inactive transients in seawater and act as a seed population for inoculating new habitats (Ruby and Nealson, 1978). This has been suggested for certain fish-associated vibrios based on their unusual ability to grow rapidly in response to nutrient addition even after prolonged incubation in seawater under starvation conditions (Jensen et al., 2003).

Intracellular associations of bacteria with protozoan and algal hosts have been described in natural and clinical settings and may represent an additional source of pathogens in marine environments. Colonization of amoeboid hosts has been observed for several human bacterial pathogens including Mycobacterium (Cirillo et al., 1997; Steinert et al., 1998), Burkholderia (Michel and Hauroder, 1997; Marolda et al., 1999; Landers et al., 2000) and Legionella species (Cianciotto and Fields, 1992; Fields, 1996). Legionella pneumophila can replicate inside amoebas in natural waters and it is currently held that adaptation to the intracellular environment of a protozoan host predisposed L. pneumophila, the agent of Legionnair’s disease, to infect mammalian cells (Cianciotto and Fields, 1992; Fields, 1996; DePaola et al., 2000; Harb et al., 2000; Swanson and Hammer, 2000). Relatively high concentrations of L. pneumophila have been found in fresh water and coastal systems (102 to 104 CFU per ml) (Fliermans et al., 1981; Ortizroque and Hazen, 1987; Fliermans, 1996). Survival of free-living L. pneumophila in seawater over several days has been demonstrated (Heller et al., 1998); however, extracellular growth in natural water has not been observed (Steinert et al., 1998; Swanson and Hammer, 2000). Whether associations of Legionella spp. or other marine pathogens with protozoan hosts promotes growth of these bacteria in marine environments remains to be determined.

Algal cells have also been shown to harbor intracellular bacterial associations (Biegala et al., 2002) and it is currently debated whether agents of harmful algal blooms (HAB) maintain bacterial symbionts that participate in toxin production (Gallacher and Smith, 1999). Bacteria found in association with cultures of HAB algae have been reported to produce a level of toxin per cell volume that is equivalent to the production of toxin in the alga (Gallacher and Smith, 1999). In addition, autonomous toxin production by free-living bacteria has been reported (Michaud et al., 2002). The relative contribution to toxin production and correlated paralytic shellfish poisoning during HABs by free-living, surface associated, or intracellular bacteria is an area of active investigation (Carmichael, 2001; Vasquez et al., 2001; Smith et al., 2002) (see also chapter [X] of this book). Overall, the role of protist and algal hosts for harboring marine pathogens in the environment remains an important but poorly understood factor to be considered in risk assessment.


Abiotic Factors

Environmental parameters such as salinity, temperature, nutrients, and solar radiation influence the survival and proliferation of pathogens directly by affecting their growth and death rates and indirectly through ecosystem interactions. The survival of contaminant pathogens in marine environments has been shown to decrease with elevated sunlight (Rozen and Belkin, 2001; Fujioka and Yoneyama, 2002; Hughes, 2003), high salinity (Anderson et al., 1979; Sinton et al., 2002), and increased temperature (Faust et al., 1975). However, elevated nutrients and particle associations have been shown to promote the survival of marine contaminants (Gerba and McLeod, 1976). There is increasing evidence that many pathogens found as pollutants in marine environments can survive harsh environmental conditions for prolonged periods of time in a spore-like, “viable but non-culturable” (VBNC) state [e.g. (Grimes et al., 1986; Rahman et al., 1996; Rigsbee et al., 1997; Steinert et al., 1997; Cappelier et al., 1999a; Cappelier et al., 1999b; Besnard et al., 2000; Asakura et al., 2002; Bates et al., 2002)]. The effects of environmental parameters on the survival of enteric bacteria are reviewed in detail in chapter [X] of this book.

In contrast to microbial contaminants, marine-indigenous pathogens are adapted to prevalent environmental conditions and their proliferation may be triggered by specific factors. For example, warm water temperatures appear to have a positive effect on the abundance of human-invasive pathogens, which tend to have mesophilic growth optima. In temperate environments, the distribution of such pathogens is typically seasonal with peaks in both environmental abundance and human infection occurring during the warmer months. This has been demonstrated for human pathogenic Aeromonas spp. (Kaper et al., 1981; Burke et al., 1984), Shewanella algae (Gram et al., 1999) and vibrios (CDC, 1999, 2000; Heidelberg et al., 2002b; Thompson et al., 2004b), including V. cholera (Jiang and Fu, 2001) V. parahaemolyticus (Kaneko and Colwell, 1978), and V. vulnificus (Wright et al., 1996). In addition, elevated sunlight can stimulate growth of marine indigenous heterotrophic bacteria by increasing nutrient availability by photochemical breakdown of complex polymers to release organic metabolites (Chrost and Faust, 1999; Tranvik and Bertilsson, 2001). Nutrient enrichment in seawater samples and sediments has been correlated to increases in the relative abundance of Vibrio populations (Eilers et al., 2000a; La Rosa et al., 2001). It remains to be established whether stimulated growth of opportunistic invasive pathogens, in response to nutrient enrichment, is a general feature of seawater environments.

Overall, the effect of salinity, temperature, and nutrients on the proliferation of marine pathogens will be determined by both the ecosystem interactions and the growth optimum of the pathogen population. To investigate whether rising water temperature, salinity changes, or coastal eutrophication may impact human exposure to pathogens in marine environments, ecological interactions influencing pathogen abundance and distribution must be better characterized.


3. ROUTES OF TRANSMISSION

Transmission of pathogens to humans through marine environments most frequently occurs by eating contaminated seafood, but can also follow other routes including seawater contact or exposure to marine aerosols and zoonoses. The potential for contracting human diseases through marine environments depends on several factors including the susceptibility of the human host, the degree of exposure to a pathogen population, and the virulence of the pathogenic agent. Individuals with medical conditions such as liver disease and diabetes, or who are immunocompromised, are most susceptible to infections (Howard and Bennett, 1993; Howard and Burgess, 1993); however, infections also occur in healthy individuals. The degree of host exposure to a marine pathogen varies with the route of transmission and has been correlated to both the environmental concentration of the pathogen and the duration of exposure. For the purposes of risk assessment for seafood consumption, an average amount of ingested seafood is assumed [e.g., 110 g oyster meat, (Miliotis et al., 2000)] and swimming related illnesses have been correlated to time spent in the water (Corbett et al., 1993). However, no explicit models appear to have been formulated for prediction of other routes of exposure (e.g., animal contact, or aerosol inhalation). Finally, the virulence of the pathogenic population determines the dose needed to establish human disease. In several cases, it has been observed that strains most closely resembling clinical isolates represent only a small subset of related co-occurring organisms suggesting that infections from marine environments may frequently be initiated by small numbers of highly virulent variants (Jackson et al., 1997).


Seafood Consumption

The most important route of infection by marine pathogens is by consumption of contaminated seafood resulting in symptoms from self-limiting gastroenteritis (typical seafood poisoning) to invasive infections that are potentially fatal. Vibrio species are the most significant risk in seafood consumption and an estimated 10,000 cases of food-borne infection occurs in the US each year (FDA, 1994; Altekruse et al., 1997). But other bacterial genera naturally found in association with fish and shellfish have also been implicated in seafood-borne diseases (e.g., Aeromonas, Clostridium, Plesiomonas). Fecal contamination from human sewage or animal sources is recognized as an additional important source of seafood-borne pathogens (e.g., Campylobacter, Escherichia, Listeria, Salmonella, Shigella, and Yersinia) (Feldhusen, 2000). However, in several cases a clear distinction cannot be made whether a pathogen is a fecal contaminant or a natural part of the marine community. For example, Salmonella, generally considered a marine contaminant, may be a natural part of marine ecosystems (Tryland, 2000; Aschfalk et al., 2002). Other genera, such as Campylobacter, are detected in the feces of marine birds (Endtz et al., 1997) and could be described as “endemic contaminants” since their presence can be detected in shellfish beds in environments not polluted by humans.

Infection by ingestion generally requires relatively large doses of pathogens (e.g. 105 to 1010 cells for most gamma proteobacterial pathogens), although some highly virulent pathogens such as Shigella or enterohemoragic E. coli can establish infections with doses as small as 10-100 cells (Canada, 2003) (Table 1). Levels of marine-indigenous pathogens in fresh seafood are usually low enough to be considered safe so that only the growth of these organisms is regarded as a hazard (e.g., during periods of improper handling) (Feldhusen, 2000). For example, non-refrigeration of oysters after harvesting can amplify the endemic Vibrio population 10,000-fold (Miliotis et al., 2000) resulting in levels that are deemed unsafe for human consumption (i.e., ≥104 cells/gram oyster (FDA, 1997).

While cooking minimizes the risk of seafood-borne infection, poisoning can occur from heat-stable bacterial toxins or compounds. Scombroid (or histamine) fish poisoning is caused when bacteria containing the enzyme histadine-decarboxylase proliferate in improperly stored fish rich in the amino acid histadine (e.g., tuna, sardines and salmon) (Burke and Tester, 2002). Bacterial transformation of histadine can produce dangerous levels of histamine, consumption of which can lead to severe allergic reactions. Several types of bacteria including Morganella morganii and Klebsiella oxytoca have been implicated in histamine production in fish (Lopez-Sabater et al., 1996). In addition, toxins produced by marine bacterial species may be concentrated by the activities of filter feeding shellfish. Although this has not been confirmed as a route of human pathogenicity in marine environments, toxin production has been observed by bacterial strains associated with HAB algae including members of the Roseobacter, Alteromonas genera, and cyanobacterial species (Gallacher and Smith, 1999; Carmichael, 2001).


Seawater Exposure

Pathogens can be transmitted to humans through seawater during accidental ingestion, inhalation, or by direct exposure of ears, eyes, nose and wounded soft tissue. Although sewage contamination has long been recognized as a significant risk factor in acquiring illnesses after seawater exposure, sewage-borne pathogens are primarily viral rather than bacterial (Cabelli et al., 1982; Griffin et al., 2001). Invasive bacterial infections acquired in marine environments have primarily been attributed to marine endemic species including gamma-proteobacterial strains related to Aeromonas, Halomonas, Pseudomonas, Shewanella, and Vibrio (Table 1). In beaches with high swimmer density, human-shed Staphylococcus or Streptococcus can cause minor wound and ear infections (Charoenca and Fujioka, 1993; Thomas and Scott, 1997). Other infections that have been reported after exposure to marine or estuarine waters include leptospirosis (Thomas and Scott, 1997) and skin granulomas caused by water-borne Mycobacterium marinum (Dobos et al., 1999). Near-drowning experiences in marine environments bring seawater into the lungs and can result in pneumonia (Ender and Dolan, 1997; Thomas and Scott, 1997). Such infections have been reported for marine indigenous pathogens including Legionella bozemanii, Francisella philomiragia, Klebsialla pneumonia and several Vibrio and Aeromonas species (Ender and Dolan, 1997).

Although the range of infectious doses for wound and skin infections is not known and the degree of exposure is difficult to estimate the danger may potentially be high. Fifty percent mortality was observed for artificially wounded rats exposed to ~107 CFU’s of marine and clinical isolates of Aeromonas hydrophila, Vibrio parahaemolyticus and V. vulnificus (Kueh et al., 1992). In the same study, similar mortalities were observed in rats exposed to 1 ml aliquots of seawater from multiple sites suggesting a high degree of indigenous seawater-associated virulence (Kueh et al., 1992).


Aerosol Exposure

The first case of Legionnaires Disease in 1976 demonstrated the importance of airborne transmission of water-borne bacterial pathogens (McDade et al., 1977). Aerosols, generated in coastal environments by wave activity, can transmit algal toxins to humans (Van Dolah, 2000) and cause viruses to become airborne (Baylor et al., 1977). Transmission of bacterial disease by marine aerosols has not been documented but should be considered as a potential route of infection. Studies have shown that Mycobacterium species are enriched in aerosols from natural waters (Wendt et al., 1980; Parker et al., 1983) and additional respiratory disease agents, which have been detected in seawater, include Francisella philomiragia, Legionella spp., Acinetobacter calcoaceticus and Klebsiella pneumoniae (Grimes, 1991; Ender and Dolan, 1997). In general, infectious doses for respiratory agents are small, e.g. 5-10 organisms for Mycobacterium tuberculosis infection. Thus, marine aerosols may be an unrecognized factor in the transmission of bacterial diseases from marine environments.


Marine Zoonoses

Zoonoses are naturally transmissible diseases from animals to humans. Warm-blooded marine mammals harbor and are afflicted by a wide variety of pathogens posing zoonotic risk to humans including Brucella, Burkholderia, Clostridium, Helicobacter, Mycobacterium, Rhodococcus, and Salmonella species (Bernardelli et al., 1996; Harper et al., 2000; Tryland, 2000; Aschfalk and Muller, 2001; Aschfalk et al., 2002) (Table 1). Tuberculosis, a chronic respiratory disease caused by Mycobacterium species including M. tuberculosis and M. bovis, has afflicted natural and captive populations of marine mammals (Bernardelli et al., 1996; Montali et al., 2001) and has been transmitted from seal to man on one reported incident (Thompson et al., 1993). Brucellosis, a systemic infection, is transmitted to humans from infected animals, meat or dairy products in many parts of the world. Brucellosis has also been observed in a wide range of marine animals including dolphins, porpoises, whales, seals, and otters (Tryland, 2000; Foster et al., 2002). The zoonotic potential of these marine Brucella species has been recognized after three incidents of infection, first of a researcher handling a marine isolate (Brew et al., 1999) and then in two cases of neurobrucellosis attributed to a marine Brucella strain in Peru (Sohn et al., 2003).

Injuries inflicted by marine animals or sustained during their handling are especially susceptible to infection by associated microorganisms and therefore emergency treatment of bites (e.g. from sharks, moray eels) includes broad-spectrum antibiotics (Erickson et al., 1992; Howard and Burgess, 1993). Handling of fish or crabs has been associated with infection by Erysipelothrix rhusopathiae, a mycoplasma-like organism common on the skin of fish, which manifests as a localized swollen purple area around a wound (Fish handler’s disease) (Thomas and Scott, 1997). Other mycoplasma-like organisms including Mycoplasma phocacerebrale have been isolated from seals during pneumonia epizootics and have been implicated in development of ‘Seal Finger’, a local infection of the hands in humans (Kirchhoff et al., 1989; Stadtlander and Madoff, 1994; Baker et al., 1998).

The transmission of disease between farmed and wild fish populations is one of several concerns regarding the sustainability of aquaculture practices (Garrett et al., 1997; Naylor et al., 2000). The zoonotic potential of farmed fish environments has also been recognized on several occasions. The fish pathogen, Streptococcus inae (Zlotkin et al., 1998; Colorni et al., 2002), caused an outbreak of infection in fish farmers in British Columbia (Weinstein et al., 1996; Weinstein et al., 1997). Additional health hazards of fish handlers include infections with Aeromonas hydrophila, Edwardsiella tarda, Erysipelothrix rhusopathiae, Mycobacterium marinum, and Vibrio species (Lehane and Rawlin, 2000). In addition, several currently emerging pathogens of fish populations are closely related to human pathogens (Fryer and Mauel, 1997; Rhodes et al., 2001; Starliper, 2001)). Recently, Serratia liquefaciens was identified as an agent of deadly systemic hospital infections in humans (Grohskopf et al., 2001) and in the same year was identified as a pathogen of farmed Atlantic salmon (Starliper, 2001).


4. INDICATORS FOR MARINE RISK ASSESSMENT

The quality of marine waters has been routinely monitored using detection of indicator organisms found in association with human pollution. Indicators are elements that can be efficiently monitored to approximate the risk of human exposure to a given environment. While the indicators themselves do not necessarily cause disease, their presence in an environment suggests a high probability of co-occurring pathogens. Although traditionally indicator organisms have been relied upon for water quality assessment, the use of physical and chemical proxies and direct detection of pathogen populations are showing promise as tools for future water quality management.


Indicators for Sewage Pollution

Sewage-associated public health risks continue to plague coastal environments worldwide. The NRDCA reports that 12,184 U.S. beach closings or advisories were issued in 2002 (of 2,922 reporting beaches) of which 87% were attributed to poor bacterial water quality (as monitored by indicators for fecal pollution) (Dorfman, 2003). In a landmark epidemiological study, Cabelli et al. (Cabelli et al., 1982) found that illness (primarily gastroenteritis and respiratory infections) associated with swimming in several marine environments increased linearly with the degree of site pollution. They further showed that levels of Gram-positive fecal enterococci and fecal coliforms were good proxies for sewage contamination. Based upon this and similar studies the current USEPAB standard for acceptably safe beaches is a monthly geometric mean of 35 enterococci per 100 ml (Dufour et al., 1986) and a median of 14 fecal coliforms per 100 ml in shellfish harvesting waters (USEPA, 1988).

The use of enterococci and fecal coliform levels as indicator organisms for marine water quality assessment has been repeatedly called into question. These indicator species have show varying degrees of specificity for detecting sewage contamination against background environmental fluctuations from animal and environmental sources (Grant et al., 2001; Boehm et al., 2002). Boehm, et al, (2002) showed that coastal enteroccoci levels are enriched by bird activity in adjacent estuaries. Alternative sewage-borne indicators, such as C. perfringens have been considered due to their stability in the marine environment (Fujioka, 1997); however, they too are found in association with marine animals [e.g. (Aschfalk and Muller, 2001)] and may be subject to environmental variability. In addition, their correlation to human illness has not been convincing (Dufour et al., 1986). Furthermore, exclusive reliance on fecal indicator bacteria for marine water quality assessment has been challenged due to their limited ability to predict viral contamination and the presence of marine-indigenous pathogens (Dumontet et al., 2000; Tamplin, 2001). While sewage indicators remain a useful tool for monitoring water pollution, continued efforts to establish alternative indicators for non-sewage related risks hold promise for future risk assessment.


Indicators for Non-Sewage Related Risk

Additional factors that have been related to human risks from seawater exposure include swimmer density, eutrophication, and thermal pollution. High swimmer density at bathing beaches has been correlated to the acquisition of ear and minor skin infections from human shed bacteria. Levels of the pathogen, Staphylococcus aureus, have been proposed as an indicator for exposure to human-shed bacteria with levels above 100 CFU per 100 ml of seawater considered unsafe (Charoenca and Fujioka, 1993; Fujioka, 1997).

Eutrophication of coastal environments may be linked to infections by marine indigenous pathogens [e.g. (Kueh et al., 1992)]. The relative abundance of Vibrio populations in seawater samples increases in response to organic nutrient enrichment, and pollution from aquaculture environments has been correlated to increased proportions of vibrios in underlying sediments (Eilers et al., 2000a; La Rosa et al., 2001). Accordingly, the prevalence of vibrios or other aerobic heterotrophs has been suggested as an indicator for nutrient-enrichment in marine environments (La Rosa et al., 2001).

That high seawater temperature bears higher risk of exposure to marine pathogens has been established in studies of shellfish (Wright et al., 1996; Motes et al., 1998; Miliotis et al., 2000), natural waters (Wright et al., 1996; Jiang and Fu, 2001; Heidelberg et al., 2002b; Louis et al., 2003; Thompson et al., 2004b), and the incidence of epidemic cholera (Colwell, 1996; Pascual et al., 2000). Remote sensing of sea surface temperature is currently being explored as a means to predict the onset of cholera outbreaks along the Indian and Bangladesh coasts (Lobitz et al., 2000). However, the validity of such measurements for marine water quality and as a predictor of local risk of infection requires further investigation.


5. DETECTION AND QUANTIFICATION

In this section an overview of the methods currently available to detect, identify, and enumerate marine pathogen (or indicator) populations is presented. At the center of the discussion will be methods with proven utility for targeting specific populations within environmental microbial communities. However, several techniques used to isolate and identify marine pathogens in clinical specimens will also be briefly evaluated. Methods used to identify and quantify microbial populations can be divided into three main groups: culture-, immunology- and nucleic acid–based. However, protocols frequently do not fall exclusively into one category but represent combinations. Because of the considerable number of published protocol and commercial kits, this overview presents the general principles that define these three main groups of methods. Where specific examples are given these have been selected because they have been (i) employed by several laboratories and/or (ii) characterized with respect to their limits of sensitivity and specificity. A summary of representative nucleic acid- and immunology-based methods for detection or quantification of marine-relevant pathogen populations is presented in Table 2. In a few cases, methods are described that have not yet been applied to pathogen detection but hold potential.

Methods for monitoring pathogen populations should be selected by evaluating the factors that mediate exposure of humans to the pathogen (e.g., abundance, virulence/infectious dose, route of exposure) and the constraints of the method (e.g., sensitivity, specificity, dynamic range, cost). Methods targeting pathogen populations must be sensitive enough to monitor populations at levels below the infectious dose, and specific enough to recognize the target group without generating false positives by cross-reacting with non-target organisms. Detection requires positive identification at or above specified threshold concentrations while enumeration requires flexibility to identify a range of population levels. For clinical purposes, detection is often sufficient, while quantification of hazardous populations is preferable for analysis of environmental samples. The methods also differ greatly in speed and cost of implementation and therefore the most accurate method may not always be the most preferable when rapid decision-making is required. The following sections present our attempt to take these considerations into account while evaluating the strengths and weaknesses of various methods.

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