The Clean Water Act (CWA), as amended by the Beaches Environmental Assessment and Coastal Health (BEACH) Act of 2000, requires coastal and Great Lakes states and territories to adopt bacterial water standards as protective as EPA's 1986 bacterial standards 2. These standards are based on concentrations of fecal indicator bacteria (FIB) such as E. coli or enterococci. However, monitoring for these indicators is not always effective for determining when streams and coastal waters are contaminated with sewage because FIB can take up residence in the environment and may even multiply under certain conditions. Another problem with current water quality standards is the known difference between fate and transport characteristics of FIB compared to pathogens. Indicator bacteria are more sensitive to inactivation through treatment processes and have also been shown to be more sensitive to inactivation by sunlight than viral and protozoan pathogens. Sunlight inactivation varies seasonally, daily and at different geographic and climatic regions 3. As a result, the concentrations of FIB in water samples measured after disinfection or measured in environmental waters are not reliable assurances that human pathogens in water have been reduced to levels that will not cause infections in swimmers 4.

Additional issues regarding the use of indicators occur with regard to the sample substrate and the sources of contamination. For over three decades, beach sands and sediments in tropical and subtropical environments have been documented to contain high concentrations of bacterial indicators. Studies conducted in Hawaii and Guam 567 and in Puerto Rico 8 have shown that in the absence of any known sources of human/animal waste, enterococci and E. coli are consistently present and recovered in high concentrations. In South Florida, river bank soils and beach sands have been implicated as the source of indicator microbes to the water column 91011, and experimentation has documented that soil moisture mediated by tidal cycles is a key factor in facilitating regrowth 12. Turnover due to current may also play an important role. Subsurface sands and sediments may serve as a refuge and a source when sands are reworked by wave action or erosion.

The significance of beach sands and other environmental sources is not necessarily limited to the sub-tropics. For example, sands and sediments have been implicated as a bacterial source at marine beaches in California 13141516 and at freshwater beaches of Lake Michigan 17, Lake Huron 18 and Lake Superior 19. Again, soil moisture has been implicated in persistence and possible regrowth of indicator microbes 20. More recently, aquatic plants including Cladophora 21 and epilithic periphyton communities 22 have also been identified as potential contributors to fecal indicator persistence and regrowth in temperate regions.

Alternative indicators are being proposed and evaluated to better identify risks to human health and to improve monitoring strategies. Ideally, these alternative indicators cannot multiply under environmental conditions, are present in low concentrations in unimpacted environmental samples, and are present in high concentrations in sewage. Proposed alternatives include bifidobacteria 23, Clostridium perfringens 24, human viruses 25 and F-RNA coliphages 26. The validation of new water quality indicators requires the correlation of the presence and abundance of the organism with human disease, often gastroenteritis. These correlations are made through epidemiology studies where study subjects (beach goers) report on disease symptoms and severity, and measurements of the organisms in the environment are conducted (Figure 2). It is expected that these techniques for identifying fecal contamination will allow for improved risk assessments and more informed monitoring.

Alternative indicators are also being proposed to help track sources of fecal pollution. Microbial source tracking methods are commonly classified as library-dependent or library-independent. A library is a database of characteristics (e.g. genetic fingerprints, antibiotic resistance profiles) of microbes from known sources. In library-dependent analyses, characteristics of isolates from contaminated waters are compared to the library to find matches, thereby identifying the source of contamination. Library-independent approaches entail analysis of water samples for source-specific markers (e.g. human-specific bacterial strains) to help identify sources. These approaches do not require building and maintenance of representative databases for each study area. Appropriate study design and application of microbial source tracking tools can be complicated, particularly for library-based methods 2930. However, source tracking technologies are rapidly advancing and can already provide useful insight for managers trying to mitigate contamination.

Non-point sources of contamination are of significant concern with respect to the transport of pathogens and their indicators into the marine environment. Point source pollution enters the environment at a distinct location through a direct route that is often easily identified, while non-point source pollution is generally diffuse and intermittent in nature, and occurs through a non-direct route. Examples include runoff from urban and agricultural areas, leaking septic systems and sewerage lines, combined sewer overflows, discharges from boats and atmospheric deposition of aerosols. We currently have a limited understanding of the actual pathogen loading of the coastal environment due to non-point source contamination, nor do we fully understand the consequences of this loading. Addressing non-point source contamination is one of the main challenges for future research. Further assessment of the effect of non-point sources on the loading of pathogens in the marine environment needs to include not only pathogen abundance, but an indication of the potential risk to human health and the ability to track the source of contamination.

Swimmers may themselves be sources of microbes in water. Bather shedding studies conducted in freshwater have long shown that humans release significant numbers of microbes, in particular enterococci and Staphylococcus aureus 313233. Elmir et al. 34 conducted the first experimental human bathing study in marine waters, and results were consistent with those found in freshwater (Figure 3). As S. aureus is isolated from human sewage in relatively low numbers (10³ CFU/100 ml) relative to enterococci (10⁴ to 10⁵ CFU/100 ml) 35, it can be used as an indicator to predict human bather impacts, which would include the combined effects of bather density, mixing, and dilution. The use of S. aureus as a potential supplemental indicator is especially significant as studies have shown an association between illness in swimmers and bather density 3036.

Given that presence and prevalence of indicators does not often correlate well with risk of infection from environmental pathogens, direct monitoring for pathogens is often suggested. Detection of pathogens from environmental samples is gaining momentum through the development of molecular technologies (Figure 4). Approaches are typically based upon measurement of specific sequences of nucleic acids (DNA or RNA), or through structural recognition of pathogens or biomarkers 38. While the detection of pathogen DNA is not necessarily indicative of viability or even infectivity, it does represent a rapid and specific method. Probably the most popular methodology involves using the polymerase chain reaction (PCR) to amplify nucleic acids to detectable levels. This technology is allowing researchers to rapidly and specifically target microbes of public health concern, including those that were previously unexamined because of our inability to culture them. New molecular assays have been introduced for detection of FIB 3940, bacterial pathogens 4142, viral pathogens 2543, and protozoan parasites 4445. Additionally, recent improvements in detection technologies are allowing simultaneous detection of multiple targets in a single assay [46474849; Figure 5]. As with new indicators, nucleic acid based detection of specific pathogens will need to go through testing to determine what level of detection is associated with unacceptable human health risk.

Despite the advances in detection technologies, sample collection and processing remains an issue for direct pathogen detection 5152. Improved filtration methods 53, electrochemical methods 49, immunochemical methods 545556 and nanotechnologies 5758 may enable better cell sorting and improve microbial isolation techniques, but each of these has its own challenges to overcome. Low recovery efficiencies associated with concentrating cells and extracting nucleic acids continue to decrease overall sensitivity for molecular targets. Isolation of microbes from complex samples is also problematic. Soil and sediment samples show high levels of heterogeneity and vary significantly with regard to physical and chemical composition, making the establishment of standardized protocols problematic. These variables represent challenges not only for quantitative recovery of nucleic acids, but for enzymatic manipulation of the resulting sample. Regardless, it is imperative that researchers calculate recovery efficiencies for their concentration and nucleic acid extraction protocols, and for amplification or other detection steps. Further, inhibition of PCR or other reactions should be quantified with an internal control, and concentration estimates should be appropriately adjusted.

It remains to be determined how direct pathogen detection and more rapid technologies will alter monitoring strategies. Given the advances made in measurements during the last two decades, monitoring programs will likely include measurements for a suite of microbes. A tiered monitoring strategy may also be a viable option 14. In a tiered approach, the first step could involve using the simplest and most practical tests for contamination, perhaps utilizing a rapid test for fecal indicators. Tier two may involve adding methods to differentiate human from animal sources of pollution, and the tier three test, if necessary, would measure for specific pathogens. Supplemental microbial measurements could also be added, including indicators of disease transmitted via other routes of infection, including respiratory routes (e.g. adenovirus, Legionella), direct human shedding (e.g. S. aureus), and oral ingestion or wound infections from indigenous microbes (e.g. Vibrio). This tiered approach would not necessarily be appropriate for characterization of transient events, unless samples are appropriately archived for each level of analysis.

Methodologies that simply identify the presence of a pathogenic species in many cases may not improve existing tools since not all strains of infectious bacteria are equally pathogenic. Strain variation can arise through several mechanisms, including mutation, genomic rearrangements, inter and intragenic recombination, or acquisition of genes from mobile genetic elements including transducing bacteriophages. Maintenance of these genetic changes occurs if they result in a selective advantage for survival in a given environmental niche 5960. Therefore, while a significant proportion of the population may not be virulent, there is always the potential for emergence of more infectious strains. This pattern of virulence is especially true of many marine bacteria including members of the Vibrio spp. For example, the majority of environmental V. cholerae strains are non-toxigenic and do not cause disease unless the cholera toxin gene is acquired via phage transduction 61. Strains of the shellfish-borne pathogen V. vulnificus can now be genetically differentiated into one group more likely to cause clinical infections and another group that is less infectious and forms the majority of environmental strains 62636465. Similarly, while V. parahaemolyticus is ubiquitous and often found in shellfish during harvest periods, relatively few genotypically distinct strains are responsible for most outbreaks of human infections 66. There also appears to be an increase or emergence of more virulent V. parahaemolyticus, with increasing numbers of human infections arising from ingestion of raw oysters containing relatively few bacteria, although increased exposure of susceptible populations may play a part as well. Regardless, rapid detection tools must not only be sensitive enough to identify low numbers of a pathogen, but also be discriminatory enough to detect the presence of specific strains of a pathogen capable of expressing virulence genes required for human infection. The application of comparative genomics with contemporary molecular pathogenesis and virulence studies will aid in the identification of genes that will serve as sensitive and accurate markers of risk 67.

Genomics is also assisting population level studies to estimate the extent to which genotypes associate with defined environmental microhabitats and the probability of genetic exchange (of virulence factors and other genes) among co-existing microbes 68. Particularly important is the identification of environmental reservoirs of virulent strains (Figure 6). Indeed, pathogenicity towards humans likely arises among indigenous marine bacteria due to adaptation to a specific marine microhabitat rather than selection for infection of the human body. This is because there is insufficient feedback from infected humans to environmental populations of the pathogen (with perhaps the notable exception of cholera in epidemic areas) to act as positive selection on virulence genes. For example, a specific V. cholerae surface protein contributes to the ability of the bacterium to attach to the chitin exoskeleton of zooplankton 70, an important environmental survival mechanism. While the same protein has also been shown to aid attachment to the human gut mucosa 70, it can be argued that the primary selective driving force for increased attachment is microhabitat adaptation. Overall, the ability to evade predation or establish zoonotic associations may predestine a microbe's ability to infect humans, although this is currently poorly understood 38.

In addition to virulence factors, it is also important to understand how bio-signaling molecules and other chemical compounds affect pathogenicity. The marine microbial community is responsible for the synthesis of highly bioactive secondary metabolites. These compounds are used to communicate (quorum sensing), defend (toxicity), or otherwise provide a competitive advantage to a select community 717273. In some cases these compounds augment the chemical behavior of other chemical or biological processes. This is often observed in cases of biofilm formation and/or incorporation of metals into metabolites 7475 where enhanced pathogenicity or antibiotic resistance has been noted 7677. For example, when a biofilm created by a given microbial community is removed, often times old generation antibiotics become effective. Previously they simply could not cross the film. Investigating the interaction of these chemicals is critical in understanding the role the microbial community plays in human health related issues.

There have been several epidemiology studies regarding the potential transmission of non-gastrointestinal illnesses associated with recreational use of water. While an increased risk of gastroenteritis for swimmers is often found, several non-enteric infections have also been associated with exposure, including respiratory illness, and ear, eye and skin infections 787980818283. In one study, 63.7% of the 683 participants reporting illnesses indicated respiratory symptoms 79. In most of these studies, the risk of non-enteric infections in swimmers increased with either exposure to urban runoff or declining water quality due to pollution or sewage.

Some pathogenic microorganisms are naturally present in freshwater environments (Aeromonas hydrophila, Naegleria fowleri, Legionella pneumophila), while other human pathogenic species are indigenous to marine and brackish waters (V. cholerae, V. vulnificus, V. parahaemolyticus, V. alginolyticus). Pathogens naturally present in water may be transmitted to humans via inhalation, contact or ingestion of water or contaminated food. Examples of sources of non-enteric pathogens in environmental waters include animal urine for Leptospira spp. and shedding from human skin for S. aureus. In addition, Khan et al. 84 have discovered Pseudomonas aeruginosa in open ocean environments. P. aerugionosa is an opportunistic pathogen that has been regarded to be widely present in terrestrial and freshwater environments. This report further illustrates the potential significance of the ocean as a reservoir for pathogenic isolates of traditionally non-indigenous bacteria.

Another potential source of non-enteric pathogens are free-living amoebae. Free living amoebae are natural reservoirs of many types of bacteria such as Legionella spp., Burkholderia pickettii, Vibrio cholerae, Myobacterium avium and Listeria monocytogenes 8586. It is known that Acanthamoeba species are natural aquatic reservoirs of several intracellular pathogens such as Legionella, Chlamydia and Mycobacterium 8788. Laboratory studies with Acanthamoeba castellanii have shown the ability of Francisella tularensis, the agent of tularemia, to survive in amoebal cysts; however, the potential for its survival under various adverse conditions in these cysts needs to be examined 89. Studies have also shown that naturally occurring marine amoebae can harbor some of these pathogens (Figure 7), but their prevalence and the extent to which they harbor other pathogens is unknown. Amoebae in cooling towers and water treatment facility biofilms are considered the primary reservoir for pathogenic legionellae, not only providing refuge for the bacteria but also enhancing the infectivity of the microbe 9293. The level of human health risk these associations represent from the marine environment is unknown, and part of this problem is potentially the lack of correlation between marine exposure and the onset of symptoms several days to a week later.

Studies on the ecology and distribution of non-enteric pathogens in the marine environment are necessary to understand their potential threat to human health. Yet one of the biggest challenges remains effective monitoring. When the source of the etiological agents for non-enteric diseases is not fecal in origin, traditional monitoring for FIB will unlikely be successful in predicting the presence or absence of these pathogens. Although for many non-enteric pathogens there are methods for their specific detection and quantification from natural samples, there are no standards relevant for predicting the level of risk for human infection. Thus, research should be targeted towards establishing these standards for non-enteric pathogens where appropriate, or else efforts should be made toward public education about infections where human behavior plays a key role.

A variety of marine species and habitats are excellent indicators or sentinels of environmental stress and potential health threats for humans. They provide information about how events in the environment may affect humans and animals. Sentinel species and habitats fall into at least three categories: (1) wildlife or habitats that tend to "sample" or concentrate contaminants, toxins, and/or pathogens from their environment and thus may provide more biologically relevant indicators of possible effects than water sampling alone; (2) wildlife with diets and/or physiologies at least partially similar to those of humans and which therefore may demonstrate early indications of potential health effects of environmental levels of contaminants, toxins and pathogens before they show up in humans (a biological early warning system); and (3) habitats that encompass key ecosystem components and are subjected to early and often high pollution exposure, thereby indicating potential effects at systems or community levels and on people.

An estimated 75% of emerging infectious diseases are zoonotic 109, and anthropogenic influence on ecosystems appears to be a common factor in the emergence and reemergence of zoonotic pathogens 110. In the marine environment proper, bacterial, viral, fungal and protozoal pathogens that can infect humans have been detected in a range of marine animals, including pinnipeds, dolphins, cetaceans and otters (Figure 9). Bacteria such as Brucella, Leptospira and Mycobacterium have been shown to infect humans handling marine mammals 112113, while others such as Clostridium, Burkholderia (formerly Pseudomonas), Salmonella and Staphylococcus have the potential to be transmitted to humans. Calicivirus and influenza A have been documented to occur in pinnipeds 114115, and Blastomyces have been detected in dolphins 116. California sea lions and ringed seals have been found to harbor G. lamblia and Cryptosporidium spp. 117118 and Giardia cysts have been found in fecal material from harp seals (Phoca groenlandica), grey seals (Halichoerus grypus), and harbour seals (Phoca vitulina) from the St. Lawrence estuary in eastern Canada 119120. Pinnipeds have also been shown to harbor Salmonella and Campylobacter species, including strains that are resistant to multiple antibiotics 121122. Shorebirds are also potentially able to transmit parasites to humans. Canada geese carry several enteric human pathogens including G. lamblia, Camplyobacter jejuni and C. parvum 123124. Therefore, it seems possible that shorebirds feeding in an area that is contaminated by a sewer outfall may be yet another source of concentrated pathogen input to either shellfishing areas or recreational areas.

Analysis of the interaction between host animals (domesticated or wildlife) and the coastal watershed, the natural reservoirs in marine habitats, and the survival, prevalence and proliferation of the pathogens are a rational area of concern for disease emergence. New methods for direct or indirect detection of microorganisms are contributing to the detection of zoonoses 125126, but there is still a lack of understanding regarding the public health significance.