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Monitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. To obtain quantitative eDNA datasets for individual species, organism-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre, genus-specific for the shortbelly rockfish assay, and broadly whale-specific for the humpback whale assay, which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples., Competing Interests: The authors have declared that no competing interests exist.

Environmental DNA (eDNA) analysis allows the simultaneous examination of organisms across multiple trophic levels and domains of life, providing critical information about the complex biotic interactions related to ecosystem change. Here we used multilocus amplicon sequencing of eDNA to survey biodiversity from an eighteen-month (2015-2016) time-series of seawater samples from Monterey Bay, California. The resulting dataset encompasses 663 taxonomic groups (at Family or higher taxonomic rank) ranging from microorganisms to mammals. We inferred changes in the composition of communities, revealing putative interactions among taxa and identifying correlations between these communities and environmental properties over time. Community network analysis provided evidence of expected predator-prey relationships, trophic linkages, and seasonal shifts across all domains of life. We conclude that eDNA-based analyses can provide detailed information about marine ecosystem dynamics and identify sensitive biological indicators that can suggest ecosystem changes and inform conservation strategies.

Fecal pollution of surface waters presents a global human health threat. New molecular indicators of fecal pollution have been developed to address shortcomings of traditional culturable fecal indicators. However, there is still little information on their fate and transport in the environment. The present study uses spatially and temporally extensive data on traditional (culturable enterococci, cENT) and molecular (qPCR-enterococci, qENT and human-associated marker, HF183/BacR287) indicator concentrations in marine water surrounding highly-urbanized San Francisco, California, USA to investigate environmental and anthropogenic processes that impact fecal pollution. We constructed multivariable regression models for fecal indicator bacteria at 14 sampling stations. The human marker was detected more frequently in our study than in many other published studies, with detection frequency at some stations as high as 97%. The odds of cENT, qENT, and HF183/BacR287 exceeding health-relevant thresholds were statistically elevated immediately following discharges of partially treated combined sewage, and cENT levels dissipated after approximately 1 day. However, combined sewer discharges were not important predictors of indicator levels typically measured in weekly monitoring samples. Instead, precipitation and solar insolation were important predictors of cENT in weekly samples, while precipitation and water temperature were important predictors of HF183/BacR287 and qENT. The importance of precipitation highlights the significance of untreated storm water as a source of fecal pollution to the urban ocean, even for a city served by a combined sewage system. Sunlight and water temperature likely control persistence of the indicators via photoinactivation and dark decay processes, respectively.

Harnessing information encoded in environmental DNA (eDNA) in marine waters has the potential to revolutionize marine biomonitoring. Whether using organism-specific quantitative PCR assays or metabarcoding in conjunction with amplicon sequencing, scientists have illustrated that realistic organism censuses can be inferred from eDNA. The next step is establishing ways to link information obtained from eDNA analyses to actual organism abundance. This is only possible by understanding the processes that control eDNA concentrations. The present study uses mesocosm experiments to study the persistence of eDNA in marine waters and explore the role of sunlight in modulating eDNA persistence. We seeded solute-permeable dialysis bags with water containing indigenous eDNA and suspended them in a large tank containing seawater. Bags were subjected to two treatments: half the bags were suspended near the water surface where they received high doses of sunlight, and half at depth where they received lower doses of sunlight. Bags were destructively sampled over the course of 87 hours. eDNA was extracted from water samples and used as template for a Scomber japonicus qPCR assay and a marine fish-specific 12S rRNA PCR assay. The latter was subsequently sequenced using a metabarcoding approach. S. japonicus eDNA, as measured by qPCR, exhibited first order decay with a rate constant ~0.01 hr -1 with no difference in decay rate constants between the two experimental treatments. eDNA metabarcoding identified 190 organizational taxonomic units (OTUs) assigned to varying taxonomic ranks. There was no difference in marine fish communities as measured by eDNA metabarcoding between the two experimental treatments, but there was an effect of time. Given the differences in UVA and UVB fluence received by the two experimental treatments, we conclude that sunlight is not the main driver of fish eDNA decay in the experiments. However, there are clearly temporal effects that need to be considered when interpreting information obtained using eDNA approaches.

Recirculating seawater is an important component of submarine groundwater discharge, yet its role in transporting microbial contaminants from beach sand to coastal water is unknown. This study investigated the extent to which recirculating seawater carries fecal indicators, Enterococcus and bird-associated Catellicoccus, through the beach subsurface. Laboratory experiments and numerical modeling were performed to characterize the transport of fecal indicators suspended in seawater through medium-grained beach sand under transient and saturated flow conditions. Enterococcus was measured both by culture (cENT) and DNA assay (tENT), and Catellicoccus (CAT) by DNA assay. There were differences between transport of tENT and CAT compared to cENT through laboratory columns containing beach sands. Under transient flow conditions, first-order attachment rate coefficients (k att ) of DNA markers were greater (∼10 h -1 ) than k att of cENT (∼1 h -1 ), although under saturated conditions k att values were similar (∼1 h -1 ). First-order detachment rate coefficients, k det , of DNA markers were greater (∼1 h -1 ) than k det of cENT (∼0.1h -1 ) under both types of flow conditions. Incorporating the rate coefficients into field-scale subsurface transport simulations showed that, in this sand type, the contribution of recirculating seawater to surf zone contamination is likely to be minimal unless bird feces are deposited close to the land-sea interface.

Most studies characterize microbial source tracking (MST) target performance using sensitivity and specificity metrics. However, it is important to also consider the temporal stability of MST targets in relation to regulated microbial pollutants. Differences among bacterial target stabilities may lead to erroneous conclusions about sources of contamination. The present study evaluates the relative stability of MST targets and fecal indicator organisms using the gull/pigeon-associated Catellicoccus marimammalium (CAT) marker and enterococci (ENT). The decay rates of CAT and ENT measured by culture (cENT) and QPCR (tENT) were compared in sand and seawater laboratory microcosms under environmentally relevant conditions (subject to tidal wetting versus no wetting in sand, and sunlit versus dark conditions in seawater). Bacterial targets were more persistent in beach sand than in seawater with decay rates on the order of 0.01-0.1 per day and 1 to 10 per day, respectively. Targets were more persistent in unwetted compared to wetted sand, and dark compared to sunlit seawater. During the first 8 days of the sand experiment, the decay rate k of CAT was greater than that of cENT. The decay rates of CAT, tENT, and cENT were similar in sand after day 8 and in dark seawater. In sunlit seawater, the decay rates were different between targets with kcENT > kCAT > ktENT. The decay rates presented here are useful for fate and transport models and also inform the use of MST marker concentrations to infer ENT sources in the environment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Billions of gallons of untreated wastewater enter the coastal ocean each year. Once sewage microorganisms are in the marine environment, they are exposed to environmental stressors, such as sunlight and predation. Previous research has investigated the fate of individual sewage microorganisms in seawater but not the entire sewage microbial community. The present study used next-generation sequencing (NGS) to examine how the microbial community in sewage-impacted seawater changes over 48 h when exposed to natural sunlight cycles and marine microbiota. We compared the results from microcosms composed of unfiltered seawater (containing naturally occurring marine microbiota) and filtered seawater (containing no marine microbiota) to investigate the effect of marine microbiota. We also compared the results from microcosms that were exposed to natural sunlight cycles with those from microcosms kept in the dark to investigate the effect of sunlight. The microbial community composition and the relative abundance of operational taxonomic units (OTUs) changed over 48 h in all microcosms. Exposure to sunlight had a significant effect on both community composition and OTU abundance. The effect of marine microbiota, however, was minimal. The proportion of sewage-derived microorganisms present in the microcosms decreased rapidly within 48 h, and the decrease was the most pronounced in the presence of both sunlight and marine microbiota, where the proportion decreased from 85% to 3% of the total microbial community. The results from this study demonstrate the strong effect that sunlight has on microbial community composition, as measured by NGS, and the importance of considering temporal effects in future applications of NGS to identify microbial pollution sources., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Microbial pollutants from coastal discharges can increase illness risks for swimmers and cause beach advisories. There is presently no predictive model for estimating the entrainment of pollution from coastal discharges into the surf zone. We present a novel, quantitative framework for estimating surf zone entrainment of pollution at a wave-dominant open beach. Using physical arguments, we identify a dimensionless parameter equal to the quotient of the surf zone width l(sz) and the cross-flow length scale of the discharge la = M(j) (1/2)/U(sz), where M(j) is the discharge's momentum flux and U(sz) is a representative alongshore velocity in the surf zone. We conducted numerical modeling of a nonbuoyant discharge at an alongshore uniform beach with constant slope using a wave-resolving hydrodynamic model. Using results from 144 numerical experiments we develop an empirical relationship between the surf zone entrainment rate α and l(sz)/(la). The empirical relationship can reasonably explain seven measurements of surf zone entrainment at three diverse coastal discharges. This predictive relationship can be a useful tool in coastal water quality management and can be used to develop predictive beach water quality models.

Salmonella enterica is one of the most important bacterial enteric pathogens worldwide. However, little is known about its distribution and diversity in the environment. The present study explored the diversity of 104 strains of Salmonella enterica isolated over 2 years from 12 coastal waterways in central California. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were used to probe species diversity. Seventy-four PFGE patterns and 38 sequence types (STs) were found, including 18 newly described STs. Nineteen of 25 PFGE patterns were indistinguishable from those of clinical isolates in PulseNet. The most common ST was consistent with S. enterica serovar Typhimurium, and other frequently detected STs were associated with the serovars Heidelberg and Enteritidis; all of these serovars are important etiologies of salmonellosis. An investigation into S. enterica biogeography was conducted at the level of ST and subspecies. At the ST and subspecies level, we found a taxon-time relationship but no taxon-area or taxon-environmental distance relationships. STs collected during wet versus dry conditions tended to be more similar; however, STs collected from waterways adjacent to watersheds with similar land covers did not tend to be similar. The results suggest that the lack of dispersal limitation may be an important factor affecting the diversity of S. enterica in the region.

Sunlight-mediated disinfection of water is of interest to both the drinking and recreational water quality community of researchers due to its potential to reduce microbial contamination and waterborne illness. Photo-inactivation of enteric bacteria has primarily been investigated using Escherichia coli and laboratory strains of model bacteria. The present study sought to document the photo-inactivation of environmental isolates of Salmonella in filter-sterilized natural seawater and freshwater and to test the hypothesis that diverse Salmonella serovars decay at similar rates both within and between water matrices. The inactivation of Salmonella enterica Typhimurium LT2, Typhimurium ST19, Heidelberg, and Mbandaka was examined in sunlit and dark microcosms. First order decay was observed in sunlit microcosms; the time until 90% inactivation was of the order of 10 min. A significant shoulder, of the order of 1 hr in length, was observed in the freshwater microcosms during which concentrations were stable. Serovar Mdandaka decayed more slowly than other serovars in both seawater and freshwater. The serovars were extremely stable in the dark microcosms showing little to no decay over 53 days. The results document intra-species variation in photo-inactivation, likely owing to differences in intracellular concentrations of photo-sensitizing molecules or molecules that quench reactive species.

Field studies in fresh and marine waters consistently show diel fluctuations in concentrations of enterococci, indicators of water quality. We investigated sunlight inactivation of Enterococcus faecalis to gain insight into photoinactivation mechanisms and cellular responses to photostress. E. faecalis bacteria were exposed to natural sunlight in clear, filtered seawater under both oxic and anoxic conditions to test the relative importance of oxygen-mediated and non-oxygen-mediated photoinactivation mechanisms. Multiple methods were used to assess changes in bacterial concentration, including cultivation, quantitative PCR (qPCR), propidium monoazide (PMA)-qPCR, LIVE/DEAD staining using propidium iodide (PI), and cellular activity, including ATP concentrations and expression of the superoxide dismutase-encoding gene, sodA. Photoinactivation, based on numbers of cultivable cells, was faster in oxic than in anoxic microcosms exposed to sunlight, suggesting that oxygen-mediated photoinactivation dominated. There was little change in qPCR signal over the course of the experiment, demonstrating that the nucleic acid targets were not damaged to a significant extent. The PMA-qPCR signal was also fairly stable, consistent with the observation that the fraction of PI-permeable cells was constant. Thus, damage to the membrane was minimal. Microbial ATP concentrations decreased in all microcosms, particularly the sunlit oxic microcosms. The increase in relative expression of the sodA gene in the sunlit oxic microcosms suggests that cells were actively responding to oxidative stress. Dark repair was not observed. This research furthers our understanding of photoinactivation mechanisms and the conditions under which diel fluctuations in enterococci can be expected in natural and engineered systems.

Despite being important etiological agents of waterborne illness, the sources, transport and decay of human viruses in recreational waters are not well understood. This study examines enterovirus and adenovirus concentrations in coastal water samples collected from four beaches impacted by microbial pollution: (1) Malibu Lagoon, Malibu; (2) Tijuana River, Imperial Beach; (3) Baja Malibu, Baja California; and (4) Punta Bandera, Baja California. Water samples were concentrated using a flocculation-based skim milk method and dead-end membrane filtration (MF). Viruses were enumerated using cell culture infectivity assays and reverse transcription quantitative polymerase chain reaction (RT-QPCR). Across concentration and quantification methods, enteroviruses were detected more often than adenoviruses. For both viruses, MF followed by (RT)QPCR yielded higher concentrations than skim milk flocculation followed by (RT)QPCR or cell culture assays. Samples concentrated by skim milk flocculation and enumerated by (RT)QPCR agreed more closely with concentrations enumerated by cell culture assays than MF followed by (RT)QPCR. The detection of viruses by MF and (RT)QPCR was positively correlated with the presence of infectious viruses. Further research is needed to determine if detection of viruses by rapid methods such as (RT)QPCR can be a useful water quality monitoring tool to assess health risks in recreational waters.

This study explores the transport of enterococci (ENT) from naturally contaminated beach sands to the groundwater table via infiltrating seawater using field, laboratory, and modeling experiments. ENT were readily mobilized and transported through the unsaturated zone during infiltration events in both the field and laboratory column experiments. Detachment mechanisms were investigated using a modified version of HYDRUS-1D. Three models for detachment kinetics were tested. Detachment kinetics that are first order with respect to the rate of change in the water content and attached surface bacterial concentrations were found to provide a best fit between predicted and observed data. From these experimental and model results we conclude that detachment mechanisms associated with the rapid increases in pore water content such as air-water interface scouring and thin film expansion are likely drivers of ENT mobilization in the investigated system. These findings suggest that through-beach transport of ENT may be an important pathway through which ENT from beach sands are transported to beach groundwater where they may be discharged to coastal waters via submarine groundwater discharge.

Enterococcus species composition was determined each hour for 72 h at a polluted marine beach in Avalon, Santa Catalina Island, CA. Species composition during the day was significantly different from that at night, based on an analysis of similarity. Enterococcus faecium and E. faecalis were more prevalent at night than during the day, while E. hirae and other Enterococcus species were more prevalent during the day than the night. Enterococcus spp. containing a yellow pigment were more common during the day than the night, suggesting that the pigmented phenotype may offer a competitive advantage under sunlit conditions. A laboratory microcosm experiment established that the pigmented E. casseliflavus isolate and a pigmented E. faecalis isolate recovered from the field site decay slower than a nonpigmented E. faecalis isolate in a solar simulator in simulated, clear seawater. This further supports the idea that the yellow carotenoid pigment in Enterococcus provides protection under sunlit conditions. The findings are in accordance with previous work with other carotenoid-containing nonphotosynthetic and photosynthetic bacteria that suggests that the carotenoid is able to quench reactive oxygen species capable of causing photoinactivation and photostress. The results suggest that using enterococcal species composition as a microbial source tracking tool may be hindered by the differential environmental persistence of pigmented and nonpigmented enterococci.

This study used molecular methods to measure concentrations of four enteric viruses (adenovirus, enterovirus, norovirus GI, and norovirus GII) and fecal source tracking markers (human, ruminant, and pig Bacteroidales) in land-based runoff from 22 tropical streams on O'ahu, Hawai'i. Each stream was sampled twice in the morning and afternoon during dry weather. Viruses and human Bacteroidales were widespread in the streams. Watershed septic tank densities were positively associated with higher occurrence of human Bacteroidales and norovirus. There were no associations between occurrence of viruses and fecal indicator concentrations. Virus concentrations and previously reported culturable Salmonella and Campylobacter were used as inputs to a quantitative microbial risk assessment (QMRA) model to estimate the risk of acquiring gastrointestinal (GI) illness from swimming in tropical marine waters adjacent to discharging streams. Monte Carlo methods were used to incorporate uncertainties in the dilution of stream discharge with seawater, swimmer ingestion volumes, pathogen concentrations, and dose-response parameters into the model. Median GI illness risk to swimmers from exposure to coastal waters adjacent to the 22 streams ranged from 0 to 21/1000. GI illness risks from viral exposures were generally orders of magnitude greater than bacterial exposures. Swimming adjacent to streams positive for norovirus or adenovirus resulted in the highest risks. The median risk adjacent to each stream was positively, significantly correlated to the concentration of Clostridium perfringens in the stream. Although a number of important assumptions were made to complete the QMRA, results suggest land-based runoff in the tropics as a potential source of GI illness risk, with pathogens coming from both human and nonhuman nonpoint sources including septic tanks.

Algae on freshwater beaches can serve as reservoirs for fecal indicator bacteria (FIB). Wrack (especially kelp) at marine beaches might sustain FIB as well. This study examines the relationship between beach wrack, FIB, and surrounding water and sediment at marine beaches along the California coast. Surveys of southern and central California beaches were conducted to observe environmental wrack-associated FIB concentrations. FIB concentrations normalized to dry weight were the highest in stranded dry wrack, followed by stranded wet and suspended 'surf' wrack. Laboratory microcosms were conducted to examine the effect of wrack on FIB persistence in seawater and sediment. Indigenous enterococci and Escherichia coli incubated in a seawater microcosm containing wrack showed increased persistence relative to those incubated in a microcosm without wrack. FIB concentrations in microcosms containing wrack-covered sand were significantly higher than those in uncovered sand after several days. These findings implicate beach wrack as an important FIB reservoir. The presence of wrack may increase water and sediment FIB levels, altering the relationship between FIB levels and actual health risk while possibly leading to beach closures. Further work will need to investigate the possibility of FIB growth on wrack and the potential for pathogen presence., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

In the last decade, the use of culture-independent methods for detecting indicator organisms and pathogens in recreational waters has increased and has led to heightened interest in their use for routine water quality monitoring. However, a thorough understanding of the persistence of genetic markers in environmental waters is lacking. In the present study, we evaluate the persistence of enterococci, enterovirus, and human-specific Bacteroidales in seawater microcosms. Two microcosms consisted of seawater seeded with human sewage. Two additional seawater microcosms were seeded with naked Enterococcus faecium DNA and poliovirus RNA. One of each replicate microcosm was exposed to natural sunlight; the other was kept in complete darkness. In the sewage microcosms, concentrations of enterococci and enterovirus were measured using standard culture-dependent methods as well as QPCR and RT-QPCR respectively. Concentrations of human-specific Bacteroidales were determined with QPCR. In the naked-genome microcosms, enterococci and enterovirus markers were enumerated using QPCR and RT-QPCR, respectively. In the sewage microcosm exposed to sunlight, concentrations of culturable enterococci fell below the detection limit within 5 days, but the QPCR signal persisted until the end of the experiment (day 28). Culturable enterococci did not persist as long as infectious enteroviruses. The ability to culture enteroviruses and enterococci was lost before detection of the genetic markers was lost, but the human-specific Bacteroidales QPCR signal persisted for a similar duration as infectious enteroviruses in the sewage microcosm exposed to sunlight. In the naked-genome microcosms, DNA and RNA from enterococci and enterovirus, respectively, persisted for over 10d and did not vary between the light and dark treatments. These results indicate differential persistence of genetic markers and culturable organisms of public health relevance in an environmental matrix and have important management implications.

Sunlight modulates concentrations of Escherichia coli and enterococci in marine waters. However, the mechanism of photoinactivation is poorly understood. Additionally, little is known about photoinactivation of other fecal indicators and human viruses in recreational waters. We sampled nearshore waters at Avalon Beach, California hourly for 72 h for reactive oxygen species (ROS), traditional indicator bacteria (E. coli and enterococci, and QPCR-based detection of enterococci), F+ (DNA and RNA) and somatic coliphages, the human-specific marker in Bacteroidales (HF marker), human enterovirus, and human adenovirus. E. coli and enterococci (regardless of measurement technique) covaried with each other and the coliphages suggesting similar sources and fates. The occurrence of the HF and enterovirus markers was correlated, but their occurrence was not positively correlated with the other indicators. Lower concentrations or occurrence of all microbes, excluding the HF and enterovirus markers, were observed during sunlit as opposed to dark hours, pointing to the importance of photoinactivation. Empirical-deterministic models for a subset of microbial indicators were created to determine field-relevant sunlight inactivation rates while accounting for time dependent sources and sinks. Photoinactivation rates of enterococci and E. coli, enterococci measured by QPCR, and somatic coliphage were estimated at 7, 6, 3, and 28 d(-1) I(-1), respectively, where I is UVB intensity in W/m(2). Average H(2)O(2) was 183 nM and the maximum singlet oxygen steady state concentration was 6.6 fM. Given the clarity of the water, direct genomic damage of bacteria and coliphage, as well as indirect endogenous damage of bacteria, were likely the most important inactivation mechanisms, but we cannot rule out a contribution by indirect mechanisms involving the H(2)O(2) and singlet oxygen produced exogenously.

Enterococci are indicator bacteria used to assess the risk of acquiring enteric disease from swimming in marine waters. Previous work identified beach sands as reservoirs of enterococci which can be transported from the sand to the sea, where they may instigate beach advisories. The present study establishes that naturally occurring enterococci can replicate in beach sands under environmentally relevant conditions. In unseeded, nonsterile microcosm experiments, it was shown that intermittent wetting of sands by seawater, like that which would occur at the high tide line, stimulates the transient replication of enterococci at rates of 0.20 to 0.63 per day (equivalent to doubling times of 1.1 to 3.5 days). Replication was not observed in control microcosms that were not subjected to wetting. Enterococci were enumerated using both culture-dependent (membrane filtration and mEI media) and culture-independent (quantitative PCR [QPCR], 23S rRNA gene based) techniques, which allowed tracking of both culturable and total enterococcus populations. Inhibition of QPCR and DNA extraction efficiencies were accounted for in the interpretation of the QPCR results. The results provide evidence that enterococci may not be an appropriate indicator of enteric disease risk at recreational beaches subject to nonpoint sources of pollution.

Submarine groundwater discharge to coastal waters can be a significant source of both contaminants and biologically limiting nutrients. Nitrogen cycling across steep gradients in salinity, oxygen and dissolved inorganic nitrogen in sandy 'subterranean estuaries' controls both the amount and form of nitrogen discharged to the coastal ocean. We determined the effect of these gradients on betaproteobacterial ammonia-oxidizing bacteria (beta-AOB) and ammonia-oxidizing archaea (AOA) in a subterranean estuary using the functional gene encoding ammonia monooxygenase subunit A (amoA). The abundance of beta-AOB was dramatically lower in the freshwater stations compared with saline stations, while AOA abundance remained nearly constant across the study site. This differing response to salinity altered the ratio of beta-AOB to AOA such that bacterial amoA was 30 times more abundant than crenarchaeal amoA at the oxic marine station, but nearly 10 times less abundant at the low-oxygen fresh and brackish stations. As the location of the brackish mixing zone within the aquifer shifted from landward in winter to oceanward in summer, the location of the transition from a beta-AOB-dominated to an AOA-dominated community also shifted, demonstrating the intimate link between microbial communities and coastal hydrology. Analysis of ammonia-oxidizing enrichment cultures at a range of salinities revealed that AOA persisted solely in the freshwater enrichments where they actively express amoA. Diversity (as measured by total richness) of crenarchaeal amoA was high at all stations and time points, in sharp contrast to betaproteobacterial amoA for which only two sequence types were found. These results offer new insights into the ecology of AOA and beta-AOB by elucidating conditions that may favour the numerical dominance of beta-AOB over AOA in coastal sediments.

Fecal indicator bacteria (FIB) concentrations in a single grab sample of water are used to notify the public about the safety of swimming in coastal waters. If concentrations are over a single-sample standard, waters are closed or placed under an advisory. Previous work has shown that notification errors occur often because FIB vary more quickly than monitoring results can be obtained (typically 24 h). Rapid detection technologies (such as quantitative polymerase chain reaction) that allow FIB quantification in hours have been suggested as a solution to notification errors. In the present study, I explore variability of enterococci (ENT) over time scales less than a day that might affect interpretation of FIB concentrations from a single grab sample, even if obtained rapidly. Five new data sets of ENT collected at 10 and 1 min periodicities for 24 and 1 h, respectively, are presented. Data sets are collected in diverse marine environments from a turbulent surf zone to a quiescent bay. ENT vary with solar and tidal cycles, as has been observed in previous studies. Over short time scales, ENT are extremely variable in each environment even the quiescent bay. Changes in ENT concentrations between consecutive samples (1 or 10 min apart) greater than the single-sample standard (104 most probable number per 100 mL) are not unusual. Variability, defined as the change in concentration between consecutive samples, is not distinct between environments. ENT change by 60% on average between consecutive samples, and by as much as 700%. Spectral analyses reveal no spectral peaks, but power-law decline of spectral density with frequency. Power-law exponents are close to 1 suggesting ENT time series share properties with 1/f noise and are fractal in nature. Since fractal time series have no characteristic time scale associated with them, it is not obvious how the fractal nature of ENT can be exploited for adaptive sampling or management. Policy makers, as well as scientists designing field campaigns for microbial source tracking and epidemiology studies, are cautioned that a single sample of water reveals little about the true water quality at a beach. Multiple samples must be taken to gain a snapshot into the patchy structure of microbial water quality and associated human health risk.

Molecular genetic markers, such as those from fecal Bacteroides microorganisms, can link microbial pollution with its source, and have been used successfully in studies of sheltered aquatic environments. Their applicability to wave-driven, open coast environments has not been tested. We assessed the contribution of a tidal outlet to surf zone water quality in coastal Orange County, California, USA by measuring three traditional culture-based fecal indicator bacteria (FIB) as well as the human-specific Bacteroides molecular marker (HF marker) at four shoreline locations. We found that total and fecal coliform levels were higher during low tides than high tides at two of the four stations, and that this effect was strongest at the mouth of the tidal lagoon and decayed with distance from the outlet. The HF marker was detected in 23% and 47% of samples from the tidal outlet and 26% and 41% of samples from an adjacent recreational beach in 2005 and 2006 respectively. Surprisingly, the station farthest from the tidal outlet had the highest occurrence of the HF marker. We found no relationship between FIB abundance and occurrence of the HF marker for individual samples, but that when the data were considered together by year, higher FIB abundance was correlated with a higher incidence of the HF marker. DNA sequences of the HF marker recovered from this site were > 99% similar to those recovered from other states and countries, suggesting low global diversity of this marker. These data provide strong support for the idea that multiple time points and physical conditions should be considered when assessing coastal water quality.

Studies of Vibrio cholerae diversity have focused primarily on pathogenic isolates of the O1 and O139 serotypes. However, autochthonous environmental isolates of this species routinely display more extensive genetic diversity than the primarily clonal pathogenic strains. In this study, genomic and metabolic profiles of 41 non-O1/O139 environmental isolates from central California coastal waters and four clinical strains are used to characterize the core genome and metabolome of V. cholerae. Comparative genome hybridization using microarrays constructed from the fully sequenced V. cholerae O1 El Tor N16961 genome identified 2,787 core genes that approximated the projected species core genome within 1.6%. Core genes are almost universally present in strains with widely different niches, suggesting that these genes are essential for persistence in diverse aquatic environments. In contrast, the dispensable genes and phenotypic traits identified in this study should provide increased fitness for certain niche environments. Environmental parameters, measured in situ during sample collection, are correlated to the presence of specific dispensable genes and metabolic capabilities, including utilization of mannose, sialic acid, citrate, and chitosan oligosaccharides. These results identify gene content and metabolic pathways that are likely selected for in certain coastal environments and may influence V. cholerae population structure in aquatic environments.

Vibrio cholerae is an autochthonous member of diverse aquatic ecosystems around the globe. Collectively, the genomes of environmental V. cholerae strains comprise a large repository of encoded functions which can be acquired by individual V. cholerae lineages through uptake and recombination. To characterize the genomic diversity of environmental V. cholerae, we used comparative genome hybridization to study 41 environmental strains isolated from diverse habitats along the central California coast, a region free of endemic cholera. These data were used to classify genes of the epidemic V. cholerae O1 sequenced strain N16961 as conserved, variably present, or absent from the isolates. For the most part, absent genes were restricted to large mobile elements and have known functions in pathogenesis. Conversely, genes present in some, but not all, California isolates were in smaller contiguous clusters and were less likely to be near genes with functions in DNA mobility. Two such clusters of variable genes encoding different selectable metabolic phenotypes (mannose and diglucosamine utilization) were transformed into the genomes of environmental isolates by chitin-dependent competence, indicating that this mechanism of general genetic exchange is conserved among V. cholerae. The transformed DNA had an average size of 22.7 kbp, demonstrating that natural competence can mediate the movement of large chromosome fragments. Thus, whether variable genes arise through the acquisition of new sequences by horizontal gene transfer or by the loss of preexisting DNA though deletion, natural transformation provides a mechanism by which V. cholerae clones can gain access to the V. cholerae pan-genome.

Beach health advisories are issued if enterococci (ENT) densities exceed the 30-d geometric mean or single-sample water quality criteria. Current ENT enumeration procedures require 1 day of incubation; therefore, beach managers make policy decisions using 1-day-old data. This is tantamount to using a model that assumes ENT density on day t is equal to ENT density on day t-1. Research has shown that ENT densities vary over time scales shorterthan a day, calling into question the usefulness of the current model for decision-making. We created Dynamic Partial Least Square Regression (DPLSR) models for ENT at water quality monitoring stations within two adjacent marine recreational sites, Huntington State Beach (HSB) and Huntington City (HCB) Beach, California, using publicly available environmental data and tested whether these models overcome the drawbacks of the current model. The DPLSR models provide a better prediction of ENT than the current models based on comparisons of root-mean-square errors of prediction and the numbers of type 1 and 2 errors. We compared outcomes in terms of predicted illness, swimmers deterred from entering the water, and net benefits to swimmers for hypothetical management scenarios where beach advisories were issued based on (a) the previously collected sample's ENT density in conjunction with the two water quality criteria, and (b) predictions from DPLSR models in conjunction with the single-sample standard. At both HSB and HCB the DPLSR scenario produced a more favorable balance between illness prevention and recreational access. The results call into question the current method of beach management and show that model-informed decision-making and elimination of the geometric mean standard will aid beach managers in achieving more favorable outcomes in terms of illness and access than are presently achieved using 1-day-old measurements, especially at beaches where water quality problems are chronic.

Marine beach water quality is typically monitored in early morning once a week without respect to tidal condition. To assess the effect of tide on this public health warning system, we analyzed enterococci (ENT) data from 60 southern California marine beaches with differing geomorphology, orientation, and proximity to runoff sources. ENT concentrations during spring tides were significantly higher (p < 0.1) than those during neap tides at 50 of the beaches, and at the majority of these, water samples were also more than twice as likely to be out of compliance with the ENT single-sample standard during spring tides compared to neap tides. When tide range (spring/neap) and tide stage (ebb/flood) conditions were considered together, spring-ebb tides yielded the highest ENT concentrations and the greatest chance of exceeding the single-sample standard at the majority of beaches. The proximity to a terrestrial runoff source, the slope of the runoff source, the slope of the beach, and the orientation of the beach had minimal influence on the tidal modulation of ENT concentrations. The presence of spring and spring-ebb tide signals at such a great percentage of beaches suggests that tide should be considered in the design and interpretation of beach monitoring program data. It also suggests that ENT delivered by tidally forced sources other than terrestrial surficial runoff are widespread. Possibilities include ENT-laden groundwater (saline and fresh) from the beach aquifer as well as ENT-enriched sands, decaying wrack, and bird feces near the high water line.

Short-lived radium isotopes (223Ra and 224Ra) are used to investigate the potential association between groundwater discharge and microbial pollution at Huntington Beach, CA. We establish the tidally driven exchange of groundwater from the surficial beach aquifer across the beach face. Groundwater is found to be a source of nutrients (silica, inorganic nitrogen, and orthophosphate) to the surf zone, and these nutrients could possibly provide an environment for enhanced growth or increased persistence of fecal indicator bacteria (FIB). Ammonium and ortho-phosphate explain up to 12-20% of the variance in FIB levels in the surf zone. Elevated levels of FIB were only found in 1 of the 26 groundwater samples. However, FIB in the surf zone covary with radium at fortnightly, diurnal, and semi-diurnal tidal periods. In addition, radium accounts for up to 38% of the variance in log-FIB levels in the surf zone. A column experiment illustrates that Enterococcus suspended in Huntington Beach saline groundwater is not significantly filtered by sand collected from the field. This work establishes a mechanism for the subterranean delivery of FIB pollution to the surf zone from the surficial aquifer and presents evidence that supports an association between groundwater discharge and FIB.

To elucidate relationships between land cover and water quality along the central California coast, we collected monthly samples from 14 coastal waterway outlets representing various degrees of human development. Sites were distributed between three salinity categories, freshwater, estuarine, and marine, to better understand land cover-water quality relationships across a range of coastal aquatic ecosystems. Samples were analyzed for fecal indicator bacteria (FIB), dissolved nutrients, stable nitrogen isotopes in particulate organic matter, and chlorophyll a (chl a). Sediment samples from 11 sites were analyzed for the concentration of the anthropogenic organic contaminant perfluorooctane sulfonate and its precursors (ΣPFOS). While the data indicated impairment by nutrient, microbial, and organic contaminants at both agricultural and urban sites, the percentage of agricultural land cover was the most robust indicator of impairment, showing significant correlations (p < 0.05) to FIB, nutrient, chl a, and ΣPFOS levels. FIB densities were strongly influenced by salinity and were highest at sites dominated by agriculture and urbanization. Nutrient levels and chl a correlated to both agricultural and urban land use metrics as well. Positive correlations among FIB, nutrients, chi a, and ΣPFOS suggest a synergy between microbial, nutrient, and organic pollution. The results emphasize the importance of land management in protecting coastal water bodies and human health, and identify nutrient, microbial, and organic pollution as prevalent problems in coastal California water bodies. [ABSTRACT FROM AUTHOR]

The decay of sewage-sourced enterococci, Escherichia coli , three human-associated microbial source tracking (MST) markers, Salmonella , Campylobacter , and norovirus GII was measured in situ in coastal, marine waters. Experiments examined the effects of sunlight intensity and season on decay. Seawater was seeded with untreated sewage, placed into permeable dialysis bags, and deployed in the coastal ocean near the water surface, and at 18 cm, and 99 cm depths, to vary solar intensity, during winter and summer seasons. Microbial decay was modeled using a log-linear or shoulder log-linear decay model. Pathogen levels were too low in sewage to obtain kinetic parameters. Human-associated MST markers all decayed with approximately the same rate constant ( k ∼ 1.5 d −1 ) in all experimental treatments, suggesting markers could be detectable up to ∼6 days after a raw sewage spill. E. coli and enterococci (culturable and molecular marker) k significantly varied with season and depth; enterococci decayed faster at shallow depths and during the summer, while E. coli decayed faster at shallow depths and during the winter. Rate constants for MST markers and culturable FIB diverged except at the deepest depth in the water column potentially complicating the use of MST marker concentrations to allocate sources of FIB contamination. [ABSTRACT FROM AUTHOR]

A correction to the article "Mechanisms for Photoinactivation of Enterococcus faecalis in Seawater," that was published in a previous issue is presented.