An Atypical Presentation of Legionnaires' Disease.

Legionnaires' disease is an atypical pneumonia caused by Legionella pneumophila. Legionella species are found in freshwater sources and are transmitted through inhalation of contaminated aerosols. Patients commonly present with fever, chills, and cough. However, in immunosuppressed patients or severe cases, the disease can lead to multiorgan failure. In recent years, the incidence of Legionnaires' disease has drastically increased and unfortunately is commonly underdiagnosed. Gold-standard diagnosis is made through sputum cultures; however, urine Legionella antigen remains the most common test used for diagnosis. Goal-directed care includes antibiotics and supportive care. This case highlights a rare and unique presentation of Legionnaires' disease presenting with an elevated 2:1 aspartate aminotransferase to alanine transaminase pattern, typically seen with alcoholic hepatitis.

Assessing the defecation practices of unsheltered individuals and their contributions to microbial water quality in an arid, urban watershed.

Outdoor defecation by people experiencing homelessness is frequently perceived as a potentially large source of human fecal pollution and a significant source of health risk in urban waterbodies with recreational contact. The goal of this study was to count the number of people experiencing homelessness and quantifies their sanitation habits in an urban river corridor setting, then use this information for estimating human fecal pollutant loading on a watershed scale. Two types of census counts were conducted including periodic point-in-time counts over six years and weekly counts of encampments. While the population census varied from count-to-count, the range of population estimates in the river corridor varied from 109 to 349 individuals during the six-year span, which mirrored the weekly counts of encampments. A face-to-face survey of people experiencing homelessness assessed the sanitation habits of the unsheltered population (N = 63), including outdoor defecation frequency and containment practices. Overall, 95 % of survey respondents reported defecating outdoors; 36 % practiced outdoor defecation between 4 and 7 days/week and 27 % practiced outdoor defecation <1 day/week. Of those that did practice outdoor defecation, 75 % contained their feces in a bucket or bag, thereby limiting fecal material contributions to the river; 6.7 % reported defecating on low ground near the river that could wash off when flood waters rise during a storm event. Only a single survey respondent reported defecating directly into the river. Based on literature values for average HF183 output for an adult human, and the average rainfall in the urban watershed, the total watershed contribution of HF183 averaged 1.2 × 1010 gene copies per storm event (95 % CI: 0.9 × 1010-1.6 × 1010) along the 41 km stretch of river in this study. This human fecal loading estimate is at least two orders of magnitude less than cumulative HF183 loading from all human sources measured at the bottom of the watershed.

Wastewater surveillance for bacterial targets: current challenges and future goals.

Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods.IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs.

Correlation between wastewater and COVID-19 case incidence rates in major California sewersheds across three variant periods.

Monitoring for COVID-19 through wastewater has been used for adjunctive public health surveillance, with SARS-CoV-2 viral concentrations in wastewater correlating with incident cases in the same sewershed. However, the generalizability of these findings across sewersheds, laboratory methods, and time periods with changing variants and underlying population immunity has not been well described. The California Department of Public Health partnered with six wastewater treatment plants starting in January 2021 to monitor wastewater for SARS-CoV-2, with analyses performed at four laboratories. Using reported PCR-confirmed COVID-19 cases within each sewershed, the relationship between case incidence rates and wastewater concentrations collected over 14 months was evaluated using Spearman's correlation and linear regression. Strong correlations were observed when wastewater concentrations and incidence rates were averaged (10- and 7-day moving window for wastewater and cases, respectively, ρ = 0.73-0.98 for N1 gene target). Correlations remained strong across three time periods with distinct circulating variants and vaccination rates (winter 2020-2021/Alpha, summer 2021/Delta, and winter 2021-2022/Omicron). Linear regression revealed that slopes of associations varied by the dominant variant of concern, sewershed, and laboratory (ß = 0.45-1.94). These findings support wastewater surveillance as an adjunctive public health tool to monitor SARS-CoV-2 community trends.

Zeta diversity patterns in metabarcoded lotic algal assemblages as a tool for bioassessment.

Assessments of the ecological health of algal assemblages in streams typically focus on measures of their local diversity and classify individuals by morphotaxonomy. Such assemblages are often connected through various ecological processes, such as dispersal, and may be more accurately assessed as components of regional-, rather than local-scale assemblages. With recent declines in the costs of sequencing and computation, it has also become increasingly feasible to use metabarcoding to more accurately classify algal species and perform regional-scale bioassessments. Recently, zeta diversity has been explored as a novel method of constructing regional bioassessments for groups of streams. Here, we model the use of zeta diversity to investigate whether stream health can be determined by the landscape diversity of algal assemblages. We also compare the use of DNA metabarcoding and morphotaxonomy classifications in these zeta diversity-based bioassessments of regional stream health. From 96 stream samples in California, we used various orders of zeta diversity to construct models of biotic integrity for multiple assemblages of diatoms, as well as hybrid assemblages of diatoms in combination with soft-bodied algae, using taxonomy data generated with both DNA sequencing as well as traditional morphotaxonomic approaches. We compared our ability to evaluate the ecological health of streams with the performance of multiple algal indices of biological condition. Our zeta diversity-based models of regional biotic integrity were more strongly correlated with existing indices for algal assemblages classified using metabarcoding compared to morphotaxonomy. Metabarcoding for diatoms and hybrid algal assemblages involved rbcL and 18S V9 primers, respectively. Importantly, we also found that these algal assemblages, independent of the classification method, are more likely to be assembled under a process of niche differentiation rather than stochastically. Taken together, these results suggest the potential for zeta diversity patterns of algal assemblages classified using metabarcoding to inform stream bioassessments.

Relationship between coliphage and Enterococcus at southern California beaches and implications for beach water quality management.

Coliphage have been suggested as an alternative to fecal indicator bacteria for assessing recreational beach water quality, but it is unclear how frequently and at what types of beaches coliphage produces a different management outcome. Here we conducted side-by-side sampling of male-specific and somatic coliphage by the new EPA dead-end hollow fiber ultrafiltration (D-HFUF-SAL) method and Enterococcus at southern California beaches over two years. When samples were combined for all beach sites, somatic and male-specific coliphage both correlated with Enterococcus. When examined categorically, Enterococcus would have resulted in approximately two times the number of health advisories as somatic coliphage and four times that of male-specific coliphage,using recently proposed thresholds of 60 PFU/100 mL for somatic and 30 PFU/100 mL for male-specific coliphage. Overall, only 12% of total exceedances would have been for coliphage alone. Somatic coliphage exceedances that occurred in the absence of an Enterococcus exceedance were limited to a single site during south swell events, when this beach is known to be affected by nearby minimally treated sewage. Thus, somatic coliphage provided additional valuable health protection information, but may be more appropriate as a supplement to FIB measurements rather than as replacement because: (a) EPA-approved PCR methods for Enterococcus allow a more rapid response, (b) coliphage is more challenging owing to its greater sampling volume and laboratory time requirements, and (c) Enterococcus' long data history has yielded predictive management models that would need to be recreated for coliphage.

Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020-21 reveals widespread transcription of antibiotic resistance genes.

Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR). Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 - August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs (as measured through PERMANOVA, P < 0.001) and that the relative abundance of many individual AMR genes/variants increased over time (as measured with MaAsLin2, Padj < 0.05). Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed. To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large population's wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds.

Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020-21 reveals widespread transcription of antibiotic resistance genes.

Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically-important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR). Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 - August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs and that the relative abundance of many individual AMR genes/variants increased over time and may be connected to antibiotic use during the COVID-19 pandemic. Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed. To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large population's wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds.

Expression Analysis in a Dispersal-Fecundity Polyphenism Identifies Growth Regulators and Effectors.

Polyphenism allows organisms to respond to varying environmental conditions by adopting alternative collections of morphological traits, often leading to different reproductive strategies. In many insects, polyphenism affecting the development of flight trades dispersal ability for increased fecundity. The soapberry bug Jadera haematoloma (Hemiptera: Rhopalidae) exhibits wing polyphenism in response to juvenile nutritional resources and cohort density. Development of full-length wings and flight-capable thoracic muscles occurs more frequently in cohorts raised under low food density conditions, and these features are correlated to reduced female fecundity. Soapberry bugs represent an example of polyphenic dispersal-fecundity trade-off. Short-wing development is not sex-limited, and morphs can also differ in male fertility. We have previously shown, via a candidate gene approach, that manipulation of insulin signaling can alter the threshold for nutritional response and that changes in the activity of this pathway underlie, at least in part, differences in the polyphenic thresholds in different host-adapted populations of J. haematoloma. We now expand the examination of this system using transcriptome sequencing across a multidimensional matrix of life stage, tissue, sex, food density, and host population. We also examine the use of wing and thorax shape as factors modeling gene expression. In addition to insulin signaling, we find that components of the TOR, Hippo, Toll, and estrogen-related receptor pathways are differentially expressed in the thorax of polyphenic morphs. The transcription factor Sox14 was one of the few genes differentially expressed in the gonads of morphs, being up-regulated in ovaries. We identify two transcription factors as potential mediators of morph-specific male fertility differences. We also find that bugs respond to nutrient limitation with expression of genes linked to cuticle structure and spermatogenesis. These findings provide a broad perspective from which to view this nutrition-dependent polyphenism.

Assessing population-level stress through glucocorticoid hormone monitoring in wastewater.

Stress is oftentimes overlooked in societies, despite its life-threatening impact. Here, we assessed the feasibility of measuring endogenous stress hormones to estimate population-level stress by wastewater-based epidemiology (WBE). Two primary glucocorticoids, cortisol and cortisone, were monitored in wastewater by liquid chromatography tandem mass spectrometry (LC-MS/MS), to assess changes in these physiological markers of stress in a student population (n = 26,000 ± 7100) on a university campus in the southwestern U.S. Daily composite samples were collected for seven consecutive days each month during the Fall (Autumn) 2017 and Spring 2018 academic semesters (n = 134). Reproducible weekly patterns were seen in stress hormone excretion, with the highest levels occurring on Mondays (124 ± 44 µg d-1 per person) and Tuesdays (127 ± 54 µg d-1 per person) and the lowest on Sundays (87 ± 32 µg d-1 per person). Stress levels on weekdays (defined by class schedules Monday-Thursday) were significantly higher than on weekends (p < 0.05). During both Fall and Spring semesters, per person stress levels of these hormones were significantly higher (p < 0.05) during the first two months of each semester, 162 ± 28 µg d-1 per person (August), 104 ± 29 µg d-1 per person (September), 180 ± 14 µg d-1 per person (January), and 114 ± 54 µg d-1 per person (February) than in the remaining measured weeks in the semester, including finals week captured in both semesters. Overall Spring semester stress levels (113 ± 45 µg d-1 per person) were significantly higher than the Fall (94 ± 42 µg d-1 per person), p < 0.01. This study is the first to demonstrate the utility of endogenous biomarkers, specifically glucocorticoid hormones, to monitor population health status (in this instance community stress) in near real-time by wastewater assessments.

SARS-CoV-2 RNA is enriched by orders of magnitude in primary settled solids relative to liquid wastewater at publicly owned treatment works.

Wastewater-based epidemiology has gained attention throughout the world for detection of SARS-CoV-2 RNA in wastewater to supplement clinical testing. Raw wastewater consists of small particles, or solids, suspended in liquid. Methods have been developed to measure SARS-CoV-2 RNA in the liquid and the solid fraction of wastewater, with some studies reporting higher concentrations in the solid fraction. To investigate this relationship further, six laboratories collaborated to conduct a study across five publicly owned treatment works (POTWs) where both primary settled solids obtained from primary clarifiers and raw wastewater influent samples were collected and quantified for SARS-CoV-2 RNA. Settled solids and influent samples were processed by participating laboratories using their respective methods and retrospectively paired based on date of collection. SARS-CoV-2 RNA concentrations, on a mass equivalent basis, were higher in settled solids than in influent by approximately three orders of magnitude. Concentrations in matched settled solids and influent were positively and significantly correlated at all five POTWs. RNA concentrations in both settled solids and influent were correlated to COVID-19 incidence rates in the sewersheds and thus representative of disease occurrence; the settled solids methods appeared to produce a comparable relationship between SARS-CoV-2 RNA concentration measurements and incidence rates across all POTWs. Settled solids and influent methods showed comparable sensitivity, N gene detection frequency, and calculated empirical incidence rate lower limits. Analysis of settled solids for SARS-CoV-2 RNA has the advantage of using less sample volume to achieve similar sensitivity to influent methods.

Assessing cross-laboratory performance for quantifying coliphage using EPA Method 1642.

AIMS: Widespread adoption of the new U.S. Environmental Protection Agency (USEPA) Method 1642 for enumeration of coliphage in recreational water requires demonstration that laboratories consistently meet internal method performance goals and yield results that are consistent across laboratories. METHODS AND RESULTS: Here we assess the performance of six laboratories processing a series of blind wastewater- and coliphage-spiked samples along with laboratory blanks. All laboratories met the method-defined recovery requirements when performance was averaged across samples, with the few failures on individual samples mostly occurring for less-experienced laboratories on the initial samples processed. Failures that occurred on later samples were generally attributed to easily correctable activities. Failure rates were higher for somatic vs. F+ coliphage, attributable to the more stringent performance criteria associated with somatic coliphage. There was no difference in failure rate between samples prepared in a marine water matrix compared to that in phosphate-buffered saline. CONCLUSIONS: Variation among laboratories was similar to that previously reported for enterococci, the current bacterial indicator used for evaluating beach water quality for public health protection. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that laboratory performance is not an inhibitor to the adoption of coliphage as a new indicator for assessing recreational health risk.

Comparative meta-analysis of organic contaminants in sewage sludge from the United States and China.

Characterizing the occurrence of organic contaminants (OCs) of environmental health concern in municipal sewage sludges is essential for safe handling and disposal of these abundant materials. This meta-analysis aimed to (i) summarize the extent of studies performed on the chemical composition of sewage sludges from China and the U.S., the world's two largest chemical producers, (ii) identify chemical groups of priority concern, (iii) quantitatively compare chemical abundance in sludge between nations, (iv) determine longitudinal contaminant accumulation trends in sludge, and (v) identify data gaps with regard to OC concentrations in sludge. A literature search was conducted on concentrations of OCs in U.S. sludges produced during treatment of domestic and industrial wastewater and compared statistically to contaminant levels in Chinese sludge abstracted from a recently established database. Longitudinal trends of OC occurrence were interpreted in the context of national chemical production, usage statistics, and regulations. A total of 105 studies on OCs in U.S. sewage sludge were found, while a total of 159 had been found in China. Among 1175 OCs monitored for, 23% of all analytes had been monitored in both countries (n = 269), 41% (n = 480) in China only, and the remaining 36% (n = 426) in the U.S. only. On average, concentrations of OCs were 4.0 times higher in U.S. than in Chinese sewage sludge, with the highest detection being observed for alkylphenol ethoxylates. Data from a new binational database on toxic OCs in sewage sludges suggest and reiterates the need for additional chemical monitoring in both countries, risk assessments for emerging OCs contained in sludges destined for application on land, and stronger enforcement of sludge disposal restrictions in China, where as much as 40% of sludge is currently being dumped improperly.

RNA Viromics of Southern California Wastewater and Detection of SARS-CoV-2 Single-Nucleotide Variants.

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the coronavirus disease 2019 (COVID-19) pandemic to track disease burden through analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the health care system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven wastewater treatment plants (WTPs), collected from August 2020 to January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single-nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater, and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically important SARS-CoV-2 variants along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. IMPORTANCE Wastewater-based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the coronavirus disease 2019 (COVID-19) pandemic to track the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) without adding burden to health care systems. In this study, we used metatranscriptomics and reverse transcription-droplet digital PCR (RT-ddPCR) to assay RNA viruses across Southern California wastewater from August 2020 to January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single-nucleotide variants through sequencing. Likewise, wastewater treatment plants (WTPs) harbored different viromes, and we detected other human pathogens, such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

Standardizing data reporting in the research community to enhance the utility of open data for SARS-CoV-2 wastewater surveillance.

SARS-CoV-2 RNA detection in wastewater is being rapidly developed and adopted as a public health monitoring tool worldwide. With wastewater surveillance programs being implemented across many different scales and by many different stakeholders, it is critical that data collected and shared are accompanied by an appropriate minimal amount of metainformation to enable meaningful interpretation and use of this new information source and intercomparison across datasets. While some databases are being developed for specific surveillance programs locally, regionally, nationally, and internationally, common globally-adopted data standards have not yet been established within the research community. Establishing such standards will require national and international consensus on what metainformation should accompany SARS-CoV-2 wastewater measurements. To establish a recommendation on minimum information to accompany reporting of SARS-CoV-2 occurrence in wastewater for the research community, the United States National Science Foundation (NSF) Research Coordination Network on Wastewater Surveillance for SARS-CoV-2 hosted a workshop in February 2021 with participants from academia, government agencies, private companies, wastewater utilities, public health laboratories, and research institutes. This report presents the primary two outcomes of the workshop: (i) a recommendation on the set of minimum meta-information that is needed to confidently interpret wastewater SARS-CoV-2 data, and (ii) insights from workshop discussions on how to improve standardization of data reporting.

A Combined Digital PCR and Next Generation DNA-Sequencing Based Approach for Tracking Nearshore Pollutant Dynamics Along the Southwest United States/Mexico Border.

Ocean currents, multiple fecal bacteria input sources, and jurisdictional boundaries can complicate pollution source tracking and associated mitigation and management efforts within the nearshore coastal environment. In this study, multiple microbial source tracking tools were employed to characterize the impact and reach of an ocean wastewater treatment facility discharge in Mexico northward along the coast and across the Southwest United States- Mexico Border. Water samples were evaluated for fecal indicator bacteria (FIB), Enterococcus by culture-based methods, and human-associated genetic marker (HF183) and Enterococcus by droplet digital polymerase chain reaction (ddPCR). In addition, 16S rRNA gene sequence analysis was performed and the SourceTracker algorithm was used to characterize the bacterial community of the wastewater treatment plume and its contribution to beach waters. Sampling dates were chosen based on ocean conditions associated with northern currents. Evidence of a gradient in human fecal pollution that extended north from the wastewater discharge across the United States/Mexico border from the point source was observed using human-associated genetic markers and microbial community analysis. The spatial extent of fecal contamination observed was largely dependent on swell and ocean conditions. These findings demonstrate the utility of a combination of molecular tools for understanding and tracking specific pollutant sources in dynamic coastal water environments.

Pathogenic Vibrio Species Are Associated with Distinct Environmental Niches and Planktonic Taxa in Southern California (USA) Aquatic Microbiomes.

Interactions between vibrio bacteria and the planktonic community impact marine ecology and human health. Many coastal Vibrio spp. can infect humans, representing a growing threat linked to increasing seawater temperatures. Interactions with eukaryotic organisms may provide attachment substrate and critical nutrients that facilitate the persistence, diversification, and spread of pathogenic Vibrio spp. However, vibrio interactions with planktonic organisms in an environmental context are poorly understood. We quantified the pathogenic Vibrio species V. cholerae, V. parahaemolyticus, and V. vulnificus monthly for 1 year at five sites and observed high abundances, particularly during summer months, with species-specific temperature and salinity distributions. Using metabarcoding, we established a detailed profile of both prokaryotic and eukaryotic coastal microbial communities. We found that pathogenic Vibrio species were frequently associated with distinct eukaryotic amplicon sequence variants (ASVs), including diatoms and copepods. Shared environmental conditions, such as high temperatures and low salinities, were associated with both high concentrations of pathogenic vibrios and potential environmental reservoirs, which may influence vibrio infection risks linked to climate change and should be incorporated into predictive ecological models and experimental laboratory systems. IMPORTANCE Many species of coastal vibrio bacteria can infect humans, representing a growing health threat linked to increasing seawater temperatures. However, their interactions with surrounding microbes in the environment, especially eukaryotic organisms that may provide nutrients and attachment substrate, are poorly understood. We quantified three pathogenic Vibrio species monthly for a duration of 1 year, finding that all three species were abundant and exhibited species-specific temperature and salinity distributions. Using metabarcoding, we investigated associations between these pathogenic species and prokaryotic and eukaryotic microbes, revealing genus and amplicon sequence variant (ASV)-specific relationships with potential functional implications. For example, pathogenic species were frequently associated with chitin-producing eukaryotes, such as diatoms in the genus Thalassiosira and copepods. These associations between high concentrations of pathogenic vibrios and potential environmental reservoirs should be considered when predicting infection risk and developing ecologically relevant model systems.

Application of ddPCR for detection of Enterococcus spp. in coastal water quality monitoring.

Droplet digital polymerase chain reaction (ddPCR) was evaluated for the detection of fecal indicator bacteria (FIB), Enterococcus spp., in San Diego County beach water samples collected under diverse conditions, from multiple pollution sources, as part of regulatory monitoring activities over 20 months. Two US EPA-approved methods, qPCR (EPA 1609.1) and Enterolert (SM9230D), were used as reference comparator methods. A total of 361 samples were assayed by both ddPCR and qPCR and yielded an acceptable Index of Agreement (IA) of 0.89, based on EPA Site-Specific analysis guidelines. A Pearson's correlation coefficient of r = 0.87 (p < 0.001), further indicated a strong relationship between the methods results. From the 361 samples, 185 split samples with ddPCR and Enterolert values within the limits of quantification, were used as a 'training' data set to derive an intrinsic copy number equation (ICE) for scaling ddPCR gene copy number to Enterolert most probable number (MPN). Of the 1993 samples that comprised the complete 'test' data set assayed by ddPCR and Enterolert, 1086 generated results that fell within the limits of quantification for Enterolert and yielded an overall IA of 0.64. Re-analysis using median as a measure of central tendency to account for significant skewing of Enterolert data yielded an IA of 0.72. Beach grouping-specific IA values ranged from 0.63 to 0.93. Pearson's correlation coefficient, r, ranged from 0.13 to 0.94 within beach groupings and generated a combined value of 0.60 for all groupings. Using the ICE, a ddPCR advisory threshold of 1413 DNA copy number/100 mL was empirically determined to be the equivalent to the California Enterolert beach action threshold of 104 MPN/100 mL, based on comparison with all 1993 paired ddPCR and Enterolert results. Using the 1413 DNA copy number/100 mL as a beach action threshold for ddPCR resulted in a 90.4% agreement with Enterolert (6.0% false negative and 3.7% false positive). Together these findings support the conclusion that ddPCR readouts align closely with Enterolert MPN for identifying FIB exceedance levels of Enterococcus spp. in coastal waters of San Diego, CA.

Metagenomics of Wastewater Influent from Southern California Wastewater Treatment Facilities in the Era of COVID-19.

Sequencing wastewater may be useful for detecting pathogens and assaying microbial water quality. We concentrated, extracted, and sequenced nucleic acids from 17 composite influent wastewater samples spanning seven southern California wastewater treatment facilities in May 2020. Bacteria were the most proportionally abundant taxonomic group present, followed by viruses and archaea.