A cryptic plasmid is among the most numerous genetic elements in the human gut.

Plasmids are extrachromosomal genetic elements that often encode fitness-enhancing features. However, many bacteria carry "cryptic" plasmids that do not confer clear beneficial functions. We identified one such cryptic plasmid, pBI143, which is ubiquitous across industrialized gut microbiomes and is 14 times as numerous as crAssphage, currently established as the most abundant extrachromosomal genetic element in the human gut. The majority of mutations in pBI143 accumulate in specific positions across thousands of metagenomes, indicating strong purifying selection. pBI143 is monoclonal in most individuals, likely due to the priority effect of the version first acquired, often from one's mother. pBI143 can transfer between Bacteroidales, and although it does not appear to impact bacterial host fitness in vivo, it can transiently acquire additional genetic content. We identified important practical applications of pBI143, including its use in identifying human fecal contamination and its potential as an alternative approach to track human colonic inflammatory states.

Separating signal from noise in wastewater data: An algorithm to identify community-level COVID-19 surges in real time.

Wastewater monitoring has provided health officials with early warnings for new COVID-19 outbreaks, but to date, no approach has been validated to distinguish signal (sustained surges) from noise (background variability) in wastewater data to alert officials to the need for heightened public health response. We analyzed 62 wk of data from 19 sites participating in the North Carolina Wastewater Monitoring Network to characterize wastewater metrics around the Delta and Omicron surges. We found that wastewater data identified outbreaks 4 to 5 d before case data (reported on the earlier of the symptom start date or test collection date), on average. At most sites, correlations between wastewater and case data were similar regardless of how wastewater concentrations were normalized and whether calculated with county-level or sewershed-level cases, suggesting that officials may not need to geospatially align case data with sewershed boundaries to gain insights into disease transmission. Although wastewater trend lines captured clear differences in the Delta versus Omicron surge trajectories, no single wastewater metric (detectability, percent change, or flow-population normalized viral concentrations) reliably signaled when these surges started. After iteratively examining different combinations of these three metrics, we developed the Covid-SURGE (Signaling Unprecedented Rises in Groupwide Exposure) algorithm, which identifies unprecedented signals in the wastewater data. With a true positive rate of 82%, a false positive rate of 7%, and strong performance during both surges and in small and large sites, our algorithm provides public health officials with an automated way to flag community-level COVID-19 surges in real time.

Genetic Determinants of Escherichia coli Survival in Beach Sand.

Escherichia coli contain a high level of genetic diversity and are generally associated with the guts of warm-blooded animals but have also been isolated from secondary habitats outside hosts. We used E. coli isolates from previous in situ microcosm experiments conducted under actual beach conditions and performed population-level genomic analysis to identify accessory genes associated with survival within the beach sand environment. E. coli strains capable of surviving had been selected for by seeding isolates originating from sand, sewage, and gull waste (n = 528; 176 from each source) into sand, which was sealed in microcosm chambers and buried for 45 days in the backshore beach of Lake Michigan. In the current work, survival-associated genes were identified by comparing the pangenome of viable E. coli populations at the end of the microcosm experiment with the original isolate collection and identifying loci enriched in the out put samples. We found that environmental survival was associated with a wide variety of genetic factors, with the majority corresponding to metabolism enzymes and transport proteins. Of the 414 unique functions identified, most were present across E. coli phylogroups, except B2 which is often associated with human pathogens. Gene modules that were enriched in surviving populations included a betaine biosynthesis pathway, which produces an osmoprotectant, and the GABA (gamma-aminobutyrate) biosynthesis pathway, which aids in pH homeostasis and nutrient use versatility. Overall, these results demonstrate that the genetic flexibility within this species allows for survival in the environment for extended periods. IMPORTANCE Escherichia coli is commonly used as an indicator of recent fecal pollution in recreational water despite its known ability to survive in secondary environments, such as beach sand. These long-term survivors from sand reservoirs can be introduced into the water column through wave action or runoff during precipitation events, thereby impacting the perception of local water quality. Current beach monitoring methods cannot differentiate long-term environmental survivors from E. coli derived from recent fecal input, resulting in inaccurate monitoring results and unnecessary beach closures. This work identified the genetic factors that are associated with long-term survivors, providing insight into the mechanistic basis for E. coli accumulation in beach sand. A greater understanding of the intrinsic ability of E. coli to survive long-term and conditions that promote such survival will provide evidence of the limitations of beach water quality assessments using this indicator.

Influence of Nutrients and the Native Community on E. coli Survival in the Beach Environment.

Previous research has identified E. coli populations that persist in freshwater beach sand distinct from fecal pollution events. This work identifies factors that influence the survival of E. coli in sand using laboratory microcosms to replicate beach conditions. Microcosms were deployed to examine the effect of genetic background, competition with native microbial community, and increased nutrient concentrations on E. coli survival. Survival was comparable between the phylotypes B1 and B2, however, deficiency of stress response greatly reduced survival. In the absence of the native community under nutrient conditions comparable to those observed in sand, E. coli cell densities remained within an order of magnitude of initial concentrations after 5 weeks of incubation. Increased nitrogen was associated with decreased decay rates in the first 2 weeks, and increased carbon appeared to provide an advantage at later time points. However, the highest survival was found with the addition of both carbon and nitrogen. Native sand seeded with fresh Cladophora maintained higher concentrations of E. coli, compared to sand containing decayed Cladophora or no Cladophora. Our findings demonstrate persistent E. coli populations in sand can be affected by the availability of carbon and nitrogen, the ability to regulate stress, and the presence of algal mats (i.e., Cladophora). Further, this work suggests that the native microbial communities may modulate survival by outcompeting E. coli for nutrients. IMPORTANCE Current monitoring for fecal pollution does not account for persistent E. coli populations in freshwater sand, which can result in higher concentrations in water when no threat to human health is present. This work examined the drivers for persistent E. coli populations in sand to aid beach management techniques. We examined the influence of nutrients, including localized sources such as stranded Cladophora, on E. coli populations. We found the major determinant of E. coli survival in freshwater beach sand was the addition of nutrients, specifically carbon and nitrogen concentrations 10-fold higher than baseline concentrations on beaches. This work provides the framework for identifying pollution sources that can promote E. coli survival in sand through the characterization of carbon and nitrogen content, which can be incorporated into beach management techniques. Through this improved knowledge, we can begin to understand E. coli fluctuations in water due to resuspension from sand into water.

SARS-CoV-2 Wastewater Surveillance for Public Health Action.

Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has garnered extensive public attention during the coronavirus disease pandemic as a proposed complement to existing disease surveillance systems. Over the past year, methods for detection and quantification of SARS-CoV-2 viral RNA in untreated sewage have advanced, and concentrations in wastewater have been shown to correlate with trends in reported cases. Despite the promise of wastewater surveillance, for these measurements to translate into useful public health tools, bridging the communication and knowledge gaps between researchers and public health responders is needed. We describe the key uses, barriers, and applicability of SARS-CoV-2 wastewater surveillance for supporting public health decisions and actions, including establishing ethics consideration for monitoring. Although wastewater surveillance to assess community infections is not a new idea, the coronavirus disease pandemic might be the initiating event to make this emerging public health tool a sustainable nationwide surveillance system, provided that these barriers are addressed.

Selective survival of Escherichia coli phylotypes in freshwater beach sand.

Escherichia coli is used as an indicator of fecal pollution at beaches despite evidence of long-term survival in sand. This work investigated the basis for survival of E. coli through field microcosm experiments and phylotypic characterization of more than >1400 E. coli isolated from sand, sewage, and gulls, enabling identification of long-surviving populations and environmental drivers of their persistence. Microcosms containing populations of E. coli from each source (n=176) were buried in the backshore of Lake Michigan for 45 & 96 days under several different nutrient treatments, including unaltered native sand, sterile autoclaved sand and baked nutrient depleted sand. Availability of carbon and nitrogen and competition with the indigenous community were major factors that influenced E. coli survival. E. coli Clermont phylotypes B1 and A were the most dominant phylotypes surviving seasonally (>6 weeks), regardless of source and nutrient treatment, whereas cryptic clade and D/E phylotypes survived over winter (>300 days). Autoclaved sand, presumably supplying nutrients through increased availability, promoted growth and the presence of the indigenous microbial community reduced this effect. Screening of 849 sand E. coli from four freshwater beaches demonstrated that B1, but also D/E, were the most common phylotypes recovered. Analysis by qPCR for the Gull2, Lachno3 and HB human markers demonstrated only 25% of the samples had evidence of gull waste and none of the samples had evidence of human waste. These findings suggest prevalence of E. coli in the sand could be attributed more to long term surviving populations than to new fecal pollution.IMPORTANCE Fecal pollution monitoring still relies upon the enumeration of E. coli, despite the fact that this organism can survive for prolonged periods and has been shown to be easily transported from sand into surrounding waters through waves and runoff, thus no longer represents recent fecal pollution events. Here, we experimentally demonstrate that regardless of host source, certain genetically distinct subgroups, or phylotypes, survive longer than others under conditions typical of Great Lakes beach sites. We found nutrients were a major driver of survival and could actually promote growth, and the presence of native microorganisms modulated these effects. These insights into the dynamics and drivers of survival will improve the interpretation of E. coli measurements at beaches and inform strategies that could focus on reducing nutrient inputs to beaches or maintaining a robust natural microbiome in beach sand.

Optical Properties of Water for Prediction of Wastewater Contamination, Human-Associated Bacteria, and Fecal Indicator Bacteria in Surface Water at Three Watershed Scales.

Relations between spectral absorbance and fluorescence properties of water and human-associated and fecal indicator bacteria were developed for facilitating field sensor applications to estimate wastewater contamination in waterways. Leaking wastewater conveyance infrastructure commonly contaminates receiving waters. Methods to quantify such contamination can be time consuming, expensive, and often nonspecific. Human-associated bacteria are wastewater specific but require discrete sampling and laboratory analyses, introducing latency. Human sewage has fluorescence and absorbance properties different than those of natural waters. To assist real-time field sensor development, this study investigated optical properties for use as surrogates for human-associated bacteria to estimate wastewater prevalence in environmental waters. Three spatial scales were studied: Eight watershed-scale sites, five subwatershed-scale sites, and 213 storm sewers and open channels within three small watersheds (small-scale sites) were sampled (996 total samples) for optical properties, human-associated bacteria, fecal indicator bacteria, and, for selected samples, human viruses. Regression analysis indicated that bacteria concentrations could be estimated by optical properties used in existing field sensors for watershed and subwatershed scales. Human virus occurrence increased with modeled human-associated bacteria concentration, providing confidence in these regressions as surrogates for wastewater contamination. Adequate regressions were not found for small-scale sites to reliably estimate bacteria concentrations likely due to inconsistent local sanitary sewer inputs.

Ecological and Technical Mechanisms for Cross-Reaction of Human Fecal Indicators with Animal Hosts.

Quantitative PCR (qPCR) assays for human/sewage marker genes have demonstrated sporadic positive results in animal feces despite their high specificities to sewage and human feces. It is unclear whether these positive reactions are caused by true occurrences of microorganisms containing the marker gene (i.e., indicator organisms) or nonspecific amplification (false positive). The distribution patterns of human/sewage indicator organisms in animals have not been explored in depth, which is crucial for evaluating a marker gene's true- or false-positive reactions. Here, we analyzed V6 region 16S rRNA gene sequences from 257 animal fecal samples and tested a subset of 184 using qPCR for human/sewage marker genes. Overall, specificities of human/sewage marker genes within sequencing data were 99.6% (BacV6-21), 96.9% (Lachno3), and 96.1% (HF183, indexed by its inferred V6 sequence). Occurrence of some true cross-reactions was associated with atypical compositions of organisms within the genera Blautia or Bacteroides For human/sewage marker qPCR assays, specificities were 96.7% (HF183/Bac287R), 96.2% (BacV6-21), 95.6% (human Bacteroides [HB]), and 94.0% (Lachno3). Select assays duplexed with either Escherichia coli or Enterococcus spp. were also validated. Most of the positive qPCR results in animals were low level and, on average, 2 orders of magnitude lower than the copy numbers of E. coli and Enterococcus spp. The lower specificity in qPCR assays compared to sequencing data was mainly caused by amplification of sequences highly similar to the marker gene and not the occurrence of the exact marker sequence in animal fecal samples.IMPORTANCE Identifying human sources of fecal pollution is critical to remediate sanitation concerns. Large financial investments are required to address these concerns; therefore, a high level of confidence in testing results is needed. Human fecal marker genes validated in this study showed high specificity in both sequencing data and qPCR results. Human marker sequences were rarely found in individual animals, and in most cases, the animals had atypical microbial communities. Sequencing also revealed the presence of closely related organisms that could account for nonspecific amplification in certain assays. Both the true cross-reactions and the nonspecific amplification had low signals well below E. coli or Enterococcus levels and likely would not impact the assay's ability to reliably detect human fecal pollution. No animal source had multiple human/sewage marker genes present; therefore, using a combination of marker genes would increase the confidence of human fecal pollution detection.

Highly Specific Sewage-Derived Bacteroides Quantitative PCR Assays Target Sewage-Polluted Waters.

The identification of sewage contamination in water has primarily relied on the detection of human-associated Bacteroides using markers within the V2 region of the 16S rRNA gene. Despite the establishment of multiple assays that target the HF183 cluster (i.e., Bacteroides dorei) and other Bacteroides organisms (e.g., Bacteroides thetaiotaomicron), the potential for more human-associated markers in this genus has not been explored in depth. We examined the Bacteroides population structure in sewage and animal hosts across the V4V5 and V6 hypervariable regions. Using near-full-length cloned sequences, we identified the sequences in the V4V5 and V6 hypervariable regions that are linked to the HF183 marker in the V2 region and found these sequences were present in multiple animals. In addition, the V4V5 and V6 regions contained human fecal marker sequences for organisms that were independent of the HF183 cluster. The most abundant Bacteroides in untreated sewage was not human associated but pipe derived. Two TaqMan quantitative PCR (qPCR) assays targeting the V4V5 and V6 regions of this organism were developed. Validation studies using fecal samples from seven animal hosts (n = 76) and uncontaminated water samples (n = 30) demonstrated the high specificity of the assays for sewage. Freshwater Bacteroides were also identified in uncontaminated water samples, demonstrating that measures of total Bacteroides do not reflect fecal pollution. A comparison of two previously described human Bacteroides assays (HB and HF183/BacR287) in municipal wastewater influent and sewage-contaminated urban water samples revealed identical results, illustrating the assays target the same organism. The detection of sewage-derived Bacteroides provided an independent measure of sewage-impacted waters.IMPORTANCEBacteroides are major members of the gut microbiota, and host-specific organisms within this genus have been used extensively to gain information on pollution sources. This study provides a broad view of the population structure of Bacteroides within sewage to contextualize the well-studied HF183 marker for a human-associated Bacteroides The study also delineates host-specific sequence patterns across multiple hypervariable regions of the 16S rRNA gene to improve our ability to use sequence data to assess water quality. Here, we demonstrate that regions downstream of the HF183 marker are nonspecific but other potential human-associated markers are present. Furthermore, we show the most abundant Bacteroides in sewage is free living, rather than host associated, and specifically found in sewage. Quantitative PCR assays that target organisms specific to sewer pipes offer measures that are independent of the human microbiome for identifying sewage pollution in water.

Host Specificity and Sensitivity of Established and Novel Sewage-Associated Marker Genes in Human and Nonhuman Fecal Samples.

Microbial source tracking (MST) methods measure fecal contamination levels and identify possible sources using quantitative PCR (qPCR) that targets host-associated fecal microorganisms. To date, most established MST assays for human sources, especially bacterial markers, have shown some nonhuman host cross-reactions. Recently developed assays, such as the crAssphage CPQ_056, Lachnospiraceae Lachno3, and Bacteroides BacV6-21, have more limited information on host sensitivity and host specificity for human or sewage sources, particularly in countries other than the United States. In this study, we rigorously evaluated six sewage-associated MST assays (i.e., Bacteroides HF183, human adenovirus [HAdV], human polyomavirus [HPyV], crAssphage CPQ_056, Lachno3, and BacV6-21) to show advantages and disadvantages of their applications for MST. A total of 29 human and 3 sewage samples and 360 nonhuman fecal samples across 14 hosts collected from a subtropical region of Australia were tested for marker host specificity, host sensitivity, and concentrations. All sewage samples were positive for all six marker genes tested in this study. Bacterial markers were more prevalent than viral markers in human feces. Testing against animal hosts showed human feces (or sewage)-associated marker gene specificity was HAdV (1.00) > HPyV (0.99) > crAssphage CPQ_056 (0.98) > HF183 (0.96) > Lachno3 (0.95) > BacV6-21 (0.90), with marker concentrations in some animal fecal samples being 3 to 5 orders of magnitude lower than those in sewage. When considering host specificity, sensitivity, and concentrations in source samples, the HF183, Lachno3, and crAssphage CPQ_056 tests were the most suitable assays in this study for sewage contamination tracking in subtropical waters of Australia.IMPORTANCE Large financial investments are required to remediate fecal contamination sources in waterways, and accurate results from field studies are crucial to build confidence in MST approaches. Host specificity and sensitivity are two main performance characteristics for consideration when choosing MST assays. Ongoing efforts for marker assay validation will improve interpretation of results and could shed light on patterns of occurrence in nontarget hosts that might explain the underlying drivers of cross-reaction of certain markers. For field applications, caution should be taken to choose appropriate MST marker genes and assays based on available host specificity and sensitivity data and background knowledge of the contaminating sources in the study area. Since many waterborne pathogens are viruses, employing both viral and bacterial markers in investigations could provide insight into contamination dynamics and ecological behavior in the environment. Therefore, combined usage of marker assays is recommended for more accurate and informative sewage contamination detection and fecal source resolution.

High levels of sewage contamination released from urban areas after storm events: A quantitative survey with sewage specific bacterial indicators.

BACKGROUND: Past studies have demonstrated an association between waterborne disease and heavy precipitation, and climate change is predicted to increase the frequency of these types of intense storm events in some parts of the United States. In this study, we examined the linkage between rainfall and sewage contamination of urban waterways and quantified the amount of sewage released from a major urban area under different hydrologic conditions to identify conditions that increase human risk of exposure to sewage. METHODS AND FINDINGS: Rain events and low-flow periods were intensively sampled to quantify loads of sewage based on two genetic markers for human-associated indicator bacteria (human Bacteroides and Lachnospiraceae). Samples were collected at a Lake Michigan estuary and at three river locations immediately upstream. Concentrations of indicators were analyzed using quantitative polymerase chain reaction (qPCR), and loads were calculated from streamflow data collected at each location. Human-associated indicators were found during periods of low flow, and loads increased one to two orders of magnitude during rain events from stormwater discharges contaminated with sewage. Combined sewer overflow (CSO) events increased concentrations and loads of human-associated indicators an order of magnitude greater than heavy rainfall events without CSO influence. Human-associated indicator yields (load per km2 of land per day) were related to the degree of urbanization in each watershed. Contamination in surface waters were at levels above the acceptable risk for recreational use. Further, evidence of sewage exfiltration from pipes threatens drinking water distribution systems and source water. While this study clearly demonstrates widespread sewage contamination released from urban areas, a limitation of this study is understanding human exposure and illness rates, which are dependent on multiple factors, and gaps in our knowledge of the ultimate health outcomes. CONCLUSIONS: With the prediction of more intense rain events in certain regions due to climate change, sewer overflows and contamination from failing sewer infrastructure may increase, resulting in increases in waterborne pathogen burdens in waterways. These findings quantify hazards in exposure pathways from rain events and illustrate the additional stress that climate change may have on urban water systems. This information could be used to prioritize efforts to invest in failing sewer infrastructure and create appropriate goals to address the health concerns posed by sewage contamination from urban areas.

Human-Associated Lachnospiraceae Genetic Markers Improve Detection of Fecal Pollution Sources in Urban Waters.

The human microbiome contains many organisms that could potentially be used as indicators of human fecal pollution. Here we report the development of two novel human-associated genetic marker assays that target organisms within the family Lachnospiraceae Next-generation sequencing of the V6 region of the 16S rRNA gene from sewage and animal stool samples identified 40 human-associated marker candidates with a robust signal in sewage and low or no occurrence in samples from nonhuman hosts. Two were chosen for quantitative PCR (qPCR) assay development using longer sequences (the V2 to V9 regions) generated from clone libraries. Validation of these assays with these markers, designated Lachno3 and Lachno12, was performed using fecal samples (n = 55) from cat, dog, pig, cow, deer, and gull sources, and the results were compared with those of established host-associated assays (the Lachno2 marker and two human Bacteroides markers, the HB and HF183/BacR287). Each of the established assays cross-reacted with samples from at least one other animal species, including animals common in urban areas. The Lachno3 and Lachno12 markers were primarily human associated; however, the Lachno12 marker demonstrated low levels of cross-reactivity with samples from select cows and nonspecific amplification with samples from pigs. This limitation may not be problematic when testing urban waters. These novel markers resolved ambiguous results from previous investigations of stormwater-impacted waters, demonstrating their utility. The complexity of the microbiome in humans and animals suggests that no single organism is strictly specific to humans, and the use of multiple complementary markers in combination will provide the highest resolution and specificity for assessing fecal pollution sources.IMPORTANCE Traditional fecal indicator bacteria do not distinguish animal from human fecal pollution, which is necessary to evaluate health risks and mitigate pollution sources. Assessing water in urban areas is challenging, since the water can be impacted by sewage, which has a high likelihood of carrying human pathogens, as well as pet and urban wildlife waste. We demonstrate that the Lachno3 and Lachno12 markers are human associated and highly specific for the detection of human fecal pollution from urban sources, offering reliable identification of fecal pollution sources in urban waters.

Patterns of Host-Associated Fecal Indicators Driven by Hydrology, Precipitation, and Land Use Attributes in Great Lakes Watersheds.

Fecal contamination from sewage and agricultural runoff is a pervasive problem in Great Lakes watersheds. Most work examining fecal pollution loads relies on discrete samples of fecal indicators and modeling land use. In this study, we made empirical measurements of human and ruminant-associated fecal indicator bacteria and combined these with hydrological measurements in eight watersheds ranging from predominantly forested to highly urbanized. Flow composited river samples were collected over low-flow ( n = 89) and rainfall or snowmelt runoff events ( n = 130). Approximately 90% of samples had evidence of human fecal pollution, with highest loads from urban watersheds. Ruminant indicators were found in ∼60-100% of runoff-event samples in agricultural watersheds, with concentrations and loads related to cattle density. Rain depth, season, agricultural tile drainage, and human or cattle density explained variability in daily flux of human or ruminant indicators. Mapping host-associated indicator loads to watershed discharge points sheds light on the type, level, and possible health risk from fecal pollution entering the Great Lakes and can inform total maximum daily load implementation and other management practices to target specific fecal pollution sources.

Human-Associated Indicator Bacteria and Human-Specific Viruses in Surface Water: A Spatial Assessment with Implications on Fate and Transport.

Hydrologic, seasonal, and spatial variability of sewage contamination was studied at six locations within a watershed upstream from water reclamation facility (WRF) effluent to define relative loadings of sewage from different portions of the watershed. Fecal pollution from human sources was spatially quantified by measuring two human-associated indicator bacteria (HIB) and eight human-specific viruses (HSV) at six stream locations in the Menomonee River watershed in Milwaukee, Wisconsin from April 2009 to March 2011. A custom, automated water sampler, which included HSV filtration, was deployed at each location and provided unattended, flow-weighted, large-volume (30-913 L) sampling. In addition, wastewater influent samples were composited over discrete 7 day periods from the two Milwaukee WRFs. Of the 8 HSV, only 3 were detected, present in up to 38% of the 228 stream samples, while at least 1 HSV was detected in all WRF influent samples. HIB occurred more often with significantly higher concentrations than the HSV in stream and WRF influent samples ( p < 0.05). HSV yield calculations showed a loss from upstream to the most-downstream sub-watershed of the Menomonee River, and in contrast, a positive HIB yield from this same sub-watershed emphasizes the complexity in fate and transport properties between HSV and HIB. This study demonstrates the utility of analyzing multiple HSV and HIB to provide a weight-of-evidence approach for assessment of fecal contamination at the watershed level, provides an assessment of relative loadings for prioritizing areas within a watershed, and demonstrates how loadings of HSV and HIB can be inconsistent, inferring potential differences in fate and transport between the two indicators of human fecal presence.

A Randomized Controlled Trial of an Intensive Nutrition Intervention Versus Standard Nutrition Care to Avoid Excess Weight Gain After Kidney Transplantation: The INTENT Trial.

OBJECTIVE: Excessive weight gain is common after kidney transplantation and increases cardiovascular risk. The aim of this randomized controlled trial was to determine whether an intensive nutrition and exercise intervention delivered alongside routine post-transplant care would reduce post-transplant weight gain. DESIGN: Single-blind, randomized controlled trial. SUBJECTS AND SETTING: Adult kidney transplant recipients at a regional transplant center were recruited during routine outpatient clinic visits in the first month after transplant. Patients with a body mass index >40 kg/m2 or <18.5 kg/m2, severe malnutrition, or ongoing medical complications were excluded. INTERVENTION: Participants were randomized to intensive nutrition intervention (individualized nutrition and exercise counselling; 12 dietitian visits; 3 exercise physiologist visits over 12 months) or to standard nutrition care (guideline based; 4 dietitian visits). MAIN OUTCOME MEASURES: The primary outcome was weight at 6 months after transplant adjusted for baseline weight, obesity, and gender, analyzed using analysis of covariance. The secondary outcomes included body composition, biochemistry, quality of life, and physical function. RESULTS: Thirty-seven participants were randomized to the intensive intervention (n = 19) or to standard care (n = 18); one intensive group participant withdrew before baseline. Weight increased between baseline, 6 and 12 months (78.0 ± 13.7 [standard deviation], 79.6 ± 13.0 kg, 81.6 ± 12.9 kg; mean change 4.6% P < .001) but at 6 months did not differ significantly between the groups: 77.0 ± 12.4 kg (intensive); 82.2 ± 13.4 kg (standard); difference in adjusted means 0.4 kg (95% confidence interval: -2.2 to 3.0 kg); analysis of covariance P = .7. No between-group differences in secondary outcomes were observed. Across the whole cohort, total body protein and physical function (gait speed, sit to stand, grip strength, physical activity, and quality of life [all but 2 domains]) improved. However, adverse changes were seen for total body fat, HbA1c, and fasting glucose across the cohort. CONCLUSIONS: Kidney transplant recipients in the first year after transplant did not benefit from an intensive nutrition intervention compared with standard nutrition care, although weight gain was relatively modest in both groups.

Distribution and Differential Survival of Traditional and Alternative Indicators of Fecal Pollution at Freshwater Beaches.

Alternative indicators have been developed that can be used to identify host sources of fecal pollution, yet little is known about how their distribution and fate compare to traditional indicators. Escherichia coli and enterococci were widely distributed at the six beaches studied and were detected in almost 95% of water samples (n = 422) and 100% of sand samples (n = 400). Berm sand contained the largest amount of E. coli (P < 0.01), whereas levels of enterococci were highest in the backshore (P < 0.01). E. coli and enterococci were the lowest in water, using a weight-to-volume comparison. The gull-associated Catellicoccus marimammalium (Gull2) marker was found in over 80% of water samples, regardless of E. coli levels, and in 25% of sand samples. Human-associated Bacteroides (HB) and Lachnospiraceae (Lachno2) were detected in only 2.4% of water samples collected under baseflow and post-rain conditions but produced a robust signal after a combined sewage overflow, despite low E. coli concentrations. Burdens of E. coli and enterococci in water and sand were disproportionately high in relation to alternative indicators when comparing environmental samples to source material. In microcosm studies, Gull2, HB, and Lachno2 quantitative PCR (qPCR) signals were reduced twice as quickly as those from E. coli and enterococci and approximately 20% faster than signals from culturable E. coli High concentrations of alternative indicators in source material illustrated their high sensitivity for the identification of fecal sources; however, differential survival and the potential for long-term persistence of traditional fecal indicators complicate the use of alternative indicator data to account for the levels of E. coli and enterococci in environmental samples. IMPORTANCE: E. coli and enterococci are general indicators of fecal pollution and may persist in beach sand, making their use problematic for many applications. This study demonstrates that gull fecal pollution is widespread at Great Lakes beaches, whereas human and ruminant contamination is evident only after major rain events. An exploration of sand as a reservoir for indicators found that E. coli was ubiquitous, while gull host markers were detected in only 25% of samples. In situ sand beach microcosms provided decay rate constants for E. coli and enterococci relative to alternative indicators, which establish comparative benchmarks that would be helpful to distinguish recent from past pollution. Overall, alternative indicators are useful for identifying sources and assessing potentially high health risk contamination events; however, beach managers should be cautious in attempting to directly link their detection to the levels of E. coli or enterococci.

Influence of Land Use, Nutrients, and Geography on Microbial Communities and Fecal Indicator Abundance at Lake Michigan Beaches.

Microbial communities within beach sand play a key role in nutrient cycling and are important to the nearshore ecosystem function. Escherichia coli and enterococci, two common indicators of fecal pollution, have been shown to persist in the beach sand, but little is known about how microbial community assemblages are related to these fecal indicator bacteria (FIB) reservoirs. We examined eight beaches across a geographic gradient and range of land use types and characterized the indigenous community structure in the water and the backshore, berm, and submerged sands. FIB were found at similar levels in sand at beaches adjacent to urban, forested, and agricultural land and in both the berm and backshore. However, there were striking differences in the berm and backshore microbial communities, even within the same beach, reflecting the very different environmental conditions in these beach zones in which FIB can survive. In contrast, the microbial communities in a particular beach zone were similar among beaches, including at beaches on opposite shores of Lake Michigan. The differences in the microbial communities that did exist within a beach zone correlated to nutrient levels, which varied among geographic locations. Total organic carbon and total phosphorus were higher in Wisconsin beach sand than in beach sand from Michigan. Within predominate genera, fine-scale sequence differences could be found that distinguished the populations from the two states, suggesting a biogeographic effect. This work demonstrates that microbial communities are reflective of environmental conditions at freshwater beaches and are able to provide useful information regarding long-term anthropogenic stress.

Identification of Specialists and Abundance-Occupancy Relationships among Intestinal Bacteria of Aves, Mammalia, and Actinopterygii.

The coalescence of next-generation DNA sequencing methods, ecological perspectives, and bioinformatics analysis tools is rapidly advancing our understanding of the evolution and function of vertebrate-associated bacterial communities. Delineation of host-microbe associations has applied benefits ranging from clinical treatments to protecting our natural waters. Microbial communities follow some broad-scale patterns observed for macroorganisms, but it remains unclear how the specialization of intestinal vertebrate-associated communities to a particular host environment influences broad-scale patterns in microbial abundance and distribution. We analyzed the V6 region of 16S rRNA genes amplified from 106 fecal samples spanning Aves, Mammalia, and Actinopterygii (ray-finned fish). We investigated the interspecific abundance-occupancy relationship, where widespread taxa tend to be more abundant than narrowly distributed taxa, among operational taxonomic units (OTUs) within and among host species. In a separate analysis, we identified specialist OTUs that were highly abundant in a single host and rare in all other hosts by using a multinomial model without excluding undersampled OTUs a priori. We show that intestinal microbes in humans and other vertebrates display abundance-occupancy relationships, but because intestinal host-associated communities have undergone intense specialization, this trend is violated by a disproportionately large number of specialist taxa. Although it is difficult to distinguish the effects of dispersal limitations, host selection, historical contingency, and stochastic processes on community assembly, results suggest that intestinal bacteria can be shared among diverse hosts in ways that resemble the distribution of "free-living" bacteria in the extraintestinal environment.

Comparison of Sewage and Animal Fecal Microbiomes by Using Oligotyping Reveals Potential Human Fecal Indicators in Multiple Taxonomic Groups.

Most DNA-based microbial source tracking (MST) approaches target host-associated organisms within the order Bacteroidales, but the gut microbiota of humans and other animals contain organisms from an array of other taxonomic groups that might provide indicators of fecal pollution sources. To discern between human and nonhuman fecal sources, we compared the V6 regions of the 16S rRNA genes detected in fecal samples from six animal hosts to those found in sewage (as a proxy for humans). We focused on 10 abundant genera and used oligotyping, which can detect subtle differences between rRNA gene sequences from ecologically distinct organisms. Our analysis showed clear patterns of differential oligotype distributions between sewage and animal samples. Over 100 oligotypes of human origin occurred preferentially in sewage samples, and 99 human oligotypes were sewage specific. Sequences represented by the sewage-specific oligotypes can be used individually for development of PCR-based assays or together with the oligotypes preferentially associated with sewage to implement a signature-based approach. Analysis of sewage from Spain and Brazil showed that the sewage-specific oligotypes identified in U.S. sewage have the potential to be used as global alternative indicators of human fecal pollution. Environmental samples with evidence of prior human fecal contamination had consistent ratios of sewage signature oligotypes that corresponded to the trends observed for sewage. Our methodology represents a promising approach to identifying new bacterial taxa for MST applications and further highlights the potential of the family Lachnospiraceae to provide human-specific markers. In addition to source tracking applications, the patterns of the fine-scale population structure within fecal taxa suggest a fundamental relationship between bacteria and their hosts.