LIPG-promoted lipid storage mediates adaptation to oxidative stress in breast cancer.

Endothelial lipase (LIPG) is a cell surface associated lipase that displays phospholipase A1 activity towards phosphatidylcholine present in high-density lipoproteins (HDL). LIPG was recently reported to be expressed in breast cancer and to support proliferation, tumourigenicity and metastasis. Here we show that severe oxidative stress leading to AMPK activation triggers LIPG upregulation, resulting in intracellular lipid droplet accumulation in breast cancer cells, which supports survival. Neutralizing oxidative stress abrogated LIPG upregulation and the concomitant lipid storage. In human breast cancer, high LIPG expression was observed in a limited subset of tumours and was significantly associated with shorter metastasis-free survival in node-negative, untreated patients. Moreover, expression of PLIN2 and TXNRD1 in these tumours indicated a link to lipid storage and oxidative stress. Altogether, our findings reveal a previously unrecognized role for LIPG in enabling oxidative stress-induced lipid droplet accumulation in tumour cells that protects against oxidative stress, and thus supports tumour progression.

Consideration of Natural Sources in a Bacteria TMDL-Lines of Evidence, Including Beach Microbial Source Tracking.

Total Maximum Daily Load (TMDL) stipulations remained unmet at a southern California beach despite a suite of management actions carried out since 2001, prompting exploration of a Natural Sources Exclusion (NSE) provision within the TMDL. Quantitative Microbial Source Tracking (MST) was employed from 2012 to 2015 to inventory sources of natural and anthropogenic fecal indicator bacteria (FIB). Data suggested FIB exceedances could be traced to gulls based on gull marker prevalence and correlations with FIB concentrations in seawater, sand, and eelgrass. In contrast, human marker concentrations and a tracer dye test did not indicate prevalent human sources. Exponential decay of gull marker in sand amended with live Catellicoccus marimammalium suggested that measured marker reflected fecal inputs versus growth outside the host. Improved water quality was coincident with a 2013 bird exclusion structure, consistent with NSE. However, load allocation needed for TMDL reconsideration was hampered by variable ratios of FIB, MST markers, and pathogens measured in seawater and in gull, cat, and raccoon feces. Quantitative Microbial Risk Assessment is a suggested path forward because such models can incorporate distributions from a combination of FIB sources and communicate criteria in terms of human health risk.

Hydrophilic interaction liquid chromatography tandem mass spectrometry analysis of malonyl-coenzyme A in breast cancer cell cultures applying online solid-phase extraction.

Cofactors such as coenzyme A and its derivatives acetyl-coenzyme A and malonyl-coenzyme A are involved in many metabolic pathways. Due to trace level concentrations in biological samples and the high reactivity of cofactors, a fast, sensitive, and selective method for quantification is mandatory. In this study, online solid-phase extraction was coupled successfully to hydrophilic interaction liquid chromatography with tandem mass spectrometry for isolation of analytes in complex matrix and quantification by external calibration. Online solid-phase extraction was carried out by application of a weak anion-exchange column, whereas hydrophilic interaction liquid chromatography separation was performed on an amide modified stationary phase. Sample preparation of the extracts before the analysis was reduced to a centrifugation and dilution step. Moreover, the applied online solid-phase extraction significantly reduced matrix effects and increased the signal-to-noise ratio. The limit of detection and the limit of quantification were in the lower nanomolar range. Finally, the applicability of this method was demonstrated on MCF-7 breast cancer cell cultures, a commonly used model system, where acetyl-coenzyme A and malonyl-coenzyme A were determined using standard addition procedure in concentrations of 1.98 µM and 41 nM, respectively.

A three-stage treatment system for highly polluted urban road runoff.

A three-stage treatment device for polluted urban road runoff was installed and tested at a highly trafficked urban road over a period of one year. In the first stage coarse material and particles from the runoff are removed by a special gutter system. The second stage eliminates particles using a hydrodynamic separator. In the third stage dissolved pollutants are adsorbed in a filter unit with lignite as filter material. Twenty-four rain events were sampled over the one year period and analyzed for dissolved and particulate copper (Cu), zinc (Zn), lead (Pb), suspended solids (SS), total organic carbon (TOC), sodium (Na), and pH value. The treatment system was able to safely retain all relevant pollutants during the investigated period, except Na. In the effluent of the treatment device Pb could never be detected, values measured for Zn were in the range of the detection limit. Cu, the element most frequently detected in the effluent, never exceeded the critical value of 50 µg/L set by the German Federal Soil Protection Act and Ordinance. The median Cu concentration in the effluent of the treatment system was 8.13 µg/L. The treatment system proved to be very effective. Highly polluted road runoff can be purified by the system to an extent that no contamination risk for soil and groundwater remains when infiltrated into the soil.

Epigenomic and transcriptional profiling identifies impaired glyoxylate detoxification in NAFLD as a risk factor for hyperoxaluria.

Epigenetic modifications (e.g. DNA methylation) in NAFLD and their contribution to disease progression and extrahepatic complications are poorly explored. Here, we use an integrated epigenome and transcriptome analysis of mouse NAFLD hepatocytes and identify alterations in glyoxylate metabolism, a pathway relevant in kidney damage via oxalate release-a harmful waste product and kidney stone-promoting factor. Downregulation and hypermethylation of alanine-glyoxylate aminotransferase (Agxt), which detoxifies glyoxylate, preventing excessive oxalate accumulation, is accompanied by increased oxalate formation after metabolism of the precursor hydroxyproline. Viral-mediated Agxt transfer or inhibiting hydroxyproline catabolism rescues excessive oxalate release. In human steatotic hepatocytes, AGXT is also downregulated and hypermethylated, and in NAFLD adolescents, steatosis severity correlates with urinary oxalate excretion. Thus, this work identifies a reduced capacity of the steatotic liver to detoxify glyoxylate, triggering elevated oxalate, and provides a mechanistic explanation for the increased risk of kidney stones and chronic kidney disease in NAFLD patients.

Presence of Bacteroidales as a predictor of pathogens in surface waters of the central California coast.

The value of Bacteroidales genetic markers and fecal indicator bacteria (FIB) to predict the occurrence of waterborne pathogens was evaluated in ambient waters along the central California coast. Bacteroidales host-specific quantitative PCR (qPCR) was used to quantify fecal bacteria in water and provide insights into contributing host fecal sources. Over 140 surface water samples from 10 major rivers and estuaries within the Monterey Bay region were tested over 14 months with four Bacteroidales-specific assays (universal, human, dog, and cow), three FIB (total coliforms, fecal coliforms, and enterococci), two protozoal pathogens (Cryptosporidium and Giardia spp.), and four bacterial pathogens (Campylobacter spp., Escherichia coli O157:H7, Salmonella spp., and Vibrio spp.). Indicator and pathogen distribution was widespread, and detection was not highly seasonal. Vibrio cholerae was detected most frequently, followed by Giardia, Cryptosporidium, Salmonella, and Campylobacter spp. Bayesian conditional probability analysis was used to characterize the Bacteroidales performance assays, and the ratios of concentrations determined using host-specific and universal assays were used to show that fecal contamination from human sources was more common than livestock or dog sources in coastal study sites. Correlations were seen between some, but not all, indicator-pathogen combinations. The ability to predict pathogen occurrence in relation to indicator threshold cutoff levels was evaluated using a weighted measure that showed the universal Bacteroidales genetic marker to have a comparable or higher mean predictive potential than standard FIB. This predictive ability, in addition to the Bacteroidales assays providing information on contributing host fecal sources, supports using Bacteroidales assays in water quality monitoring programs.

Systematic review and meta-analysis of decay rates of waterborne mammalian viruses and coliphages in surface waters.

Surface waters are essential natural resources. They are also receiving waters for a variety of anthropogenic waste streams that carry a myriad of pollutants including pathogens. Watershed and fate and transport models can help inform the spatial and temporal extent of microbial pollution from point and non-point sources and thus provide useful information for managing surface waters. Viruses are particularly important water-related pathogens because they often have a low infectious dose, which means that ingestion of even a small volume of water containing a low concentration of virions has the potential to cause disease. We conducted a systematic review of the literature, following best practices, to gather decay rate constants (k) of mammalian waterborne viruses (enteroviruses, adenoviruses, noroviruses, astroviruses, rotaviruses, and hepatitis A viruses) and coliphages in raw surface waters to aid in the parameterization of virus fate and transport models. We identified 562 k values from the literature, with the largest number identified for enteroviruses and coliphages and the smallest for astrovirus, hepatitis A virus, and norovirus. Average k values for each virus varied from 0.07 to 0.9 per day, in order from smallest to largest: Norwalk virus (i.e., noroviruses) < Human astrovirus < Mastadenovirus (i.e., adenoviruses) < Hepatovirus A (i.e., hepatitis A viruses) < Rotavirus A < coliphages < Enterovirus. A meta-analysis investigated how k varied among viruses for experiments conducted with different virus serotypes or species at different temperatures, salinities, and sunlight exposures, and for experiments that enumerated viruses using different methodologies. Virus species or serotype did not affect k among decay experiments. k values were generally larger for experiments conducted at higher temperatures, in sunlight, and in estuarine waters, and enumerated using culture methods. k values were statistically different between virus types with Norwalk virus, Hepatovirus A, and Mastadenovirus having smaller k values than other viruses, controlling for experimental condition and enumeration method. While F+ coliphage k values were similar to those of Enterovirus, Human astrovirus, and Rotavirus A, they were different from those of the other mammalian viruses. This compilation of coliphage and mammalian virus k values provides essential information for researchers and risk assessors who model virus fate and transport in surface waters and identifies avenues for future research to fill knowledge gaps.

Effects of salinity and transparent exopolymer particles on formation of aquatic aggregates and their association with norovirus.

Human noroviruses (NoVs) are responsible for 50% of food-related disease outbreaks and are notably associated with shellfish consumption. Despite the detrimental health impacts of human NoV-contaminated seafood to public health, there is a lack of knowledge on the physicochemical conditions that govern NoV transmission in aquatic ecosystems. In the present study, we investigated the propensity for NoVs to associate with aquatic aggregates, which have been shown to efficiently deliver nano-sized particles to shellfish. Specific physicochemical conditions characteristic of shellfish cultivation waters, specifically salinity and transparent exopolymer particles (TEP), were targeted in this study for investigating aggregate formation and NoV association dynamics. Murine norovirus (MNV) was used in aggregation experiments as a model surrogate for NoVs. Results demonstrate increased aggregate formation as a function of increasing salinity and TEP concentrations, as well as greater numbers of MNV genomes incorporated into aggregates under conditions that favor aggregation. As aggregate formation was enhanced in waters representing optimal conditions for shellfish production, specifically saline and high TEP waters, the implications to virus transport and shellfish food safety are profound: more aggregates implies increased scavenging of virus particles from surrounding waters and therefor greater risk for bivalve contamination with nano-sized pathogens. These novel data provide insight into where and when NoVs are most likely to be ingested by shellfish via contaminated aggregates, thereby informing best management and water quality monitoring practices aimed at providing safe seafood to consumers.

Celecoxib use and circulating oxylipins in a colon polyp prevention trial.

Drugs that inhibit cyclooxygenase (COX)-2 and the metabolism of arachidonic acid (ARA) to prostaglandin E2 are potent anti-inflammatory agents used widely in the treatment of joint and muscle pain. Despite their benefits, daily use of these drugs has been associated with hypertension, cardiovascular and gastrointestinal toxicities. It is now recognized that ARA is metabolized to a number of bioactive oxygenated lipids (oxylipins) by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP450) enzymes. Currently, the contribution of individual variability in ARA metabolism in response to the COX-2 inhibitors and potential adverse effects remains poorly understood. Using patient samples from the randomized, placebo-controlled phase III selenium/celecoxib (Sel/Cel) trial for the prevention of colorectal adenomatous polyps, we analyzed plasma concentrations of 74 oxylipins in a subset of participants who received celecoxib (n = 90) or placebo (n = 95). We assessed the effect of celecoxib (with and without low dose aspirin) on circulating oxylipins and systolic blood pressure (SBP). Individual CYP450- and LOX- but not COX-derived metabolites were higher with celecoxib than placebo (P<0.05) and differences were greater among non-aspirin users. LOX derived 5- and 8-HETE were elevated with celecoxib and positively associated with systolic blood pressure (P = 0.011 and P = 0.019 respectively). 20-HETE, a prohypertensive androgen-sensitive CYP450 metabolite was higher with celecoxib absent aspirin and was positively associated with SBP in men (P = 0.040) but not women. Independent of celecoxib or aspirin, LOX derived metabolites from ARA were strongly associated with SBP including 5- and 8-HETE. These findings support oxylipins, particularly the ARA LOX-derived, in blood pressure control and indicate that pharmacologic inhibition of COX-2 has effects on LOX and CYP450 ARA metabolism that contribute to hypertension in some patients.

Oxalic acid quantification in mouse urine and primary mouse hepatocyte cell culture samples by ion exclusion chromatography-mass spectrometry.

Due to medical relevance and a direct correlation with some diseases, accurate quantification of oxalic acid in different complex matrices is required. Effective chromatographic separation of this strong carboxylic acid was achieved by ion exclusion chromatography (IELC). Sensitive and selective detection was carried out by means of electrospray ionization-tandem mass spectrometry. Furthermore, it was shown that the isobaric interference of lactic acid is chromatographically resolved. Structurally similar compounds like glyoxylic acid and glycolic acid were baseline separated as well. The application of stable isotope dilution analysis with 13C2 oxalic acid facilitated precise quantification. The developed method was validated with a reference oxalate sample of human urine diluted to a range of 10-500µM. Finally, the applicability of this method was demonstrated on complex matrices, like mouse urine and supernatants of primary mouse hepatocyte cell cultures.

Regional Assessment of Human Fecal Contamination in Southern California Coastal Drainages.

Host-associated genetic markers that allow for fecal source identification have been used extensively as a diagnostic tool to determine fecal sources within watersheds, but have not been used in routine monitoring to prioritize remediation actions among watersheds. Here, we present a regional assessment of human marker prevalence among drainages that discharge to the U.S. southern California coast. Approximately 50 samples were analyzed for the HF183 human marker from each of 22 southern California coastal drainages under summer dry weather conditions, and another 50 samples were targeted from each of 23 drainages during wet weather. The HF183 marker was ubiquitous, detected in all but two sites in dry weather and at all sites during wet weather. However, there was considerable difference in the extent of human fecal contamination among sites. Similar site ranking was produced regardless of whether the assessment was based on frequency of HF183 detection or site average HF183 concentration. However, site ranking differed greatly between dry and wet weather. Site ranking also differed greatly when based on enterococci, which do not distinguish between pollution sources, vs. HF183, which distinguishes higher risk human fecal sources from other sources, indicating the additional value of the human-associated marker as a routine monitoring tool.

Human fecal and pathogen exposure pathways in rural Indian villages and the effect of increased latrine coverage.

Efforts to eradicate open defecation and improve sanitation access are unlikely to achieve health benefits unless interventions reduce microbial exposures. This study assessed human fecal contamination and pathogen exposures in rural India, and the effect of increased sanitation coverage on contamination and exposure rates. In a cross-sectional study of 60 villages of a cluster-randomized controlled sanitation trial in Odisha, India, human and domestic animal fecal contamination was measured in community tubewells and ponds (n = 301) and via exposure pathways in homes (n = 354), using Bacteroidales microbial source tracking fecal markers validated in India. Community water sources were further tested for diarrheal pathogens (rotavirus, adenovirus and Vibrio cholerae by quantitative PCR; pathogenic Escherichia coli by multiplex PCR; Cryptosporidium and Giardia by immunomagnetic separation and direct fluorescent antibody microscopy). Exposure pathways in intervention and control villages were compared and relationships with child diarrhea examined. Human fecal markers were rarely detected in tubewells (2.4%, 95%CI: 0.3-4.5%) and ponds (5.6%, 95%CI: 0.8-10.3%), compared to homes (35.4%, 95%CI: 30.4-40.4%). In tubewells, V. cholerae was the most frequently detected pathogen (19.8%, 95%CI: 14.4-25.2%), followed by Giardia (14.8%, 95%CI: 10.0-19.7%). In ponds, Giardia was most often detected (74.5%, 95%CI: 65.7-83.3%), followed by pathogenic E. coli (48.1%, 95%CI: 34.8-61.5%) and rotavirus (44.4%, 95%CI: 34.2-54.7%). At village-level, prevalence of fecal pathogen detection in community drinking water sources was associated with elevated prevalence of child diarrhea within 6 weeks of testing (RR 2.13, 95%CI: 1.25-3.63) while within homes, higher levels of human and animal fecal marker detection were associated with increased risks of subsequent child diarrhea (P = 0.044 and 0.013, respectively). There was no evidence that the intervention, which increased functional latrine coverage and use by 27 percentage points, reduced human fecal contamination in any tested pathway, nor the prevalence of pathogens in water sources. In conclusion, the study demonstrates that (1) improved sanitation alone may be insufficient and further interventions needed in the domestic domain to reduce widespread human and animal fecal contamination observed in homes, (2) pathogens detected in tubewells indicate these sources are microbiologically unsafe for drinking and were associated with child diarrhea, (3) domestic use of ponds heavily contaminated with multiple pathogens presents an under-recognized health risk, and (4) a 27 percentage point increase in improved sanitation access at village-level did not reduce detectable human fecal and pathogen contamination in this setting.

Validation of Bacteroidales quantitative PCR assays targeting human and animal fecal contamination in the public and domestic domains in India.

We compared host-associated Bacteroidales qPCR assays developed in the continental United States and Europe for the purpose of measuring the effect of improved sanitation on human fecal exposure in rural Indian communities where both human and animal fecal loading are high. Ten candidate Bacteroidales qPCR assays were tested against fecal samples (human, sewage, cow, buffalo, goat, sheep, dog and chicken) from a test set of 30 individual human, 5 sewage, and 60 pooled animal samples collected in coastal Odisha, India. The two universal/general Bacteroidales assays tested (BacUni, GenBac3) performed equally well, achieving 100% sensitivity on the test set. Across the five human-associated assays tested (HF183 Taqman, BacHum, HumM2, BacH, HF183 SYBR), we found low sensitivity (17 to 49%) except for HF183 SYBR (89%), and moderate to high cross-reactivity with dog (20 to 80%) and chicken fecal samples (60 to 100%). BacHum had the highest accuracy (67%), amplified all sewage samples within the range of quantification (ROQ), and did not cross-react with any fecal samples from cows, the most populous livestock animal in India. Of the ruminant- and cattle-associated assays tested (BacCow, CowM2), BacCow was more sensitive in detecting the full range of common Indian livestock animal fecal sources, while CowM2 only detected cow sources with 50% sensitivity. Neither assay cross-reacted with human sources. BacCan, the dog-associated assay tested, showed no cross-reactivity with human sources, and high sensitivity (90%) for dog fecal samples. Overall, our results indicate BacUni, BacHum, HumM2, BacCan and BacCow would be the most suitable MST assays to distinguish and quantify relative amounts of human-associated and livestock/domestic animal-associated contributions to fecal contamination in Odisha, India.

Human and Animal Fecal Contamination of Community Water Sources, Stored Drinking Water and Hands in Rural India Measured with Validated Microbial Source Tracking Assays.

We examined pathways of exposure to fecal contamination of human and animal origin in 24 villages in Odisha, India. In a cross-sectional study during the monsoon season, fecal exposure via community water sources (N = 123) and in the home (N = 137) was assessed using human- and nonhuman-associated Bacteroidales microbial source tracking (MST) markers and fecal coliforms (FCs). Detection rates and marker concentrations were examined to pinpoint pathways of human fecal exposure in the public and domestic domains of disease transmission in study communities. Human fecal markers were detected much more frequently in the domestic domain (45% of households) than in public domain sources (8% of ponds; 4% of groundwater drinking sources). Animal fecal markers were widely detected in both domains (74% of ponds, 96% of households, 10% of groundwater drinking sources), indicating ubiquitous risks of exposure to animal feces and zoonotic pathogens. This study confirms an often suggested contamination link from hands to stored water in the home in developing countries separately for mothers' and children's hands and both human and animal fecal contamination. In contrast to MST markers, FCs provided a poor metric to assess risks of exposure to fecal contamination of human origin in this rural setting.

Spatial and hydrologic variation of Bacteroidales, adenovirus and enterovirus in a semi-arid, wastewater effluent-impacted watershed.

Bacteroidales and viruses were contemporaneously measured during dry and wet weather conditions at a watershed-scale in a semi-arid watershed impacted by a mixture of agricultural runoff, municipal wastewater effluent and municipal runoff. The results highlight the presence of municipal wastewater effluent as a confounding factor for microbial source tracking (MST) studies, and thus data were segregated into groups based on whether they were impacted by wastewater effluent. In semi-arid environments such as the Calleguas Creek watershed, located in southern California, the relative contribution of municipal wastewater effluent is dependent on hydrology as storm events lead to conditions where agricultural and municipal stormwater dominate receiving waters (rather than municipal wastewater, which is the case during dry weather). As such, the approach to data segregation was dependent on hydrology/storm conditions. Storm events led to significant increases in ruminant- and dog-associated Bacteroidales concentrations, indicating that overland transport connects strong non-human fecal sources with surface waters. Because the dataset had a large number of non-detect samples, data handling included the Kaplan-Meir estimator and data were presented graphically in a manner that reflects the potential effect of detection limits. In surface water samples with virus detections, Escherichia coli concentrations were often below (in compliance with) the recreational water quality criteria. In fact, sites downstream of direct inputs of municipal wastewater effluent exhibited the lowest concentrations of E. coli, but the highest concentrations of human-associated Bacteroidales and highest detection rates of human viruses. The toolkit, comprised of the four Bacteroidales assays and human virus assays used, can be successfully applied to inform watershed managers seeking to comply with recreational water quality criteria. However, care should be taken when analyzing data to account for the effect of non-detect samples, sources with differing microbial viability, and diverging hydrologic conditions.

Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study.

The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.

Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources.

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation.

Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods.

Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1-3.3% and 1.9-7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1-2 log10copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories.

Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study.

A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman(®), HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman(®) was found to be the most effective marker of human fecal contamination in this California-based study.

Multi-laboratory evaluations of the performance of Catellicoccus marimammalium PCR assays developed to target gull fecal sources.

Here we report results from a multi-laboratory (n = 11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR(®) Green qPCR method, and two TaqMan(®) qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions.