Assessing the impact of rainwater harvesting infrastructure and gardening trends on microbial indicator organism presence in harvested rainwater and garden soils.

AIM: To assess the microbial water quality of harvested rainwater infrastructure used to supplement household water uses for homegrown produce. METHODS AND RESULTS: Using a co-created community science methodology, between 2017 and 2020, a total of 587 harvested rainwater samples and 147 garden soil samples irrigated with harvested rainwater were collected from four Arizona communities and analyzed for coliform, Escherichia coli, and/or Salmonella. Participants also completed a home description survey regarding their home and surrounding area, water harvesting infrastructure, and gardening habits. CONCLUSION: Chi-Square tests revealed that the quality of harvested rainwater is affected by proximity to a waste disposal or incineration facility, animal presence, cistern treatment, and cistern age (P < 0.05), while soil samples were associated with community (P < 0.05). Coliform and E. coli concentrations in both sample types were greater in the monsoon season.

The efficacy of hydrogen sulfide (H2S) tests for detecting microbial contamination in rooftop-harvested rainwater.

As climate change strains the world's freshwater resources, access to safe and clean water becomes limited. The use of alternative water sources, such as rooftop-harvested rainwater, has become one mechanism to address freshwater scarcity in the American Southwest, particularly when it comes to home gardening. The University of Arizona's Project Harvest, in partnership with the Sonora Environmental Research Institute, Inc., is a multi-year, co-created citizen science project aimed at increasing current understanding of harvested rainwater quality. Citizens in four Arizona, USA, communities (Hayden/Winkelman, Globe/Miami, Dewey-Humboldt, and Tucson) submitted harvested rainwater samples over 3 years. The harvested rainwater samples were then analyzed using IDEXX Colilert® for total coliforms and E. coli and using Hach PathoScreen™ test for sulfate-reducing bacteria (SRB). This study design allows for the validation of a low-cost, at-home alternative methodology for testing rainwater for bacteria that may indicate fecal contamination. In total, 226 samples were tested using both methodologies, revealing a positive correlation (r=0.245; p<0.002) between total coliform MPN and SRB MPN, but no discernable correlation between E. coli MPN and SRB MPN. This work indicates a potential value of SRB testing for harvested rainwater if cost, laboratory access, and fecal contamination are of concern.

Crayfish (Cherax quadricarinatus) susceptibility to acute hepatopancreatic necrosis disease (AHPND).

Acute hepatopancreatic necrosis disease (AHPND) is an OIE-listed enteric disease that has continued to plague the shrimp aquaculture industry since its first discovery in 2009. AHPND is one of the biggest disease threats to the shrimp aquaculture industry along with white spot disease (WSD) which has severely impacted both crayfish and shrimp aquaculture. AHPND is caused by specific marine Vibrio spp. which carry plasmid-borne binary toxins PirAVp and PirBVp. This research investigated if crayfish are susceptible to AHPND-causing Vibrio parahaemolyticus (VpAHPND) to discern the potential risk that AHPND may pose to the crayfish aquaculture industry. Susceptibility was investigated by challenging Cherax quadricarinatus (Australian red claw crayfish) and Penaeus vannamei (Pacific white shrimp) with VpAHPND in a cohabitation immersion bioassay. Upon termination of the bioassay, crayfish survival was significantly higher than shrimp survival (87% vs. 33%). Hepatopancreas dissected from experimentally challenged animals were screened for the binary toxin genes pirAVp and pirBVp by real-time and duplex conventional PCR assays, and also were examined by H&E histology for the detection of characteristic AHPND pathology. Although AHPND toxin genes pirAVp and pirBVp were detected in a subset of crayfish samples, histopathology did not reveal any pathognomonic lesions that are characteristic of AHPND in any crayfish samples examined. These findings suggest that crayfish are likely resistant to AHPND.

Review of water quality criteria for water reuse and risk-based implications for irrigated produce under the FDA Food Safety Modernization Act, produce safety rule.

Questions related to the safety of alternative water sources, such as recycled water or reclaimed water (including grey water, produced water, return flows, and recycled wastewater), for produce production have been largely un-explored at the detail warranted for protection of public health. Additionally, recent outbreaks of Escherichia coli (E. coli) in fresh produce, in which agricultural water was suspected as the source, coupled with heightened media coverage, have elevated fruit and vegetable safety into the forefront of public attention. Exacerbating these concerns, new Federal regulations released by the U.S. Food and Drug Administration (FDA) as part of implementation of the FDA Food Safety Modernization Act (FSMA), require testing of agricultural water quality for generic E. coli. Here, we present a review of water quality criteria - including surface water, groundwater recreational water, and water reuse - in an attempt to better understand implications of new FDA regulations on irrigated produce. In addition, a Quantitative Microbial Risk Assessment (QMRA) was conducted to estimate risks from pathogen contamination of food crops eaten fresh under the context of FDA regulations to provide perspective on current water reuse regulations across the country. Results indicate that irrigation water containing 126 CFU/100 mL of E. coli correspond to a risk of GI illness (diarrhea) of 9 cases in 100,000,000 persons (a 0.000009% risk) for subsurface irrigation, 1.1 cases in 100,000 persons (a 0.0011% risk) for furrow irrigation, and 1.1 cases in 1000 persons (a 0.11% risk) for sprinkler irrigation of lettuce. In comparison to metrics in states that currently regulate the use of recycled water for irrigation of food crops eaten fresh, the FDA FSMA water quality metrics are less stringent and therefore the use of recycled water presents a reduced risk to consumers than the FDA regulations. These findings, while limited to a one-time exposure event of lettuce irrigated with water meeting FSMA water quality regulations, highlight the need for additional assessments to determine if the scientific-basis of the regulation is protective of public health.

Microbial Ecology and Water Chemistry Impact Regrowth of Opportunistic Pathogens in Full-Scale Reclaimed Water Distribution Systems.

Need for global water security has spurred growing interest in wastewater reuse to offset demand for municipal water. While reclaimed (i.e., nonpotable) microbial water quality regulations target fecal indicator bacteria, opportunistic pathogens (OPs), which are subject to regrowth in distribution systems and spread via aerosol inhalation and other noningestion routes, may be more relevant. This study compares the occurrences of five OP gene markers ( Acanthamoeba spp., Legionella spp., Mycobacterium spp., Naegleria fowleri, Pseudomonas aeruginosa) in reclaimed versus potable water distribution systems and characterizes factors potentially contributing to their regrowth. Samples were collected over four sampling events at the point of compliance for water exiting treatment plants and at five points of use at four U.S. utilities bearing both reclaimed and potable water distribution systems. Reclaimed water systems harbored unique water chemistry (e.g., elevated nutrients), microbial community composition, and OP occurrence patterns compared to potable systems examined here and reported in the literature. Legionella spp. genes, Mycobacterium spp. genes, and total bacteria, represented by 16S rRNA genes, were more abundant in reclaimed than potable water distribution system samples ( p ≤ 0.0001). This work suggests that further consideration should be given to managing reclaimed water distribution systems with respect to nonpotable exposures to OPs.

Metagenomic Characterization of Antibiotic Resistance Genes in Full-Scale Reclaimed Water Distribution Systems and Corresponding Potable Systems.

Water reclamation provides a valuable resource for meeting nonpotable water demands. However, little is known about the potential for wastewater reuse to disseminate antibiotic resistance genes (ARGs). Here, samples were collected seasonally in 2014-2015 from four U.S. utilities' reclaimed and potable water distribution systems before treatment, after treatment, and at five points of use (POU). Shotgun metagenomic sequencing was used to profile the resistome (i.e., full contingent of ARGs) of a subset ( n = 38) of samples. Four ARGs ( qnrA, blaTEM, vanA, sul1) were quantified by quantitative polymerase chain reaction. Bacterial community composition (via 16S rRNA gene amplicon sequencing), horizontal gene transfer (via quantification of intI1 integrase and plasmid genes), and selection pressure (via detection of metals and antibiotics) were investigated as potential factors governing the presence of ARGs. Certain ARGs were elevated in all ( sul1; p ≤ 0.0011) or some ( blaTEM, qnrA; p ≤ 0.0145) reclaimed POU samples compared to corresponding potable samples. Bacterial community composition was weakly correlated with ARGs (Adonis, R2 = 0.1424-0.1734) and associations were noted between 193 ARGs and plasmid-associated genes. This study establishes that reclaimed water could convey greater abundances of certain ARGs than potable waters and provides observations regarding factors that likely control ARG occurrence in reclaimed water systems.

Culture-based Methods for Detection of Antibiotic Resistance in Agroecosystems: Advantages, Challenges, and Gaps in Knowledge.

Various culture-based methodologies are used in assessment of antibiotic resistance in samples collected in agroecosystems. Culture-based methods commonly involve isolating target bacteria on general or selective media and assessing growth in response to specific concentrations of antibiotics. The advantages of culture-based methods are multifold. In particular, isolation of bacteria is key to understanding phenotypic characteristics of isolates and their resistance patterns, and most national and international antibiotic resistance monitoring projects are isolate based. This review covers current knowledge of bacterial groups and antibiotics commonly targeted in resistance studies using bacterial culture and discusses the range in methods used, data interpretation, and factors supporting and confounding the use of culture-based methods in assessment of antibiotic resistance. Gaps in knowledge related to study design and resistance databases are discussed. Finally, a case is made for the integration of culture-based and molecular methods to better inform our understanding of antibiotic resistance in agroecosystems.

Antibiotics and Antibiotic Resistance in Agroecosystems: State of the Science.

We propose a simple causal model depicting relationships involved in dissemination of antibiotics and antibiotic resistance in agroecosystems and potential effects on human health, functioning of natural ecosystems, and agricultural productivity. Available evidence for each causal link is briefly summarized, and key knowledge gaps are highlighted. A lack of quantitative estimates of human exposure to environmental bacteria, in general, and antibiotic-resistant bacteria, specifically, is a significant data gap hindering the assessment of effects on human health. The contribution of horizontal gene transfer to resistance in the environment and conditions that might foster the horizontal transfer of antibiotic resistance genes into human pathogens also need further research. Existing research has focused heavily on human health effects, with relatively little known about the effects of antibiotics and antibiotic resistance on natural and agricultural ecosystems. The proposed causal model is used to elucidate gaps in knowledge that must be addressed by the research community and may provide a useful starting point for the design and analysis of future research efforts.

Antibiotics in Agroecosystems: Introduction to the Special Section.

The presence of antibiotic drug residues, antibiotic resistant bacteria, and antibiotic resistance genes in agroecosystems has become a significant area of research in recent years and is a growing public health concern. While antibiotics are used in both human medicine and agricultural practices, the majority of their use occurs in animal production where historically they have been used for growth promotion, in addition to the prevention and treatment of disease. The widespread use of antibiotics and the application of animal wastes to agricultural lands play major roles in the introduction of antibiotic-related contamination into the environment. Overt toxicity in organisms directly exposed to antibiotics in agroecosystems is typically not a major concern because environmental concentrations are generally lower than therapeutic doses. However, the impacts of introducing antibiotic contaminants into the environment are unknown, and concerns have been raised about the health of humans, animals, and ecosystems. Despite increased research focused on the occurrence and fate of antibiotics and antibiotic resistance over the past decade, standard methods and practices for analyzing environmental samples are limited and future research needs are becoming evident. To highlight and address these issues in detail, this special collection of papers was developed with a framework of five core review papers that address the (i) overall state of science of antibiotics and antibiotic resistance in agroecosystems using a causal model, (ii) chemical analysis of antibiotics found in the environment, (iii) need for background and baseline data for studies of antibiotic resistance in agroecosystems with a decision-making tool to assist in designing research studies, as well as (iv) culture- and (v) molecular-based methods for analyzing antibiotic resistance in the environment. With a focus on the core review papers, this introduction summarizes the current state of science for analyzing antibiotics and antibiotic resistance in agroecosystems, discusses current knowledge gaps, and develops future research priorities. This introduction also contains a glossary of terms used in the core reivew papers of this special section. The purpose of the glossary is to provide a common terminology that clearly characterizes the concepts shared throughout the narratives of each review paper.

Isolation of Bacteroides from fish and human fecal samples for identification of unique molecular markers.

Bacteroides molecular markers have been used to identify human fecal contamination in natural waters, but recent work in our laboratory confirmed cross-amplification of several human-specific Bacteroides spp. assays with fecal DNA from fish. For identification of unique molecular markers, Bacteroides from human (n = 4) and fish (n = 7) fecal samples were cultured and their identities were further confirmed using Rapid ID 32A API strips. The 16S rDNA from multiple isolates from each sample was PCR amplified, cloned, and sequenced to identify unique markers for development of more stringent human-specific assays. In human feces, Bacteroides vulgatus was the dominant species (75% of isolates), whereas in tilapia feces, Bacteroides eggerthii was dominant (66%). Bacteroides from grass carp, channel catfish, and blue catfish may include Bacteroides uniformis, Bacteroides ovatus, or Bacteroides stercoris. Phylogenic analyses of the 16S rRNA gene sequences showed distinct Bacteroides groupings from each fish species, while human sequences clustered with known B. vulgatus. None of the fish isolates showed significant similarity to Bacteroides sequences currently deposited in NCBI (National Center for Biotechnology Information). This study expands the current sequence database of cultured fish Bacteroides. Such data are essential for identification of unique molecular markers in human Bacteroides that can be utilized in differentiating fish and human fecal contamination in water samples.

Mitigating risks and maximizing sustainability of treated wastewater reuse for irrigation.

Scarcity of freshwater for agriculture has led to increased utilization of treated wastewater (TWW), establishing it as a significant and reliable source of irrigation water. However, years of research indicate that if not managed adequately, TWW may deleteriously affect soil functioning and plant productivity, and pose a hazard to human and environmental health. This review leverages the experience of researchers, stakeholders, and policymakers from Israel, the United-States, and Europe to present a holistic, multidisciplinary perspective on maximizing the benefits from municipal TWW use for irrigation. We specifically draw on the extensive knowledge gained in Israel, a world leader in agricultural TWW implementation. The first two sections of the work set the foundation for understanding current challenges involved with the use of TWW, detailing known and emerging agronomic and environmental issues (such as salinity and phytotoxicity) and public health risks (such as contaminants of emerging concern and pathogens). The work then presents solutions to address these challenges, including technological and agronomic management-based solutions as well as source control policies. The concluding section presents suggestions for the path forward, emphasizing the importance of improving links between research and policy, and better outreach to the public and agricultural practitioners. We use this platform as a call for action, to form a global harmonized data system that will centralize scientific findings on agronomic, environmental and public health effects of TWW irrigation. Insights from such global collaboration will help to mitigate risks, and facilitate more sustainable use of TWW for food production in the future.

Assessing environmental impacts of treated wastewater through monitoring of fecal indicator bacteria and salinity in irrigated soils.

To assess the potential for treated wastewater irrigation to impact levels of fecal indicator bacteria (FIB) and salinity in irrigated soils, levels of Escherichia coli, Enterococcus, and environmental covariates were measured in a treated wastewater holding pond (irrigation source water), water leaving the irrigation system, and in irrigated soils over 2 years in a municipal parkland in Arizona. Higher E. coli levels were measured in the pond in winter (56 CFU 100 mL(-1)) than in summer (17 CFU 100 mL(-1)); however, in the irrigation system, levels of FIB decreased from summer (26 CFU 100 mL(-1)) to winter (4 CFU 100 mL(-1)), possibly related to low winter water use and corresponding death of residual bacteria within the system. For over 2 years, no increase in FIB was found in irrigated soils, though highest E. coli levels (700 CFU g(-1) soil) were measured in deeper (20-25 cm) soils during summer. Measurements of water inputs vs. potential evapotranspiration indicate that irrigation levels may have been sufficient to generate bacterial percolation to deeper soil layers during summer. No overall increase in soil salinity resulting from treated wastewater irrigation was detected, but distinct seasonal peaks as high as 4 ds m(-1) occurred during both summers. The peaks significantly declined in winter when surface ET abated and more favorable water balances could be maintained. Monitoring of seasonal shifts in irrigation water quality and/or factors correlated with increases and decreases in FIB will aid in identification of any public health or environmental risks that could arise from the use of treated wastewater for irrigation.

False-positive identification of Escherichia coli in treated municipal wastewater and wastewater-irrigated soils.

The increasing use of treated wastewater for irrigation heightens the importance of accurate monitoring of water quality. Chromogenic media, because they are easy to use and provide rapid results, are often used for detection of Escherichia coli in environmental samples, but unique levels of organic and inorganic compounds alter the chemistry of treated wastewater, potentially hindering the accurate performance of chromogenic media. We used MI agar and molecular confirmatory methods to assess false-positive identification of E. coli in treated wastewater samples collected from municipal utilities, an irrigation holding pond, irrigated soils, and in samples collected from storm flows destined for groundwater recharge. False-positive rates in storm flows (4.0%) agreed closely with USEPA technical literature but were higher in samples from the pond, soils, and treatment facilities (33.3%, 38.0%, and 48.8%, respectively). Sequencing of false-positive isolates confirmed that most were, like E. coli, of the family Enterobacteriaceae, and many of the false-positive isolates were reported to produce the ß-D-glucuronidase enzyme targeted by MI agar. False-positive identification rates were inversely related to air temperature, suggesting that seasonal variations in water quality influence E. coli identification. Knowledge of factors contributing to failure of chromogenic media will lead to manufacturer enhancements in media quality and performance and will ultimately increase the accuracy of future water quality monitoring programs.

Seasonal variation in accurate identification of Escherichia coli within a constructed wetland receiving tertiary-treated municipal effluent.

As the reuse of municipal wastewater escalates worldwide as a means to extend increasingly limited water supplies, accurate monitoring of water quality parameters, including Escherichia coli (E. coli), increases in importance. Chromogenic media are often used for detection of E. coli in environmental samples, but the presence of unique levels of organic and inorganic compounds alters reclaimed water chemistry, potentially hindering E. coli detection using enzyme-based chromogenic technology. Over seven months, we monitored E. coli levels using m-Coli Blue 24 broth in a constructed wetland filled with tertiary-treated municipal effluent. No E. coli were isolated in the wetland source waters, but E. coli, total coliforms, and heterotrophic bacteria increased dramatically within the wetland on all sampling dates, most probably due to fecal inputs from resident wildlife populations. Confirmatory testing of isolates presumptive for E. coli revealed a 41% rate of false-positive identification using m-Coli Blue 24 broth over seven months. Seasonal differences were evident, as false-positive rates averaged 35% in summer, but rose sharply to 75% in the late fall and winter. Corrected E. coli levels were significantly correlated with electrical conductivity, indicating that water chemistry may be controlling bacterial survival within the wetland. This is the first study to report that accuracy of chromogenic media for microbial enumeration in reclaimed water may show strong seasonal differences, and highlights the importance of validation of microbiological results from chromogenic media for accurate analysis of reclaimed water quality.

Increasing Environmental Health Literacy through Contextual Learning in Communities at Risk.

Environmental health literacy (EHL) has recently been defined as the continuum of environmental health knowledge and awareness, skills and self-efficacy, and community action. In this study, an interdisciplinary team of university scientists, partnering with local organizations, developed and facilitated EHL trainings with special focus on rainwater harvesting and water contamination, in four communities with known environmental health stressors in Arizona, USA. These participatory trainings incorporated participants' prior environmental health risk knowledge and personal experiences to co-create training content. Mixed methods evaluation was conducted via pre-post participant surveys in all four trainings (n = 53). Participants who did not demonstrate baseline environmental science knowledge pre-training demonstrated significant knowledge increase post-training, and participants who demonstrated low self-efficacy (SE) pre-training demonstrated a significant increase in SE post-training. Participants overall demonstrated a significant increase in specific environmental health skills described post-training. The interdisciplinary facilitator-scientist team also reported multiple benefits, including learning local knowledge that informed further research, and building trust relationships with community members for future collaboration. We propose contextual EHL education as a valuable strategy for increasing EHL in environmental health risk communities, and for building academia-community partnerships for environmental health research and action.

Impacts of solids retention time on trace organic compound attenuation and bacterial resistance to trimethoprim and sulfamethoxazole.

Bacteria can grow in the presence of trimethoprim and sulfamethoxazole by expressing antibiotic resistance genes or by acquiring thymine or thymidine from environmental reservoirs to facilitate DNA synthesis. The purpose of this study was to evaluate whether activated sludge serves as a reservoir for thymine or thymidine, potentially impacting the quantification of antibiotic resistant bacteria. This study also assessed the impacts of varying solids retention time (SRT) on trimethoprim and sulfamethoxazole removal during wastewater treatment and single and multi-drug resistance. When assayed in the presence of the antibiotics at standard clinical concentrations, up to 40% increases in the relative prevalence of resistant bacteria were observed with (1) samples manually augmented with reagent-grade thymidine, (2) samples manually augmented with sonicated biomass (i.e., cell lysate), (3) samples manually augmented with activated sludge filtrate, and (4) activated sludge samples collected from reactors with longer SRTs. These observations suggest that longer SRTs may select for antibiotic resistant bacteria and/or result in false positives for antibiotic resistance due to higher concentrations of free thymine, thymidine, or other extracellular constituents.

A tool box strategy using Bacteroides genetic markers to differentiate human from non-human sources of fecal contamination in natural water.

Bacteroides genetic markers have been widely used to identify fecal pollution of water originating from human and animal sources. Many of the assays currently used for detecting human-specific Bacteroides produce false positive results. The focus of this study was to develop a microbial source tracking (MST) tool box strategy for differentiating Bacteroides from human and animal sources. Bacteroides 16S rRNA gene sequences from fish and selected animals were aligned against human fecal Bacteroides isolates to compare and characterize the variable regions within the 16S rRNA gene sequence. Conserved sequences between 4 variable regions were deleted and the truncated sequences were combined to develop a hyper-variable genomic segment (HVGS). The cladogram created from truncated sequences show a clear separation of Bacteroides from human feces and those from animal sources. The proposed strategy was field tested by collecting water samples from central Arizona source waters and three different recreational ponds. PCR using HF134 and HF183 primer sets was performed and sequences from positive reactions were aligned against human Bacteroides sequences to identify the source of contamination. Based on PCR results, the source of fecal contamination was presumptively identified as either human or from another source. For samples testing positive using the HF183 primer set (8/13), fecal contamination was presumed to be from human sources, but to confirm the results, PCR products were sequenced and aligned against the four variable regions and then incorporated within the truncated cladogram. As expected, the sequences from water samples with human fecal contamination grouped in a separate clade. A variability matrix, developed after exclusion of conserved sequences among the four regions, was utilized to establish discrete groupings for sequences within the truncated cladogram, generally differentiating Bacteroides isolates from varying host animals, but most importantly, separating Bacteroides from human feces from Bacteroides from other animals. The proposed strategy offers a new tool box method for MST and a step-wise methodology essential for identifying human sources of fecal pollution.

Microbial community activities during establishment, performance, and decline of bench-scale passive treatment systems for mine drainage.

Permeable reactive barrier (PRB) technology, in which sulfate-reducing bacteria (SRB) facilitate precipitation of metal sulfides, is a promising approach for remediation of sulfate- and metal-laden mine drainage. While PRBs are easily established, they often decline for reasons not well understood. SRB depend on or compete with multiple dynamic microbial populations within a PRB; as a result, performance depends on the changing PRB chemical composition and on succession and competition within the microbial community. To investigate these interactions, we constructed and monitored eight bench-scale PRBs to define periods of establishment, performance, and decline. We then conducted short-term batch studies, using substrate-supplemented column materials, on Days 0 (pre-establishment), 27 (establishment), 41 (performance), and 99 (decline) to reveal potential activities of cellulolytic bacteria, fermenters + anaerobic respirers, SRB, and methanogens. PRBs showed active sulfate reduction, with sulfate removal rates (SRR) of approximately 1-3 mol/m3/d, as well as effective removal of Zn2+. Potential activities of fermentative + anaerobic respiratory bacteria were initially high but diminished greatly during establishment and dropped further during performance and decline. In contrast, potential SRB activity rose during establishment, peaked during performance, and diminished as performance declined. Potential methanogen activity was low; in addition, SRB-methanogen substrate competition was shown not to limit SRB activity. Cellulolytic bacteria showed no substrate limitation at any time. However, fermenters experienced substrate limitation by Day 0, SRB by Day 27, and methanogens by Day 41, showing the dependence of each group on upstream populations to provide substrates. All potential activities, except methanogenesis, were ultimately limited by cellulose hydrolysis; in addition, all potential activities except methanogenesis declined substantially by Day 99, showing that long-term substrate deprivation strongly diminished the intrinsic capacity of the PRB community to perform.

Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief.

Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections.

Lack of specificity for PCR assays targeting human Bacteroides 16S rRNA gene: cross-amplification with fish feces.

Methods focused on members of the genus Bacteroides have been increasingly utilized in microbial source-tracking studies for identifying and quantifying sources of nonpoint fecal contamination. We present results using standard and real-time PCR to show cross-amplification of Bacteroides 16S rRNA gene molecular assays targeting human fecal pollution with fecal DNA from freshwater fish species. All except one of the presumptively human-specific assays amplified fecal DNA from at least one fish species, and one real-time PCR assay amplified DNA from all fish species tested. Sequencing of PCR amplicons generated from fish fecal DNA using primers from the real-time assay revealed no mismatches to the human-specific probe sequences, but the nucleotide sequences of clones from fish fecal samples differed markedly from those of human feces, suggesting that the fish-related bacteria may be different strains. Our results strongly demonstrate the potential for cross-amplification of human-specific PCR assays with fish feces, and may call into question the results of studies in which these Bacteroides-specific molecular markers are used to quantify human fecal contamination in waters where fish contribute to fecal inputs.