Efficacy of Peracetic Acid and Chlorine on the Reduction of Shiga Toxin-producing Escherichia coli and a Nonpathogenic E. coli Strain in Preharvest Agricultural Water.

Produce-borne outbreaks of Shiga toxin-producing Escherichia coli (STEC) linked to preharvest water emphasize the need for efficacious water treatment options. This study quantified reductions of STEC and generic E. coli in preharvest agricultural water using commercially available sanitizers. Water was collected from two sources in Virginia (pond, river) and inoculated with either a seven-strain STEC panel or environmental generic E. coli strain TVS 353 (∼9 log10 CFU/100 mL). Triplicate inoculated water samples were equilibrated to 12 or 32°C and treated with peracetic acid (PAA) or chlorine (Cl) [low (PAA:6ppm, Cl:2-4 ppm) or high (PAA:10 ppm, Cl:10-12 ppm) residual concentrations] for an allotted contact time (1, 5, or 10 min). Strains were enumerated, and a log-linear model was used to characterize how treatment combinations influenced reductions. All Cl treatment combinations achieved a ≥3 log10 CFU/100 mL reduction, regardless of strain (3.43 ± 0.25 to 7.05 ± 0.00 log10 CFU/100 mL). Approximately 80% (19/24) and 67% (16/24) of PAA treatment combinations achieved a ≥3 log10 CFU/100 mL for STEC and E. coli TVS 353, respectively. The log-linear model showed contact time (10 > 5 > 1 min) and sanitizer type (Cl > PAA) had the greatest impact on STEC and E. coli TVS 353 reductions (p < 0.001). E. coli TVS 353 in water samples was more resistant to sanitizer treatment (p < 0.001) indicating applicability as a good surrogate. Results demonstrated Cl and PAA can be effective agricultural water treatment strategies when sanitizer chemistry is managed. These data will assist with the development of in-field validation studies and may identify suitable candidates for the registration of antimicrobial pesticide products for use against foodborne pathogens in preharvest agricultural water treatment.

Ranking Food Safety Priorities of the Fresh Produce Industry in the United States.

A broad understanding of community member food safety priorities in the fresh produce supply chain does not currently exist. This information is essential to improve food safety knowledge and practices effectively and efficiently throughout the fresh produce industry; therefore, the goal of this study was to identify and rank community produce safety priorities in the United States. Survey questions were designed and approved by food safety experts for participants to rank 24 fresh produce safety priorities. The anonymous survey was distributed online via Qualtrics™ to fresh produce community members from November 2020 to May 2021. A score was calculated for each priority by summing weighted ranking scores across responses. Descriptive statistics and logistic regression were used to determine frequencies and distribution of response and identify factors (e.g., role in produce safety, size/location of organization/operation) that influenced rankings. A total of 281 respondents represented fourteen different roles in the fresh produce industry, with most identified as growers (39.5%). Produce operations were distributed across the U.S. and annual produce sales ranged from below $25,000 to over $5,000,000. Health and hygiene, training, postharvest sanitation, traceability, and harvest sanitation were ranked as the top five food safety priorities. These findings provide insight into community member priorities in fresh produce safety and can be used to inform intervention efforts, ranging from specialized training for produce growers and packers, industry-driven research projects, and gaps in risk communication strategies.

Sanitizer Type and Contact Time Influence Salmonella Reductions in Preharvest Agricultural Water Used on Virginia Farms.

No Environmental Protection Agency (EPA) chemical treatments for preharvest agricultural water are currently labeled to reduce human health pathogens. The goal of this study was to examine the efficacy of peracetic acid- (PAA) and chlorine (Cl)-based sanitizers against Salmonella in Virginia irrigation water. Water samples (100 mL) were collected at three time points during the growing season (May, July, September) and inoculated with either the 7-strain EPA/FDA-prescribed cocktail or a 5-strain Salmonella produce-borne outbreak cocktail. Experiments were conducted in triplicate for 288 unique combinations of time point, residual sanitizer concentration (low: PAA, 6 ppm; Cl, 2-4 ppm or high: PAA, 10 ppm; Cl, 10-12 ppm), water type (pond, river), water temperature (12°C, 32°C), and contact time (1, 5, 10 min). Salmonella were enumerated after each treatment combination and reductions were calculated. A log-linear model was used to characterize how treatment combinations influenced Salmonella reductions. Salmonella reductions by PAA and Cl ranged from 0.0 ± 0.1 to 5.6 ± 1.3 log10 CFU/100 mL and 2.1 ± 0.2 to 7.1 ± 0.2 log10 CFU/100 mL, respectively. Physicochemical parameters significantly varied by untreated water type; however, Salmonella reductions did not (p = 0.14), likely due to adjusting the sanitizer amounts needed to achieve the target residual concentrations regardless of source water quality. Significant differences (p < 0.05) in Salmonella reductions were observed for treatment combinations, with sanitizer (Cl > PAA) and contact time (10 > 5 > 1 min) having the greatest effects. The log-linear model also revealed that outbreak strains were more treatment-resistant. Results demonstrate that certain treatment combinations with PAA- and Cl-based sanitizers were effective at reducing Salmonella populations in preharvest agricultural water. Awareness and monitoring of water quality parameters are essential for ensuring adequate dosing for the effective treatment of preharvest agricultural water.

Combined Infection Control Interventions Protect Essential Food Workers from Occupational Exposures to SARS-CoV-2 in the Agricultural Environment.

Essential food workers experience elevated risks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection due to prolonged occupational exposures in food production and processing areas, shared transportation (car or bus), and employer-provided shared housing. Our goal was to quantify the daily cumulative risk of SARS-CoV-2 infection for healthy susceptible produce workers and to evaluate the relative reduction in risk attributable to food industry interventions and vaccination. We simulated daily SARS-CoV-2 exposures of indoor and outdoor produce workers through six linked quantitative microbial risk assessment (QMRA) model scenarios. For each scenario, the infectious viral dose emitted by a symptomatic worker was calculated across aerosol, droplet, and fomite-mediated transmission pathways. Standard industry interventions (2-m physical distancing, handwashing, surface disinfection, universal masking, ventilation) were simulated to assess relative risk reductions from baseline risk (no interventions, 1-m distance). Implementation of industry interventions reduced an indoor worker's relative infection risk by 98.0% (0.020; 95% uncertainty interval [UI], 0.005 to 0.104) from baseline risk (1.00; 95% UI, 0.995 to 1.00) and an outdoor worker's relative infection risk by 94.5% (0.027; 95% UI, 0.013 to 0.055) from baseline risk (0.487; 95% UI, 0.257 to 0.825). Integrating these interventions with two-dose mRNA vaccinations (86 to 99% efficacy), representing a worker's protective immunity to infection, reduced the relative infection risk from baseline for indoor workers by 99.9% (0.001; 95% UI, 0.0002 to 0.005) and outdoor workers by 99.6% (0.002; 95% UI, 0.0003 to 0.005). Consistent implementation of combined industry interventions, paired with vaccination, effectively mitigates the elevated risks from occupationally acquired SARS-CoV-2 infection faced by produce workers. IMPORTANCE This is the first study to estimate the daily risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection across a variety of indoor and outdoor environmental settings relevant to food workers (e.g., shared transportation [car or bus], enclosed produce processing facility and accompanying breakroom, outdoor produce harvesting field, shared housing facility) through a linked quantitative microbial risk assessment framework. Our model has demonstrated that the elevated daily SARS-CoV-2 infection risk experienced by indoor and outdoor produce workers can be reduced below 1% when vaccinations (optimal vaccine efficacy, 86 to 99%) are implemented with recommended infection control strategies (e.g., handwashing, surface disinfection, universal masking, physical distancing, and increased ventilation). Our novel findings provide scenario-specific infection risk estimates that can be utilized by food industry managers to target high-risk scenarios with effective infection mitigation strategies, which was informed through more realistic and context-driven modeling estimates of the infection risk faced by essential food workers daily. Bundled interventions, particularly if they include vaccination, yield significant reductions (>99%) in daily SARS-CoV-2 infection risk for essential food workers in enclosed and open-air environments.

Distributions of waterborne pathogens in raw wastewater based on a 14-month, multi-site monitoring campaign.

The California State Water Resources Control Board is the first regulatory body in the United States to develop statewide regulations for direct potable reuse (DPR). To support this effort, a pathogen monitoring campaign was undertaken to develop and implement an optimized standard operating protocol to better characterize the concentration of human pathogens in raw wastewater. Methods to detect relevant viral and protozoan pathogens in raw wastewater were optimized and implemented during a 14-month monitoring campaign. Over 120 samples were collected from five wastewater treatment plants treating a quarter of California's population. Samples were analyzed for two protozoa (Cryptosporidium and Giardia) using microscopy methods, three enteric viruses (enterovirus, adenovirus, and norovirus) using culture and/or molecular methods, and male-specific coliphage using culture methods. The method recovery efficiency was measured in every protozoa sample and every other virus sample to confirm minimum recovery efficiencies were achieved and to correct the concentrations for pathogen losses during sample processing. The results from this study provide the industry with a large, high-quality dataset as demonstrated by the high degree of method sensitivity, method recovery, and QA/QC steps. Such high-quality data on pathogen concentrations in raw wastewater are critical for confirming the level of treatment needed to reduce pathogen concentrations down to acceptable levels for potable water in DPR projects.

Complex Interactions Between Weather, and Microbial and Physicochemical Water Quality Impact the Likelihood of Detecting Foodborne Pathogens in Agricultural Water.

Agricultural water is an important source of foodborne pathogens on produce farms. Managing water-associated risks does not lend itself to one-size-fits-all approaches due to the heterogeneous nature of freshwater environments. To improve our ability to develop location-specific risk management practices, a study was conducted in two produce-growing regions to (i) characterize the relationship between Escherichia coli levels and pathogen presence in agricultural water, and (ii) identify environmental factors associated with pathogen detection. Three AZ and six NY waterways were sampled longitudinally using 10-L grab samples (GS) and 24-h Moore swabs (MS). Regression showed that the likelihood of Salmonella detection (Odds Ratio [OR] = 2.18), and eaeA-stx codetection (OR = 6.49) was significantly greater for MS compared to GS, while the likelihood of detecting L. monocytogenes was not. Regression also showed that eaeA-stx codetection in AZ (OR = 50.2) and NY (OR = 18.4), and Salmonella detection in AZ (OR = 4.4) were significantly associated with E. coli levels, while Salmonella detection in NY was not. Random forest analysis indicated that interactions between environmental factors (e.g., rainfall, temperature, turbidity) (i) were associated with likelihood of pathogen detection and (ii) mediated the relationship between E. coli levels and likelihood of pathogen detection. Our findings suggest that (i) environmental heterogeneity, including interactions between factors, affects microbial water quality, and (ii) E. coli levels alone may not be a suitable indicator of food safety risks. Instead, targeted methods that utilize environmental and microbial data (e.g., models that use turbidity and E. coli levels to predict when there is a high or low risk of surface water being contaminated by pathogens) are needed to assess and mitigate the food safety risks associated with preharvest water use. By identifying environmental factors associated with an increased likelihood of detecting pathogens in agricultural water, this study provides information that (i) can be used to assess when pathogen contamination of agricultural water is likely to occur, and (ii) facilitate development of targeted interventions for individual water sources, providing an alternative to existing one-size-fits-all approaches.

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.

U.S. farmers' opinions on the use of nontraditional water sources for agricultural activities.

Water is a key resource for agricultural production in the United States. Due to projected changes in water availability across the country, long-term sustainability of agricultural production may rely on finding alternatives to traditional water sources. The aim of this study was to assess farmers' opinions on the use of nontraditional water sources (e.g., agricultural runoff, treated wastewater, recycled water, produced water, untreated surface water, and brackish surface and groundwater) for agricultural activities. A survey was distributed to farmers (n = 746) in the Mid-Atlantic and Southwest regions of the United States (U.S.) about water availability and nontraditional irrigation water perceptions. Chi-square, Fisher's exact tests, f-tests, and multinomial and ordinal logistic regression analyses were conducted. Of farmers surveyed, 80% (431/543) considered the use of nontraditional water sources to be at least moderately important and 61% (444/727) would use nontraditional water if given the option. Each of the following factors individually increased the likelihood that a farmer considered nontraditional water very important for agriculture: Farmers who lived in the Southwest region compared to the Mid-Atlantic, farmers who were concerned about water availability compared with those who were not, farmers with a graduate or professional degree compared to those with less education, farmers with access to nontraditional water, and farmers with some knowledge of nontraditional water compared to those with no reported knowledge. Concern about water availability and knowledge of nontraditional water sources were significantly associated with willingness to use these water sources (p < 0.001 for both). Water quality, food safety and health risks were the main concerns regarding nontraditional water use across both regions. Willingness to use nontraditional water increased significantly if the water quality was proven to be as good or better than farmers' current water sources (63% vs. 84%; p < 0.001). Projects focused on nontraditional water use in agriculture should be regionally tailored as our data found significant differences between farmers in two distinct U.S. regions.

Understanding grower perceptions and attitudes on the use of nontraditional water sources, including reclaimed or recycled water, in the semi-arid Southwest United States.

The use of nontraditional water sources, including reclaimed or recycled water, has become a desirable option to meet increasing demands in water stressed regions. In the Southwest United States, utilization of alternative water sources is becoming increasingly common, including use for landscape irrigation, environmental enhancement, cooling and power generation, potable reuse, and as a source water for agricultural irrigation. While much research has gone into identifying public perception towards water reuse schemes, little attention has been given to understanding grower attitudes, perceptions, and knowledge on the use of nontraditional water, including reclaimed water, in agriculture and how that may influence grower acceptance and production practices. This unique study utilized a needs assessment survey of growers (n = 521) within the Southwest region of the United States to gain an understanding of industry attitudes and needs regarding nontraditional water in agriculture. Results indicate that the majority of survey respondents were concerned with water availability (67.49%) yet less than half (48.30%) thought using a nontraditional water source in agriculture was 'very important'. Interestingly, respondents rated irrigation of 'food crops' third (42.20%) among agricultural activities for which they would be willing to use nontraditional water sources, behind irrigation of forage crops (61.60%) and dust control (61.60%). The importance of the use of nontraditional water sources in agriculture was influenced mostly by farm size (p = 0.007) and primary water source (p = 0.016), and the level of education was significant in respondent's level of concern over water availability (p = 0.021). Information on the quality of nontraditional water sources, showing that it is as good or better than respondents current sources, was found to shift rejection and uncertainty towards acceptance by 16.04%. The results of this study provide insight into perceived risks, willingness to use, drivers and constraints to grower adoption, and preferred methods of education regarding water reuse in agriculture. These findings can be used by water managers and planners to aid in the adoption of nontraditional waters, including reclaimed or recycled water, in agriculture thus extending water resources, securing food supplies, and protecting public health.

Overview of Monitoring Techniques for Evaluating Water Quality at Potable Reuse Treatment Facilities.

Needless to say, the safety of treated water for potable reuse must be definitively ensured. Numerous methods are available for assessing water quality; it's important to understand their challenges and limitations.

Reducing uncertainty in estimating virus reduction by advanced water treatment processes.

Treatment of wastewater for potable reuse requires the reduction of enteric viruses to levels that pose no significant risk to human health. Advanced water treatment trains (e.g., chemical clarification, reverse osmosis, ultrafiltration, advanced oxidation) have been developed to provide reductions of viruses to differing levels of regulatory control depending upon the levels of human exposure and associated health risks. Importance in any assessment is information on the concentration and types of viruses in the untreated wastewater, as well as the degree of removal by each treatment process. However, it is critical that the uncertainty associated with virus concentration and removal or inactivation by wastewater treatment be understood to improve these estimates and identifying research needs. We reviewed the critically literature to assess to identify uncertainty in these estimates. Biological diversity within families and genera of viruses (e.g. enteroviruses, rotaviruses, adenoviruses, reoviruses, noroviruses) and specific virus types (e.g. serotypes or genotypes) creates the greatest uncertainty. These aspects affect the methods for detection and quantification of viruses and anticipated removal efficiency by treatment processes. Approaches to reduce uncertainty may include; 1) inclusion of a virus indicator for assessing efficiency of virus concentration and detection by molecular methods for each sample, 2) use of viruses most resistant to individual treatment processes (e.g. adenoviruses for UV light disinfection and reoviruses for chlorination), 3) data on ratio of virion or genome copies to infectivity in untreated wastewater, and 4) assessment of virus removal at field scale treatment systems to verify laboratory and pilot plant data for virus removal.

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.

A unified method to process biosolids samples for the recovery of bacterial, viral, and helminths pathogens.

For land application, biosolids are classified as Class A or Class B based on the levels of bacterial, viral, and helminths pathogens in residual biosolids. The current EPA methods for the detection of these groups of pathogens in biosolids include discrete steps. Therefore, a separate sample is processed independently to quantify the number of each group of the pathogens in biosolids. The aim of the study was to develop a unified method for simultaneous processing of a single biosolids sample to recover bacterial, viral, and helminths pathogens. At the first stage for developing a simultaneous method, nine eluents were compared for their efficiency to recover viruses from a 100 gm spiked biosolids sample. In the second stage, the three top performing eluents were thoroughly evaluated for the recovery of bacteria, viruses, and helminthes. For all three groups of pathogens, the glycine-based eluent provided higher recovery than the beef extract-based eluent. Additional experiments were performed to optimize performance of glycine-based eluent under various procedural factors such as, solids to eluent ratio, stir time, and centrifugation conditions. Last, the new method was directly compared with the EPA methods for the recovery of the three groups of pathogens spiked in duplicate samples of biosolids collected from different sources. For viruses, the new method yielded up to 10% higher recoveries than the EPA method. For bacteria and helminths, recoveries were 74% and 83% by the new method compared to 34% and 68% by the EPA method, respectively. The unified sample processing method significantly reduces the time required for processing biosolids samples for different groups of pathogens; it is less impacted by the intrinsic variability of samples, while providing higher yields (P = 0.05) and greater consistency than the current EPA methods.

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.

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.

PCR inhibitor levels in concentrates of biosolid samples predicted by a new method based on excitation-emission matrix spectroscopy.

Biosolids contain a wide variety of organic contaminants that are known for their ability to inhibit PCR. During sample processing, these contaminants are coconcentrated with microorganisms. Elevated concentrations of these compounds in concentrates render samples unsuitable for molecular applications. Glycine-based elution and recovery methods have been shown to generate samples with fewer PCR inhibitory compounds than the current U.S. EPA-recommended method for pathogen recovery from biosolids. Even with glycine-based methods, PCR inhibitors still persist in concentrations that may interfere with nucleic acid amplification. This results in considerable loss of time and resources and increases the probability of false negatives. A method to estimate the degree of inhibition prior to application of molecular methods is desirable. Here we report fluorescence excitation-emission matrix (EEM) profiling as a tool for predicting levels of molecular inhibition in sample concentrates of biosolids.

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.

Optimization of a reusable hollow-fiber ultrafilter for simultaneous concentration of enteric bacteria, protozoa, and viruses from water.

The detection and identification of pathogens from water samples remain challenging due to variations in recovery rates and the cost of procedures. Ultrafiltration offers the possibility to concentrate viral, bacterial, and protozoan organisms in a single process by using size-exclusion-based filtration. In this study, two hollow-fiber ultrafilters with 50,000-molecular-weight cutoffs were evaluated to concentrate microorganisms from 2- and 10-liter water samples. When known quantities (10(5) to 10(6) CFU/liter) of two species of enteric bacteria were introduced and concentrated from 2 liters of sterile water, the addition of 0.1% Tween 80 increased Escherichia coli strain K-12 recoveries from 70 to 84% and Salmonella enterica serovar Enteritidis recoveries from 36 to 72%. An E. coli antibiotic-resistant strain, XL1-Blue, was recovered at a level (87%) similar to that for strain K-12 (96%) from 10 liters of sterile water. When E. coli XL1-Blue was introduced into 10 liters of nonsterile Rio Grande water with higher turbidity levels (23 to 29 nephelometric turbidity units) at two inoculum levels (9 x 10(5) and 2.4 x 10(3) per liter), the recovery efficiencies were 89 and 92%, respectively. The simultaneous addition of E. coli XL1-Blue (9 x 10(5) CFU/liter), Cryptosporidium parvum oocysts (10 oocysts/liter), phage T1 (10(5) PFU/liter), and phage PP7 (10(5) PFU/liter) to 10 liters of Rio Grande surface water resulted in mean recoveries of 96, 54, 59, and 46%, respectively. Using a variety of surface waters from around the United States, we obtained recovery efficiencies for bacteria and viruses that were similar to those observed with the Rio Grande samples, but recovery of Cryptosporidium oocysts was decreased, averaging 32% (the site of collection of these samples had previously been identified as problematic for oocyst recovery). Results indicate that the use of ultrafiltration for simultaneous recovery of bacterial, viral, and protozoan pathogens from variable surface waters is ready for field deployment.

Effects of pH and magnetic material on immunomagnetic separation of Cryptosporidium oocysts from concentrated water samples.

In this study, we examined the effect that magnetic materials and pH have on the recoveries of Cryptosporidium oocysts by immunomagnetic separation (IMS). We determined that particles that were concentrated on a magnet during bead separation have no influence on oocyst recovery; however, removal of these particles did influence pH values. The optimal pH of the IMS was determined to be 7.0. The numbers of oocysts recovered from deionized water at pH 7.0 were 26.3% higher than those recovered from samples that were not at optimal pH. The results indicate that the buffers in the IMS kit did not adequately maintain an optimum pH in some water samples. By adjusting the pH of concentrated environmental water samples to 7.0, recoveries of oocysts increased by 26.4% compared to recoveries from samples where the pH was not adjusted.