Jean E. McLain

Irrigation Waters as a Source of Pathogenic Microorganisms in Produce. A Review

There is increasing evidence that consumption of raw fresh produce is a major factor contributing to human gastrointestinal illness. A wide variety of pathogens contribute to food-borne illnesses, including bacteria (e.g., Salmonella, pathogenic Escherichia coli), protozoa (e.g., Cryptosporidium, Giardia), and viruses (e.g., noroviruses). Large-scale production of produce typically requires some form of irrigation during the growing season. There is a rapidly growing body of research documenting and elucidating the pathways of produce contamination by water-borne pathogens. However, many gaps still exist in our knowledge and understanding. The purpose of this review is to provide a comprehensive approach to the issue, including the most recent research. Topics covered include: temporal and spatial variability, and regional differences, in pathogen and indicator organism concentrations in water; direct and circumstantial evidence for contaminated water as a source of food-borne pathogens; fate and transport of pathogens and indicator organisms in irrigation systems, and the role of environmental microbial reservoirs; and current standards for irrigation water quality, and risk assessment. A concerted effort by researchers and practitioners is needed to maintain food safety of fresh produce in an increasingly intensive food production system and limited and declining irrigation water resources.

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.

US agricultural nitrous oxide emissions: context, status, and trends

The use of commercial nitrogen (N) fertilizers has led to enormous increases in US agricultural productivity. However, N losses from agricultural systems have resulted in numerous deleterious environmental impacts, including a continuing increase in atmospheric nitrous oxide (N2O), a greenhouse gas (GHG) and an important catalyst of stratospheric ozone depletion. Although associated with about 7% of total US GHG emissions, agricultural systems account for 75% of total US N2O emissions. Increased productivity in the crop and livestock sectors during the past 30 to 70 years has resulted in decreased N2O emissions per unit of production, but N2O emissions from US agriculture continue to increase at a rate of approximately 0.46 teragrams of carbon dioxide equivalents per year (2002–2009). This rate is lower than that during the late 20th century. Improvements in agricultural productivity alone may be insufficient to lead to reduced emissions; implementing strategies specifically targeted at reducing N2O emiss...

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.

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.

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.

Soil persistence and fate of carbamazepine, lincomycin, caffeine, and ibuprofen from wastewater reuse.

The reuse of treated wastewater for groundwater recharge is an effective way to provide advanced treatment and water storage. Contaminants such as human drugs have been identified as a potential problem for use of this water. Gilbert, Arizona maintains a 28.3-ha facility designed to recharge 15,150 m d through recharge basins constructed on native soil. The facility averages an infiltration rate of >5 cm d, resulting in the potential of pharmaceutical compounds leaching to groundwater. One 4-ha basin was selected for spatial sampling of four pharmaceutically active compounds (PhACs). The compounds were carbamazepine, lincomycin, ibuprofen, and caffeine. Soils were extracted and analyzed using pressurized liquid extraction and liquid chromatography-mass spectrometry-mass spectrometry. The concentration of ibuprofen was below detection limits in all samples. Lincomycin exhibited no net accumulation from year to year but had significantly higher concentrations from depths of 0 to 5 cm than from depths >10 cm. Carbamazepine had the lowest concentration at 0 to 5 cm (0.18 ng g soil), providing evidence that there is potential degradation of carbamazepine in surface soils. Carbamazepine also exhibited significant accumulation from year to year. Caffeine exhibited net accumulation and had higher concentrations in surface samples. The accumulation of PhACs in the soil beneath recharge basins indicates that PhACs are being removed from the infiltrating water and that, regarding ibuprofen and lincomycin, the treatment is sustainable due to the lack of accumulation. Regarding carbamazepine and caffeine, further investigations are needed to determine possible management and environmental conditions that could prevent accumulation.

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.

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 β-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.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, ...

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

PCR device amplifies DNA directly from tissue samples and achieves smartphone-based quantification at early thermal cycles. 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.