A comparison of methods to detect low levels of Salmonella enterica in surface waters to support antimicrobial resistance surveillance efforts performed in multiple laboratories.

Developing effective and sensitive detection methods for antimicrobial resistant Salmonella enterica from surface water is a goal of the National Antimicrobial Resistance Monitoring System (NARMS). There are no specified methods for recovery of S. enterica in surface waters in the U.S. A multi-laboratory evaluation of four methods - bulk water enrichment (BW), vertical Modified Moore Swab (VMMS), modified Standard Method 9260.B2 (SM), and dead-end ultrafiltration (DEUF) - was undertaken to recover S. enterica from surface water. In Phase 1, one-liter volumes of water were collected from the same site on five different dates. Water was shipped and analyzed at four different laboratory locations (A, B, C, and D) for recovery of 1) inoculated fluorescent S. Typhimurium strain (ca. 30 CFU/L) and 2) Salmonella present in the water sampled. At each location, BW, VMMS, or SM recovery was performed on five separate 1 L water samples. Twenty 1 L water samples were subjected to each recovery method, and overall, sixty 1 L samples were assayed for Salmonella. Inoculated, fluorescent Salmonella Typhimurium and environmental Salmonella spp. were recovered from 65 % (39/60) and 45 % (27/60) of water samples, respectively. BW, VMMS, and SM recovered fluorescent S. Typhimurium from 60 %, 60 %, and 75 % of inoculated samples, respectively. Analysis by Chi-squared test determined laboratory location had a significant (p < 0.05) effect on fluorescent S. Typhimurium recovery compared to method or date of water collection. In Phase 2, recovery of inoculated fluorescent S. Typhimurium from 1 L samples by SM and DEUF was compared at laboratory locations B and D. SM and DEUF recovered fluorescent S. Typhimurium from 100 % (20/20) and 95 % (19/20) of inoculated water samples, respectively; laboratory location (p > 0.05) did not affect Salmonella recovery. Uniform laboratory methodology and training should be prioritized in conducting Salmonella recovery from surface water in laboratories.

Sorption and desorption behavior of four antibiotics at concentrations simulating wastewater reuse in agricultural and forested soils.

Due to rises in antibiotic resistance, fate and transport of antibiotics in soil systems requires greater understanding to determine potential risks to human and animal health. Adsorption coefficients (Kd and Kf) are standard measures for determining sorption capacity and partitioning behavior of organic contaminants in solid matrices. Frequently, sorption studies use higher antibiotic concentrations (mg L-1) and larger spiked water volume to mass of soil (>5:1), which may not reflect sorption behaviors of antibiotics at low concentrations (ng L-1 - µg L-1) in natural soils. The aim of this study was to determine sorption and desorption behaviors of four antibiotics commonly found in soils due to wastewater reuse using parameters replicating typical soil conditions. Concentrations (µg L-1) of sulfamethoxazole (SMX), trimethoprim (TMP), lincomycin (LIN) and ofloxacin (OFL) were equilibrated with four soil types at a 2:1 ratio of spiked water volume to mass of soil, which better represents field conditions. Log Kf and log Kfoc value ranges in this study were 1.88-1.95 and 3.2-4.7 for TMP, 0.43-1.4 and 2.7-3.2 for SMX, and 0.65-1.4 and 2.0-4.1 for LIN, respectively. Ofloxacin adsorbed tightly to soil particles, and adsorption coefficients could not be calculated. Sorption values were higher than previous studies that used similar soil types but had higher ratios of spiking solution to mass of soil (>5:1). Overall, OFL and TMP are expected to strongly interact with soil particles and be less mobile, while SMX and LIN are expected to be more mobile due to weaker sorption interactions.

Assessment of Soil to Mitigate Antibiotics in the Environment Due to Release of Wastewater Treatment Plant Effluent.

With low levels of human antibiotics in the environment due to release of wastewater treatment plant (WWTP) effluent, concern is rising about impacts on human health and antibiotic resistance development. Furthermore, WWTP effluent may be released into waterways used as drinking water sources. The aim of this study was to analyze three antibiotics important to human health (sulfamethoxazole, ofloxacin, and trimethoprim) in soil and groundwater at a long-term wastewater reuse system that spray irrigates effluent. Soil samples were collected (i) at a site that had not received irrigation for 7 mo (approximate background concentrations), and then at the same site after (ii) one irrigation event and (iii) 10 wk of irrigation. Water samples were collected three times per year to capture seasonal variability. Sulfamethoxazole was typically at the highest concentrations in effluent (22 ± 3.7 µg L) with ofloxacin and trimethoprim at 2.2 ± 0.6 and 1.0 ± 0.02 µg L, respectively. In the soil, ofloxacin had the highest background concentrations (650 ± 204 ng kg), whereas concentrations of sulfamethoxazole were highest after continuous effluent irrigation (730 ± 360 ng kg). Trimethoprim was only quantified in soil after 10 wk of effluent irrigation (190 ± 71 ng kg). Groundwater concentrations were typically <25 ng L with high concentrations of 660 ± 20 and 67 ± 7.0 ng L for sulfamethoxazole and ofloxacin, respectively. Given that antibiotics interacted with the soil profile and groundwater concentrations were frequently about 1000-fold lower than effluent, soil may be an adequate tertiary treatment for WWTP effluent leading to improved water quality and protection of human health.

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.

How Should We Be Determining Background and Baseline Antibiotic Resistance Levels in Agroecosystem Research?

Although historically, antibiotic resistance has occurred naturally in environmental bacteria, many questions remain regarding the specifics of how humans and animals contribute to the development and spread of antibiotic resistance in agroecosystems. Additional research is necessary to completely understand the potential risks to human, animal, and ecological health in systems altered by antibiotic-resistance-related contamination. At present, analyzing and interpreting the effects of human and animal inputs on antibiotic resistance in agroecosystems is difficult, since standard research terminology and protocols do not exist for studying background and baseline levels of resistance in the environment. To improve the state of science in antibiotic-resistance-related research in agroecosystems, researchers are encouraged to incorporate baseline data within the study system and background data from outside the study system to normalize the study data and determine the potential impact of antibiotic-resistance-related determinants on a specific agroecosystem. Therefore, the aims of this review were to (i) present standard definitions for commonly used terms in environmental antibiotic resistance research and (ii) illustrate the need for research standards (normalization) within and between studies of antibiotic resistance in agroecosystems. To foster synergy among antibiotic resistance researchers, a new surveillance and decision-making tool is proposed to assist researchers in determining the most relevant and important antibiotic-resistance-related targets to focus on in their given agroecosystems. Incorporation of these components within antibiotic-resistance-related studies should allow for a more comprehensive and accurate picture of the current and future states of antibiotic resistance in the environment.

Uptake of Three Antibiotics and an Antiepileptic Drug by Wheat Crops Spray Irrigated with Wastewater Treatment Plant Effluent.

With rising demands on water supplies necessitating water reuse, wastewater treatment plant (WWTP) effluent is often used to irrigate agricultural lands. Emerging contaminants, like pharmaceuticals and personal care products (PPCPs), are frequently found in effluent due to limited removal during WWTP processes. Concern has arisen about the environmental fate of PPCPs, especially regarding plant uptake. The aim of this study was to analyze uptake of sulfamethoxazole, trimethoprim, ofloxacin, and carbamazepine in wheat ( L.) plants that were spray-irrigated with WWTP effluent. Wheat was collected before and during harvest, and plants were divided into grain and straw. Subsamples were rinsed with methanol to remove compounds adhering to surfaces. All plant tissues underwent liquid-solid extraction, solid-phase extraction cleanup, and liquid chromatography-tandem mass spectrometry analysis. Residues of each compound were present on most plant surfaces. Ofloxacin was found throughout the plant, with higher concentrations in the straw (10.2 ± 7.05 ng g) and lower concentrations in the grain (2.28 ± 0.89 ng g). Trimethoprim was found only on grain or straw surfaces, whereas carbamazepine and sulfamethoxazole were concentrated within the grain (1.88 ± 2.11 and 0.64 ± 0.37 ng g, respectively). These findings demonstrate that PPCPs can be taken up into wheat plants and adhere to plant surfaces when WWTP effluent is spray-irrigated. The presence of PPCPs within and on the surfaces of plants used as food sources raises the question of potential health risks for humans and animals.