J. Brett Sallach

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.

Challenges in the Measurement of Antibiotics and in Evaluating Their Impacts in Agroecosystems: A Critical Review

Large quantities of antibiotics are used in agricultural production, resulting in their release to agroecosystems through numerous pathways, including land application of contaminated manure, runoff from manure-fertilized fields, and wastewater irrigation of croplands. Antibiotics and their transformation products (TPs) exhibit a wide range of physico-chemical and biological properties and thus present substantive analytical challenges. Advances in the measurement of these compounds in various environmental compartments (plants, manure, soil, sediment, and water) have uncovered a previously unrealized landscape of antibiotic residues. These advanced multiresidue methods, designed to measure sub-ng g concentrations in complex mixtures, remain limited by the inherent intricacy of the sample matrices and the difficultly in eliminating interferences that affect antibiotic detection. While efficient extraction methods combined with high sensitivity analysis by liquid chromatography/mass spectrometry can provide accurate quantification of antibiotics and their TPs, measured concentrations do not necessarily reflect their bioavailable fractions and effects in the environment. Consequently, there is a need to complement chemical analysis with biological assays that can provide information on bioavailability, biological activity, and effects of mixtures. Enzyme-linked immunosorbent assays (ELISA), often used as screening tools for antibiotic residues, may be useful for detecting the presence of structurally related antibiotic mixtures but not their effects. Other tools, including bioreporter assays, hold promise in measuring bioavailable antibiotics and could provide insights on their biological activity. Improved assessment of the ecological and human health risks associated with antibiotics in agroecosystems requires continued advances in analytical accuracy and sensitivity through improvements in sample preparation, instrumentation, and screening technologies.

Concomitant uptake of antimicrobials and Salmonella in soil and into lettuce following wastewater irrigation

The use of wastewater for irrigation may introduce antimicrobials and human pathogens into the food supply through vegetative uptake. The objective of this study was to investigate the uptake of three antimicrobials and Salmonella in two lettuce cultivars. After repeated subirrigation with synthetic wastewater, lettuce leaves and soil were collected at three sequential harvests. The internalization frequency of Salmonella in lettuce was low. A soil horizon-influenced Salmonella concentration gradient was determined with concentrations in bottom soil 2 log CFU/g higher than in top soil. Lincomycin and sulfamethoxazole were recovered from lettuce leaves at concentrations as high as 822 ng/g and 125 ng/g fresh weight, respectively. Antimicrobial concentrations in lettuce decreased from the first to the third harvest suggesting that the plant growth rate may exceed antimicrobial uptake rates. Accumulation of antimicrobials was significantly different between cultivars demonstrating a subspecies level variation in uptake of antibiotics in lettuce.

Effects of soil texture and drought stress on the uptake of antibiotics and the internalization of Salmonella in lettuce following wastewater irrigation

Treated wastewater is expected to be increasingly used as an alternative source of irrigation water in areas facing fresh water scarcity. Understanding the behaviors of contaminants from wastewater in soil and plants following irrigation is critical to assess and manage the risks associated with wastewater irrigation. The objective of this study was to evaluate the effects of soil texture and drought stress on the uptake of antibiotics and the internalization of human pathogens into lettuce through root uptake following wastewater irrigation. Lettuce grown in three soils with variability in soil texture (loam, sandy loam, and sand) and under different levels of water stress (no drought control, mild drought, and severe drought) were irrigated with synthetic wastewater containing three antibiotics (sulfamethoxazole, lincomycin and oxytetracycline) and one Salmonella strain a single time prior to harvest. Antibiotic uptake in lettuce was compound-specific and generally low. Only sulfamethoxazole was detected in lettuce with increasing uptake corresponding to increasing sand content in soil. Increased drought stress resulted in increased uptake of lincomycin and decreased uptake of oxytetracycline and sulfamethoxazole. The internalization of Salmonella was highly dependent on the concentration of the pathogen in irrigation water. Irrigation water containing 5 Log CFU/mL Salmonella resulted in limited incidence of internalization. When irrigation water contained 8 Log CFU/mL Salmonella, the internalization frequency was significantly higher in lettuce grown in sand than in loam (p = 0.009), and was significantly higher in lettuce exposed to severe drought than in unstressed lettuce (p = 0.049). This work demonstrated how environmental factors affected the risk of contaminant uptake by food crops following wastewater irrigation.

Sequestration of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin by activated carbon eliminates bioavailability and the suppression of immune function in mice

The effectiveness of activated carbon in reducing the bioavailability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was examined from the context of using in situ sorbent amendments to remediate soils/sediments contaminated with polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs). This technology has gained rapid acceptance based on observations that activated carbon amendments predictably lower PCDD/F concentrations in water and bioaccumulation by simple aquatic organisms and earthworms; it has been assumed that bioavailability to mammals is similarly reduced, although this has been disproven for other sorbent materials. In the present study TCDD was absorbed to a microporous activated carbon (TCDD-AC) using the incipient wetness method. An aqueous suspension of TCDD-AC and an equivalent dosage of TCDD in corn oil were administered by oral gavage to B6C3F1 mice. The relative bioavailability of TCDD-AC was determined by quantifying and comparing the hepatic induction of cyp1A1 (messenger ribonucleic acid) and suppression of the immunoglobulin M antibody-forming cell immune response by the 2 forms of TCDD. A concentration-dependent response was observed for both assays when TCDD in corn oil was administered to mice. However, when equivalent masses of TCDD were administered as TCDD-AC, no induction of cyp1A1 or suppression of the immunoglobulin M antibody-forming cell response was observed. The absence of these 2 sensitive aryl hydrocarbon receptor-mediated responses in mice provides the first direct evidence that activated carbon can sequester TCDD in a form that eliminates its bioavailability to mammals. These results support the premise that activated carbon can be used to reduce the bioeffective dose of TCDD delivered to mammals and that activated carbon amendments may provide a low-cost alternative to traditional remediation technologies. Environ Toxicol Chem 2017;36:2671-2678.

Sequestration of TCDD by activated carbon eliminates bioavailability and the suppression of immune function in mice.

The effectiveness of activated carbon in reducing the bioavailability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was examined from the context of using in situ sorbent amendments to remediate soils/sediments contaminated with polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs). This technology has gained rapid acceptance based on observations that activated carbon amendments predictably lower PCDD/F concentrations in water and bioaccumulation by simple aquatic organisms and earthworms; it has been assumed that bioavailability to mammals is similarly reduced, although this has been disproven for other sorbent materials. In the present study TCDD was absorbed to a microporous activated carbon (TCDD-AC) using the incipient wetness method. An aqueous suspension of TCDD-AC and an equivalent dosage of TCDD in corn oil were administered by oral gavage to B6C3F1 mice. The relative bioavailability of TCDD-AC was determined by quantifying and comparing the hepatic induction of cyp1A1 (messenger ribonucleic acid) and suppression of the immunoglobulin M antibody-forming cell immune response by the 2 forms of TCDD. A concentration-dependent response was observed for both assays when TCDD in corn oil was administered to mice. However, when equivalent masses of TCDD were administered as TCDD-AC, no induction of cyp1A1 or suppression of the immunoglobulin M antibody-forming cell response was observed. The absence of these 2 sensitive aryl hydrocarbon receptor-mediated responses in mice provides the first direct evidence that activated carbon can sequester TCDD in a form that eliminates its bioavailability to mammals. These results support the premise that activated carbon can be used to reduce the bioeffective dose of TCDD delivered to mammals and that activated carbon amendments may provide a low-cost alternative to traditional remediation technologies. Environ Toxicol Chem 2017;36:2671-2678.

Development and comparison of four methods for the extraction of antibiotics from a vegetative matrix.

Studies have shown the potential for antibiotic uptake into food crops from irrigation water and soils containing pharmaceuticals. The objective of the present study was to develop and compare methods quantifying uptake of antibiotics in food crops. Four methods were evaluated: freeze-and-thaw cell lysing, mechanical maceration, tissue sonication, and microwave-assisted solvent extraction. Four antibiotics (ciprofloxacin, lincomycin, oxytetracycline, and sulfamethoxazole) were tested representing 4 classes of antibiotics. The methods were evaluated based on method detection limits, analyte recoveries, and sample preparation time. The 2 most viable methods, freeze-and-thaw lysing and mechanical maceration, were used on replicate lettuce (Lactuca sativa) samples grown using irrigation water spiked with 3 of the antibiotic contaminants. Only lincomycin and sulfamethoxazole were detected in lettuce samples at concentrations as high as 1757 ng/g and 425 ng/g, with detection limits of 57 ng/g and 35 ng/g, respectively. Freeze-and-thaw cell lysing provided the highest level of extraction efficiency on environmental samples and required the least amount of sample preparation while providing adequate detection limits and reproducible analyte recovery.

TCDD administered on activated carbon eliminates bioavailability and subsequent shifts to a key murine gut commensal

Activated carbon (AC) is an increasingly attractive remediation alternative for the sequestration of dioxins at contaminated sites globally. However, the potential for AC to reduce the bioavailability of dioxins in mammals and the residing gut microbiota has received less attention. This question was partially answered in a recent study examining 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hallmark toxic responses in mice administered with TCDD sequestered by AC or freely available in corn oil by oral gavage. Results from that study support the use of AC to significantly reduce the bioavailability of TCDD to the host. Herein, we examined the bioavailability of TCDD sequestered to AC on a key murine gut commensal and the influence of AC on the community structure of the gut microbiota. The analysis included qPCR to quantify the expression of segmented filamentous bacteria (SFB) in the mouse ileum, which has responded to TCDD-induced host toxicity in previous studies and community structure via sequencing the 16S ribosomal RNA (rRNA) gene. The expression of SFB 16S rRNA gene and functional genes significantly increased with TCDD administered with corn oil vehicle. Such a response was absent when TCDD was sequestered by AC. In addition, AC appeared to have a minimal influence on murine gut community structure and diversity, affecting only the relative abundance of Lactobacillaceae and two other groups. Results of this study further support the remedial use of AC for eliminating bioavailability of TCDD to host and subsequent influence on the gut microbiome.

Activated carbons of varying pore structure eliminate the bioavailability of 2,3,7,8-tetrachlorodibenzo-p-dioxin to a mammalian (mouse) model

The use of activated carbon (AC) as an in situ sorbent amendment to sequester polychlorinated-dibenzo-p-dioxins and furans (PCDD/Fs) present in contaminated soils and sediments has recently gained attention as a novel remedial approach. This remedy could be implemented at much lower cost while minimizing habitat destruction as compared to traditional remediation technologies that rely on dredging/excavation and landfilling. Several prior studies have demonstrated the ability of AC amendments to reduce pore water concentrations and hence bioaccumulation of PCDD/Fs in invertebrate species. However, our recent study was the first to show that AC had the ability to sequester 2,3,7,8‑tetrachlorodibenzo‑p‑dioxin (TCDD) in a form that eliminated bioavailability to a mammalian (mouse) model. Here we show that three commercially available ACs, representing a wide range of pore size distributions, were equally effective in eliminating the bioavailability of TCDD based upon two sensitive bioassays, hepatic induction of cyp1A1 mRNA and immunoglobulin M antibody-forming cell response. These results provide direct evidence that a wide range of structurally diverse commercially available ACs may be suitable for use as in situ sorbent amendments to provide a cost-effective remedy for PCDD/F contaminated soils and sediments. Potentially, adaption of this technology would minimize habitat destruction and be protective of ecosystem and human health.