bConsumption of vegetables represents a route of direct human exposure to bacteria found in soil. The present study evaluated the complement of bacteria resistant to various antibiotics on vegetables often eaten raw (tomato, cucumber, pepper, carrot, radish, lettuce) and how this might vary with growth in soil fertilized inorganically or with dairy or swine manure. Vegetables were sown into field plots immediately following fertilization and harvested when of marketable quality. Vegetable and soil samples were evaluated for viable antibiotic-resistant bacteria by plate count on Chromocult medium supplemented with antibiotics at clinical breakpoint concentrations. DNA was extracted from soil and vegetables and evaluated by PCR for the presence of 46 gene targets associated with plasmid incompatibility groups, integrons, or antibiotic resistance genes. Soil receiving manure was enriched in antibiotic-resistant bacteria and various antibiotic resistance determinants. There was no coherent corresponding increase in the abundance of antibiotic-resistant bacteria enumerated from any vegetable grown in manure-fertilized soil. Numerous antibiotic resistance determinants were detected in DNA extracted from vegetables grown in unmanured soil. A smaller number of determinants were additionally detected on vegetables grown only in manured and not in unmanured soil. Overall, consumption of raw vegetables represents a route of human exposure to antibiotic-resistant bacteria and resistance determinants naturally present in soil. However, the detection of some determinants on vegetables grown only in freshly manured soil reinforces the advisability of pretreating manure through composting or other stabilization processes or mandating offset times between manuring and harvesting vegetables for human consumption.

No abstract

The World Health Organization has identified antibiotic resistance as one of the top three threats to global health. There is concern that the use of antibiotics as growth promoting agents in livestock production contributes to the increasingly problematic development of antibiotic resistance. Many antibiotics are excreted at high rates, and the land application of animal manures represents a significant source of environmental exposure to these agents. To evaluate the long-term effects of antibiotic exposure on soil microbial populations, a series of field plots were established in 1999 that have since received annual applications of a mixture of sulfamethazine (SMZ), tylosin (TYL), and chlortetracycline (CTC). During the first 6 yr (1999-2004) soils were treated at concentrations of 0, 0.01 0.1, and 1.0 mg kg soil, in subsequent years at concentrations of 0, 0.1, 1.0, and 10 mg kg soil. The lower end of this concentration range is within that which would result from an annual application of manure from medicated swine. Following ten annual applications, the fate of the drugs in the soil was evaluated. Residues of SMZ and TYL, but not CTC were removed much more rapidly in soil with a history of exposure to 10 mg/kg drugs than in untreated control soil. Residues of C-SMZ were rapidly and thoroughly mineralized to CO in the historically treated soils, but not in the untreated soil. A SMZ-degrading sp. was isolated from the treated soil. Overall, these results indicate that soil bacteria adapt to long-term exposure to some veterinary antibiotics resulting in sharply reduced persistence. Accelerated biodegradation of antibiotics in matrices exposed to agricultural, wastewater, or pharmaceutical manufacturing effluents would attenuate environmental exposure to antibiotics, and merits investigation in the context of assessing potential risks of antibiotic resistance development in environmental matrices.

Animal manures recycled onto crop production land carry antibiotic-resistant bacteria. The present study evaluated the fate in soil of selected genes associated with antibiotic resistance or genetic mobility in field plots cropped to vegetables and managed according to normal farming practice. Referenced to unmanured soil, fertilization with swine or dairy manure increased the relative abundance of the gene targets sul1, erm(B), str(B), int1, and IncW repA. Following manure application in the spring of 2012, gene copy number decayed exponentially, reaching background levels by the fall of 2012. In contrast, gene copy number following manure application in the fall of 2012 or spring of 2013 increased significantly in the weeks following application and then declined. In both cases, the relative abundance of gene copy numbers had not returned to background levels by the fall of 2013. Overall, these results suggest that under conditions characteristic of agriculture in a humid continental climate, a 1-year period following a commercial application of raw manure is sufficient to ensure that an additional soil burden of antibiotic resistance genes approaches background. The relative abundance of several gene targets exceeded background during the growing season following a spring application or an application done the previous fall. Results from the present study reinforce the advisability of treating manure prior to use in crop production systems.

No abstract

The consumption of crops fertilized with human waste represents a potential route of exposure to antibiotic-resistant fecal bacteria. The present study evaluated the abundance of bacteria and antibiotic resistance genes by using both culture-dependent and molecular methods. Various vegetables (lettuce, carrots, radish, and tomatoes) were sown into field plots fertilized inorganically or with class B biosolids or untreated municipal sewage sludge and harvested when of marketable quality. Analysis of viable pathogenic bacteria or antibiotic-resistant coliform bacteria by plate counts did not reveal significant treatment effects of fertilization with class B biosolids or untreated sewage sludge on the vegetables. Numerous targeted genes associated with antibiotic resistance and mobile genetic elements were detected by PCR in soil and on vegetables at harvest from plots that received no organic amendment. However, in the season of application, vegetables harvested from plots treated with either material carried gene targets not detected in the absence of amendment. Several gene targets evaluated by using quantitative PCR (qPCR) were considerably more abundant on vegetables harvested from sewage sludge-treated plots than on vegetables from control plots in the season of application, whereas vegetables harvested the following year revealed no treatment effect. Overall, the results of the present study suggest that producing vegetable crops in ground fertilized with human waste without appropriate delay or pretreatment will result in an additional burden of antibiotic resistance genes on harvested crops. Managing human exposure to antibiotic resistance genes carried in human waste must be undertaken through judicious agricultural practice.

We investigated the prevalence and diversity of Escherichia coli strains isolated from surface waters from multiple watersheds within the South Nation River basin in eastern Ontario, Canada. The basin is composed of mixed but primarily agricultural land uses. From March 2004 to November 2007, a total of 2,004 surface water samples were collected from 24 sampling sites. E. coli densities ranged from undetectable to 1.64 ؋ 10 5 CFU 100 ml ؊1 and were correlated with stream order and proximity to livestock production systems. The diversity of 21,307 E. coli isolates was characterized using repetitive extragenic palindromic PCR (rep-PCR), allowing for the identification of as many as 7,325 distinct genotypes, without capturing all of the diversity. The community was temporally and spatially dominated by a few dominant genotypes (clusters of more than 500 isolates) and several genotypes of intermediary abundance (clustering between 10 and 499 isolates). Simpson diversity indices, assessed on a normalized number of isolates per sample, ranged from 0.050 to 0.668. Simpson indices could be statistically discriminated on the basis of year and stream order, but land use, discharge, weather, and water physical-chemical properties were not statistically important discriminators. The detection of Campylobacter species was associated with statistically lower Simpson indices (greater diversity; P < 0.05). Waterborne E. coli isolates from genotypes of dominant and intermediary abundance were clustered with isolates obtained from fecal samples collected in the study area over the same period, and 90% of the isolates tested proved to share genotypes with fecal isolates. Overall, our data indicated that the densities and distribution of E. coli in these mixed-use watersheds were linked to stream order and livestock-based land uses. Waterborne E. coli populations that were distinct from fecal isolates were detected and, on this basis, were possibly naturalized E. coli strains.Escherichia coli is ubiquitously distributed in fecal material from humans and warm-blooded animals (38). The detection of E. coli in water is an implicit indicator of recent fecal contamination and therefore of the risk of cooccurrence of enteric pathogens that can cause illness in susceptible populations (62). Many jurisdictions evaluate and mandate compliance with drinking and recreational water quality standards on the basis of the presence and abundance of E. coli (14,44). For example, Canadian recreational water quality standards stipulate that E. coli densities in excess of a geometric mean of 200 CFU per 100 ml indicate that the water is unsuitable for swimming and bathing (23).In a background of increasing occurrence of microbial contamination of surface water, a variety of methods for elucidating the sources of fecal contamination have been developed, and these microbial source tracking (MST) methods are recommended components of fecal pollution abatement strategies (16,57). So-called library-dependent MST methods compare environmental isolates to co...

Canada's National Agri-Environmental Standards Initiative sought to develop an environmental benchmark for low-level waterborne pathogen occurrence in agricultural watersheds. A field study collected 902 water samples from 27 sites in four intensive agricultural watersheds across Canada from 2005 to 2007. Four of the sites were selected as reference sites away from livestock and human fecal pollution sources in each watershed. Water samples were analyzed for Campylobacter spp., Salmonella spp., Escherichia coli O157:H7, Cryptosporidium spp., Giardia spp., and the water quality indicator E. coli. The annual mean number of pathogen species was higher at agricultural sites (1.54 ± 0.07 species per water sample) than at reference sites (0.75 ± 0.14 species per water sample). The annual mean concentration of E. coli was also higher at agricultural sites (491 ± 96 colony-forming units [cfu] 100 mL(-1)) than at reference sites (53 ± 18 cfu 100 mL(-1)). The feasibility of adopting existing E. coli water quality guideline values as an environmental benchmark was assessed, but waterborne pathogens were detected at agricultural sites in 80% of water samples with low E. coli concentrations (<100 cfu 100 mL(-1)). Instead, an approach was developed based on using the natural background occurrence of pathogens at reference sites in agricultural watersheds to derive provisional environmental benchmarks for pathogens at agricultural sites. The environmental benchmarks that were derived were found to represent E. coli values lower than geometric mean values typically found in recreational water quality guidelines. Additional research is needed to investigate environmental benchmarks for waterborne pathogens within the context of the "One World, One Health" perspective for protecting human, domestic animal, and wildlife health.