Composting reduces the risks of resistome in beef cattle manure at the transcriptional level

Transcriptomic evidence is needed to determine whether composting is more effective than conventional stockpiling in mitigating the risk of resistome in livestock manure. The objective of this study is to compare composting and stockpiling for their effectiveness in reducing the risk of antibiotic resistance in beef cattle manure. Samples collected from the center and the surface of full-size manure stockpiling and composting piles were subject to metagenomic and metatranscriptomic analyses. While the distinctions in resistome between stockpiled and composted manure were not evident at the DNA level, the advantages of composting over stockpiling were evident at the transcriptomic level in terms of the abundance of antibiotic resistance genes (ARGs), the number of ARG subtypes, and the prevalence of high-risk ARGs (i.e., mobile ARGs associated with zoonotic pathogens). DNA and transcript contigs show that the pathogen hosts of high-risk ARGs included Escherichia coli O157:H7 and O25b:H4, Klebsiella pneumoniae, and Salmonella enterica. Although the average daily temperatures for the entire composting pile exceeded 55°C throughout the field study, more ARG and ARG transcripts were removed at the center of the composting pile than at the surface. This work demonstrates the advantage of composting over stockpiling in reducing ARG risk in active populations in beef cattle manure

Effects of Nutrient Level and Growth Rate on the Conjugation Process That Transfers Mobile Antibiotic Resistance Genes in Continuous Cultures

Bacteria in the effluent of wastewater treatment plants (WWTPs) can transfer antibiotic resistance genes (ARGs) to the bacteria in receiving water through conjugation; however, there is a lack of quantitative assessment of this phenomenon in continuous cultures. Our objective was to determine the effects of background nutrient levels in river water column and growth rates of bacteria on the conjugation frequency of ARGs from effluent bacteria to river bacteria, as well as on the resulting resistance level (i.e., MICs) of the river bacteria. Chemostats were employed to simulate the discharge points of WWTPs into rivers, where effluent bacteria (donor cells) meet river bacteria (recipient cells). Both donor and recipient cells were Escherichia coli cells, and the donor cells were constructed by filter mating with bacteria in the effluent of a local WWTP. Results showed that higher bacterial growth rate (0.45 h21 versus 0.15 h21) led to higher conjugation frequencies (1024 versus 1026 transconjugant per recipient). The nutrient level also significantly affected the conjugation frequency, albeit to a lesser extent than the growth rate. The MIC against tetracycline increased from 2 mg/L in the recipient to 64 to 128 mg/L in transconjugants. In comparison, the MIC only increased to as high as 8 mg/L in mutants. Whole-genome sequencing showed that the tet-containing plasmid in both the donor and the transconjugant cells also occur in other fecal bacterial genera. The quantitative information obtained from this study can inform hazard identification related to the proliferation of wastewater-associated ARGs in surface water

Microbes in beach sands : integrating environment, ecology and public health

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Reviews in Environmental Science and Bio/Technology 13 (2014): 329-368, doi:10.1007/s11157-014-9340-8.Beach sand is a habitat that supports many microbes, including viruses, bacteria, fungi and protozoa (micropsammon). The apparently inhospitable conditions of beach sand environments belie the thriving communities found there. Physical factors, such as water availability and protection from insolation; biological factors, such as competition, predation, and biofilm formation; and nutrient availability all contribute to the characteristics of the micropsammon. Sand microbial communities include autochthonous species/phylotypes indigenous to the environment. Allochthonous microbes, including fecal indicator bacteria (FIB) and waterborne pathogens, are deposited via waves, runoff, air, or animals. The fate of these microbes ranges from death, to transient persistence and/or replication, to establishment of thriving populations (naturalization) and integration in the autochthonous community. Transport of the micropsammon within the habitat occurs both horizontally across the beach, and vertically from the sand surface and ground water table, as well as at various scales including interstitial flow within sand pores, sediment transport for particle-associated microbes, and the large-scale processes of wave action and terrestrial runoff. The concept of beach sand as a microbial habitat and reservoir of FIB and pathogens has begun to influence our thinking about human health effects associated with sand exposure and recreational water use. A variety of pathogens have been reported from beach sands, and recent epidemiology studies have found some evidence of health risks associated with sand exposure. Persistent or replicating populations of FIB and enteric pathogens have consequences for watershed/beach management strategies and regulatory standards for safe beaches. This review summarizes our understanding of the community structure, ecology, fate, transport, and public health implications of microbes in beach sand. It concludes with recommendations for future work in this vastly under-studied area.2015-05-0

Direct and Indirect Effects of Agrochemicals on Bacterial Pathogens and Fecal Indicator Bacteria

The presence of agrochemical residues in both urban and agricultural water bodies has become ubiquitous, often producing deleterious effects in the impacted watershed including reductions in biodiversity, alterations in species interactions, and toxicity to non-target organisms. While these effects have been studied on metazoan consumers, the consequences of agrochemical contamination on microorganisms, such as bacteria, protozoa, and viruses, are poorly understood. Agrochemicals could act directly on microorganisms, including pathogens, by either facilitating their survival or decreasing their abundance. Further, a multitude of indirect effects of agrochemicals on microorganisms are possible, whereby agrochemicals alter predation, competition, or parasitism on or available nutrient to microbes. The primary method by which agrochemicals enter water bodies is through stormwater and agricultural runoff, which can also introduce agriculturally-associated zoonotic pathogens. Presently, regulatory standards utilize fecal indicator bacteria (FIB) to predict the presence of pathogens in contaminated watersheds. However, if agrochemicals have different effects on FIB and bacterial pathogens, then these regulatory standards might be confounded by the presence of pesticide residues in impacted water bodies. Additionally, if agrochemicals promote the survival of zoonotic pathogens, then the presence of pesticide residues could potentially increase risks to human health. The studies in this dissertation investigated both the direct and indirect effects of agrochemicals on the growth and survival of FIBs ( Escherichia coli and Enterococcus faecalis), zoonotic bacterial pathogens (E. coli O157:H7, and Salmonella enterica), and two virus groups (human polyomaviruses and adenoviruses). The agrochemicals utilized in these experiments are among the most prominently used in their respective pesticide classes and included the herbicide atrazine, the insecticide malathion, the fungicide chlorothalonil and inorganic fertilizer containing phosphate and fixed nitrogen. Initially, complex mesocosms containing zooplankton, phytoplankton, leaf litter, and vertebrate and invertebrate species were used to examine net (direct and indirect) effects of agrochemicals on FIB in sediments. Subsequent studies utilized experiments in simplified microcosms to detect direct or indirect effects (i.e., predation, competition or effects on nutrient resources) on FIBs and pathogens. In complex mesocosms, atrazine and fertilizer significantly increased FIB densities in the sediment; however, because of the complexity of the mesocosms, it was not possible to determine whether these results were the product of direct or indirect agrochemical effects. Simplified microcosms, limited to predominantly direct effects, as well as in vitro growth curves, revealed no direct effects of any agrochemical treatment on either growth or survival of FIB or bacterial pathogens. When algal communities were allowed to establish, however, atrazine significantly reduced both phytoplankton and E. coli densities in the water column, but increased E. coli densities within the sediments. These effects on E. coli were indirect because they required the presence of algal species. To investigate indirect effects of predation on FIBs and E. coli O157:H7, we manipulated the presence and absence of an obligate heterotroph, Tetrahymena pyriformis, a facultative heterotroph, Ochromonas danica, and natural protozoan populations. In both laboratory and greenhouse microcosm experiments, the fungicide chlorothalonil significantly reduced all protozoan populations, which resulted in increased densities of FIBs and E. coli O157:H7 because of reduced predation. Atrazine was not found to have any significant direct effect on the densities of T. pyriformis or natural protozoans; however, atrazine did significantly reduce O. danica densities in greenhouse experiments. In laboratory experiments with O. danica, atrazine treatments resulted in decreased densities of E. coli O157:H7. Presumably, atrazine prevented or reduced photosynthesis forcing O. danica to increase its predation on E. coli thus shifting its trophic level. These studies reveal that agrochemicals can have a significant effect on microbial communities, but that these effects are often indirect and mediated through alterations of nutrient resources and predation. Atrazine application reduced FIB and pathogen densities in the water column via reduction of phytoplankton and increased predation by O. danica. These data suggest that the net effects of atrazine is deleterious to FIB survival in the water column and that application of this herbicide could result in an ecosystem service, reducing the abundance of zoonotic pathogens and lessening the risk to human health. However, elevation of FIB densities was observed in the sediments when atrazine was applied. The potential resuspension of increased sediment bacteria may negate or out-weigh the deleterious effects of atrazine on bacteria in the water column. Chlorothalonil application decreased protozoan densities, lessening the stress of predation on the bacterial targets and increasing FIB and E. coli O157:H7 densities. The use of chlorothalonil may therefore have negative implications for human health risks, as the reduction in predation seems to facilitate the survival of zoonotic waterborne pathogens. Understanding the net effects of agrochemicals is important for public health, as pesticide applications can act to either maintain or diminish potential bacterial and protozoan pathogens of humans. These studies show that indirect effects of agrochemicals on non-target microbes tend to be more prominent than direct effects and can significantly impact the fate of bacterial pathogens in aquatic environments

Stockpiling versus Composting: Effectiveness in Reducing Antibiotic-Resistant Bacteria and Resistance Genes in Beef Cattle Manure

Manure storage methods can affect the concentration and prevalence of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in cattle manure prior to land application. The objective of this study was to compare stockpiling and composting with respect to their effectiveness in reducing ARB and ARGs in beef cattle manure in a field-scale study. Field experiments were conducted in different seasons with different bulking agents for composting. For both the winter-spring cycle and the summer-fall cycle, ARB concentrations declined below the limit of quantification rapidly in both composting piles and stockpiles; however, ARB prevalence was significantly greater in the composting piles than in the stockpiles. This was likely due to the introduction of ARB from bulking agents. There was no significant change in ARG concentrations between initial and final concentrations for either manure storage treatment during the winter-spring cycle, but a significant reduction of the ARGs erm (B), tet (O), and tet (Q) over time was observed for both the composting pile and stockpile during the summer-fall cycle. Results from this study suggest that (i) bulking agent may be an important source of ARB and ARGs for composting; (ii) during cold months, the heterogeneity of the temperature profile in composting piles could result in poor ARG reduction; and (iii) during warm months, both stockpiling and composting can be effective in reducing ARG abundance. IMPORTANCE Proper treatment of manure is essential to reduce the spread of antibiotic resistance and protect human health. Stockpiling and composting are two manure storage methods which can reduce antibiotic-resistant bacteria and resistance genes, although few field-scale studies have examined the relative efficiency of each method. This study examined the ability of both methods in both winter-spring and summer-fall cycles, while also accounting for heterogeneity within field-scale manure piles. This study determined that bulking agents used in composting could contribute antibiotic-resistant bacteria and resistance genes. Additionally, seasonal variation could hinder the efficacy of composting in colder months due to heterogeneity in temperature within the pile; however, in warmer months, either method of manure storage could be effective in reducing the spread of antibiotic resistance

Foreshore beach sand as a reservoir and source of total phosphorus in Lake Ontario

<p>Many regions around the Great Lakes have been designated Areas of Concern as a result of consistent water quality problems from pollutants like phosphorus and <i>Escherichia coli</i>, which cause eutrophication, beach postings and Beneficial Use Impairments. While foreshore beach sand is a potential reservoir for <i>E. coli</i>, there is less understanding of whether it might also be a reservoir and source of phosphorus for adjacent beach waters. We measured levels of <i>E. coli,</i> total phosphorus and soluble reactive phosphorus at Sunnyside and Rouge Beaches in the Toronto and Region Area of Concern, and stormwater outfalls in the adjacent Humber and Rouge Rivers within their beachsheds. Additionally, we used microbial source tracking assays to detect human and gull fecal contamination. Soluble reactive phosphorus concentrations were highest in stormwater outfalls, with concentrations as high as 556 µg l⁻¹ at an outfall in the Sunnyside beachshed, and 4780 µg l⁻¹ at an outfall in the Rouge beachshed. In contrast, the highest total phosphorus concentrations were typically found in foreshore beach sand pore water and were more associated with gull fecal contamination. Beach sand total phosphorus levels were as high as 10,600 µg l⁻¹ at Sunnyside Beach, although the highest total phosphorus concentration measured (25,600 µg l⁻¹) was in a Rouge River outfall. Concentrations of total phosphorus in outfalls were significantly correlated with concentrations of <i>E. coli</i> in both beachsheds and the human microbial source tracking marker in the Sunnyside beachshed outfalls. These results indicate that stormwater outfalls with sewage cross-contamination can deliver high concentrations of total phosphorus, soluble reactive phosphorus and fecal bacterial contamination to associated beachsheds. Further, similar to <i>E. coli</i>, foreshore beach sand can act as a reservoir of total phosphorus and a source for adjacent water bodies via wave action or groundwater discharge. High phosphorus inputs from beach sand could contribute localized changes to microbial communities and unique eutrophication effects along beach shorelines.</p

The Human Health Implications of Antibiotic Resistance in Environmental Isolates from Two Nebraska Watersheds

One Health field-based approaches are needed to connect the occurrence of antibiotics present in the environment with the presence of antibiotic resistance genes (ARGs) in Gram-negative bacteria that confer resistance to antibiotics important in for both veterinary and human health. Water samples from two Nebraska watersheds influenced by wastewater effluent and agricultural runoff were tested for the presence of antibiotics used in veterinary and human medicine. The water samples were also cultured to identify the bacteria present. Of those bacteria isolated, the Gram-negative rods capable of causing human infections had antimicrobial susceptibility testing and whole-genome sequencing (WGS) performed to identify ARGs present. Of the 211 bacterial isolates identified, 37 belonged to pathogenic genera known to cause human infections. Genes conferring resistance to beta-lactams, aminoglycosides, fosfomycins, and quinolones were the most frequently detected ARGs associated with horizontal gene transfer (HGT) in the watersheds. WGS also suggest recent HGT events involving ARGs transferred between watershed isolates and bacteria of human and animal origins. The results of this study demonstrate the linkage of antibiotics and bacterial ARGs present in the environment with potential human and/or veterinary health impacts. IMPORTANCE One health is a transdisciplinary approach to achieve optimal health for humans, animals, plants and their shared environment, recognizing the interconnected nature of health in these domains. Field based research is needed to connect the occurrence of antibiotics used in veterinary medicine and human health with the presence of antibiotic resistance genes (ARGs). In this study, the presence of antibiotics, bacteria and ARGs was determined in two watersheds in Nebraska, one with agricultural inputs and the other with both agricultural and wastewater inputs. The results presented in this study provide evidence of transfer of highly mobile ARG between environment, clinical, and animal-associated bacteria.This article is published as Donner, Linsey, Zachery R. Staley, Jonathan Petali, Jodi Sangster, Xu Li, Wayne Mathews, Daniel Snow, Adina Howe, Michelle Soupir, and Shannon Bartelt-Hunt. "The Human Health Implications of Antibiotic Resistance in Environmental Isolates from Two Nebraska Watersheds." Microbiology Spectrum (2022): e02082-21. DOI: 10.1128/spectrum.02082-21. Copyright 2022 Donner et al. Attribution 4.0 International (CC BY 4.0). Posted with permission