Modeling Antibiotic Concentrations in the Vicinity of Antibiotic-Producing Bacteria at the Micron Scale.

It is generally thought that antibiotics confer upon the producing bacteria the ability to inhibit or kill neighboring microorganisms, thereby providing the producer with a significant competitive advantage. Were this to be the case, the concentrations of emitted antibiotics in the vicinity of producing bacteria might be expected to fall within the ranges of MICs that are documented for a number of bacteria. Furthermore, antibiotic concentrations that bacteria are punctually or chronically exposed to in environments harboring antibiotic-producing bacteria might fall within the range of minimum selective concentrations (MSCs) that confer a fitness advantage to bacteria carrying acquired antibiotic resistance genes. There are, to our knowledge, no available in situ measured antibiotic concentrations in the biofilm environments that bacteria typically live in. The objective of the present study was to use a modeling approach to estimate the antibiotic concentrations that might accumulate in the vicinity of bacteria that are producing an antibiotic. Fick's law was used to model antibiotic diffusion using a series of key assumptions. The concentrations of antibiotics within a few microns of single producing cells could not reach MSC (8 to 16 µg/L) or MIC (500 µg/L) values, whereas the concentrations around aggregates of a thousand cells could reach these concentrations. The model outputs suggest that single cells could not produce an antibiotic at a rate sufficient to achieve a bioactive concentration in the vicinity, whereas a group of cells, each producing the antibiotic, could do so. IMPORTANCE It is generally assumed that a natural function of antibiotics is to provide their producers with a competitive advantage. If this were the case, sensitive organisms in proximity to producers would be exposed to inhibitory concentrations. The widespread detection of antibiotic resistance genes in pristine environments suggests that bacteria are indeed exposed to inhibitory antibiotic concentrations in the natural world. Here, a model using Fick's law was used to estimate potential antibiotic concentrations in the space surrounding producing cells at the micron scale. Key assumptions were that per-cell production rates drawn from the pharmaceutical manufacturing industry are applicable in situ, that production rates were constant, and that produced antibiotics are stable. The model outputs indicate that antibiotic concentrations in proximity to aggregates of a thousand cells can indeed be in the minimum inhibitory or minimum selective concentration range.

Metagenome meta-analysis reveals an increase in the abundance of some multidrug efflux pumps and mobile genetic elements in chemically polluted environments.

Many human activities contaminate terrestrial and aquatic environments with numerous chemical pollutants that not only directly alter the environment but also affect microbial communities in ways that are potentially concerning to human health, such as selecting for the spread of antibiotic-resistance genes (ARGs) through horizontal gene transfer. In the present study, metagenomes available in the public domain from polluted (with antibiotics, with petroleum, with metal mining, or with coal-mining effluents) and unpolluted terrestrial and aquatic environments were compared to examine whether pollution has influenced the abundance and composition of ARGs and mobile elements, with specific focus on IS26 and class 1 integrons (intI1). When aggregated together, polluted environments had a greater relative abundance of ARGs than unpolluted environments and a greater relative abundance of IS26 and intI1. In general, chemical pollution, notably with petroleum, was associated with an increase in the prevalence of ARGs linked to multidrug efflux pumps. Included in the suite of efflux pumps were mexK, mexB, mexF, and mexW that are polyspecific and whose substrate ranges include multiple classes of critically important antibiotics. Also, in some instances, ß-lactam resistance (TEM181 and OXA-541) genes increased, and genes associated with rifampicin resistance (RNA polymerases subunits rpoB and rpoB2) decreased in relative abundance. This meta-analysis suggests that different types of chemical pollution can enrich populations that carry efflux pump systems associated with resistance to multiple classes of medically critical antibiotics.IMPORTANCEThe United Nations has identified chemical pollution as being one of the three greatest threats to environmental health, through which the evolution of antimicrobial resistance, a seminally important public health challenge, may be favored. While this is a very plausible outcome of continued chemical pollution, there is little evidence or research evaluating this risk. The objective of the present study was to examine existing metagenomes from chemically polluted environments and evaluate whether there is evidence that pollution increases the relative abundance of genes and mobile genetic elements that are associated with antibiotic resistance. The key finding is that for some types of pollution, particularly in environments exposed to petroleum, efflux pumps are enriched, and these efflux pumps can confer resistance to multiple classes of medically important antibiotics that are typically associated with Pseudomonas spp. or other Gram-negative bacteria. This finding makes clear the need for more investigation on the impact of chemical pollution on the environmental reservoir of ARGs and their association with mobile genetic elements that can contribute to horizontal gene transfer events.

Characterization of Escherichia coli and Other Enterobacterales Resistant to Extended-Spectrum Cephalosporins Isolated from Dairy Manure in Ontario, Canada.

Extended-spectrum cephalosporins (ESCs) resistance genes, such as blaCTX-M, blaCMY, and blaSHV, have been found regularly in bacteria from livestock. However, information on their distribution in dairy cattle in Canada and on the associated genome sequences of ESC-resistant Enterobacterales is sparse. In this study, the diversity and distribution of ESC-resistant Escherichia coli throughout manure treatments in six farms in Southern Ontario were assessed over a one-year period, and their ESC-resistance plasmids were characterized. The manure samples were enriched using selective media. The resulting isolates were screened via polymerase chain reaction for blaCTX-M, blaCMY, and blaSHV. No E. coli carrying blaSHV were detected. Escherichia coli (n = 248) carrying blaCTX-M or blaCMY underwent whole-genome sequencing using an Illumina MiSeq/NextSeq. These isolates were typed using multilocus sequence typing (MLST) and their resistance gene profiles. A subset of E. coli (n = 28) were sequenced using Oxford Nanopore Technologies. Plasmids were assembled using Unicycler and characterized via the resistance genes pattern, replicon type, plasmid MLST, phylogenetic analysis, and Mauve alignments. The recovery of ESC-resistant Enterobacterales (18 species, 8 genera) was drastically reduced in manure outputs. However, multiple treatment stages were needed to attain a significant reduction. 62 sequence types were identified, with ST10, ST46, ST58, ST155, ST190, ST398, ST685, and ST8761 being detected throughout the treatment pipeline. These STs overlapped with those found on multiple farms. The ESC-resistance determinants included CTX-M-1, -14, -15, -17, -24, -32, -55, and CMY-2. The plasmids carrying blaCTX-M were more diverse than were the plasmids carrying blaCMY. Known "epidemic plasmids" were detected for both blaCTX-M and blaCMY. IMPORTANCE The increase in antimicrobial resistance is of concern for human and animal health, especially when resistance is conferred to extended-spectrum cephalosporins, which are used to treat serious infections in both human and veterinary medicine. Bacteria carrying extended-spectrum cephalosporin resistance genes, including blaCTX-M and blaCMY, are frequently found in dairy manure. Manure treatment influences the loads and diversity of bacteria, including those carrying antimicrobial resistance genes, such as Enterobacterales and Escherichia coli. Any bacteria that survive the treatment process are subsequently applied to the environment. Enterobacterales carrying blaCTX-M or blaCMY can contaminate soil and crops consumed by humans and animals, thereby increasing the potential for antimicrobial resistance genes to integrate into the human gut microflora through horizontal gene transfer. This furthers the dissemination of resistance. Therefore, it is imperative to understand the effects manure treatments have on ESC-resistance in environmentally applied manure.

Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Potential selection and maintenance of manure-originated multi-drug resistant plasmids at sub-clinical concentrations for tetracycline family antibiotics.

The goal of this study was to (a) determine the minimum selection concentrations of tetracycline family antibiotics necessary to maintain plasmids carrying tetracycline-resistant genes and (b) correlate these results to environmental hotspot concentrations reported in previous studies. This study used two plasmids (pT295A and pT413A) originating from dairy manure in a surrogate Escherichia coli host CV601. The minimum selection concentrations of antibiotics tested in nutrient-rich medium were determined as follows: 0.1 mg/L for oxytetracycline, 0.45 mg/L for chlortetracycline, and 0.13-0.25 mg/L for tetracycline. Mixing oxytetracycline and chlortetracycline had minimum selection concentration values increased 2-fold compared to those in single antibiotic tests. Minimum selection concentrations found in this study were lower than reported environmental hotspot concentrations, suggesting that tetracycline family antibiotics were likely to be the driver for the selection and maintenance of these plasmids. Relatively high plasmid loss rates (>90%) were observed when culturing a strain carrying a tetracycline-resistant plasmid in antibiotic-free nutrient-rich and nutrient-defined media. Overall, results suggested that these plasmids can be maintained at concentrations environmentally relevant in wastewater treatment plants, sewage, manure, and manured soil; however, they are unstable and easily lost in the absence of antibiotics.

Responses of the Soil Bacterial Community, Resistome, and Mobilome to a Decade of Annual Exposure to Macrolide Antibiotics.

Biosolids that are applied to agricultural soil as an organic fertilizer are frequently contaminated with pharmaceutical residues that have persisted during wastewater treatment and partitioned into the organic phase. Macrolide antibiotics, which serve as a critically important human medicine, have been detected within biosolids. To determine the impacts of macrolide antibiotics on soil bacteria, every year for a decade, a series of replicated field plots received an application of a mixture of erythromycin, clarithromycin, and azithromycin at a realistic (0.1 mg kg soil-1) or an unrealistically high (10 mg kg soil-1) dose or were left untreated. The effects of repeated antibiotic exposure on the soil bacterial community, resistome, mobilome, and integron gene cassette content were evaluated by 16S rRNA and integron gene cassette amplicon sequencing, as well as whole-metagenome sequencing. At the unrealistically high dose, the overall diversity of the resistome and mobilome was altered, as 21 clinically important antibiotic resistance genes predicted to encode resistance to 10 different antibiotic drug classes were increased and 20 mobile genetic element variants (tnpA, intI1, tnpAN, and IS91) were increased. In contrast, at the realistic dose, no effect was observed on the overall diversity of the soil bacterial community, resistome, mobilome, or integron gene cassette-carrying genes. Overall, these results suggest that macrolide antibiotics entrained into soil at concentrations anticipated with biosolid applications would not result in major changes to these endpoints. IMPORTANCE Biosolids, produced from the treatment of sewage sludge, are rich in plant nutrients and are a valuable alternative to inorganic fertilizer when applied to agricultural soil. However, the use of biosolids in agriculture, which are frequently contaminated with pharmaceuticals, such as macrolide antibiotics, may pose a risk to human health by selecting for antibiotic resistance genes that could be transferred to plant-based food destined for human consumption. The consequences of long-term, repeated macrolide antibiotic exposure on the diversity of the soil bacterial community, resistome, and mobilome were evaluated. At unrealistically high concentrations, macrolide antibiotics alter the overall diversity of the resistome and mobilome, enriching for antibiotic resistance genes and mobile genetic elements of concern to human health. However, at realistic antibiotic concentrations, no effect on these endpoints was observed, suggesting that current biosolids land management practices are unlikely to pose a risk to human health due to macrolide antibiotic contamination alone.

Contamination of hay and haylage with enteric bacteria and selected antibiotic resistance genes following fertilization with dairy manure or biosolids.

The present study evaluated if enteric bacteria or antibiotic resistance genes carried in fecal amendments contaminate the hay at harvest, representing a potential route of exposure to ruminants that consume the hay. In the field experiments, dairy manure was applied to a hay field for three successive growing seasons, and biosolids were applied to a hay field for one growing season. Various enteric bacteria in the amendments were enumerated by viable plate count, and selected gene targets were quantified by qPCR. Key findings include the following: at harvest, hay receiving dairy manure or biosolids did not carry more viable enteric bacteria than hay from unamended control plots. The fermentation of hay did not result in a detectable increase in viable enteric bacteria. The application of dairy manure or biosolids resulted in a few gene targets being more abundant in hay during the first harvest. Fermentation of hay resulted in an increase in the abundance of gene targets, but this occurred with hay from both the amended and control plots. Overall, the application of fecal amendments resulted in an increase in the abundance of some gene targets associated with antibiotic resistance in the first cut hay.

Effects of litter from antimicrobial-fed broiler chickens on soil bacterial community structure and diversity.

This study examined changes in soil bacterial community composition and diversity in response to fertilization with litter from chickens fed a diet without antibiotics and with bambermycin, penicillin, bacitracin, salinomycin, or mix of salinomycin and bacitracin. Litter (27.5 T/ha) was applied to 24 agricultural plots in the Fraser Valley of British Columbia. Nonfertilized plots were used as a negative control. Soil samples collected from the studied plots were used to quantify Escherichia coli by plate counts, and Clostridium perfringens by qPCR. The 16S rRNA gene sequencing was performed for microbiota analysis. Following litter application in December, the population size of E. coli was 5.4 log CFU/g; however, regardless of treatments, the results revealed 5.2 and 1.4 log CFU/g of E. coli in soil sampled in January and March, respectively. Fertilization with litter from antibiotic-treated birds increased (P < 0.05) the relative abundance of Proteobacteria, Actinobacteria, and Firmicutes in soil, but decreased Acidobacteria and Verrucomicrobia groups. The alpha diversity parameters were higher (P < 0.05) in nonfertilized soil compared to the fertilized ones, suggesting that litter application was a major factor in shaping the soil bacterial communities. These results may help develop efficient litter management strategies like composting, autoclaving, or anaerobic digestion of poultry litter before application to land for preservation of soil health and crop productivity.

Fate of Clostridia and other spore-forming Firmicute bacteria during feedstock anaerobic digestion and aerobic composting.

Pathogenic spore-forming Firmicutes are commonly present in animal and human wastes that are used as fertilizers in crop production. Pre-treatments of organic waste prior to land application offer the potential to abate enteric microorganisms, and therefore reduce the risk of contamination of crops or adjacent water resources with pathogens carried in these materials. The inactivation and reduction of gram-positive spore formers such as Clostridium spp., Clostridioides spp. and Bacillus spp. from animal and human waste can be challenging given the recalcitrance of the spores these bacteria produce. Given the significance of these organisms to human and animal health, information concerning spore-forming bacteria inactivation during anaerobic digestion (AD) and aerobic composting (AC) is required as the basis for recommending safe organic waste management practices. In this review, an assessment of the inactivation of spore-forming Firmicutes during AD and AC was conducted to provide guidance for practical management of organic matrices of animal or human origin. Temperature and pH may be the main factors contributing to the inactivation of spore-forming Firmicutes during batch lab-scale AD (log reduction <0.5-5 log). In continuous digesters, wet AD systems do not effectively inactivate spore-forming Firmicutes even under thermopholic conditions (log reduction -1.09 - 0.98), but dry AD systems could be a feasible management practice to inactivate spore-forming Firmicutes from organic materials with high solid content (log reduction 1.77-3.1). In contrast, composting is an effective treatment to abate spore-forming Firmicutes (log reduction 1.7-6.5) when thermophilic conditions last at least six consecutive days. Temperature, moisture content and composting scale are the key operating conditions influencing the inactivation of spore-forming Firmicutes during composting. Where possible, undertaking AD with subsequent composting to ensure the biosafety of digestate before its downstream processing and recycling is recommended to abate recalcitrant bacteria in digestate.

On-Farm Anaerobic Digestion of Dairy Manure Reduces the Abundance of Antibiotic Resistance-Associated Gene Targets and the Potential for Plasmid Transfer.

The present study investigated the impact of on-farm anaerobic digestion on the abundance of enteric bacteria, antibiotic resistance-associated gene targets, and the horizontal transfer potential of extended-spectrum ß-lactamase (ESBL) genes. Samples of raw and digested manure were obtained from six commercial dairy farms in Ontario, Canada. Digestion significantly abated populations of viable coliforms in all six farms. Conjugative transfer of plasmids carrying ß-lactamase genes from manure bacteria enriched overnight with buffered peptone containing 4 mg/liter cefotaxime into a ß-lactam-sensitive green fluorescent protein (GFP)-labeled Escherichia coli recipient strain was evaluated in patch matings. Digestion significantly decreased the frequency of the horizontal transfer of ESBL genes. Twenty-five transconjugants were sequenced, revealing six distinct plasmids, ranging in size from 40 to 180 kb. A variety of ESBL genes were identified: blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, and blaPER-1. blaCTX-M-15 was the most prevalent ESBL gene detected on plasmids harbored by transconjugants. Various mobile genetic elements were found located proximal to resistance genes. Ten gene targets, including sul1, str(A), str(B), erm(B), erm(F), intI1, aadA, incW, blaPSE, and blaOXA-20, were quantified by quantitative PCR on a subset of 18 raw and 18 digested samples. Most targets were significantly more abundant in raw manure; however, erm(B) and erm(F) targets were more abundant in digested samples. Overall, on-farm digestion of dairy manure abated coliform bacteria, a number of antibiotic resistance-associated gene targets, and the potential for in vitro conjugation of plasmids conferring resistance to extended-spectrum ß-lactams and other classes of antibiotics into E. coli CV601. IMPORTANCE Using livestock manure for fertilization can entrain antibiotic-resistant bacteria into soil. Manure on some dairy farms is anaerobically digested before being land applied. Recommending the widespread implementation of the practice should be founded on understanding the impact of this treatment on various endpoints of human health concern. Although lab-scale anaerobic treatments have shown potential for reducing the abundance of antibiotic resistance genes, there are very few data from commercial farms. Anaerobic digestion of manure on six dairy farms efficiently abated coliform bacteria, E. coli, and a majority of antibiotic resistance-associated gene targets. In addition, the conjugation potential of plasmids carrying ESBL genes into introduced E. coli strain CV601 was reduced. Overall, anaerobic digestion abated coliform bacteria, the genes that they carry, and the potential for ESBL-carrying plasmid transfer.

Resistance determinants and their genetic context in enterobacteria from a longitudinal study of pigs reared under various husbandry conditions.

Pigs are major reservoirs of resistant Enterobacteriaceae that can reach humans through consumption of contaminated meat or vegetables grown in manure-fertilized soil. Samples were collected from sows during lactation and their piglets at five time points spanning the production cycle. Cefotaxime-resistant bacteria were quantified and isolated from feed, feces, manures and carcasses of pigs reared with penicillin-using or antibiotic-free husbandries. The isolates were characterized by antibiotic susceptibility testing, whole genome sequencing and conjugation assays. The extended spectrum ß-lactamase (ESBL) phenotype was more frequent in isolates originating from antibiotic-free animals, while the bacteria isolated from penicillin-using animals were on average resistant to a greater number of antibiotics. The ESBL-encoding genes identified were bla CTX-M-1, bla CTX-M-15 and bla CMY-2 and they co-localised on plasmids with various genes encoding resistance to ß-lactams, co-trimoxazole, phenicols and tetracycline, all antibiotics used in pig production. Groups of genes conferring the observed resistance and the mobile elements disseminating multidrug resistance were determined. The observed resistance to ß-lactams was mainly due to the complementary actions of penicillin-binding proteins, an efflux pump and ß-lactamases. Most resistance determinants were shared by animals raised with or without antimicrobials. This suggests a key contribution of indigenous enterobacteria maternally transmitted along the sow lineage, regardless of antimicrobial use. It is unclear if the antimicrobial resistance observed in the enterobacteria populations of the commercial pig herds studied were present before the use of antibiotics, or the extent to which historical antimicrobial use exerted a selective pressure defining the resistant bacterial populations in farms using penicillin prophylaxis.Importance: Antimicrobial resistance is a global threat that needs to be fought on numerous fronts along the One Health continuum. Vast quantities of antimicrobials are used in agriculture to ensure animal welfare and productivity, and are arguably a driving force for the persistence of environmental and food-borne resistant bacteria. This study evaluated the impact of conventional, organic and other antibiotic-free husbandry practices on the frequency and nature of antimicrobial resistance genes and multidrug resistant enterobacteria. It provides knowledge about the relative contribution of specific resistance determinants to observed antibiotic resistance. It also showed the clear co-selection of genes coding for extended-spectrum beta-lactamases and genes coding for the resistance to antibiotics commonly used for prophylaxis or in curative treatments in pig operations.

Real-time quantitative PCR assay development and application for assessment of agricultural surface water and various fecal matter for prevalence of Aliarcobacter faecis and Aliarcobacter lanthieri.

BACKGROUND: Aliarcobacter faecis and Aliarcobacter lanthieri are recently identified as emerging human and animal pathogens. In this paper, we demonstrate the development and optimization of two direct DNA-based quantitative real-time PCR assays using species-specific oligonucleotide primer pairs derived from rpoB and gyrA genes for A. faecis and A. lanthieri, respectively. Initially, the specificity of primers and amplicon size of each target reference strain was verified and confirmed by melt curve analysis. Standard curves were developed with a minimum quantification limit of 100 cells mL- 1 or g- 1 obtained using known quantities of spiked A. faecis and A. lanthieri reference strains in autoclaved agricultural surface water and dairy cow manure samples. RESULTS: Each species-specific qPCR assay was validated and applied to determine the rate of prevalence and quantify the total number of cells of each target species in natural surface waters of an agriculturally-dominant and non-agricultural reference watershed. In addition, the prevalence and densities were determined for human and various animal (e.g., dogs, cats, dairy cow, and poultry) fecal samples. Overall, the prevalence of A. faecis for surface water and feces was 21 and 28%, respectively. The maximum A. faecis concentration for water and feces was 2.3 × 107 cells 100 mL- 1 and 1.2 × 107 cells g- 1, respectively. A. lanthieri was detected at a lower frequency (2%) with a maximum concentration in surface water of 4.2 × 105 cells 100 mL- 1; fecal samples had a prevalence and maximum density of 10% and 2.0 × 106 cells g- 1, respectively. CONCLUSIONS: The results indicate that the occurrence of these species in agricultural surface water is potentially due to fecal contamination of water from livestock, human, or wildlife as both species were detected in fecal samples. The new real-time qPCR assays can facilitate rapid and accurate detection in < 3 h to quantify total numbers of A. faecis and A. lanthieri cells present in various complex environmental samples.

Novel virulence, antibiotic resistance and toxin gene-specific PCR-based assays for rapid pathogenicity assessment of Arcobacter faecis and Arcobacter lanthieri.

BACKGROUND: Arcobacter faecis and A. lanthieri are two newly classified species of genus Arcobacter. The prevalence and distribution of virulence, antibiotic resistance and toxin (VAT) genes in these species are required to assess their potential pathogenic health impacts to humans and animals. This study (i) developed species- and gene-specific primer pairs for the detection of six virulence, two antibiotic resistance, and three toxin genes in two target species; (ii) optimized eight single-tube multiplex and three monoplex PCR protocols using the newly developed species- and gene-specific primers; and (iii) conducted specificity and sensitivity evaluations as well as validation of eleven mono- and multiplex PCR assays by testing A. faecis (n= 29) and A. lanthieri (n= 10) strains isolated from various fecal and agricultural water sources to determine the prevalence and distribution of VAT genes and assess the degree of pathogenicity within the two species. RESULTS: Detection of all ten and eleven target VAT genes, and expression of cytolethal distending toxin (cdtA, cdtB and cdtC) genes in A. faecis and A. lanthieri reference strains with high frequency in field isolates suggest that they are potentially pathogenic strains. These findings indicate that these two species can pose a health risk to humans and animals. CONCLUSIONS: The study results show that the developed mono- and multiplex PCR (mPCR) assays are simple, rapid, reliable and sensitive for the simultaneous assessment of the potential pathogenicity and antibiotic resistance profiling of tet(O) and tet(W) genes in these two newly discovered species. Also, these assays can be useful in diagnostic and analytical laboratories to determine the pathotypes and assessment of the virulence and toxin factors associated to human and animal infections.

Long-Term Exposure of Agricultural Soil to Veterinary Antibiotics Changes the Population Structure of Symbiotic Nitrogen-Fixing Rhizobacteria Occupying Nodules of Soybeans (Glycine max).

Antibiotics are entrained in agricultural soil through the application of manures from medicated animals. In the present study, a series of small field plots was established in 1999 that receive annual spring applications of a mixture of tylosin, sulfamethazine, and chlortetracycline at concentrations ranging from 0.1 to 10 mg · kg-1 soil. These antibiotics are commonly used in commercial swine production. The field plots were cropped continuously for soybeans, and in 2012, after 14 annual antibiotic applications, the nodules from soybean roots were sampled and the occupying bradyrhizobia were characterized. Nodules and isolates were serotyped, and isolates were distinguished using 16S rRNA gene and 16S to 23S rRNA gene intergenic spacer region sequencing, multilocus sequence typing, and RSα fingerprinting. Treatment with the antibiotic mixture skewed the population of bradyrhizobia dominating the nodule occupancy, with a significantly larger proportion of Bradyrhizobium liaoningense organisms even at the lowest dose of 0.1 mg · kg-1 soil. Likewise, all doses of antibiotics altered the distribution of RSα fingerprint types. Bradyrhizobia were phenotypically evaluated for their sensitivity to the antibiotics, and there was no association between in situ treatment and a decreased sensitivity to the drugs. Overall, long-term exposure to the antibiotic mixture altered the composition of bradyrhizobial populations occupying nitrogen-fixing nodules, apparently through an indirect effect not associated with the sensitivity to the drugs. Further work evaluating agronomic impacts is warranted.IMPORTANCE Antibiotics are entrained in agricultural soil through the application of animal or human waste or by irrigation with reused wastewater. Soybeans obtain nitrogen through symbiotic nitrogen fixation. Here, we evaluated the impact of 14 annual exposures to antibiotics commonly used in swine production on the distribution of bradyrhizobia occupying nitrogen-fixing nodules on soybean roots in a long-term field experiment. By means of various sequencing and genomic fingerprinting techniques, the repeated exposure to a mixture of tylosin, sulfamethazine, and chlortetracycline each at a nominal soil concentration of 0.1 mg · kg-1 soil was found to modify the diversity and identity of bradyrhizobia occupying the nodules. Nodule occupancy was not associated with the level of sensitivity to the antibiotics, indicating that the observed effects were not due to the direct toxicity of the antibiotics on bradyrhizobia. Altogether, these results indicate the potential for long-term impacts of antibiotics on this agronomically important symbiosis.

Enrichment of antibiotic resistance genes in soil receiving composts derived from swine manure, yard wastes, or food wastes, and evidence for multiyear persistence of swine Clostridium spp.

The impact of amendment with swine manure compost (SMC), yard waste compost (YWC), or food waste compost (FWC) on the abundance of antibiotic resistance genes in soil was evaluated. Following a commercial-scale application of the composts in a field experiment, soils were sampled periodically for a decade, and archived air-dried. Soil DNA was extracted and gene targets quantified by qPCR. Compared with untreated control soil, all 3 amendment types increased the abundance of gene targets for up to 4 years postapplication. The abundance of several gene targets was much higher in soil amended with SMC than in soil receiving either YWC or FWC. The gene target ermB remained higher in the SMC treatment for a decade postapplication. Clostridia were significantly more abundant in the SMC-amended soil throughout the decade following application. Eight percent of Clostridium spp. isolates from the SMC treatment carried ermB. Overall, addition of organic amendments to soils has the potential to increase the abundance of antibiotic resistance genes. Amendments of fecal origin, such as SMC, will in addition entrain bacteria carrying antibiotic resistance genes. Environmentally recalcitrant clostridia, and the antibiotic resistance genes that they carry, will persist for many years under field conditions following the application of SMC.

Spectral Counting Approach to Measure Selectivity of High-Resolution LC-MS Methods for Environmental Analysis.

Advances in high-resolution mass spectrometers have allowed for the development of nontargeted screening methods, where data sets can be archived and retrospectively mined as new environmental contaminants are identified. We have developed a spectral counting approach to calculate the selectivities of LC-MS acquisition modes taking mass accuracy, sample matrix, and the analyte properties into account. The selectivities of high-resolution MS (HRMS) alone or in combination with all-ion-fragmentation (AIF), data-independent-acquisition (DIA), and data-dependent-acquisition (DDA) modes, performed on a Q-Exactive Orbitrap were compared by retrospectively screening surface water samples for 95 pharmaceuticals. Samples were reanalyzed using targeted LC-MS/MS to confirm the accuracy of each acquisition method and to quantitate the 29 putatively detected drugs. LC-HRMS provided the lowest calculated selectivities and accordingly produced the highest number of false positives (6). In contrast, DDA provided the highest selectivities, yielding only one false positive; however, it was bias toward the most intense signals resulting in the detection of only 10 compounds. AIF had lower selectivities than traditional LC-MS/MS, produced one false positive and did not detect 6 confirmed compounds. Because of the high-quality archived data, DIA selectivities were better than traditional LC-MS/MS, showed no bias toward the most intense signals, achieved low limits of detection, and confidently detected the greatest number of pharmaceuticals (22) with only one false positive. This spectral counting method can be used across different instrument platforms or samples and provides a robust and empirical estimation of selectivities to give more confident detection of trace analytes.

Novel Antibiotic Resistance Determinants from Agricultural Soil Exposed to Antibiotics Widely Used in Human Medicine and Animal Farming.

Antibiotic resistance has emerged globally as one of the biggest threats to human and animal health. Although the excessive use of antibiotics is recognized as accelerating the selection for resistance, there is a growing body of evidence suggesting that natural environments are "hot spots" for the development of both ancient and contemporary resistance mechanisms. Given that pharmaceuticals can be entrained onto agricultural land through anthropogenic activities, this could be a potential driver for the emergence and dissemination of resistance in soil bacteria. Using functional metagenomics, we interrogated the "resistome" of bacterial communities found in a collection of Canadian agricultural soil, some of which had been receiving antibiotics widely used in human medicine (macrolides) or food animal production (sulfamethazine, chlortetracycline, and tylosin) for up to 16 years. Of the 34 new antibiotic resistance genes (ARGs) recovered, the majority were predicted to encode (multi)drug efflux systems, while a few share little to no homology with established resistance determinants. We characterized several novel gene products, including putative enzymes that can confer high-level resistance against aminoglycosides, sulfonamides, and broad range of beta-lactams, with respect to their resistance mechanisms and clinical significance. By coupling high-resolution proteomics analysis with functional metagenomics, we discovered an unusual peptide, PPPAZI 4, encoded within an alternative open reading frame not predicted by bioinformatics tools. Expression of the proline-rich PPPAZI 4 can promote resistance against different macrolides but not other ribosome-targeting antibiotics, implicating a new macrolide-specific resistance mechanism that could be fundamentally linked to the evolutionary design of this peptide.IMPORTANCE Antibiotic resistance is a clinical phenomenon with an evolutionary link to the microbial pangenome. Genes and protogenes encoding specialized and potential resistance mechanisms are abundant in natural environments, but understanding of their identity and genomic context remains limited. Our discovery of several previously unknown antibiotic resistance genes from uncultured soil microorganisms indicates that soil is a significant reservoir of resistance determinants, which, once acquired and "repurposed" by pathogenic bacteria, can have serious impacts on therapeutic outcomes. This study provides valuable insights into the diversity and identity of resistance within the soil microbiome. The finding of a novel peptide-mediated resistance mechanism involving an unpredicted gene product also highlights the usefulness of integrating proteomics analysis into metagenomics-driven gene discovery.

Waterborne Viruses and F-Specific Coliphages in Mixed-Use Watersheds: Microbial Associations, Host Specificities, and Affinities with Environmental/Land Use Factors.

From the years 2008 to 2014, a total of 1,155 water samples were collected (spring to fall) from 24 surface water sampling sites located in a mixed-used but predominantly agricultural (i.e., dairy livestock production) river basin in eastern Ontario, Canada. Water was analyzed for viable F-specific DNA (F-DNA) and F-specific RNA (F-RNA) (genogroup I [GI] to GIV) coliphage and a suite of molecularly detected viruses (norovirus [GI to GIV], torque teno virus [TTV], rotavirus, kobuvirus, adenovirus, astrovirus, hepatitis A, and hepatitis E). F-DNA and F-RNA coliphage were detected in 33 and 28% of the samples at maximum concentrations of 2,000 and 16,300 PFU · 100 ml-1, respectively. Animal TTV, human TTV, kobuvirus, astrovirus, and norovirus GIII were the most prevalent viruses, found in 23, 20, 13, 12, and 11% of samples, respectively. Viable F-DNA coliphage was found to be a modest positive indicator of molecularly detected TTV. F-RNA coliphage, unlike F-DNA coliphage, was a modest positive predictor of norovirus and rotavirus. There were, however, a number of significant negative associations among F-specific coliphage and viruses. F-DNA coliphage densities of >142 PFU · 100 ml-1 delineated conditions when ∼95% of water samples contained some type of virus. Kobuvirus was the virus most strongly related to detection of any other virus. Land use had some associations with virus/F-specific coliphage detection, but season and surface water flow were the variables that were most important for broadly delineating detection. Higher relative levels of detection of human viruses and human F-RNA coliphage were associated with higher relative degrees of upstream human land development in a catchment. IMPORTANCE: This study is one of the first, to our knowledge, to evaluate relationships among F-specific coliphages and a large suite of enteric viruses in mixed-use but agriculturally dominated surface waters in Canada. This study suggested that relationships between viable F-specific coliphages and molecularly detected viruses do exist, but they are not always positive. Caution should be employed if viable F-specific coliphages are to be used as indicators of virus presence in surface waters. This study elucidates relative effects of agriculture, wildlife, and human activity on virus and F-specific coliphage detection. Seasonal and meteorological attributes play a strong role in the detection of most virus and F-specific coliphage targets.

Evidence of Naturalized Stress-Tolerant Strains of Escherichia coli in Municipal Wastewater Treatment Plants.

UNLABELLED: Escherichia coli has been proposed to have two habitats-the intestines of mammals/birds and the nonhost environment. Our goal was to assess whether certain strains of E. coli have evolved toward adaptation and survival in wastewater. Raw sewage samples from different treatment plants were subjected to chlorine stress, and ∼59% of the surviving E. coli strains were found to contain a genetic insertion element (IS30) located within the uspC-flhDC intergenic region. The positional location of the IS30 element was not observed across a library of 845 E. coli isolates collected from various animal hosts or within GenBank or whole-genome reference databases for human and animal E. coli isolates (n = 1,177). Phylogenetics clustered the IS30 element-containing wastewater E. coli isolates into a distinct clade, and biomarker analysis revealed that these wastewater isolates contained a single nucleotide polymorphism (SNP) biomarker pattern that was specific for wastewater. These isolates belonged to phylogroup A, possessed generalized stress response (RpoS) activity, and carried the locus of heat resistance, features likely relevant to nonhost environmental survival. Isolates were screened for 28 virulence genes but carried only the fimH marker. Our data suggest that wastewater contains a naturalized resident population of E. coli We developed an endpoint PCR targeting the IS30 element within the uspC-flhDC intergenic region, and all raw sewage samples (n = 21) were positive for this marker. Conversely, the prevalence of this marker in E. coli-positive surface and groundwater samples was low (≤5%). This simple PCR assay may represent a convenient microbial source-tracking tool for identification of water samples affected by municipal wastewater. IMPORTANCE: The results of this study demonstrate that some strains of E. coli appear to have evolved to become naturalized populations in the wastewater environment and possess a number of stress-related genetic elements likely important for survival in this nonhost environment. The presence of non-host-adapted strains in wastewater challenges our understanding of using E. coli as a microbial indicator of wastewater treatment performance, suggesting that the E. coli strains present in human and animal feces may be very different from those found in treated wastewater.

An evaluation of logic regression-based biomarker discovery across multiple intergenic regions for predicting host specificity in Escherichia coli.

Several studies have demonstrated that E. coli appears to display some level of host adaptation and specificity. Recent studies in our laboratory support these findings as determined by logic regression modeling of single nucleotide polymorphisms (SNP) in intergenic regions (ITGRs). We sought to determine the degree of host-specific information encoded in various ITGRs across a library of animal E. coli isolates using both whole genome analysis and a targeted ITGR sequencing approach. Our findings demonstrated that ITGRs across the genome encode various degrees of host-specific information. Incorporating multiple ITGRs (i.e., concatenation) into logic regression model building resulted in greater host-specificity and sensitivity outcomes in biomarkers, but the overall level of polymorphism in an ITGR did not correlate with the degree of host-specificity encoded in the ITGR. This suggests that distinct SNPs in ITGRs may be more important in defining host-specificity than overall sequence variation, explaining why traditional unsupervised learning phylogenetic approaches may be less informative in terms of revealing host-specific information encoded in DNA sequence. In silico analysis of 80 candidate ITGRs from publically available E. coli genomes was performed as a tool for discovering highly host-specific ITGRs. In one ITGR (ydeR-yedS) we identified a SNP biomarker that was 98% specific for cattle and for which 92% of all E. coli isolates originating from cattle carried this unique biomarker. In the case of humans, a host-specific biomarker (98% specificity) was identified in the concatenated ITGR sequences of rcsD-ompC, ydeR-yedS, and rclR-ykgE, and for which 78% of E. coli originating from humans carried this biomarker. Interestingly, human-specific biomarkers were dominant in ITGRs regulating antibiotic resistance, whereas in cattle host-specific biomarkers were found in ITGRs involved in stress regulation. These data suggest that evolution towards host specificity may be driven by different natural selection pressures on the regulome of E. coli among different animal hosts.