Comparison of IncK-blaCMY-2 Plasmids in Extended-Spectrum Cephalosporin-Resistant Escherichia coli Isolated from Poultry and Humans in Denmark, Finland, and Germany.

Escherichia coli carrying IncK-blaCMY-2 plasmids mediating resistance to extended-spectrum cephalosporins (ESC) has been frequently described in food-producing animals and in humans. This study aimed to characterize IncK-blaCMY-2-positive ESC-resistant E. coli isolates from poultry production systems in Denmark, Finland, and Germany, as well as from Danish human blood infections, and further compare their plasmids. Whole-genome sequencing (Illumina) of all isolates (n = 46) confirmed the presence of the blaCMY-2 gene. Minimum inhibitory concentration (MIC) testing revealed a resistant phenotype to cefotaxime as well as resistance to ≥3 antibiotic classes. Conjugative transfer of the blaCMY-2 gene confirmed the resistance being on mobile plasmids. Pangenome analysis showed only one-third of the genes being in the core with the remainder being in the large accessory gene pool. Single nucleotide polymorphism (SNP) analysis on sequence type (ST) 429 and 1286 isolates showed between 0-60 and 13-90 SNP differences, respectively, indicating vertical transmission of closely related clones in the poultry production, including among Danish, Finnish, and German ST429 isolates. A comparison of 22 ST429 isolates from this study with 80 ST429 isolates in Enterobase revealed the widespread geographical occurrence of related isolates associated with poultry production. Long-read sequencing of a representative subset of isolates (n = 28) allowed further characterization and comparison of the IncK-blaCMY-2 plasmids with publicly available plasmid sequences. This analysis revealed the presence of highly similar plasmids in ESC-resistant E. coli from Denmark, Finland, and Germany pointing to the existence of common sources. Moreover, the analysis presented evidence of global plasmid transmission and evolution. Lastly, our results indicate that IncK-blaCMY-2 plasmids and their carriers had been circulating in the Danish production chain with an associated risk of spreading to humans, as exemplified by the similarity of the clinical ST429 isolate to poultry isolates. Its persistence may be driven by co-selection since most IncK-blaCMY-2 plasmids harbor resistance factors to drugs used in veterinary medicine.

A novel metagenomic approach uncovers phage genes as markers for increased disinfectant tolerance in mixed Listeria monocytogenes communities.

Listeria monocytogenes is an important human pathogen with a high mortality rate. Consumption of contaminated ready-to-eat food is the main mode of transmission to humans. Disinfectant-tolerant L. monocytogenes have emerged, which are believed to have increased persistence potential. Elucidating the mechanisms of L. monocytogenes disinfectant tolerance has been the focus of previous studies using pure cultures. A limitation of such approach is the difficulty to identify strains with reduced susceptibility due to inter-strain variation and the need to screen large numbers of strains and genes. In this study, we applied a novel metagenomic approach to detect genes associated with disinfectant tolerance in mixed L. monocytogenes planktonic communities. Two communities, consisting of 71 and 80 isolates each, were treated with the food industry disinfectants benzalkonium chloride (BC, 1.75 mg/L) or peracetic acid (PAA, 38 mg/L). The communities were subjected to metagenomic sequencing and differences in individual gene abundances between biocide-free control communities and biocide-treated communities were determined. A significant increase in the abundance of Listeria phage-associated genes was observed in both communities after treatment, suggesting that prophage carriage could lead to an increased disinfectant tolerance in mixed L. monocytogenes planktonic communities. In contrast, a significant decrease in the abundance of a high-copy emrC-harbouring plasmid pLmN12-0935 was observed in both communities after treatment. In PAA-treated community, a putative ABC transporter previously found to be necessary for L. monocytogenes resistance to antimicrobial agents and virulence, was among the genes with the highest weight for differentiating treated from control samples. The undertaken metagenomic approach in this study can be applied to identify genes associated with increased tolerance to other antimicrobials in mixed bacterial communities.

Predicting Listeria monocytogenes virulence potential using whole genome sequencing and machine learning.

Contamination with food-borne pathogens, such as Listeria monocytogenes, remains a big concern for food safety. Hence, rigorous and continuous microbial surveillance is a standard procedure. At this point, however, the food industry and authorities only focus on detection of Listeria monocytogenes without characterization of individual strains into groups of more or less concern. As whole genome sequencing (WGS) gains increasing interest in the industry, this methodology presents an opportunity to obtain finer resolution of microbial traits such as virulence. Within this study, we therefore aimed to explore the use of WGS in combination with Machine Learning (ML) to predict L. monocytogenes virulence potential on a sub-species level. The WGS datasets used in this study for ML model training consisted of i) national surveillance isolates (n = 169, covering 38 MLST types) and ii) publicly available isolates acquired through the GenomeTrakr network (n = 2880, spanning 80 MLST types). We used the clinical frequency, i.e., ratio of the number of clinical isolates to total amount of isolates, as estimate for virulence potential. The predictive performance of input features from three different genomic levels (i.e., virulence genes, pan-genome genes, and single nucleotide polymorphisms (SNPs)) and six machine learning algorithms (i.e., Support Vector Machine with a linear kernel, Support Vector Machine with a radial kernel, Random Forrest, Neural Networks, LogitBoost, and Majority Voting) were compared. Our machine learning models predicted sub-species virulence potential with nested cross-validation F1-scores up to 0.88 for the majority voting classifier trained on national surveillance data and using pan-genome genes as input features. The validation of the pre-trained ML models based on 101 previously in vivo studied isolates resulted in F1-scores up to 0.76. Furthermore, we found that the more rapid and less computationally intensive raw read alignment yields comparably accurate models as de novo assembly. The results of our study suggest that a majority voting classifier trained on pan-genome genes is the best and most robust choice for the prediction of clinical frequency. Our study contributes to more rapid and precise characterization of L. monocytogenes virulence and its variation on a sub-species level. We further demonstrated a possible application of WGS data in the context of microbial hazard characterization for food safety. In the future, predictive models may assist case-specific microbial risk management in the food industry. The python code, pre-trained models, and prediction pipeline are deposited at (https://github.com/agmei/LmonoVirulenceML).

Implementing risk-based approaches to improve drinking water quality in small water supplies in the Nordic region - barriers and solutions.

Small water supplies face similar problems worldwide, regardless of ownership or management type. Non-compliance with water quality regulations is more frequent in small supplies than in large ones, as are waterborne disease outbreaks. The new European Union Drinking Water Directive requires risk-based approach (RBA) to secure water safety as is recommended in the World Health Organization's Guidelines for drinking water quality through 'water safety plans'. This is already in regulation in the Nordic countries, although less used in small supplies. In this research, we explore the challenges, barriers and possible solutions to implementing RBA and improving compliance in small supplies. This was achieved by conducting and analysing interviews with 53 stakeholders from all eight Nordic countries to produce recommendations for action by the different implicated actors. Our findings suggest the centrality of governmental policy, including support for continuous training, provision of simple RBA guidelines and increasing cooperation in the water sector. The Nordic experience reflects global challenges with small water supplies and the trend towards systematic preventive management epitomized in the framework for drinking water safety advocated by the World Health Organization since 2004.

Prevalence and Transmission of Extended-Spectrum Cephalosporin (ESC) Resistance Genes in Escherichia coli Isolated from Poultry Production Systems and Slaughterhouses in Denmark.

The emergence of extended-spectrum cephalosporin (ESC)-resistant Escherichia coli is a global concern. This study aimed to assess the prevalence and transmission of ESC-resistant E. coli in the Danish broiler production system. Samples from two vertically integrated Production Systems (1 and 2) and two slaughterhouses (A and B) were analyzed (n = 943) for the occurrence of ESC-resistant E. coli from 2015 to 2018. ESC-resistant E. coli isolates were whole-genome sequenced (WGS) for characterization of the multi-locus sequence type (MLST), antibiotic resistance genes, virulence genes, and plasmid replicon types. An ad hoc core genome (cg) MLST based on 2513 alleles was used to examine the genetic relatedness among isolates. The prevalence of ESC-resistant E. coli in the conventional Production System 1 was 2.7%, while in Production System 2 the prevalence was 26.7% and 56.5% for samples from the conventional and organic production, respectively. The overall prevalence of ESC-resistant E. coli in broiler thigh and fecal samples ranged from 19.3% in Slaughterhouse A to 22.4% in Slaughterhouse B. In total, 162 ESC-resistant E. coli were isolated and shown to belong to 16 different sequence types (STs). The most prevalent STs were ST2040 (n = 85) and ST429 (n = 22). Seven ESC resistance genes were detected: blaCMY-2 (n = 119), blaTEM-52B (n = 16), blaCTX-M-1 (n = 5), blaTEM-52C (n = 3), blaCTX-M-14 (n = 1), blaSHV-12 (n = 1), and up-regulation of ampC (n = 16), with an unknown resistance gene in one isolate (n = 1). The carriage of blaCMY-2 in 119 isolates was primarily associated with IncI1 (n = 87), and IncK plasmids (n = 31). Highly similar blaCMY-2 carrying E. coli isolates from ST429 were found in production systems as well as in slaughterhouses. In conclusion, findings from this study indicate that ESC-resistant E. coli are transferred vertically from farms in the production systems to slaughterhouses with the potential to enter the food supply.

Application of hydraulic modelling and quantitative microbial risk assessment (QMRA) for cloudburst management in cities with combined sewer systems.

Urban cloudburst management may include the intentional temporary storage of flood water in green recreational areas. In cities with combined sewers, this will expose the population visiting the area to sewage and increase the risk of diarrhoeal disease. We present a unique approach to estimate the risk of diarrhoeal disease after urban flooding. The exposure scenario was: rainwater mixed with sewage flows into a park; sewage with pathogens deposit on the grass; after discharge, a baby plays on the grass and is exposed to the pathogens in the deposited sewage by hand-to-mouth transfer. The work included modelling the transport of sewage into four parks intended to be flooded during future cloudbursts. A flood simulation experiment was conducted to estimate the deposition of pathogens from sewage to grass and transfer from grass to hand. Hand-to-mouth transfer, based on literature values, was used to estimate the ingested dose of pathogens. The probability of illness was estimated by QMRA. The estimated average probability of illness varied between 0.03 and 17%. If the probability of illness is considered unacceptable, the cloudburst plans should be changed, or interventions, e.g. informing the public about the risk or restricting access to the flooded area, should be implemented.

Proteolytic activity and heat resistance of the protease AprX from Pseudomonas in relation to genotypic characteristics.

AprX is an alkaline metalloprotease produced by Pseudomonas spp. and encoded by its initial gene of the aprX-lipA operon. The intrinsic diversity among Pseudomonas spp. regarding their proteolytic activity is the main challenge for the development of accurate methods for spoilage prediction of ultra-high temperature (UHT) treated milk in the dairy industry. In the present study, 56 Pseudomonas strains were characterized by assessing their proteolytic activity in milk before and after lab-scale UHT treatment. From these, 24 strains were selected based on their proteolytic activity for whole genome sequencing (WGS) to identify common genotypic characteristics that correlated with the observed variations in proteolytic activity. Four groups (A1, A2, B and N) were determined based on operon aprX-lipA sequence similarities. These alignment groups were observed to significantly influence the proteolytic activity of the strains, with an average proteolytic activity of A1 > A2 > B > N. The lab-scale UHT treatment did not significantly influence their proteolytic activity, indicating a high thermal stability of proteases among strains. Amino acid sequence variation of biologically-relevant motifs in the AprX sequence, namely the Zn2+-binding motif at the catalytic domain and the C-terminal type I secretion signaling mechanism, were found to be highly conserved within alignment groups. These motifs could serve as future potential genetic biomarkers for determination of alignment groups and thereby strain spoilage potential.

Domestic water supply in rural Greenland - sufficiency, affordability and accessibility.

Global efforts are still under way to ensure sustainable development goal 6 of providing enough clean water to sustain public health in many regions, and especially in the Arctic where the remoteness of communities and the harsh climate make water provision especially challenging. This study aimed to examine the sufficiency, accessibility, and affordability of water supplies in rural Greenland. The state of the water supply was investigated using quantitative data on infrastructure and demographics. Qualitative data on water-related practices and perceptions were collected through fieldwork and interviews in a selection of settlements. Generally, the supply of drinking water was found to be sufficient and affordable for most. However, access was severely constrained by the lack of piping to rural homes (20% were piped). The daily water consumption of residents from un-piped households was between 13 and 23 L/d/cap, i.e. within the basic access level according to WHO, which is in theory not sufficient to sustain public health. Several health risks could be caused by the low daily consumption in un-piped homes, and water saving practices induced by it - i.e. the use of shared handwashing basins, and household water storage, which could lead to degradation of water quality at the point-of-use.

Quality and safety aspects in fermentation of winged kelp (Alaria esculenta) and sugar kelp (Saccharina latissima) by the natural microbiota with or without addition of a Lactiplantibacillus plantarum starter culture.

Nourishment of the growing human population requires new and alternative food sources, preferable produced without occupying new land areas. Cultivation of seaweed presents an opportunity, however, a major obstacle is sustainable preservation. Fermentation has been used for centuries to preserve vegetables, e.g., to produce kimchi based on cabbage. This study investigated changes in the microbiota, characteristics (pH, organic acids and water soluble carbohydrates) and food safety of raw shredded Alaria esculenta and Saccharina latissima during fermentation by the natural microbiota with or without addition of a Lactiplantibacillus plantarum starter culture. The Lb. plantarum fermented products retained a high Shannon diversity index, indicating a partially unsuccessful fermentation. Lb. plantarum performed better in A. esculenta causing pH to drop to below 4.6, a critical limit for control of growth of Clostridium botulinum, within 2 days compared to 7 days for S. latissima. Natural fermentation by the endogenous microbiota resulted in unsafe products with high final pH values (4.8-5.2), presence of unwanted organic acids, such as butyric acid, and in the case of A. esculenta sustenance of inoculated Listeria monocytogenes. Fermentation of A. esculenta and S. latissima by Lb. plantarum is a promising preservation method. However, future work is needed to optimise the process, by investigation of the use of different starter cultures, seaweed pre-treatments (blanching, freezing, etc.) and adjuvants (i.e., addition of sugars, minerals and similar) to promote growth of the starter culture and ensure the fermented products are safe to eat.

Comparative genomic analyses of ß-lactamase (blaCMY-42)-encoding plasmids isolated from wastewater treatment plants in Canada.

Wastewater treatment plants (WWTPs) are useful environments for investigating the occurrence, diversity, and evolution of plasmids encoding clinically relevant antibiotic resistance genes (ARGs). Our objective was to isolate and sequence plasmids encoding meropenem resistance from bacterial hosts within Canadian WWTPs. We used two enrichment culture approaches for primary plasmid isolation, followed by screening for antibiotic resistance, conjugative mobility, and stability in enteric bacteria. Isolated plasmids were sequenced using Illumina MiSeq and Sanger sequencing methods. Bioinformatics analyses resolved a multi-resistance IncF/MOBF12 plasmid, pFEMG (209 357 bp), harbouring resistance genes to ß-lactam (blaCMY-42, blaTEM-1ß, and blaNDM-5), macrolide (mphA-mrx-mphR), tetracycline (tetR-tetB-tetC-tetD), trimethoprim (dfrA12), aminoglycoside (aadA2), and sulfonamide (sul1) antibiotic classes. We also isolated an IncI1/MOBP12 plasmid pPIMR (172 280 bp) carrying similar ß-lactamase and a small multi-drug efflux resistance gene cluster (blaCMY-42-blc-sugE) to pFEMG. The co-occurrence of different ARGs within a single 24 552 bp cluster in pFEMG - interspersed with transposons, insertion sequence elements, and a class 1 integron - may be of significant interest to human and veterinary medicine. Additionally, the presence of conjugative and plasmid maintenance genes in the studied plasmids corresponded to observed high conjugative transfer frequencies and stable maintenance. Extensive investigation is required to further understand the fitness trade-offs of plasmids with different types of conjugative transfer and maintenance modules.

Predicting the effect of salt on heat tolerance of Listeria monocytogenes in meat and fish products.

Listeria monocytogenes is a potentially fatal foodborne pathogen that can be found in various ready-to-eat (RTE) products. It tolerates adverse conditions such as high salt concentrations and refrigerated storage, thus, the elimination of the pathogen in food processing often relies on heat processing. The objective of this study was to create a model to predict the effect of salt on heat tolerance of L. monocytogenes in meat and seafood products during heat treatments conducted at 57 to 65 °C to reduce numbers by ≥3 log10 cycles. Salt concentrations, up to 6% in the water phase (WPS%), were applied to cover a variety of lightly salted RTE meat and seafood products. The experimental work involved samples of ground pork tenderloin, ground chicken breast fillet and skinned, ground salmon fillet adjusted to different WPS% i.e., 3.6 and 5.2 WPS% for pork samples, 2.0, 3.0, 3.5 and 6.0 WPS% for chicken samples and 3.0 and 6.0 WPS% for salmon samples. All samples were inoculated with late-stationary phase L. monocytogenes cultures. For pork samples, a two-strain mixture of a pork isolate (MS22254) and an environmental isolate (MS22246) was applied. For chicken and salmon samples, a seafood isolate (MS22258) and isolate MS22246 was applied as single cultures. Samples were vacuum-packed in sterile bags, immerged in water bath, and held at constant temperatures of 57, 60 and 65 °C for pork samples and 58, 61 and 62.5 °C for chicken and salmon samples. For survivor curves, where at least 3 log10-reduction were obtained, heat tolerance was expressed as decimal reduction times, D-values. D-values were observed to increase with increasing WPS%. The effect of salt on heat tolerance of L. monocytogenes was defined as the relative increase (RI-value) in D-value obtained when salt had been added to the food. The effect of WPS% on RI-values was independent of heating temperatures, foods and strains. For secondary modelling, RI-values were transformed using the natural logarithm, ln(RI) and fitted to a linear model as a function of WPS%. Model validation, with 56 independent values collected from the scientific literature, resulted in bias and accuracy factors of 0.89 and 1.26, respectively, suggesting acceptable performance with tendency to slightly under-predict. The developed predictive model can be used to guide the design of heat processes for manufacturers of lightly preserved and mildly processed meat and seafood products requiring more than 3 log10 reduction of L. monocytogenes to ensure safety.

Fate and distribution of determinants of antimicrobial resistance in lateral flow sand filters used for treatment of domestic wastewater.

Residuals of antimicrobial products from anthropogenic uses can create a selective environment in domestic wastewater treatment systems and receiving environments and contribute to the spread of antimicrobial resistance (AMR). On-site wastewater treatment systems are widely used for domestic wastewater management in rural and remote regions, but the fate of determinants of AMR in these types of environments has received little attention. In this study, the mechanisms responsible for the attenuation of determinants of AMR in lateral flow sand filters were explored using a combination of lab, field and modeling investigations. The degradation kinetics and adsorption potential in the sand filter medium of three antibiotic resistance genes (ARGs; sul1, tetO, and ermB) and culturable bacteria resistant to sulfamethoxazole, tetracycline, and erythromycin were measured using lab experiments. The spatial distribution of ARGs and antibiotic resistant bacteria were also assessed in field scale sand filters, and mechanistic modeling was conducted to characterize filtration processes. The results indicated that the primary mechanisms responsible for AMR attenuation within the sand filters were degradation and filtration. The spatial distribution of AMR determinants illustrated that attenuation was occurring along the entire length of each filter. This study provides new insights on primary mechanisms of AMR attenuation in on-site wastewater treatment systems and supports the use of conservative design guidelines and separation distances for reducing AMR transmission.

Characterisation and chemometric evaluation of 17 elements in ten seaweed species from Greenland.

Several Greenland seaweed species have potential as foods or food ingredients, both for local consumption and export. However, knowledge regarding their content of beneficial and deleterious elements on a species specific and geographical basis is lacking. This study investigated the content of 17 elements (As, Ca, Cd, Cr, Cu, Fe, Hg, I, K, Mg, Mn, Na, Ni, P, Pb, Se and Zn) in 77 samples of ten species (Agarum clathratum, Alaria esculenta, Ascophyllum nodosum, Fucus distichus, Fucus vesiculosus, Hedophyllum nigripes, Laminaria solidungula, Palmaria palmata, Saccharina latissima and Saccharina longicruris). Element profiles differed between species but showed similar patterns within the same family. For five species, different thallus parts were investigated separately, and showed different element profiles. A geographic origin comparison of Fucus species indicated regional differences. The seaweeds investigated were especially good sources of macrominerals (K > Na > Ca > Mg) and trace minerals, such as Fe. Iodine contents were high, especially in macroalgae of the family Laminariaceae. None of the samples exceeded the EU maximum levels for Cd, Hg or Pb, but some exceeded the stricter French regulations, especially for Cd and I. In conclusion, these ten species are promising food items.

Quantitative Microbial Risk Assessment Based on Whole Genome Sequencing Data: Case of Listeria monocytogenes.

The application of high-throughput DNA sequencing technologies (WGS) data remain an increasingly discussed but vastly unexplored resource in the public health domain of quantitative microbial risk assessment (QMRA). This is due to challenges including high dimensionality of WGS data and heterogeneity of microbial growth phenotype data. This study provides an innovative approach for modeling the impact of population heterogeneity in microbial phenotypic stress response and integrates this into predictive models inputting a high-dimensional WGS data for increased precision exposure assessment using an example of Listeria monocytogenes. Finite mixture models were used to distinguish the number of sub-populations for each of the stress phenotypes, acid, cold, salt and desiccation. Machine learning predictive models were selected from six algorithms by inputting WGS data to predict the sub-population membership of new strains with unknown stress response data. An example QMRA was conducted for cultured milk products using the strains of unknown stress phenotype to illustrate the significance of the findings of this study. Increased resistance to stress conditions leads to increased growth, the likelihood of higher exposure and probability of illness. Neglecting within-species genetic and phenotypic heterogeneity in microbial stress response may over or underestimate microbial exposure and eventual risk during QMRA.

Status of risk-based approach and national framework for safe drinking water in small water supplies of the Nordic water sector.

Reliable safe water supply is a pillar of society and a key to public health. The Nordic countries have an abundance of clean fresh water as a source for drinking water supplies. They have followed developments in safeguarding water, both the recommendations of the World Health Organization framework for safe drinking water and European legislation. Worldwide, including the Nordic countries, small water supplies are less compliant with water safety regulation. The forthcoming EU directive on drinking water require risk-based approaches and improved transparency on water quality. This research looks at the Nordic frameworks for safe water supply, with emphasis on risk-based approaches and smaller systems. We analyzed the legal frameworks for safe water, the structure of the water sector across the Nordic countries and explored how prepared these countries are to meet these requirements. Our findings show that, while legal requirements are mostly in place, delivery of information to the public needs to be improved. Most Nordic countries are in the process of implementing risk-based management in large and medium size water supplies, whereas small supplies are lagging. We conclude that a key to success is increased training and support for small supplies. We suggest wider adoption of the Nordic model of cooperation with benchmarking of safe water for all to transfer knowledge between the countries. This work provides insights into challenges and opportunities for the Nordic countries and provides insights relevant to countries worldwide in their effort towards realization of SDG Target 6.1.

Lateral flow sand filters are effective for removal of antibiotic resistance genes from domestic wastewater.

The ability of lateral flow sand filters, used as on-site wastewater treatment systems (OWTS), to remove antibiotic resistance genes (ARGs), antibiotic resistant bacteria (ARB), and other relevant genetic markers (HF183, 16S rRNA, and int1) was assessed. Municipal wastewater was settled in a septic tank prior to loading into six pilot-scale lateral flow sand filters comprised of three different sand media types, at 5 and 30% slopes. The sand filters were sampled bi-weekly for: 9 ARGs and 3 other complimentary gene markers (sul1, sul2, qnrS, tetO, ermB, blaTEM, blaCTX-M, mecA, vanA, int1, HF183, 16S rRNA), and conventional microbial and water quality indicators, from July to November in 2017, and four times in the summer of 2018. The sand filters were observed to attenuate 7 of the ARGs to mostly below 2 log gene copies per mL. Log reductions ranging from 2.9 to 5.4 log were observed for the removal of absolute abundances of ARGs from septic tank effluent in 5 of the 6 sand filters. The fine-grained filter on the 5% slope did not perform as well for ARG attenuation due to hydraulic failure. The apportionment of cell-associated versus cell-free DNA was determined for the gene markers and this indicated that the genes were primarily carried intracellularly. Average log reductions of ARB with resistance to either sulfamethoxazole, erythromycin, or tetracycline were approximately 2.3 log CFU per mL within the filters compared to the septic tank effluent. This field study provides in-depth insights into the attenuation of ARB, ARGs, and their genetic compartmentalization in variably saturated sand OWTS. Overall, this type of OWTS was found to pose little risk of antimicrobial resistance contamination spread into surrounding environments when proper hydraulic function was maintained.

Sources of Antibiotic Resistance Genes in a Rural River System.

The increasing prevalence of antibiotic resistance genes (ARGs) in the environment is problematic due to the risk of horizontal gene transfer and development of antibiotic resistant pathogenic bacteria. Using a suite of monitoring tools, this study aimed to investigate the sources of ARGs in a rural river system in Nova Scotia, Canada. The monitoring program specifically focused on the relative contribution of ARGs from a single tertiary-level wastewater treatment plant (WWTP) in comparison to contributions from the upgradient rural, sparsely developed, watershed. The overall gene concentration significantly ( < 0.05) increased downstream from the WWTP, suggesting that tertiary-level treatment still contributes ARGs to the environment. As a general trend, ARG concentrations upstream were found to decrease as proximity to human-impacted areas decreased; however, many ARGs remained above detection limits in headwater river samples, which suggested their ubiquitous presence in this watershed in the absence of obvious pollution sources. Significant correlations with ARGs were found for human fecal marker, and some antibiotics, suggesting that these markers may be useful for prediction and understanding of ARG levels and sources in rural rivers.

Effect of hillslope position and manure application rates on the persistence of fecal source tracking indicators in an agricultural soil.

The influence of liquid dairy manure (LDM) application rates (12.5 and 25 kL ha) and soil type on the decay rates of library-independent fecal source tracking markers (host-associated and mitochondrial DNA) and persistent (>58 d) population structure was examined in a field study. The soils compared were an Aquic Haplorthod and a Typic Haplorthod in Nova Scotia, Canada, that differed according to landscape position and soil moisture regime. Soil type and LDM application rate did not influence decay rates (0.045-0.057 d). population structure, in terms of the occurrence of abundance of strain types, varied according to soil type ( = 0.012) but did not vary by LDM application rate ( = 0.121). Decay of ruminant-specific (BacR), bovine-specific (CowM2), and mitochondrial DNA (AcytB) markers was analyzed for 13 d after LDM application. The decay rates of BacR were greater under high-LDM application rates (0.281-0.358 d) versus low-LDM application rates (0.212-0.236 d) but were unaffected by soil type. No decay rates could be calculated for the CowM2 marker because it was undetectable within 6 d after manure application. Decay rates for AcytB were lower for the Aquic Haplorthod (0.088-0.100 d), with higher moisture status compared with the Typic Haplorthod (0.135 d). Further investigation into the decay of fecal source tracking indicators in agricultural field soils is warranted to assess the influence of soil type and agronomic practice on the differential decay of relevant markers and the likelihood of transport in runoff.

Characterizing spatial structure of sediment E. coli populations to inform sampling design.

Escherichia coli can persist in streambed sediments and influence water quality monitoring programs through their resuspension into overlying waters. This study examined the spatial patterns in E. coli concentration and population structure within streambed morphological features during baseflow and following stormflow to inform sampling strategies for representative characterization of E. coli populations within a stream reach. E. coli concentrations in bed sediments were significantly different (p = 0.002) among monitoring sites during baseflow, and significant interactive effects (p = 0.002) occurred among monitoring sites and morphological features following stormflow. Least absolute shrinkage and selection operator (LASSO) regression revealed that water velocity and effective particle size (D 10) explained E. coli concentration during baseflow, whereas sediment organic carbon, water velocity and median particle diameter (D 50) were important explanatory variables following stormflow. Principle Coordinate Analysis illustrated the site-scale differences in sediment E. coli populations between disconnected stream segments. Also, E. coli populations were similar among depositional features within a reach, but differed in relation to high velocity features (e.g., riffles). Canonical correspondence analysis resolved that E. coli population structure was primarily explained by spatial (26.9­31.7 %) over environmental variables (9.2­13.1 %). Spatial autocorrelation existed among monitoring sites and morphological features for both sampling events, and gradients in mean particle diameter and water velocity influenced E. coli population structure for the baseflow and stormflow sampling events, respectively. Representative characterization of streambed E. coli requires sampling of depositional and high velocity environments to accommodate strain selectivity among these features owing to sediment and water velocity heterogeneity.

Evaluation of statistical models for predicting Escherichia coli particle attachment in fluvial systems.

Modeling surface water Escherichia coli fate and transport requires partitioning E. coli into particle-attached and unattached fractions. Attachment is often assumed to be a constant fraction or is estimated using simple linear models. The objectives of this study were to: (i) develop statistical models for predicting E. coli attachment and virulence marker presence in fluvial systems, and (ii) relate E. coli attachment to a variety of environmental parameters. Stream water samples (n = 60) were collected at four locations in a rural, mixed-use watershed between June and October 2012, with four storm events (>20 mm rainfall) being captured. The percentage of E. coli attached to particles (>5 µm) and the occurrences of virulence markers were modeled using water quality, particle concentration, particle size distribution, hydrology and land use factors as explanatory variables. Three types of statistical models appropriate for highly collinear, multidimensional data were compared: least angle shrinkage and selection operator (LASSO), classification and regression trees using the general, unbiased, interaction detection and estimation (GUIDE) algorithm, and multivariate adaptive regression splines (MARS). All models showed that E. coli particle attachment and the presence of E. coli virulence markers in the attached and unattached states were influenced by a combination of water quality, hydrology, land-use and particle properties. Model performance statistics indicate that MARS models outperform LASSO and GUIDE models for predicting E. coli particle attachment and virulence marker occurrence. Validating the MARS modeling approach in multiple watersheds may allow for the development of a parameterizing model to be included in watershed simulation models.