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.

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.

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.

Recruited fibroblasts reconstitute the peri-islet membrane: a longitudinal imaging study of human islet grafting and revascularisation.

AIMS/HYPOTHESIS: Rapid and adequate islet revascularisation and restoration of the islet-extracellular matrix (ECM) interaction are significant factors influencing islet survival and function of the transplanted islets in individuals with type 1 diabetes. Because the ECM encapsulating the islets is degraded during islet isolation, understanding the process of revascularisation and engraftment after transplantation is essential and needs further investigation. METHODS: Here we apply a longitudinal and high-resolution imaging approach to investigate the dynamics of the pancreatic islet engraftment process up to 11 months after transplantation. Human and mouse islet grafts were inserted into the anterior chamber of the mouse eye, using a NOD.ROSA-tomato.Rag2-/- or B6.ROSA-tomato host allowing the investigation of the expansion of host vs donor cells and the contribution of host cells to aspects such as promoting the encapsulation and vascularisation of the graft. RESULTS: A fibroblast-like stromal cell population of host origin rapidly migrates to ensheath the transplanted islet and aid in the formation of a basement membrane-like structure. Moreover, we show that the vessel network, while reconstituted by host endothelial cells, still retains the overall architecture of the donor islets. CONCLUSIONS/INTERPRETATION: In this transplantation situation the fibroblast-like stromal cells appear to take over as main producers of ECM or act as a scaffold for other ECM-producing cells to reconstitute a peri-islet-like basement membrane. This may have implications for our understanding of long-term graft rejection and for the design of novel strategies to interfere with this process.

A long-amplicon quantitative PCR assay with propidium monoazide to enumerate viable Listeria monocytogenes after heat and desiccation treatments.

The objective of this study was to develop a qPCR method for specific enumeration of viable Listeria monocytogenes in food processing facilities and heat treated products. Primers specific for L. monocytogenes were designed to amplify a short (199 bp) or long (1561 bp) fragment of the listeriolysin (hly) gene. The short- and long-amplicon qPCR methods with and without propidium monoazide (PMA) treatment of the cells were tested for their ability to discriminate between viable (no heat) and heat-killed cells (90 °C, 10 min). The PMA-qPCR methods were subsequently used to assess the survival of L. monocytogenes during desiccation (33% RH, 15 °C) on stainless steel surfaces for ten days with and without prior biofilm formation. The long-amplicon qPCR method had a limit of quantification (LOQ) of 1.32 log CFU/reaction (efficiency 92%, R2 = 0.991), while the LOQ for the short-amplicon qPCR method was 1.44 log CFU/reaction (efficiency 102%, R2 = 0.991). PMA was essential for detection of viable cells, and the long-amplicon PMA-qPCR significantly (p < 0.05) reduced the signal from heat-killed cells compared to the short-amplicon method. L. monocytogenes survival during desiccation without biofilm formation was accurately enumerated with the long-amplicon PMA-qPCR method. However, when L. monocytogenes had formed biofilm prior to desiccation, the long-amplicon PMA-qPCR accurately measured the log fold inactivation but underestimated the number of viable cells even with use of an optimized DNA extraction method. This long-amplicon PMA-qPCR method can aid in the detection and enumeration of viable L. monocytogenes cells to further the understanding of its survival and persistence in food processing facilities. The developed method was demonstrated to work on both heat and desiccation treated cells and highlights the importance of amplicon size in viability-qPCR.

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.

The Reliability of the Segmental Assessment of Trunk Control (SATCo) in Children with Cerebral Palsy.

AIMS: To assess the live-versus-video, intrarater interday and interrater interday reliability of the test Segmental Assessment of Trunk Control (SATCo), which seeks to estimate the degree of sitting trunk control in children with cerebral palsy (CP). METHOD: Thirty-one children with CP between 9 months and 16 years of age (22 males, mean age 8y 10mo [SD 3y 5mo], Gross Motor Function Classification System level I [n = 13], II [n = 4], III [n = 4], IV [n = 3], and V [n = 7]) were included. Children were tested twice by two raters and tests were video recorded. Wilcoxon Signed-Rank Test, ICC [2,1] and a descriptive measure for absolute reliability were applied. RESULTS: No systematic differences were found between live-versus-video, between raters or days (p > 0.05) except for one analysis. All ICC values were excellent (ICC ≥ 0.9) except for one analysis for which it was good (ICC = 0.73). Complete agreement between scores was seen in 75% of all cases while 22% differed by one segmental level. Only 3% showed disagreement above one segmental level. CONCLUSIONS: SATCo is a clinically applicable assessment tool. Relative reliability is excellent and absolute agreement is good. Modifications regarding testing method could potentially improve the reliability and the value of the test in research and in clinical practice.

Deficiency in plasmacytoid dendritic cells and type I interferon signalling prevents diet-induced obesity and insulin resistance in mice.

AIMS/HYPOTHESIS: Obesity is associated with glucose intolerance and insulin resistance and is closely linked to the increasing prevalence of type 2 diabetes. In mouse models of diet-induced obesity (DIO) and type 2 diabetes, an increased fat intake results in adipose tissue expansion and the secretion of proinflammatory cytokines. The innate immune system not only plays a crucial role in obesity-associated chronic low-grade inflammation but it is also proposed to play a role in modulating energy metabolism. However, little is known about how the modulation of metabolism by the immune system may promote increased adiposity in the early stages of increased dietary intake. Here we aimed to define the role of type I IFNs in DIO and insulin resistance. METHODS: Mice lacking the receptor for IFN-α (IFNAR-/-) and deficient in plasmacytoid dendritic cells (pDCs) (B6.E2-2 fl/fl .Itgax-cre) were fed a diet with a high fat content or normal chow. The mice were analysed in vivo and in vitro using cellular, biochemical and molecular approaches. RESULTS: We found that the development of obesity was inhibited by an inability to respond to type I IFNs. Furthermore, the development of obesity and insulin resistance in this model was associated with pDC recruitment to the fatty tissues and liver of obese mice (a 4.3-fold and 2.7-fold increase, respectively). Finally, we demonstrated that the depletion of pDCs protects mice from DIO and from developing obesity-associated metabolic complications. CONCLUSIONS/INTERPRETATION: Our results provide genetic evidence that pDCs, via type I IFNs, regulate energy metabolism and promote the development of obesity.

Longitudinal three-dimensional visualisation of autoimmune diabetes by functional optical coherence imaging.

AIMS/HYPOTHESIS: It is generally accepted that structural and functional quantitative imaging of individual islets would be beneficial to elucidate the pathogenesis of type 1 diabetes. We here introduce functional optical coherence imaging (FOCI) for fast, label-free monitoring of beta cell destruction and associated alterations of islet vascularisation. METHODS: NOD mouse and human islets transplanted into the anterior chamber of the eye (ACE) were imaged with FOCI, in which the optical contrast of FOCI is based on intrinsic variations of the index of refraction resulting in a faster tomographic acquisition. In addition, the phase sensitivity allows simultaneous label-free acquisition of vascularisation. RESULTS: We demonstrate that FOCI allows longitudinal quantification of progressive autoimmune insulitis, including the three-dimensional quantification of beta cell volume, inflammation and vascularisation. The substantially increased backscattering of islets is dominated by the insulin-zinc nanocrystals in the beta cell granules. This translates into a high specificity for the functional beta cell volume of islets. Applying FOCI to a spontaneous mouse model of type 1 diabetes, we quantify the modifications of the pancreatic microvasculature accompanying the progression of diabetes and reveal a strong correlation between increasing insulitis and density of the vascular network of the islet. CONCLUSIONS/INTERPRETATION: FOCI provides a novel imaging technique for investigating functional and structural diabetes-induced alterations of the islets. The label-free detection of beta cell volume and infiltration together with vascularisation offers a unique extension to study ACE-transplanted human islets. These results are contributing to a deeper understanding of human islet transplant rejection and label-free in vivo monitoring of drug efficacy.

Local and Systemic Changes in Pain Sensitivity After 4 Weeks of Calf Muscle Stretching in a Nonpainful Population: A Randomized Trial.

BACKGROUND: Stretching is often used in clinical practice for a variety of purposes, including pain therapy. The possible mechanism behind the effect of stretching remains to be clarified. AIM: To investigate whether 4 weeks of unilateral stretching of the calf muscles would affect local and central pain sensitivity. METHOD: This study was a randomized assessor-blinded clinical study. Healthy participants (age 18 to 40) were included and randomized. Participants in the intervention group were instructed to perform 2 stretching exercises targeting the calf muscles; 3 times 30 seconds, 7 days a week for 4 weeks on the dominant leg. Participants in the control group were instructed not to do any stretching for 4 weeks. Pressure pain threshold (PPT) and temporal summation (TS) of pressure pain were measured on the stretched calf, the contra-lateral calf, and contra-lateral lower arm using a computerized cuff algometer. Analyses of variance on the per-protocol population (defined as participants that adhered to the protocol) were used to assess group differences in the changes from baseline. RESULT: Forty healthy volunteers were included, of which 34 participants adhered to the protocol (15 intervention group/19 control group). No statistically significant group differences in the changes from baseline were found regarding PPT and TS measurements for the stretched calf, the contra-lateral calf, and the arm. CONCLUSION: Four weeks of regular stretching of the calf muscles does not affect pressure pain sensitivity, suggesting that pressure pain sensitivity is unaffected by stretching in a healthy population. The mechanisms underlying any benefits of regular stretching remain to be explained.

Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis.

Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm(2) relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses.

Comparison of the Prevalences and Diversities of Listeria Species and Listeria monocytogenes in an Urban and a Rural Agricultural Watershed.

Foods and related processing environments are commonly contaminated with the pathogenic Listeria monocytogenes. To investigate potential environmental reservoirs of Listeria spp. and L. monocytogenes, surface water and point source pollution samples from an urban and a rural municipal water supply watershed in Nova Scotia, Canada, were examined over 18 months. Presumptive Listeria spp. were cultured from 72 and 35% of rural and urban water samples, respectively, with 24% of the positive samples containing two or three different Listeria spp. The L. innocua (56%) and L. welshimeri (43%) groups were predominant in the rural and urban watersheds, respectively. Analysis by the TaqMan assay showed a significantly (P < 0.05) higher prevalence of L. monocytogenes of 62% versus 17% by the culture-based method. Both methods revealed higher prevalences in the rural watershed and during the fall and winter seasons. Elevated Escherichia coli (≥ 100 CFU/100 ml) levels were not associated with the pathogen regardless of the detection method. Isolation of Listeria spp. were associated with 70 times higher odds of isolating L. monocytogenes (odds ratio = 70; P < 0.001). Serogroup IIa was predominant (67.7%) among the 285 L. monocytogenes isolates, followed by IVb (16.1%), IIb (15.8%), and IIc (0.4%). L. monocytogenes was detected in cow feces and raw sewage but not in septic tank samples. Pulsotyping of representative water (n = 54) and local human (n = 19) isolates suggested genetic similarities among some environmental and human L. monocytogenes isolates. In conclusion, temperate surface waters contain a diverse Listeria species population and could be a potential reservoir for L. monocytogenes, especially in rural agricultural watersheds.

Role of sigB and osmolytes in desiccation survival of Listeria monocytogenes in simulated food soils on the surface of food grade stainless steel.

This research aimed to determine whether the SigB (σ(B)) regulon and osmolytes impact the survival of the foodborne pathogen, Listeria monocytogenes, during desiccation in simulated food soils with varying salt and nutrient contents on food grade stainless steel (SS) surfaces. L. monocytogenes 568 (Lm568, serotype 1/2a), its isogenic sigB mutant (ΔsigB) and the back-complemented ΔsigB were desiccated in BHI, TSB with 1% glucose (TSB-glu), peptone physiological saline (PPS) and minimal media (MM) for 21 days at 43% relative humidity (RH) and 15 °C on SS. The effect of food related osmolytes (proline, betaine and carnitine) on desiccation survival was studied by (a) pre-culturing strains in MM with an osmolyte followed by desiccation in MM and (b) by desiccating strains in MM with an osmolyte. Desiccation survival of L. monocytogenes was positively correlated to the nutrient and osmolyte concentrations in the desiccation substrates. Initial Lm568 levels of 8 Log(CFU/cm(2)) decreased by 0.9 Log(CFU/cm(2)) in BHI and 1.1-2.9 Log(CFU/cm(2)) in TSB-glu, PPS and MM after 21 days. Comparatively, the initial survival of ΔsigB was reduced in PS and MM, while no differences were observed among the three strains in BHI and TSB-glu. Pre-culture in osmolyte containing MM enhanced (p < 0.05) desiccation survival of all strains. Desiccation in osmolyte-containing MM improved desiccation survival of all strains, albeit the protection was less than that observed after pre-culture with the osmolytes. Complementation of the ΔsigB mutant restored the wildtype phenotype. In conclusion, this work shows the protective effect of osmolytes in desiccation survival of L. monocytogenes, while the σ(B) regulon only improved the initial survival in nutrient and osmolyte poor environments.

Fecal Contamination in the Surface Waters of a Rural- and an Urban-Source Watershed.

Surface waters are commonly used as source water for drinking water and irrigation. Knowledge of sources of fecal pollution in source watersheds benefits the design of effective source water protection plans. This study analyzed the relationships between enteric pathogens ( O157:H7, spp., and spp. [, and ]), water quality (turbidity, temperature, and ), and human and ruminant-cow and mitochondrial DNA (mtDNA)-based fecal source tracking (FST) markers in two source watersheds. Water samples ( = 329) were collected at 10 sites (five in each watershed) over 18 mo. The human marker (HF183) occurred in 9 to 10% of the water samples at nine sampling sites; while a forested site in the urban watershed tested negative. Ruminant-cow markers (BacR and CowM2) only appeared in the rural watershed (6%). The mtDNA markers (HcytB and AcytB) showed the same pattern but were less sensitive due to lower fecal concentrations. Higher prevalences ( < 0.05) of spp. (41 vs. 16% for the rural and urban watershed, respectively) and O157:H7 (12 vs. 3%) were observed in the rural watershed, while spp. levels were comparable (23-28%). Densities of ≥100 colony-forming units (CFU) 100 mL increased the odds ( < 0.05) of detecting the enteric bacterial pathogens. The water turbidity levels (nephelometric turbidity units [NTU] ≥ 1.0) similarly predicted ( < 0.05) pathogen presence. Storm events increased ( < 0.01) pathogen and fecal marker concentrations in the waterways. The employment of multiple FST methods suggested failing onsite wastewater systems contribute to human fecal pollution in both watersheds.

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.

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).

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.

Imaging dynamics of CD11c⁺ cells and Foxp3⁺ cells in progressive autoimmune insulitis in the NOD mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: The aim of this study was to visualise the dynamics and interactions of the cells involved in autoimmune-driven inflammation in type 1 diabetes. METHODS: We adopted the anterior chamber of the eye (ACE) transplantation model to perform non-invasive imaging of leucocytes infiltrating the endocrine pancreas during initiation and progression of insulitis in the NOD mouse. Individual, ACE-transplanted islets of Langerhans were longitudinally and repetitively imaged by stereomicroscopy and two-photon microscopy to follow fluorescently labelled leucocyte subsets. RESULTS: We demonstrate that, in spite of the immune privileged status of the eye, the ACE-transplanted islets develop infiltration and beta cell destruction, recapitulating the autoimmune insulitis of the pancreas, and exemplify this by analysing reporter cell populations expressing green fluorescent protein under the Cd11c or Foxp3 promoters. We also provide evidence that differences in morphological appearance of subpopulations of infiltrating leucocytes can be correlated to their distinct dynamic behaviour. CONCLUSIONS/INTERPRETATION: Together, these findings demonstrate that the kinetics and dynamics of these key cellular components of autoimmune diabetes can be elucidated using this imaging platform for single cell resolution, non-invasive and repetitive monitoring of the individual islets of Langerhans during the natural development of autoimmune diabetes.