Harmonisation of in-silico next-generation sequencing based methods for diagnostics and surveillance.

Improvements in cost and speed of next generation sequencing (NGS) have provided a new pathway for delivering disease diagnosis, molecular typing, and detection of antimicrobial resistance (AMR). Numerous published methods and protocols exist, but a lack of harmonisation has hampered meaningful comparisons between results produced by different methods/protocols vital for global genomic diagnostics and surveillance. As an exemplar, this study evaluated the sensitivity and specificity of five well-established in-silico AMR detection software where the genotype results produced from running a panel of 436 Escherichia coli were compared to their AMR phenotypes, with the latter used as gold-standard. The pipelines exploited previously known genotype-phenotype associations. No significant differences in software performance were observed. As a consequence, efforts to harmonise AMR predictions from sequence data should focus on: (1) establishing universal minimum to assess performance thresholds (e.g. a control isolate panel, minimum sensitivity/specificity thresholds); (2) standardising AMR gene identifiers in reference databases and gene nomenclature; (3) producing consistent genotype/phenotype correlations. The study also revealed limitations of in-silico technology on detecting resistance to certain antimicrobials due to lack of specific fine-tuning options in bioinformatics tool or a lack of representation of resistance mechanisms in reference databases. Lastly, we noted user friendliness of tools was also an important consideration. Therefore, our recommendations are timely for widespread standardisation of bioinformatics for genomic diagnostics and surveillance globally.

F74 plasmids are major vectors of virulence genes in bovine NTEC2.

Necrotoxigenic Escherichia coli 2 (NTEC2) are defined as E. coli producing the toxin known as cytotoxic necrotizing factor 2 (CNF2), a potent toxin primarily found in bovine but also in humans. NTEC2 are mostly associated with bovine, and cnf2 is known to be carried by pVir-like plasmids. In this study, we looked for NTEC2 in a collection of E. coli collected between 2011 and 2018 in French bovine. Thirty-two isolates, collected from both sick (n = 19) and healthy (n = 13) animals, were identified and characterized using whole-genome sequencing. One F74 plasmid of this bacterial collection was long-read sequenced: its size was 138 121 bp and it carried the cnf2, F17cA-eG, cdtB, iutA, iucC and ompP virulence factors (VFs), but no resistance gene. A large variety of genetic backgrounds was observed, but all cnf2-carrying plasmids belonged to the IncF family, and most of them (78·1%) were of the F74 group. Similar F74 plasmids were also reported from bovine in the United Kingdom and the United States, as identified in the publically available databases. Consequently, these F74 plasmids, which are widely disseminated among E. coli from cattle in the French territory, are vectors of virulence determinants that largely went unnoticed until now.

OASIS evaluation of the French surveillance network for antimicrobial resistance in diseased animals (RESAPATH): success factors underpinning a well-performing voluntary system.

Antimicrobial resistance is a One Health issue requiring the development of surveillance systems in the human, environmental and animal sectors. In the European Economic Area, the surveillance of antimicrobial resistance in zoonotic pathogens and indicator bacteria in healthy food-producing animals is required legally, while countries are also expected to extend their surveillance to diseased animals in the frame of national action plans. In this context, evaluating existing antimicrobial resistance surveillance systems in animal health is important to improve systems in place, but also to help other countries learn from these experiences, understand success factors and anticipate challenges. With this aim, the French surveillance network for antimicrobial resistance in bacteria from diseased animals (RESAPATH) was evaluated using the Outil d'Analyse des Systèmes d'Information en Santé (OASIS) assessment tool. Key performance factors included (i) a strong and inclusive central institutional organisation defining clear and well-accepted surveillance objectives, scope and procedures, (ii) strong skills in epidemiology and microbiology and (iii) a win-win approach enabling the voluntary participation of 71 field laboratories and where free annual proficiency testing plays a pivotal role. The main area for improvement of RESAPATH was its time-consuming data management system.

Overall changes in the transcriptome of Escherichia coli O26:H11 induced by a subinhibitory concentration of ciprofloxacin.

AIMS: The goal was to explore the effects of subinhibitory concentration (SIC) (0·5 MIC = 20 µg l-1 ) of ciprofloxacin on the transcriptome of enterohaemorrhagic Escherichia coli O26:H11 isolate by 60 minutes of exposure. MATERIALS AND RESULTS: We used a combination of comparative genomic and transcriptomic (RNAseq) analyses. The whole genome of the E. coli O26:H11 #30934 strain of bovine origin was sequenced and assembled. This genome was next used as reference for the differential gene expression analysis. A whole-genome-based analysis of 36 publicly available E. coli O26:H11 genomes was performed to define the core and the accessory transcriptome of E. coli O26:H11. Using RNAseq and RT-qPCR analysis we observed overexpression of the SOS response and of T3SS effectors, together with the inhibition of specific motility-associated genes. Among the large set of transposases present, only three were activated, suggesting moderate transposition of genes with low doses of ciprofloxacin. Our results illustrated that transcriptional repressors, such as the CopG family protein, belonging to the core genome of E. coli O26:H11, are altered in response to fluoroquinolone exposure. The gene ontology enrichment analysis showed SIC of ciprofloxacin induced binding functions and catalytic activities, including mostly transferase and hydrolase proteins. The amino acid pathways involved in metabolic processes were significantly enhanced after the treatment. CONCLUSIONS: Although the core genome of E. coli O26:H11 constituted only 54·5% of the whole genome, we demonstrated that most differentially expressed genes were associated with the core genome of E. coli O26:H11, and that effects on the mobile genetic element, phage, and plasmid-related genes were rare. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time the effect of low dose of ciprofloxacin on the core transcriptome of E. coli O26:H11 was described. The effects on the main biological functions and protein classes including transcriptional regulators were illustrated.

Antimicrobial resistance in bacteria isolated from diseased horses in France.

BACKGROUND: Horses are one of the potential reservoirs of antimicrobial resistance (AMR) determinants that could be transferred to human subjects. OBJECTIVE: To describe the AMR patterns of major bacteria isolated from diseased horses in France. STUDY DESIGN: Retrospective observational study. METHODS: Data collected between 2012 and 2016 by RESAPATH, the French national surveillance network for AMR, were analysed. Only antimicrobials relevant in veterinary and human medicine for the isolated bacteria were considered. Mono- and multidrug resistance were calculated. The resistance proportions of major equine diseases were assessed and compared. Where data permitted, resistance trends were investigated using nonlinear analysis (generalised additive models). RESULTS: A total of 12,695 antibiograms were analysed. The five most frequently isolated bacteria were Streptococcus spp., Escherichia coli, Pseudomonas spp., Staphylococcus aureus, Pantoea spp. and Klebsiella spp. The highest proportions of resistance to gentamicin were found for S. aureus (22.1%) and Pseudomonas spp. (26.9%). Klebsiella spp. and E. coli had the highest proportions of resistance to trimethoprim-sulfamethoxazole (15.5 and 26.2%, respectively). Proportions of resistance to tetracycline were among the highest for all the bacteria considered. Resistance to third-generation cephalosporins was below 10% for all Enterobacteriaceae. The highest proportions of multidrug resistance (22.5%) were found among S. aureus isolates, which is worrying given their zoonotic potential. From 2012 to 2016, resistance proportions decreased in Pseudomonas spp. isolates, but remained the same for S. aureus. For Streptococcus spp. and E. coli, resistance proportions to trimethoprim-sulfamethoxazole increased. MAIN LIMITATIONS: Since antibiograms are not systematic analyses, any selection bias could impact the results. CONCLUSIONS: Such studies are essential to estimate the magnitude of the potential threat of AMR to public health, to design efficient control strategies and to measure their effectiveness. These findings may also guide the initial empirical treatment of horse diseases.

Antimicrobial resistance patterns of bacteria isolated from dogs with otitis.

Antimicrobial-resistant bacteria in dogs can be transmitted to humans and close contact between dogs and people might foster dissemination of resistance determinants. The aim of our study was to describe the antimicrobial resistance (AMR) pattern of the major causative agents of canine otitis - one of the most common diseases in dogs - isolated in France. Data collected between 2012 and 2016 by the French national surveillance network for AMR, referred to as RESAPATH, were analysed. Resistance trends were investigated using non-linear analysis (generalised additive models). A total of 7021 antibiograms were analysed. The four major causative agents of canine otitis in France were coagulase-positive staphylococci, Pseudomonas aeruginosa, Proteus mirabilis and streptococci. Since 2013, resistance to fluoroquinolones has been on the decrease in both P. aeruginosa and Staphylococcus pseudintermedius isolates. For P. aeruginosa, 19.4% of isolates were resistant to both enrofloxacin and gentamicin. The levels of multidrug resistance (acquired resistance to at least one antibiotic in three or more antibiotic classes) ranged between 11.9% for P. mirabilis and 16.0% for S. pseudintermedius. These results are essential to guide prudent use of antibiotics in veterinary medicine. They will also help in designing efficient control strategies and in measuring their effectiveness.

Antimicrobial resistance trends in Escherichia coli isolated from diseased food-producing animals in France: A 14-year period time-series study.

Antimicrobial resistance (AMR) among bacteria isolated from food-producing animals is a growing concern with implications for public health. AMR surveillance is essential to identify resistance trends and help in the design of effective and efficient control strategies. The aim of the study was to describe the antimicrobial susceptibility of pathogenic Escherichia coli isolated from three livestock productions in France (cattle, swine and poultry). The trend in resistance to the most commonly prescribed antibiotics in animal health was analysed as follows: amoxicillin (penicillin), spectinomycin or streptomycin (aminoglycoside), tetracycline and trimethoprim-sulfamethoxazole/Enrofloxacin and ceftiofur were also taken into account as members of critically important antimicrobial families in human and veterinary medicine, that is fluoroquinolones and third-generation cephalosporins, respectively. Data collected between 2002 and 2015 by the French national surveillance network of AMR referred to as RESAPATH were analysed. Resistance trends were investigated using non-linear analysis (generalized additive models) applied to time-series stratified by livestock production and antibiotic. Irrespective of the species and the antibiotic considered, resistance signals over time showed no significant annual cycle. Resistance to third-generation cephalosporins emerged during the period of the study, with a peak at 22% [20.5; 24.0] in poultry in 2010, decreasing afterwards, while it remained consistently below 10% for the other species. The proportion of resistance to fluoroquinolones was broadly similar between species and remained under 30%, with a slight decreasing trend after 2009. Resistances to tetracycline and amoxicillin remained high, between 90% and 40% over time in cattle and swine. After 2010, there was a decrease in resistance to these antibiotics for all species, especially to tetracycline for poultry with a drop from 84% in 2009 to 43% in 2015. These results contribute to risk assessment and constitute objective evidence on which to evaluate the efficacy of control measures implemented to limit AMR occurrence.

Extended-spectrum ß-lactamase/AmpC- and carbapenemase-producing Enterobacteriaceae in animals: a threat for humans?

There has been a great and long-term concern that extended-spectrum ß-lactamase (ESBL)/AmpC- and carbapenemase-producing Enterobacteriaceae occurring in animals may constitute a public-health issue. A large number of factors with complex interrelations contribute to the spread of those bacteria among animals and humans. ESBL/AmpC- or carbapenemase-encoding genes are most often located on mobile genetic elements favouring their dissemination. Some shared reservoirs of ESBL/AmpC or carbapenemase genes, plasmids or clones have been identified and suggest cross-transmissions. Even though exposure to animals is regarded as a risk factor, evidence for a direct transfer of ESBL/AmpC-producing bacteria from animals to humans through close contacts is limited. Nonetheless, the size of the commensal ESBL/AmpC reservoir in non-human sources is dramatically rising. This may constitute an indirect risk to public health by increasing the gene pool from which pathogenic bacteria can pick up ESBL/AmpC/carbapenemase genes. The extent to which food contributes to potential transmission of ESBL/AmpC producers to humans is also not well established. Overall, events leading to the occurrence of ESBL/AmpC- and carbapenemase-encoding genes in animals seem very much multifactorial. The impact of animal reservoirs on human health still remains debatable and unclear; nonetheless, there are some examples of direct links that have been identified.

A simplified method of performance indicators development for epidemiological surveillance networks--application to the RESAPATH surveillance network.

BACKGROUND: Develop and calculate performance indicators allows to continuously follow the operation of an epidemiological surveillance network. This is an internal evaluation method, implemented by the coordinators in collaboration with all the actors of the network. Its purpose is to detect weak points in order to optimize management. A method for the development of performance indicators of epidemiological surveillance networks was developed in 2004 and was applied to several networks. Its implementation requires a thorough description of the network environment and all its activities to define priority indicators. Since this method is considered to be complex, our objective consisted in developing a simplified approach and applying it to an epidemiological surveillance network. METHODS: We applied the initial method to a theoretical network model to obtain a list of generic indicators that can be adapted to any surveillance network. RESULTS: We obtained a list of 25 generic performance indicators, intended to be reformulated and described according to the specificities of each network. It was used to develop performance indicators for RESAPATH, an epidemiological surveillance network of antimicrobial resistance in pathogenic bacteria of animal origin in France. CONCLUSION: This application allowed us to validate the simplified method, its value in terms of practical implementation, and its level of user acceptance. Its ease of use and speed of application compared to the initial method argue in favor of its use on broader scale.

Development and testing of texture discriminators for the analysis of trabecular bone in proximal femur radiographs.

PURPOSE: Texture analysis of femur radiographs may serve as a potential low cost technique to predict osteoporotic fracture risk and has received considerable attention in the past years. A further application of this technique may be the measurement of the quality of specific bone compartments to provide useful information for treatment of bone fractures. Two challenges of texture analysis are the selection of the best suitable texture measure and reproducible placement of regions of interest (ROIs). The goal of this in vitro study was to automatically place ROIs in radiographs of proximal femur specimens and to calculate correlations between various different texture analysis methods and the femurs' anchorage strength. METHODS: Radiographs were obtained from 14 femoral specimens and bone mineral density (BMD) was measured in the femoral neck. Biomechanical testing was performed to assess the anchorage strength in terms of failure load, breakaway torque, and number of cycles. Images were segmented using a framework that is based on the usage of level sets and statistical in-shape models. Five ROIs were automatically placed in the head, upper and lower neck, trochanteric, and shaft compartment in an atlas subject. All other subjects were registered rigidly, affinely, and nonlinearly, and the resulting transformation was used to map the five ROIs onto the individual femora. RESULTS: In each ROI, texture features were extracted using gray level co-occurence matrices (GLCM), third-order GLCM, morphological gradients (MGs), Minkowski dimensions (MDs), Minkowski functionals (MFs), Gaussian Markov random fields, and scaling index method (SIM). Coefficients of determination for each texture feature with parameters of anchorage strength were computed. In a stepwise multiregression analysis, the most predictive parameters were identified in different models. Texture features were highly correlated with anchorage strength estimated by the failure load of up to R2=0.61 (MF and MG features, p<0.01) and were partially independent of BMD. The correlations were dependent on the choice of the ROI and the texture measure. The best predictive multiregression model for failure load R2adj=0.86 (p<0.001) included a set of recently developed texture methods (MF and SIM) but excluded bone mineral density and commonly used texture measures. CONCLUSIONS: The results suggest that texture information contained in trabecular bone structure visualized on radiographs may predict whether an implant anchorage can be used and may determine the local bone quality from preoperative radiographs.