c Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-producing Escherichia coli (VTEC). In Denmark, the Statens Serum Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week period in the fall of 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, multilocus sequence type (MLST) typing, and determination of phylogenetic relationship, and a specific VirulenceFinder for detection of E. coli virulence genes was developed as part of this study. In total, 46 suspected VTEC isolates were characterized in parallel during the study. VirulenceFinder proved successful in detecting virulence genes included in routine typing, explicitly verocytotoxin 1 (vtx1), verocytotoxin 2 (vtx2), and intimin (eae), and also detected additional virulence genes. VirulenceFinder is also a robust method for assigning verocytotoxin (vtx) subtypes. A real-time clustering of isolates in agreement with the epidemiology was established from WGS, enabling discrimination between sporadic and outbreak isolates. Overall, WGS typing produced results faster and at a lower cost than the current routine. Therefore, WGS typing is a superior alternative to conventional typing strategies. This approach may also be applied to typing and surveillance of other pathogens. Bacterial pathogens still pose a major threat to public health, and in order to limit their spread and prevent infectious disease outbreaks, accurate and rapid diagnostics and classification of isolates are of great importance. In current routine practice, isolation and identification are mostly performed at clinical microbiological laboratories, and verification and further characterization are performed for a few selected pathogens at national, or regional, reference laboratories, using a variety of species-specific methods. Typing and surveillance of bacterial pathogens rely mainly on well-established, standardized phenotypic and molecular typing methods, such as serotyping and pulsed-field gel electrophoresis (PFGE) (1, 2). However, to obtain sufficient discrimination between isolates, it is typically necessary to combine typing results from several different typing techniques, both phenotypic and genotypic. As a result, it is laborious, time-consuming, and expensive to perform proper typing for surveillance and outbreak detection.As the cost of whole-genome sequencing (WGS) has decreased and benchtop sequencing machines enable fast turnaround, it has become increasingly attractive...

c Accurate and rapid typing of pathogens is essential for effective surveillance and outbreak detection. Conventional serotyping of Escherichia coli is a delicate, laborious, time-consuming, and expensive procedure. With whole-genome sequencing (WGS) becoming cheaper, it has vast potential in routine typing and surveillance. The aim of this study was to establish a valid and publicly available tool for WGS-based in silico serotyping of E. coli applicable for routine typing and surveillance. A FASTA database of specific O-antigen processing system genes for O typing and flagellin genes for H typing was created as a component of the publicly available Web tools hosted by the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org). All E. coli isolates available with WGS data and conventional serotype information were subjected to WGS-based serotyping employing this specific SerotypeFinder CGE tool. SerotypeFinder was evaluated on 682 E. coli genomes, 108 of which were sequenced for this study, where both the whole genome and the serotype were available. In total, 601 and 509 isolates were included for O and H typing, respectively. The O-antigen genes wzx, wzy, wzm, and wzt and the flagellin genes fliC, flkA, fllA, flmA, and flnA were detected in 569 and 508 genome sequences, respectively. SerotypeFinder for WGS-based O and H typing predicted 560 of 569 O types and 504 of 508 H types, consistent with conventional serotyping. In combination with other available WGS typing tools, E. coli serotyping can be performed solely from WGS data, providing faster and cheaper typing than current routine procedures and making WGS typing a superior alternative to conventional typing strategies. Escherichia coli is usually a harmless commensal, but some strains have evolved the capability to cause disease in humans and/or animals by specific particular pathogenic mechanisms. In some cases, infection can be fatal (1).Serotyping is a method for classification of E. coli that has existed since the 1940s and has since been developed into standardized procedures (2-4). Performance of serotyping requires a high level of expertise and access to cross-absorbed antisera. It is a time-consuming and laborious procedure. O:K:H serotyping is based on a combination of the three immunogenic structures: the lipopolysaccharide (LPS) (O antigen), the capsular antigen (K), and the flagellar (H) antigen.Since few laboratories are able to perform K typing, O:H serotyping has become the gold standard for characterization of pathogenic E. coli. O:H serotyping is crucial in the detection of outbreaks, for epidemiological surveillance, for taxonomic differentiation of E. coli, for detecting pathogenic serotypes within the species, and for clonal and evolutionary studies. In contrast to several more recently developed molecular typing methods, such as pulsed-field gel electrophoresis (PFGE), ribotyping and to some extent multilocus sequence typing (MLST), serotyping provides information that is directly associated with the antigenic response an...

BackgroundAntibiotic resistance is a major health problem, as drugs that were once highly effective no longer cure bacterial infections. WGS has previously been shown to be an alternative method for detecting horizontally acquired antimicrobial resistance genes. However, suitable bioinformatics methods that can provide easily interpretable, accurate and fast results for antimicrobial resistance associated with chromosomal point mutations are still lacking.MethodsPhenotypic antimicrobial susceptibility tests were performed on 150 isolates covering three different bacterial species: Salmonella enterica, Escherichia coli and Campylobacter jejuni. The web-server ResFinder-2.1 was used to identify acquired antimicrobial resistance genes and two methods, the novel PointFinder (using BLAST) and an in-house method (mapping of raw WGS reads), were used to identify chromosomal point mutations. Results were compared with phenotypic antimicrobial susceptibility testing results.ResultsA total of 685 different phenotypic tests associated with chromosomal resistance to quinolones, polymyxin, rifampicin, macrolides and tetracyclines resulted in 98.4% concordance. Eleven cases of disagreement between tested and predicted susceptibility were observed: two C. jejuni isolates with phenotypic fluoroquinolone resistance and two with phenotypic erythromycin resistance and five colistin-susceptible E. coli isolates with a detected pmrB V161G mutation when assembled with Velvet, but not when using SPAdes or when mapping the reads.ConclusionsPointFinder proved, with high concordance between phenotypic and predicted antimicrobial susceptibility, to be a user-friendly web tool for detection of chromosomal point mutations associated with antimicrobial resistance.