Center For Genomic Epidemiology Research Articles

WGS-based surveillance of third-generation cephalosporin-resistant Escherichia coli from bloodstream infections in Denmark.

To evaluate a genome-based surveillance of all Danish third-generation cephalosporin-resistant Escherichia coli (3GC-R Ec ) from bloodstream infections between 2014 and 2015, focusing on horizontally transferable resistance mechanisms. A collection of 552 3GC-R Ec isolates were whole-genome sequenced and characterized by using the batch uploader from the Center for Genomic Epidemiology (CGE) and automatically analysed using the CGE tools according to resistance profile, MLST, serotype and fimH subtype. Additionally, the phylogenetic relationship of the isolates was analysed by SNP analysis. The majority of the 552 isolates were ESBL producers (89%), with bla CTX-M-15 being the most prevalent (50%) gene, followed by bla CTX-M-14 (14%), bla CTX-M-27 (11%) and bla CTX-M-101 (5%). ST131 was detected in 50% of the E. coli isolates, with the remaining isolates belonging to 73 other STs, including globally disseminated STs (e.g. ST10, ST38, ST58, ST69 and ST410). Five of the bloodstream isolates were carbapenemase producers, carrying bla OXA-181 (3) and bla OXA-48 (2). Phylogenetic analysis revealed 15 possible national outbreaks during the 2 year period, one caused by a novel ST131/ bla CTX-M-101 clone, here observed for the first time in Denmark. Additionally, the analysis revealed three individual cases with possible persistence of closely related clones collected more than 13 months apart. Continuous WGS-based national surveillance of 3GC-R Ec , in combination with more detailed epidemiological information, can improve the ability to follow the population dynamics of 3GC-R Ec , thus allowing for the detection of potential outbreaks and the effects of changing treatment regimens in the future.

Comparative Phylogenomic Analysis of Escherichia coli ST131 Isolates and IncF-type Plasmids from North Carolina Community Hospitals and Other Regions of the United States

Background Escherichia coli sequence type 131 (ST131), associated with CTX-M-15-type extended-spectrum β-lactamases (ESBLs), predominates globally among multidrug-resistant (MDR) E. coli. Among MDR E. coli ST131 isolates from North Carolina and other US regions, we explored genetic associations of the H30Rx and H30R1 subclones with IncF-type plasmid variants.MethodsSequence data from 144 clinical E. coli isolates, including 39 MDR ST131 isolates from our 7 NC community hospitals and previous 104 ST131 isolates from other US regions, were analyzed and compared. Specific genes were sought using a bioinformatic pipeline at the Center for Genomic Epidemiology and BLAST. Evolutionary analyses were performed using MEGA7. Plasmid incompatibility groups and its STs were identified using PlasmidFinder and pMLST. Comparisons of the completed sequences of 2 ST131 reference plasmids with 144 ST131 draft genome assemblies were performed using CGView Comparison Tool, and circular comparison maps were generated. Among 31 blaCTX-M-containing contigs, blaCTX-M gene insertion sites were examined.ResultsIn the core genome phylogeny, the H30R1 and H30Rx clades were placed within a larger H30R clade (Figure 1). pMLST revealed association of IncF[F2:A1:B-] plasmids (mostly blaCTX-M-15-positive) with H30Rx, and IncF[F1:A2:B20] plasmids (all blaCTX-M-15-negative) with H30R1. The ST131-associated IncF[F2:A1:B-] plasmids from NC generally resembled those from other US regions (Figure 2). However, in contrast to the others, some of the NC plasmids lacked regions previously described as gisland 1 h and/or the gColla-region, h in addition to gisland 2 h (Figure 3). Among the 31 NC ST131 isolates producing CTX-M (26 H30Rx and 5 H30R1 isolates), blaCTX-M-15 was inserted at the same chromosomal site in 3 H30Rx isolates, at different chromosomal sites in 3 H30Rx isolates, and on IncF plasmids in 1 H30Rx and 1 H30R1 isolates, suggesting that the blaCTX-M-containing gene cassette has moved around among and/or within these strains.ConclusionOur genomic and plasmid analysis of community hospital-source ST131 isolates demonstrated fixation and ongoing evolution of specific plasmids within the separate but closely related H30Rx and H30R1 sublineages.Disclosures J. R. Johnson, Crucell/Janssen: Consultant, Consulting fee Merck: Grant Investigator, Grant recipient Tetraphase: Grant Investigator, Research grant Actavis/Allergan: Grant Investigator, Grant recipient

MetaPhinder-Identifying Bacteriophage Sequences in Metagenomic Data Sets.

Bacteriophages are the most abundant biological entity on the planet, but at the same time do not account for much of the genetic material isolated from most environments due to their small genome sizes. They also show great genetic diversity and mosaic genomes making it challenging to analyze and understand them. Here we present MetaPhinder, a method to identify assembled genomic fragments (i.e.contigs) of phage origin in metagenomic data sets. The method is based on a comparison to a database of whole genome bacteriophage sequences, integrating hits to multiple genomes to accomodate for the mosaic genome structure of many bacteriophages. The method is demonstrated to out-perform both BLAST methods based on single hits and methods based on k-mer comparisons. MetaPhinder is available as a web service at the Center for Genomic Epidemiology https://cge.cbs.dtu.dk/services/MetaPhinder/, while the source code can be downloaded from https://bitbucket.org/genomicepidemiology/metaphinder or https://github.com/vanessajurtz/MetaPhinder.

Implementation of Whole Genome Sequencing (WGS) for Identification and Characterization of Shiga Toxin-Producing Escherichia coli (STEC) in the United States.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen capable of causing severe disease in humans. Rapid and accurate identification and characterization techniques are essential during outbreak investigations. Current methods for characterization of STEC are expensive and time-consuming. With the advent of rapid and cheap whole genome sequencing (WGS) benchtop sequencers, the potential exists to replace traditional workflows with WGS. The aim of this study was to validate tools to do reference identification and characterization from WGS for STEC in a single workflow within an easy to use commercially available software platform. Publically available serotype, virulence, and antimicrobial resistance databases were downloaded from the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org) and integrated into a genotyping plug-in with in silico PCR tools to confirm some of the virulence genes detected from WGS data. Additionally, down sampling experiments on the WGS sequence data were performed to determine a threshold for sequence coverage needed to accurately predict serotype and virulence genes using the established workflow. The serotype database was tested on a total of 228 genomes and correctly predicted from WGS for 96.1% of O serogroups and 96.5% of H serogroups identified by conventional testing techniques. A total of 59 genomes were evaluated to determine the threshold of coverage to detect the different WGS targets, 40 were evaluated for serotype and virulence gene detection and 19 for the stx gene subtypes. For serotype, 95% of the O and 100% of the H serogroups were detected at > 40x and ≥ 30x coverage, respectively. For virulence targets and stx gene subtypes, nearly all genes were detected at > 40x, though some targets were 100% detectable from genomes with coverage ≥20x. The resistance detection tool was 97% concordant with phenotypic testing results. With isolates sequenced to > 40x coverage, the different databases accurately predicted serotype, virulence, and resistance from WGS data, providing a fast and cheaper alternative to conventional typing techniques.