Abstract
Whole-genome sequencing (WGS) is becoming the de facto standard for bacterial typing and outbreak surveillance of resistant bacterial pathogens. However, interoperability for WGS of bacterial outbreaks is poorly understood. We hypothesized that harmonization of WGS for outbreak surveillance is achievable through the use of identical protocols for both data generation and data analysis. A set of 30 bacterial isolates, comprising of various species belonging to the Enterobacteriaceae family and Enterococcus genera, were selected and sequenced using the same protocol on the Illumina MiSeq platform in each individual centre. All generated sequencing data were analysed by one centre using BioNumerics (6.7.3) for (i) genotyping origin of replications and antimicrobial resistance genes, (ii) core-genome multi-locus sequence typing (cgMLST) for Escherichia coli and Klebsiella pneumoniae and whole-genome multi-locus sequencing typing (wgMLST) for all species. Additionally, a split k-mer analysis was performed to determine the number of SNPs between samples. A precision of 99.0% and an accuracy of 99.2% was achieved for genotyping. Based on cgMLST, a discrepant allele was called only in 2/27 and 3/15 comparisons between two genomes, for E. coli and K. pneumoniae, respectively. Based on wgMLST, the number of discrepant alleles ranged from 0 to 7 (average 1.6). For SNPs, this ranged from 0 to 11 SNPs (average 3.4). Furthermore, we demonstrate that using different de novo assemblers to analyse the same dataset introduces up to 150 SNPs, which surpasses most thresholds for bacterial outbreaks. This shows the importance of harmonization of data-processing surveillance of bacterial outbreaks. In summary, multi-centre WGS for bacterial surveillance is achievable, but only if protocols are harmonized.
Highlights
The dissemination of antimicrobial resistance (AMR) has grown to an issue of worldwide proportions
As the implementation of Whole-g enome sequencing (WGS) for routine outbreak surveillance is dependent on standardized methodology, we evaluated the technical variation in phylogenetic comparison using a commercially available whole-genome multi-locus sequencing typing (wgMLST) tool in BioNumerics and an open-s ource reference-f ree SNP-b ased tool called SKA [13]
In total 30 resistant bacterial isolates were selected based on their extended-spectrum beta-lactamase (ESBL) or carbapenemase activity, or based on ciprofloxacin or vancomycin resistance phenotype
Summary
The dissemination of antimicrobial resistance (AMR) has grown to an issue of worldwide proportions. Routine surveillance by molecular typing can aid in the fight against AMR, as outlined by the global action plan of the World Health Organization [1]. Reports show that the number of infections by resistant micro-organisms have been on the rise in recent years. Infections by multi-drug-r esistant (MDR) bacteria are associated with an increase in economic burden [2] and negative patient outcomes such as morbidity and mortality [3, 4].
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