Dataset Information

Methodology for Whole-Genome Sequencing of Methicillin-Resistant Staphylococcus aureus Isolates in a Routine Hospital Microbiology Laboratory.

ABSTRACT: There is growing evidence for the value of bacterial whole-genome sequencing in hospital outbreak investigations. Our aim was to develop methods that support efficient and accurate low-throughput clinical sequencing of methicillin-resistant Staphylococcus aureus (MRSA) isolates. Using a test panel of 25 MRSA isolates previously associated with outbreak investigations, we devised modifications to library preparation that reduced the processing time by 1?hour. We determined the maximum number of isolates that could be sequenced per run using an Illumina MiniSeq platform and a 13-hour (overnight) run time, which equated to 21 MRSA isolates and 3 controls (no template, positive, and negative). Repeatability and reproducibility assays based on this sequencing methodology demonstrated 100% accuracy in assigning species and sequence type (ST) and in detecting mecA Established genetic relatedness between isolates was recapitulated. Quality control (QC) metrics were evaluated over nine sequencing runs. Of the test panel MRSA genomes, 168/173 (97%) passed QC metrics based on the correct species assigned, detection of mecA and ST, and depth/coverage metrics. An evaluation of contamination in these 9 runs showed that positive and negative controls and test MRSA sequence files contained <0.14% and <0.48% of fragments that matched another species, respectively. Deliberate contamination experiments confirmed that this was insufficient to impact data interpretation. These methods support reliable and reproducible clinical MRSA sequencing with a turnaround time (from DNA extraction to availability of data files) of 24?hours.

SUBMITTER: Raven KE

PROVIDER: S-EPMC6535593 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Publications

Methodology for Whole-Genome Sequencing of Methicillin-Resistant <i>Staphylococcus aureus</i> Isolates in a Routine Hospital Microbiology Laboratory.

Raven Kathy E KE Blane Beth B Leek Danielle D Churcher Carol C Kokko-Gonzales Paula P Pugazhendhi Dhamayanthi D Fraser Louise L Betley Jason J Parkhill Julian J Peacock Sharon J SJ

Journal of clinical microbiology 20190524 6

There is growing evidence for the value of bacterial whole-genome sequencing in hospital outbreak investigations. Our aim was to develop methods that support efficient and accurate low-throughput clinical sequencing of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) isolates. Using a test panel of 25 MRSA isolates previously associated with outbreak investigations, we devised modifications to library preparation that reduced the processing time by 1 hour. We determined the maximum numb ...[more]

PMID: 30894439

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Project description:As genotyping of S. aureus is important for epidemiologic research and for hygiene management, methods are required for standardized fast and easily applicable evaluation of closely related epidemic strains with high prevalence in hospitals. In this single centre matched control study we compared a new commercially available DNA microarray (IdentiBAC) with standard spa-typing for S. aureus genotyping. Included in the study was a subgroup of 46 MRSA and matched 46 MSSA nasal isolates of the Saarland University Medical Center collected during a state-wide admission prevalence screening. Microarray (MA) and also spa-typing could easily differentiate the genetically diverse MSSA group. However, due to the predominance of CC5/t003 in the MRSA group a sufficient subtyping required analysis of more complex genetic profiles as was shown here by the MA comprising a total number of 334 different hybridization probes. The genetic repertoire of the MRSA group was characterized by more virulence genes as compared to the MSSA group. The standard evaluation of MA results by the original software into CCs, agr-, SCCmec- and capsule-types was substituted in the present study by implementation of multivariate subtyping of closely related CC5 isolates using three different bioinformatic methods (splits graph, cluster dendrogram, and principal component analysis). Each method used was applicable for standardized and highly discriminative subtyping with high concordance. We propose that the identified S. aureus subtypes with characteristic virulence gene profiles are presumably associated also with virulence and pathogenicity in vivo; however, this remains to be analyzed in future studies. MA was superior to spa-typing for epidemiologic and presumably also provide functional respectively virulence associated characterization of S. aureus isolates. This is of specific importance for the hospital setting. In future, MA could become a new standard test for S. aureus typing in combination with multivariate bioinformatic analysis.

Dataset Information

Methodology for Whole-Genome Sequencing of Methicillin-Resistant Staphylococcus aureus Isolates in a Routine Hospital Microbiology Laboratory.

Publications

Methodology for Whole-Genome Sequencing of Methicillin-Resistant <i>Staphylococcus aureus</i> Isolates in a Routine Hospital Microbiology Laboratory.

Similar Datasets

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