Healthcare-associated outbreak of meticillin-resistant Staphylococcus aureus bacteraemia: Role of a cryptic variant of an epidemic clone

Background: New strains of meticillin-resistant Staphylococcus aureus (MRSA) may be associated with changes in rates of disease or clinical presentation. Conventional typing techniques may not detect new clonal variants that underlie changes in epidemiology or clinical phenotype. Aim: To investigate the role of clonal variants of MRSA in an outbreak of MRSA bacteraemia at a hospital in England. Methods: Bacteraemia isolates of the major UK lineages (EMRSA-15 and -16) from before and after the outbreak were analysed by whole-genome sequencing in the context of epidemiological and clinical data. For comparison, EMRSA-15 and -16 isolates from another hospital in England were sequenced. A clonal variant of EMRSA-16 was identified at the outbreak hospital and a molecular signature test designed to distinguish variant isolates among further EMRSA-16 strains. Findings: By whole-genome sequencing, EMRSA-16 isolates during the outbreak showed strikingly low genetic diversity (P < 1 × 10-6, Monte Carlo test), compared with EMRSA-15 and EMRSA-16 isolates from before the outbreak or the comparator hospital, demonstrating the emergence of a clonal variant. The variant was indistinguishable from the ancestral strain by conventional typing. This clonal variant accounted for 64/72 (89%) of EMRSA-16 bacteraemia isolates at the outbreak hospital from 2006. Conclusions: Evolutionary changes in epidemic MRSA strains not detected by conventional typing may be associated with changes in disease epidemiology. Rapid and affordable technologies for whole-genome sequencing are becoming available with the potential to identify and track the emergence of variants of highly clonal organisms. © 2013 The Authors

A Modified RNA-Seq Approach for Whole Genome Sequencing of RNA Viruses from Faecal and Blood Samples

<div><p>To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.</p></div

Coverage across the genome for two Hepatitis C samples sequenced directly from RNA.

<p>Coverage across the genome for two Hepatitis C samples sequenced directly from RNA.</p

Coverage profiles of one Norovirus sample from amplicon and direct RNA sequencing.

<p>A – Coverage across the genome for one Norovirus sample sequenced from PCR amplicons (others similar). Green and orange dotted lined mark the locations of the PCR primers used to generate the amplicons. B – coverage across the genome for the same Norovirus sample sequenced directly from RNA.</p

A comparison of workflows and consumable costs for various viral sequencing approaches.

<p>The ‘Custom’ protocol refers to the modified RNA-Seq method used in this study to create larger insert fragments. Alternative methods include Amplicon-seq and hybridisation capture (SureSelect Target Enrichment for Illumina Paired-End mRNA-Seq Library Prep; version 1.1). Failure rates are determined by failure to sequence at least one amplicon (<86% of the genome). The failure rate for SureSelect is not given as it was not performed in our study. Consumable costs are list price per sample and exclude sequencing.</p>*<p>failure rate based on that observed with Sanger Sequencing.</p>**<p>estimated cost for probes only. Extra cost incurred for Agilent library preparation kit plus additional reagents recommended by Agilent.</p>***<p>linear amplification during SPIA reverse transcription has not been accounted for.</p

Schematic representation of different strategies for viral genome resequencing.

<p>A) Total RNA library: all the RNA species present in the sample are sequenced, no assumption on which genome is present, B) Hybridisation capture of a mRNA library: a good reference genome is needed to design the probes for capture, C) PCR enrichment: the desired genome is amplified from cDNA, a reference genome is needed to design specific oligos. Red lines, genomes of interest; Blue segments, Illumina adapters; Black lines, other RNA species.</p