Comparison of Whole-Genome Sequencing Methods for Analysis of Three Methicillin-Resistant Staphylococcus aureus Outbreaks

Abstract

Whole-genome sequencing (WGS) can provide excellent resolution in global and local epidemiological investigations of Staphylococcus aureus outbreaks. A variety of sequencing approaches and analytical tools have been used; it is not clear which is ideal. We compared two WGS strategies and two analytical approaches to the standard method of SmaI restriction digestion pulsed-field gel electrophoresis (PFGE) for typing S. aureus. Forty-two S. aureus isolates from three outbreaks and 12 reference isolates were studied. Near-complete genomes, assembled de novo with paired-end and long-mate-pair (8 kb) libraries were first assembled and analyzed utilizing an in-house assembly and analytical informatics pipeline. In addition, pairedend data were assembled and analyzed using a commercial software package. Single nucleotide variant (SNP) analysis was performed using the in-house pipeline. Two assembly strategies were used to generate core genome multilocus sequence typing (cgMLST) data. First, the near-complete genome data generated with the inhouse pipeline were imported into the commercial software and used to perform cgMLST analysis. Second, the commercial software was used to assemble paired-end data, and resolved assemblies were used to perform cgMLST. Similar isolate clustering was observed using SNP calling and cgMLST, regardless of data assembly strategy. All methods provided more discrimination between outbreaks than did PFGE. Overall, all of the evaluated WGS strategies yielded statistically similar results for S. aureus typing.KEYWORDS MRSA, PFGE, Staphylococcus aureus, molecular typing, whole-genome sequencing M ethicillin-resistant Staphylococcus aureus (MRSA) infections are associated with high morbidity and mortality. MRSA transmission poses a challenge to hospital infection prevention and control practitioners and public health professionals. The Centers for Disease Control and Prevention's Active Bacterial Surveillance Report estimated that there were 72,444 cases of invasive MRSA infection in 2014, the majority of which were health care associated (HCA) (1). Proactive screening strategies (molecular and culture based) are emphasized in many institutions and are mandatory in some states. Despite these measures, HCA-MRSA outbreaks continue to occur. Thorough investigation of outbreaks is essential for confirming that an outbreak is occurring, understanding transmission patterns and reservoirs, and intervening to interrupt out-

Whole genome sequencing options for bacterial strain typing and epidemiologic analysis based on single nucleotide polymorphism versus gene-by-gene–based approaches

“…cgMLST) comparisons. While both typing approaches may in some cases provide comparable epidemiologic discrimination [66], they vary in their overall suitability for different epidemiologic or infection control settings. At present, strain typing for public health is often performed by cgMLST, with its capability for readily scaling analyses from small to large isolate numbers, national and international data sharing, and standardized strain nomenclature [55].…”

Contribution of whole-genome sequencing to understanding of the epidemiology and control of meticillin-resistant Staphylococcus aureus

“…Two of the most widely used NGS platforms, also known as second generation sequencers or short read sequencers, include the Illumina sequencing systems (Illumina, Eindhoven, The Netherlands) and the ion semiconductor systems (Thermo Fisher Scientific) [12,13,16]. In recent years, most studies reporting NGS of bacterial pathogens have used Illumina short read sequencers.…”

“…Some of the more frequently used bioinformatics approaches and software for microbial genome assembly and subsequent analysis have been recently reviewed [20,21,12]. Many software packages used for the analysis of WGS data are freely available and several commercially available and easy to use software packages including BioNumerics (Applied Maths, Ghent, Belgium) and SeqSphere (Ridom GmBH, Münster, Germany) are widely used for this purpose [12,14,16]…”

“…Studies have shown the value of WGS in teasing out some of the subtleties of general and hospital outbreak evolution and development [11,16,23,[44][45][46][47][48][49][50]. The isolates from three separate outbreaks were studied, including one in a hospital, resulting in a total of 42 isolates; 15 of 16 isolates from a burns unit formed a single cluster but 12 isolates from a post-surgical unit were more diverse; the authors concluded that those with less than eight SNPs should be considered related, and those between nine to 29, as being possibly unrelated [16].…”

Microbiome at the Frontier of Personalized Medicine

“…18,19,20 These methods have been tested across various species and range from SNP-based tests to use of whole genome comparisons. 21,22 This has also given rise to a large number of publically-available phylogenetic reconstruction algorithms that analyze genomic data for mutational signatures of relatedness in order to track relationships between different pathogens. 23,24,25 These algorithms use the evolutionary principle of descent with modification to assess which strains descended from a very recent common ancestor.…”