Whole-Genome Sequencing for High-Resolution Investigation of Methicillin-Resistant Staphylococcus aureus Epidemiology and Genome Plasticity

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) infections pose a major challenge in health care, yet the limited heterogeneity within this group hinders molecular investigations of related outbreaks. Pulsed-field gel electrophoresis (PFGE) has been the gold standard approach but is impractical for many clinical laboratories and is often replaced with PCR-based methods. Regardless, both approaches can prove problematic for identifying subclonal outbreaks. Here, we explore the use of whole-genome sequencing for clinical laboratory investigations of MRSA molecular epidemiology. We examine the relationships of 44 MRSA isolates collected over a period of 3 years by using whole-genome sequencing and two PCR-based methods, multilocus variablenumber tandem-repeat analysis (MLVA) and spa typing. We find that MLVA offers higher resolution than spa typing, as it resolved 17 versus 12 discrete isolate groups, respectively. In contrast, whole-genome sequencing reproducibly cataloged genomic variants (131,424 different single nucleotide polymorphisms and indels across the strain collection) that uniquely identified each MRSA clone, recapitulating those groups but enabling higher-resolution phylogenetic inferences of the epidemiological relationships. Importantly, whole-genome sequencing detected a significant number of variants, thereby distinguishing between groups that were considered identical by both spa typing (minimum, 1,124 polymorphisms) and MLVA (minimum, 193 polymorphisms); this suggests that these more conventional approaches can lead to false-positive identification of outbreaks due to inappropriate grouping of genetically distinct strains. An analysis of the distribution of variants across the MRSA genome reveals 47 mutational hot spots (comprising ϳ2.5% of the genome) that account for 23.5% of the observed polymorphisms, and the use of this selected data set successfully recapitulates most epidemiological relationships in this pathogen group.

Transmission of MDR MRSA between primates, their environment and personnel at a United States primate centre

“…20 We have continued to isolate MRSA over the last year from the primates and have only identified ST188 and ST3268 in the colony. All the primate MRSA isolates were MDR carrying both acquired genes and resistance-associated mutations ( Table 1).…”

Large-scale genomic sequencing of extraintestinal pathogenic Escherichia coli strains

“…Although the trio of related isolates was obtained within several weeks of one another, collection of the paired isolates representing a true genomic clone occurred more than a year apart and, in the absence of a clear epidemiological link, perhaps indicating an environmental reservoir or a dominant strain within the larger community (Manges et al 2008). Indeed, rates of nosocomial transmission of lineages as assayed by conventional, lower-resolution typing technologies (Hilty et al 2012) may have led to an overestimation of the frequency of such events, as has been observed for other bacteria (Miller et al 2014;SenGupta et al 2014). However, given our finding of multiple polyclonal E. coli infections, it should be noted that our conclusions may be influenced by incomplete clinical sampling of the multiple strains present in some infections (Lindsay 2014), reflecting a limitation of current clinical microbiological procedures.…”

Application of Whole-Genome Sequencing for Bacterial Strain Typing in Molecular Epidemiology

“…WGS and analysis can be completed rapidly and are likely to become more rapid (57), potentially enabling real-time epidemiological investigations: the methods implemented here are compatible with a turnaround time of 3 working days, and the computational analysis methods implemented here are scalable to encompass even large numbers of specimens. Although present barriers to the universal adoption of WGS by clinical laboratories include relatively high costs of instrumentation and a lack of bioinformatic expertise (52,58), these beneficial capabilities of WGS will potentially enable epidemiological investigation of bacterial outbreaks in real time and with an unprecedented ability to define strain relationships, significantly impacting the practice of infection control and patient outcomes and justifying the additional efforts required for its implementation.…”

“…The digital nature of sequencing data allows sharing of sequence information among laboratories, potentially enabling a system capable of identifying large-scale outbreaks analogous to existing PFGE databases such as PulseNet (6). Genomic-sequencing libraries are prepared with protocols similar to current PCR-based strain typing protocols (52), requiring less technical skill than PFGE. WGS can enable exploration of isolates' virulence genes, antibiotic resistance mechanisms, and other medically relevant factors (56), concordantly with molecular epidemiology investigation.…”