A pilot study of rapid benchtop sequencing of Staphylococcus aureus and Clostridium difficile for outbreak detection and surveillance

Abstract

Objectives: To investigate the prospects of newly available benchtop sequencers to provide rapid whole-genome data in routine clinical practice. Next-generation sequencing has the potential to resolve uncertainties surrounding the route and timing of person-to-person transmission of healthcare-associated infection, which has been a major impediment to optimal management.

Design: The authors used Illumina MiSeq benchtop sequencing to undertake case studies investigating potential outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile.

Setting: Isolates were obtained from potential outbreaks associated with three UK hospitals.

Participants: Isolates were sequenced from a cluster of eight MRSA carriers and an associated bacteraemia case in an intensive care unit, another MRSA cluster of six cases and two clusters of C difficile. Additionally, all C difficile isolates from cases over 6 weeks in a single hospital were rapidly sequenced and compared with local strain sequences obtained in the preceding 3 years.

Main outcome measure: Whole-genome genetic relatedness of the isolates within each epidemiological cluster.

Results: Twenty-six MRSA and 15 C difficile isolates were successfully sequenced and analysed within 5 days of culture. Both MRSA clusters were identified as outbreaks, with most sequences in each cluster indistinguishable and all within three single nucleotide variants (SNVs). Epidemiologically unrelated isolates of the same spa-type were genetically distinct (≥21 SNVs). In both C difficile clusters, closely epidemiologically linked cases (in one case sharing the same strain type) were shown to be genetically distinct (≥144 SNVs). A reconstruction applying rapid sequencing in C difficile surveillance provided early outbreak detection and identified previously undetected probable community transmission.

Conclusions: This benchtop sequencing technology is widely generalisable to human bacterial pathogens. The findings provide several good examples of how rapid and precise sequencing could transform identification of transmission of healthcare-associated infection and therefore improve hospital infection control and patient outcomes in routine clinical practice.

Figure 1

Clusters and samples. All clusters of cases occurred in the Oxford University Hospitals NHS Trust between July and October 2011, apart from MRSA cluster 2 where samples were obtained by the Health Protection Agency from an outbreak in southern England between July and September 2011. CDI, Clostridium difficile infection: ≥3 unformed stools in 24 h, enzyme immunoassay positive, culture positive.

Figure 2

Staphylococcus aureus cluster genetic and epidemiological relationships. (A and B) MRSA cluster 1 and related MSSA isolates. (C and D) MRSA cluster 2. Panel A shows all isolates from MRSA cluster 1 (white circles) and all S aureus isolates from the following month (grey circles, all methicillin sensitive). The left panels show the genetic relationships between cases as maximum likelihood trees, labelled with the number of single nucleotide variants at which samples differ. Genetically indistinguishable samples are shown in the same circle. The right panels show time spent on the same ward as a horizontal line for each case in both clusters. In panel B, the dashed line indicates time on the same ward and the solid line time in the same bay. In panel D, the solid line indicates time spent on the same ward. The timing of the first positive sample for each case is indicated with a cross. Case H subsequently developed a bloodstream infection.

Figure 3

Clostridium difficile cluster genetic and epidemiological relationships. (A and B) C difficile cluster 1. (C and D) C difficile cluster 2. The left panels show the genetic relationships between cases as maximum likelihood trees, labelled with the number of single nucleotide variants at which samples differ. Genetically indistinguishable cases are shown in the same circle. The right-hand panels (B and D) show time spent on the same ward as a horizontal line for each case. The timing of the first positive sample for each case is indicated with a cross.

Figure 4

Potential for whole-genome sequencing to enhance Clostridium difficile surveillance. Genetic relationships between cases are shown as a maximum likelihood tree, labelled with the number of single nucleotide variants (SNVs) at which samples differ. Genetically indistinguishable cases are shown in the same circle. Cases from an existing database of sequenced isolates are shown in grey circles with the date of isolation. Four cases arising during a 6-week surveillance reconstruction are shown in white circles. Three additional cases sequenced as part of the reconstruction differed at >3000 SNVs from the cases shown and are not shown in the figure. The maximum likelihood tree shown is consistent with data; however, homoplasy was observed at one of the variant sites, 2464157, in the CD630 reference genome.