Whole Genome Sequencing as a unified platform for outbreak identification, resistance prediction and virulence profiling in Staphylococcus aureus

Lead Research Organisation: University of Oxford
Department Name: Clinical Medicine

Abstract

Infections due to the bacteria Staphylococcus aureus continue to present a threat to health. At present, it takes several days and a number of different techniques to grow the bacteria from a specimen, confirm that it is S. aureus and test for susceptibility to antibiotics. If a particularly aggressive infection is suspected or it is thought that the patient is part of an outbreak, specialised tests are needed which are usually done at a reference laboratory. All of these steps take time and result in delays to the patient getting the right treatment.

Whole genome sequencing (WGS) is a new technology which, by decoding the entire genetic content of a bacterium, can give information about the type of organism, which antibiotics can be used to treat it and whether it is likely to cause severe disease. Having the whole genome can also tell us whether the bacteria from one person are related to the bacteria from someone else. This has been likened to the DNA detection techniques used to solve crime. WGS is becoming as fast and inexpensive as current traditional techniques, but is likely to be able to provide all the information in a single test. However, at present there is little data available to help us interpret whole genome information or use it in the direct care of patients.

The purpose of this study is to develop WGS to the point where it could be used as a single rapid test to help in the treatment and prevention of disease caused by S. aureus.

In the first part of the study, samples will be collected from definite outbreaks which have already been investigated and where an infection has been shown to have passed rapidly from person to person. I will compare the information from WGS to the results from routine testing and information about contact between patients. I will use this to develop a method based on the similarities or differences in the whole genomes to determine if bacteria have been transmitted from patient to patient.

In the second part of the study, I will compare the resistance genes identified in bacteria by WGS and whether the bacteria are resistant when tested in the routine laboratory. I also expect that by looking closely at the whole genomes of a number of bacteria I may discover genes that have not previously been fully investigated. These genes will be tested by "copying" them from the original bacteria and transferring them into harmless laboratory strains using small, transferable pieces of DNA called plasmids. The gene can also be removed from the original bacteria and replaced with a "normal" copy of the gene. The antibiotic resistance of the altered bacteria can then be compared with the original bacteria to prove that the genetic change has caused a change in antibiotic resistance. This is important in identifying mechanisms of resistance and possible targets to develop new antibiotics.

The final part of the study will look at genes known as "virulence factors". These are genes which may be associated with severe or invasive disease. Normally, specialised tests performed at a reference laboratory are needed to identify these but they can be specifically looked for in the whole genome sequence. All the bacteria collected will be screened for the most important known virulence factors. In addition, a number of possible virulence factors have been described but their clinical importance is not known. This study will collect a large number of S. aureus bacteria both from people with infections and those carrying it harmlessly. All the bacteria will be screened for these possible factors. It will then be possible to compare how frequently these possible factors occur in disease and carriage bacteria, identifying the most important genes for future study.

Technical Summary

The overall aim of the project is to develop whole genome sequencing (WGS) as a unified platform for outbreak identification, resistance prediction and virulence profiling of Staphylococcus aureus.

The first objective is to evaluate WGS as a real-time method for the detection of S. aureus outbreaks. Outbreaks with strong epidemiological links will be used to establish cut-off values for the number of single nucleotide variants (SNVs) which can reliably identify outbreak isolates. For each outbreak, isolates will be collected from cases and controls and whole genome sequenced using the Illumina HiSeq platform. Phylogenetic trees will be constructed based on WGS and used to infer relatedness.

These results will then be applied in real-time, using putative outbreaks and rapid sequencing (Illumina MiSeq platform) of case and control isolates in parallel with the routine investigation to compare the turnaround times.

The second objective is to use WGS in the prediction and investigation of resistance phenotypes. All isolates plus 500 bacteraemias will be tested for susceptibility to anti-staphylococcal agents. Whole genome sequences will be screened using BLAST for the genes associated with resistance to these agents and the frequency of critical (false susceptibility by genetic testing), major (false resistance by genetic testing) and minor errors will be determined.
The phenotypic effect of any novel variants which appear to confer resistance will be will be assessed by plasmid expression and allelic exchange in the susceptible S. aureus Newman strain.

The final objective is to investigate the prevalence of virulence factors. Isolates will be screened using a BLAST search for a panel of virulence factors and compared with the clinical presentation. Isolates will also be screened for putative virulence factors, to compare prevalence in disease vs carriage to identify factors for future study.

Planned Impact

Potential beneficiaries include

1) patients with Staphylococcal infection
-more rapid information regarding resistance and virulence will result in patients getting appropriate treatment more quickly.
(timescale 3-5 years)

2) other patients
-an improved understanding of how patient to patient transmission occurs will result in more effective interventions and more rapid containment of outbreaks, reducing the number of people adversely affected by an outbreak. (timescale 3-5 years)

3) decision making bodies
-at the regional level reference laboratories are likely to adopt this technology as an alternative to current typing methods.
(timescale 2-3 years)
-the results will also be of interest to policy makers in the Department of Health and Health Protection, to inform decisions regarding allocation of resources to public health.
(timescale 3-5 years)


4) clinicians
- more rapid information regarding resistance will benefit clinical management decisions, meaning that fewer ineffective drugs are used.
(timescale 3-5 years)
-rapid identification of transmission events will also enable more targetted use of isolation facilities and decolonisation regimens.
(timescale 3-5 years)

Publications

10 25 50

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Das S (2016) Natural mutations in a Staphylococcus aureus virulence regulator attenuate cytotoxicity but permit bacteremia and abscess formation. in Proceedings of the National Academy of Sciences of the United States of America

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Eyre DW (2017) WGS to predict antibiotic MICs for Neisseria gonorrhoeae. in The Journal of antimicrobial chemotherapy

 
Description AMR surveillance in low and middle income countries
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
URL http://amr.lshtm.ac.uk/2016/11/17/report-amr-surveillance-low-middle-income-countries/
 
Title S. aureus resistance database 
Description Database of antimicrobial susceptibility testing results for almost 1000 clinical S. aureus isolates linked to whole genome sequences. Sequences are uploaded to the European Nucleotide Archive Sequence Read Archive, and the phenotype database is held in Oxford and available to researchers as requested. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Data used to develop a rapid AST prediction app (https://www.ncbi.nlm.nih.gov/pubmed/26686880) and as a test set for GWAS studies (https://www.ncbi.nlm.nih.gov/pubmed/27572646) 
URL http://www.ebi.ac.uk/ena/data/view/PRJEB5261
 
Description Annual group workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Stimulated interest / discussion and potential collaborations

Discusion re potential use of the data in the routine setting
Year(s) Of Engagement Activity 2012,2013
URL http://modmedmicro.nsms.ox.ac.uk/events/?ytd_1=2013
 
Description Conference proceeding (ECCMID 2014) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation paper presentation
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Helpful discussions regarding impact of data, positive feedback from several audience members.

Entered discussions with potential collaborators
Year(s) Of Engagement Activity 2014
 
Description Conference proceeding (ISSSI 2014) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Lots of helpful feedback

.
Year(s) Of Engagement Activity 2014
 
Description Establishment of Public/Patient Involvement working group 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Participants in your research and patient groups
Results and Impact Talk and discussion stimulated establishment of patient/public directed working group.

Feedback from working group has raised research questions/interest to be considered in future paroject applications. Also some e-mail questions/ discussion regarding my work from interested parties.
Year(s) Of Engagement Activity 2014
URL http://modmedmicro.nsms.ox.ac.uk/wp-content/uploads/2014/04/CGordon-public-27Mar14-Small.pdf
 
Description Investigating S. aureus outbreaks using Whole Genome Sequencing, oral presentation, ECCMID 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral presentation at ECCMID 2015, session on clinical applications of sequencing technology
Year(s) Of Engagement Activity 2015
 
Description Invited speaker, BSAC Antimicrobial Resistance Mechanisms Workshop, Birmingham UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited speaker, BSAC annual ARM workshop.
Presentation on use of Whole Genome Sequencing for resistance prediction in S. aureus, followed by debate on benefits / pitfalls of using this approach versus standard phenotyping
Year(s) Of Engagement Activity 2014
URL http://bsac.org.uk/wp-content/uploads/2014/01/ARM-2014-FINAL-PROGRAMME.pdf
 
Description Visit / presentation to KEMRI-Wellcome Trust unit, Kilifi, Kenya 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Visit and lab talk to KEMRI-Wellcome Trust Research Programme, Kenya, to explore possibility of future work. Presented my work at research seminar (attendance approx 100) and discussed with relevant staff members re possibility of future collaborations.
Year(s) Of Engagement Activity 2016