1. A framework for the future: Building molecular tools to understand the epidemiology of Clostridium difficile in Scotland

2. Talk Plan Highlight extent of problem in 2009 Progress made – first SIRN grant –Evaluation of Molecular tools Conclusions reached regarding application Progress new grant –Aims to link epidemiological data generated from whole genomic sequencing with patient healthcare informatics

3. Scale of problem Scottish C. difficile reference laboratory set up 2007

4. Typing methods In 2007, numerous typing methods –Toxinotyping, Pulse Field Electrophoresis – North Americans (NAP), Restriction Endonuclease activity (REA) –Ribotyping Based on amplification of 16s-23S ribosomal RNA intergenic regions 2. Image analysis 1. PCR amplification O’Neill GL et al Anaerobe 1996; 2; 205-209 3. Comparison with BioNumerics® database Ribotype 001 – represented first banding pattern observed, 002.....

5. Ribotyping is informative 002005 014 015 020023 078 20 30 10 Prevalence as a percentage of ten most frequently recovered ribotypes Pre/Post 2009 001 027106 Problems with ribotyping Lack of discriminatory power –Difficulties - band calling Size of band /not sequence –Exchangeability between labs –Basis of variation /epidemiology

6. Funding enabled Determine strength and weaknesses of several typing methods –Multi-locus sequence analysis (MLST) Amplification and sequencing of 7 amplicons –Multi-locus Variable Number Tandem Repeat Analysis (MLVA) Amplification and sizing of 7 unique regions encoding tandem repeat sequences –Whole Genome Single nucleotide polymorpism (SNP) analysis Sequencing and analysis of entire genome

7. MLST adkatpAdxrglyArecAsodAtpitype 00100001 00101002 Involves amplification of 7 amplicons –C.difficile adk, atpA, dxr, glyA, recA, sodA, tpi –Sequence and determine varients in each amplicon Determine population based on relative similarities/ differences in these alleles

8. Sequence based/epidemiologically accurate No significant advantage over ribotyping –Lacked sufficient discrimination for use in outbreaks 96 isolates typed using MLST

9. Multilocus Variable Number Tandem Repeat Analysis (MLVA) VNTR = Variable-Number Tandem Repeat Locus 1 Strain A: VNTR array 4x3 atgggtaatccgtcgACgCACgCACgCgccaatcgatacgat Strain B: VNTR array 4x5 atgggtaatccgtcgACgCACgCACgCACgCACgCgccaatcgatacgat Locus 2 Strain A: VNTR array 3x4 ggtaccggtaaagcgcACCACCACCACCttgacactgccggttg Strain B: VNTR array 3x6 ggtaccggtaaagcgcACCACCACCACCACCACCttgacactgccggttg

10. Data analysis – relative relatedness Using this approach – determine minimum total distance between strains

11. MLVA analysis performed on the top 10 ribotypes in Scotland (n = 748)

12. MLVA analysis of 027 isolates reveals strong geographical clustering over time

13. RibotypeMLVA Locus (Van den Berg et al, 2007) A6B7C6E7F3G8H9 0010.8930.7290.9100.1190.2300.5060.119 0020.8980.8270.9160.5870.4360.7110.240 0050.9210.8820.9300.7850.5180.7600.038 0140.8880.8160.8780.8160.5820.5510.255 0150.9220.9090.9360.0820.1600.8060.161 0200.9340.9200.9270.6560.2990.8650.156 023-0.7900.9070.8210.4570.8890.296 0270.9290.7730.8700.1730.4970.7960.024 078---0.5490.1280.7900.128 1060.8890.8360.9230.5130.0960.3670.083 Diversity of MLVA loci in the ten most common ribotypes in Scotland. Simpson’s Index of Diversity for the seven MLVA loci in the ten most common ribotypes in Scotland. High CDI values indicate accurate measurement of a highly variable locus.

14. Whole Genome Sequence Analysis Using SNP (single nucleotide polymorphism)

15. Scottish Isolates included in global analysis of 027 ribotypes He et al. Nature genetics 2013

16. GLA 020 GLA 022 GLA 017 GLA 021 GLA 004 GLA 010 GLA 008 GLA 013/019 GLA 014 GLA 002/009 GLA 012 GLA 018 GLA 006 GLA 007 GLA 003 GLA 005 GLA 015 GLA 001 GLA 016 Correlation between whole genome and MVLA analysis MLVA sufficiently discriminating to allow tracking within an outbreak situation

17. Conclusions PCR-ribotyping valuable typing tool –lacks sufficient discriminatory power to analyse outbreaks. MLVA as a supplemental subtyping method –Cost effective –is highly discriminatory/discern outbreaks. –Application limited for some ribotypes Whole Genome SNP analysis –validated and supported data generated by MLVA –Opportunity to use in limited analysis of 078 outbreaks Prediction of phenotypes (antibiotic resistance)

18. IMPORTANCE OF DATA LINKAGE

19. 2009 Antibiotic policy change Reduction in use of –Fluoroquinolones –Third generation cephalosporins –Clindamycin –Co-amoxyclav 20 30 10 001 027106 002005 014 015 020023 078

20. Modelling impact of policy change MoxifloxacinLevofloxacinErythromycin Policy Change0.250.500.31 Epidemic167.8925.3854.48 Non-epidemic0.380.50.49 Resistant vs sensitive Unclear what driving this enhanced resistance Possible treatment of a particular population of patients Identifiable if we could link with patient data

21. Project Objectives To develop a fully automated pipeline to allow rapid SNP analysis of genomic DNA from C. difficile. To use this pipeline to evaluate –Relationship between community- and hospital-associated C. difficile strains –Further differentiate epidemiology of ribotype 078 in Scotland To link patient health data and C. difficile WGS –To identify risk factors that could be reduced through modification of clinical practice Going forward

22. Where are we? Have identified 500 strains to be sequenced –100 078 strains –134 community associated strains isolated pre 2009 –270 matched community/hospital associated strains isolated post 2009 350/500 have been sequenced Automated SNP analysis pipeline generated Permissions have been sort and granted to allow patient linkage Plan in next 12 months to begin to link the data together

23. Long term aim: To develop a unique Scottish electronic resource that can be interrogated by researchers focussed on this infection

24. Acknowledgments SSSCDRL John Coia Derek Brown Health Protection Scotland Camilla Wuiff A-Lan Banks University of Glasgow Jan Lindstrom Cosmika Goswami Umer Ijaz Chris Quince University of Dundee Charis Marwick Peter Doonan Peter Davey Nichosa De Souza University of Strathclyde Marion Bennie The Wellcome Trust Sanger Institute Trevor Lawley Miao He Sequencing undertaken Glasgow Polyomics facility