1. Presenter- Janet Nale (jyn1@le.ac.uk)
ISOLATION AND CHARACTERISATION OF TEMPERATE BACTRIOPHAGES OF THE HYPERVIRULENT Clostridium difficile 027 STRAIN
Presenter- Janet Nale
Dr Martha Clokie
Department of Infection, Immunity and Inflammation, University of Leicester, United Kingdom

2. Clostridium difficile
Janet2010
Cause of pseudomembranous colitis and nosocomial diarrhoea
Morbidity and mortality of CDI is still high in England
(6000 fatal cases in 2009)
C.difficile 027
Binary toxin gene
39 bp,18 bp and 1 bp mutation in tcdC (negative toxin regulator)
Existence of subtypes

3. Fawley et al., 2008

4. Examples of how temperate bacteriophage can moderate disease
Horizontal gene transfer
Lysogenic conversion
In C. difficile
Interact with the pathogenic locus
Evidence of regulation of toxin production (Goh et al., 2005)
May encode additional toxins which may influence the pathogenicity outbreak strain or sporulation (Zimmer et al., 2002)
toxin genes

5. My previous findings Phage carriage was surprisingly varied in
C. difficile 027 strains obtained from UHL
Nale et al., in prep

6. Aims
To isolate and characterise temperate bacteriophages of C. difficile 027 sub-clades
from a range of clinical strains isolated from UK hospitals.
To relate phage carriage to the disease severity shown in the sub-clades.
To determine the role of temperate bacterio-
phages in the pathogenicity of C. difficile 027.

7. 1 2
Methods 3 4

8. Results

9. Phage morphologies A B C D E F G H Siphovirus Myovirus 4 (4%) 3 (3%)
Defective myovirus
57 (63%)
PT-LPs 1 2 A B C D E F G H
PT-LPs (27%)
Twizzles
1(1%)
Bar ~ 70 nm

10. MLVA type
PFGE pulovar type
Number of samples
Morphology of phage isolated
Exceptions 1 I 3
Defective myovirus - 2 4 7 5 6 8 9 10 11 12 13
96L sipho and myo 14 15
91 myo 16 IV
PT- LPs
16L Myo 17 18
II, III
Siphovirus
51L Defective myo 19 20 21 22
1 no phage 23 V
PT-LPs

11. Phage genome sizes 49.04 kb 23.11 kb 9.2 kb 6.55 kb 82L 48LM 91LM 37L

12. Conclusion/Significance of study
Future work
Phage concentration/ Genome amplification
Sequencing phage for novel toxins
Analyse phage genomes using restriction endonuclease analysis
Effects of sporulation and biofilm formation on severity of CDI caused by these strains
Conclusion/Significance of study
Phage carriage is highly variable in C. difficile 027 strains but correlates to the sub-clades
May suggest the role temperate bacteriophages in the pathogenicity of C. difficile 027
Phage detection may be a useful marker to identify strains which are associated with increased severity.

13. Acknowledgements Dr Martha Clokie - Supervisor
Prof Mark Wilcox - Providing the bacterial isolates
Grace and John Styler – Financial support
SfAM –Student conference grant
Members of Lab 105 Department of Infection, Immunity and Inflammation University of Leicester
REFERENCES
Fawley, W. N., Freeman, J., Smith, C., Harmanus, C., van den Berg, R. J., Kuijper, E. J. & Wilcox, M. H. (2008). Use of Highly Discriminatory Fingerprinting to Analyze Clusters of Clostridium difficile Infection Cases Due to Epidemic Ribotype 027 Strains. J Clin Microbiol 46,
Goh, S., Chang, B. J. & Riley, T. V. (2005). Effect of phage infection on toxin production by Clostridium difficile. J Med Microbiol 54,
Zimmer, M., Scherer, S. & Loessner, M. J. (2002). Genomic analysis of Clostridium perfringens bacteriophage phi3626, which integrates into guaA and possibly affects sporulation. J Bacteriol 184,

14. Thanks for listening