Presenter- Janet Nale (jyn1@le.ac.uk) ISOLATION AND CHARACTERISATION OF TEMPERATE BACTRIOPHAGES OF THE HYPERVIRULENT Clostridium difficile 027 STRAIN.

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Presentation transcript:

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

Clostridium difficile http://www.hpa.org.uk/web/HPAwebFile/HPAweb_C/1267551242367 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

Fawley et al., 2008

Examples of how temperate bacteriophage can moderate disease Horizontal gene transfer Lysogenic conversion http://www.sharkchic.com.au/WildBillPhage.html 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

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

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 bacteriophages in the pathogenicity of C. difficile 027.

1 2 Methods 3 4

Results

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 25 (27%) Twizzles 1(1%) Bar ~ 70 nm

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

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

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.

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, 954-960. Goh, S., Chang, B. J. & Riley, T. V. (2005). Effect of phage infection on toxin production by Clostridium difficile. J Med Microbiol 54, 129-135. 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, 4359-4368.

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