1. Shigella sonnei Antimicrobial Resistance, Phage Typing And Molecular Characterisation Niall DeLappe 1, D.Morris 2, S.Fanning 3, M.Daly 3, T.Chiesty 4, F.O’Holloran 3,G.Corbett-Feeney 1,2, M.Cormican 1,2. 1 Interim National Salmonella Reference Laboratory, UCHG. 2 National University of Ireland, Galway 3 Cork Institute of Technology. 4 LEP, PHLS, Colindale

2. Abstract

3. Dysentery  Amoebic and Bacillary  HistoricallyAdysenteriae15 serotypes Bboydii18 serotypes Cflexneri 6 serotypes & 2 variants Dsonnei 1 serotype (5 biotypes)  Evolved from different ancestral strains of E.coli.  Virulence plasmid, Pinv (140 MDa) directs the entry of bacterium into host epithelial cells.  produce various enterotoxins Introduction

4. Shigella sonnei  Evolved from E.coli O62  Acquired O antigen from Pleisiomonas shigelloides O17 by lateral transfer  Gene cluster for O antigen is located on Pinv.  IncidenceUSA199825,000 cases 199918,000 cases UK approx. 900 cases/year  Spread by person-to-person contact and contaminated food or water  Low infectious dose (10 cells)

5. Isolates analysed in this study  Galwayn = 42 (clusters of 4,2 &2 related)  Mayon = 9 (cluster of 3 related)  Roscommonn = 8 (cluster of 2 related)  Dublinn = 6 (unknown)  Corkn = 2 (unknown )

6. Materials and Methods  Pulsed Field Gel Electrophoresis- XbaI  20 h at 5-50s, 16 h at 5-20s  Phage typing (8 isolates)  Sensitivity testing  NCCLS Disk Diffusion  Plasmid profiling  alkaline lysis method of Kado & Liu  Conjugation-liquid mating  Integron analysis(14 isolates)  PCR

7. Number of Isolates Galway34 Mayo8 Roscommon4 Location of predominant strain

8. 19981999 Seasonal Variation of Predominant strain PFGE A, ASSu *(Ampicillin,Streptomycin,Sulphonamide) Results

9. B A A B 1 B 4 A A A A B 4 B 3 B 1 B B 5 B B B 5 B B B 4 Pulsed Field Gel Electrophoresis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Restriction patterns interpreted by criteria of Tenover et al 48.5 97.0 145.5 194.0 242.5 291.0 339.5 Kb

10. PFGE analysed by Bionumerics RDNC PT9 PT6 PT50 PT6 PT9 PT6

11. Phage Typing of S. sonnei isolates (performed by LEP, Colindale, London)  PT 9 PFGE A(n=2)  PT6 PFGE B (n=1) PFGE B 1 (n=1) PFGE B 4 (n=1) PFGE B 5 (n=1)  PT50 PFGE B 3 (n=1)  RDNC PFGE B (n=1) Phage type 6 accounts for approximately 80% of U.K. infections. PT9 is quite rare.

12. Conjugation of S.sonnei isolates 1 Isolated on Trptone Soya Agar, Oxoid 2 Isolated on Mueller Hinton Agar, BBL A = Ampicillin 100  g/ml agar Na = Nalidixic acid 30  g/ml agar T = Tetracycline 20  g/ml agar Tm = Trimethoprim 5  g/ml agar

13. Plasmid Profiles B 4 A A A A A A A 1 A 1 B A A B 1 B 4 pDu 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 8 6 4 2 3 5 Kb Most isolates had multiple plasmids ranging in size from 2kb to >50kb

14. Antibiotics tested: Amp10, C 30, S10, Su300, Tet30, W5, Na30, Nt300, K30, Cip5 Oxoid Antibiotic Resistance Patterns

15. Conclusions  Majority of isolates tested were from 1 PFGE group, A  Good concurrence between PFGE,phage typing and resistance typing  High rate of resistance to antibiotics with several being encoded by conjugative plasmids or integrons  It would prove interesting to analyse isolates from other countries