Shigella sonneiShigella sonnei Antimicrobial Resistance, Phage Typing And Antimicrobial Resistance, Phage Typing And

Molecular CharacterisationMolecular Characterisation

Niall DeLappeNiall DeLappe11, D.Morris, D.Morris22, S.Fanning, S.Fanning33, M.Daly, M.Daly33, T.Chiesty, T.Chiesty44,,

F.O’HolloranF.O’Holloran3 3 ,G.Corbett-Feeney,G.Corbett-Feeney1,2 1,2 , M.Cormican, M.Cormican1 ,21 ,2..

11Interim National Salmonella Reference Laboratory, UCHG.Interim National Salmonella Reference Laboratory, UCHG.22National University of Ireland, GalwayNational University of Ireland, Galway

33Cork Institute of Technology.Cork Institute of Technology.44LEP, PHLS, ColindaleLEP, PHLS, Colindale

Shigella sonnei is a leading cause of gastroenteritis in both developing and industrialised

countries. Various phenotypic and genotypic methods have been used to type S. sonnei

isolates, including phage typing, antimicrobial resistance typing (ART), pulsed field gel

electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD) and

plasmid profiling. Sixty-seven S. sonnei isolates from various counties in Ireland were

analysed using PFGE and ART , while phage typing (n=8), plasmid profiling (n=28), and

integron analysis (n=14) was carried out on subsets of strains. PFGE divided the isolates

into two major clusters, A (n= 53) and B (n=14). There were seven different resistance

profiles detected, with resistance to ampicillin, streptomycin(S) and sulphonamide (Su)

(associated exclusively with PFGE A), being the most common (76%). Resistance to S,

Su, tetracycline and trimethoprim was common within PFGE B (78%). Two PFGE A

strains phage typed were PT9, while the PFGE B strains typed as PT6 (n=4), and PT50

(n=1) and RDNC (n=1). There were several different plasmid profiles among the strains

analysed. Two different plasmid profiles were observed among PFGE A, ASSu strains.

Integron analysis of a subset of isolates (n=14) revealed that at least 1 isolate contained a

complete class 1 integron structure while 10 isolates contained a gene encoding integrase

2. Six isolates which were resistant to trimethoprim by phenotypic methods contained an

amplicon for a DHFR gene which confers resistance to trimethoprim. Our data

demonstrate very limited diversity among S. sonnei in Ireland by PFGE, but greater

discrimination can be achieved by a use of a variety of typing methods.

AbstractAbstract

Dysentery

Amoebic and Bacillary

Historically A dysenteriae 15 serotypesB boydii 18 serotypesC flexneri 6 serotypes & 2 variantsD sonnei 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

IntroductionIntroduction

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.

Incidence USA 1998 25,000 cases 1999 18,000 cases

UK approx. 900 cases/year

Spread by person-to-person contact and contaminated food or water Low infectious dose (10 cells)

Isolates analysed in this study

Galway n = 42 (clusters of 4,2 &2 related)

Mayo n = 9 (cluster of 3 related)

Roscommon n = 8 (cluster of 2 related)

Dublin n = 6 (unknown)

Cork n = 2 (unknown )

Materials and MethodsMaterials 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

Number of Isolates

Galway 34Mayo 8Roscommon 4

Location of predominant strain

0

5

10

15

20

No. of Isolates

Feb Apr June Aug Oct Dec Feb Apr June

1998 1999

Seasonal Variation of Predominant strain PFGE A, ASSu

*(Ampicillin,Streptomycin,Sulphonamide)

ResultsResults

B A A B1 B4 A A A A B4 B3 B1 B B5 B B B5 B B B4

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

PFGE analysed by Bionumerics

RDNC

PT9

PT6PT50

PT6

PT9

PT6

Phage Typing of S. sonnei isolates (performed by LEP, Colindale, London)

PT 9 PFGE A (n=2) PT6 PFGE B (n=1) PFGE B1 (n=1)

PFGE B4 (n=1)

PFGE B5 (n=1)

PT50 PFGE B3 (n=1)

RDNC PFGE B (n=1)

Phage type 6 accounts for approximately 80% of U.K. infections.PT9 is quite rare.

Conjugation of S.sonnei isolates

1 Isolated on Trptone Soya Agar , Oxoid2 Isolated on Mueller Hinton Agar, BBLA = Ampicillin 100g/ml agarNa = Nalidixic acid 30g/ml agarT = Tetracycline 20g/ml agarTm = Trimethoprim 5g/ml agar

Isolate no Resistance profile

Transconjugant Res. profile

Selected on:

3331 ASSu ASSu A + Na 1 3331 ASSu A A + Na 1 3599 A A A + Na 1 Dublin 3 SSuTTm SSuT T + Na 1 Cork 2 ASSuTTm ASuT A + Na 1 3219 ASuTm A A + Na 1 3219 ASuTm ASuTm Tm + Na 2

Plasmid Profiles

B4 A A A A A A A1 A1 B A A B1 B4 pDu

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

108

6

4

2

3

5

Kb

Most isolates had multiple plasmids ranging in size from 2kb to >50kb

ASSu

SSuTTm

ASuTm

ASSuTTm

SSuTTmNa

STm

A

Antibiotics tested: Amp10, C 30, S10, Su300, Tet30, W5, Na30, Nt300, K30, Cip5Oxoid

Antibiotic Resistance Patterns

ConclusionsConclusions

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