Salmonella enterica Bredeney: Third commonest cause of human infection in Ireland in 1999 C. A. O’ Hare, M. Cormican. G. Corbett-Feeney, S. Fanning and J. Moore.

Salmonella enterica is an important human pathogen. In 1999 three serotypes predominated amongst strains submitted for typing to the Interim National Salmonella Reference Laboratory. As in Europe generally, S. Typhimurium (42%) and S. Enteritidis (33%) predominate. Salmonella Bredeney is not a common human pathogen in most countries. However isolates of this serotype accounted for 8% and 17% of the total number of Salmonella enterica isolates received in 1998 and 1999 respectively. We have performed a detailed analysis of a collection of 112 isolates of S. Bredeney from humans (n=52), animals (n=40) and food (n=20) sources from throughout the island of Ireland. Antimicrobial susceptibility testing was performed by the disk diffusion method of the National Committee for Clinical Laboratory Standards (NCCLS). Activity of ten antibiotics were assessed (ampicillin 10  g, chloramphenicol 30  g, sulphonamides 300  g, streptomycin 10  g, Abstract

tetracycline 30  g, trimethoprim 5  g, nalidixic acid 30  g, kanamycin 30  g, ciprofloxacin 5  g and nitrofurantion 300  g (Oxoid). Pulsed field gel electrophoresis was performed with digestion of whole chromosomal DNA with XbaI, and DNA amplification fingerprinting (DAF) were performed on all isolates. Resistance to sulphonamide and trimethoprim (SuTm) was observed in one human isolate (1%), 20 animal isolates (18%) and 3 food (3%) isolates. Eight distinct PFGE patterns (3 or more band differences) were observed with a single pattern accounting for 86% of strains. Nine distinct patterns were observed by DAF. Groupings identified by DAF were in close agreement with those identified by PFGE. Resistance to SuTm was associated with PGFE type I and DAF types I and II. These results indicate that most isolates of S. Bredeney from human, food and animal sources are closely related. It is interesting however that antimicrobial resistance was observed much more frequently in isolates from animal than from human sources. Abstract

v Salmonella enterica: an important human and animal pathogen In humans it is most commonly associated with self-limiting gastroenteritis- Infection may be invasive and life-threatening in vulnerable groups v Serotypes most often associated with human infection in Ireland S. Typhimurium (42% isolates, 1999) S. Enteritidis (33% isolates, 1999) S. Bredeney (12% isolates, 1999) v In Ireland, S. Bredeney isolate numbers rose from less than 2% in 1997 to 12% in No corresponding increase was reported elsewhere in Europe. v S. Bredeney in USA is a rare cause of food poisoning. Less than 0.1% of isolates to CDC annually. Introduction

v Only three major outbreaks reported since S. Bredeney was typed by Kauffmann in v1983: Brazil (ref) v1995: Austrailia (ref) v1998: Alabama, USA (ref) v Italian study (ref) found that S. Bredeney isolates had a higher MIC to mercury based disinfectants than other serovars v Objective of study. To investigate the degree of diversity of S. Bredeney strains from Ireland using molecular and non-molecular methods. Introduction

v Bacterial strains: 112 isolates of S. Bredeney from Ireland and Northern Ireland. Sources included human, animal and food v Antibiotic sensitivity testing: Disk diffusion methods of the NCCLS (1). v Pulse field gel electrophoresis: Plugs cut with XbaI. 1.3% agarose gel ran for 17h at 160V with a ramp switch of 5-50s at 8 o C. v Plasmid DNA prepared by the method of Kado and Lui (2). Plasmid DNA ran on 0.7% gel at 70V for 2h. v DNA amplification fingerprinting: 10-mer primer P1254 used to PCR. samples ran on 1% agarose gel at 100V for 90min. Materials and Methods

Isolate source Number of isolates (n=total no)antibiotic resistant* Human (n=52)1 Poultry 1 (n=27)16 Porcine (n=1)1 Bovine 2 (n=9)3 Deer (n=3)2 Food 3 (n=20)4 * 23/25 isolates had resistance to sulphonamides and trimethoprim (SuTm) 1 one isolate tet only, 1, 2 one amp only, 3 one ASSuTTm Table 1: Origin of Salmonella Bredeney isolates

Figure 1: DNA Amplification fingerprinting (DAF) patterns of Salmonella Bredeney isolates Lanes: M= molecular weight markers kb 1. S. Typhimurium DT104 control 7. B039 DAF II13. B061 DAF V 2. B003 DAF I8. B070 DAF II14. B072 DAF V 3. B037 DAF I 9. B053 DAF III15. B058 DAF V 4. B062 DAF I10. B056 DAF III16. B052 DAF V 5. B024 DAF VII11. B054 DAF IV17. B023 DAF VI 6. B001 DAF II12. B067 DAF III18. B071 DAF IX

Figure 2: PFGE types I to VIII of Irish Salmonella Bredeney isolates Lanes: 1. Concatamer ladder 2. PFGE type I 3. PFGE type I 4. PFGE type II 5. PFGE type III 6. PFGE type I 7. PFGE type IV 8. PFGE type V 9. PFGE type VI 10.PFGE type VII 11.Concatamer  ladder

Table 2: Distribution (n) of PFGE types among sources of Salmonella Bredeney PFGE typeHuman Poultry Bovine Porcine Deer Food I II III IV V VI VII none* *Samples gave the appearance of smearing/degradation on PFGE analysis. Repeated in triplicate.

Figure 3: Plasmid analysis of S. Bredeney Lanes: 1. Clinical isolate 2. Clinical isolate 3. Clinical isolate 4. Clinical isolate 5. Poultry isolate (SuTm)* 6. Poultry isolate 7. E.coli 36R891 marker 8. Clinical isolate 9. Food isolate (SuTm)* 10 Food isolate 11.Clinical isolate *Resistance to Sulphonamides and tetracycline

 Seven different PFGE types observed (I to VII)  1 PFGE type accounted for most isolates (n=97) and predominates in humans, animals and food  Nine DAF patterns observed (I to IX). Very good agreement between PFGE and DAF patterns.  Antimicrobial resistance much more common in poultry.  Isolates resistant to ampicillin only or ASSuTTm, were assigned to PFGE types IV, VII or were untypable by PFGE (DAF V) Results

v Small plasmids of similar size (>20kb) were observed in eight out of ten isolates of PFGE Type I. v Observed plasmid carriage was not always associated with antibiotic resistance. Results

Bibliography 1. National Committee for Clinical Laboratory Standards (NCCLS) (1997). Performance standards for antimicrobial susceptibility tests, sixth edition: approved standard. M2-A6. Villanova, PA: NCCLS. MARTIN - TO BE DONE TOMORROW