Salmonella Reference Laboratory Epidemiology of Salmonella enterica from C. O’ Hare 1, N. Delappe 1, G. Doran 1, D. Morris 2, D. Kilmartin 2, G. Corbett-Feeney 1 and M. Cormican 1. 1 National Salmonella Reference Laboratory, Medical Microbiology, UCHG, Galway. 2 Department of Bacteriology, NUI, Galway.

Salmonella infection is a significant global public health problem. There are over 2000 serotypes of Salmonella enterica, the bacterial species associated with Salmonellosis. The Salmonella enterica serotypes associated with gastroenteritidis in humans are often present in the gastrointestinal tract of animals including animals intended for human comsumption. The primary source of human infection is by transfer from animals to humans through the food chain, although person-to-person transfer can occur. Most cases of human infection with Salmonella are associated with an acute self-limiting gastrointestinal illness, characterised by diarrhoea, abdominal cramps, fever and vomiting. However, in a proportion of cases, especially among the very young, aged or immunosuppressed the bacteria may invade the blood steam and cause very serious life-threatening infection. The NSRL provides an overview of the epidemiology and burden of disease caused by Salmonella infection in Ireland today. Another important role of the Laboratory is the extended sensitivity testing and phage typing of Salmonella isolates. Global levels of antimicrobial resistance is of increasing concern. Laboratory susceptibilty testing of isolates, is important both as a guide to the therapy of individual patients and as an indicator of the nature and extent of the problem of resistance Introduction

Abstract The National Salmonella Reference Laboratory (NSRL) was established in January 2000 with funding through the Western Health Board from the Department of Health and Children. The laboratory assists in the epidemiology and surveillance of Salmonella enterica isolates by the routine testing of extended antimicrobial susceptibilities, phage typing, serotyping and molecular analysis of submitted isolates. By assembly of all Salmonella enterica isolates in a single laboratory, the NSRL facilitates recognition and confirmation of links between individual cases of infection, even where outbreaks are widely dispersed Between 1998 and 2002, a total of 4038 isolates (2693 human and 1345 non human) of Salmonella enterica were received By the NRSL (banks of Salmonella were kept prior to 2000) Serotyping, antimicrobial susceptibility testing and phage typing were performed by standard methods.

Abstract From 1998 to 2002 the relative frequency of isolation of the serotype S. Typhimurium from both clinical and non-clinical Sources declined from 80% to 27% while the frequency of isolation of S. Enteritidis increased from 8% to 20.5%. S. Typhimurium phage type DT104 and the closely related type DT104b accounted for between 90% to 50% of S Typhimurium isolates between Phage type 4 (PT4) declined from 85% of all S. Enteritidis isolates in 1998 to 21% in 2002, while PT1 increased from non-detected in 1998 to 36% of isolates in Resistance to multiple antimicrobial agents was very commonly observed in S Typhimurium but rare in S. Enteritidis. Non-human isolates included 1084 isolates from food and animals, and 262 isolates from environmental samples. Of isolates received from cattle and pigs, S. Typhimurium was the most prominent serotype at 57%. By contrast, from isolates submitted from poultry S Kentucky was the most prevalent serotype (21%), and S Typhimurium accounted for only 10%. As in humans, DT104 in S. Typhimurium predominated

Materials and Methods  Clincal Salmonella isolates N= 714, 438, 638, 509, 394  Non-clinical Salmonella isolates N= 7, 16, 212, 573, 537  Serotyping (Kauffmann and White)  Susceptibility Testing (1)(NCCLS) WHONET  Phage typing (2)(PHLS, Colindale)  Molecular analysis PFGE (3)(PULSENET) Plasmid analysis (refs) Bionumerics

Results: Serotyping Top Serotypes of Salmonella enterica Clinical Non-clinical Typhimurium1306 (48%) 325 (24%) Enteritidis831 (31%) 68 (5%) Bredeney107 (4%) 123 (9%) Kentucky46 (2%) 174 (13%) Dublin42 (2%) 21 (2%) Agona21 (1%) 75 (6%) Virchow42 (2%) 7 (1%) Typhi 1 9 (<1%) 0 Livingstone (6%) Other (11%) 552 (41%) 1 S. Typhi is a human pathogen only 2 Primarily found in poultry 3 Other serotypes which singly represent >2%

Results: Salmonella Typhimurium phage types Clinical Non-clinical

Results: Salmonella Enteritidis phage types Clinical Non-clinical

Results: Pulse Field Gel Electrophoresis PGFE has been standardised for Salmonella and E. coli by PULSENET (CDC, Atlanta, USA). Restriction enzymes used are XbaI and BlnI

Discussion Over the period among samples from both Clinical and non-clinical sources  In general, isolate numbers have be declining in recent years in line with published data from other EU countries.  Numbers of S. Typhimurium received are decreasing (80% to 27%) while S. Enteritidis are increasing (8% to 21%) Non-clinical isolates encompass a much broader range of serotypes 78% of clinical S. Typhimurium DT104/DT104b 51% of non-clinical

Discussion 65% of all S. Typhimurium harboured the ACSSuT multi-resistance phenotype Of these 84% were either phage type DT104 or DT104b 2% of S. Typhimurium were susceptible to the 15 antibiotics tested, by comparison to 33% of S. Enteritidis PT4 and PT1 are the most common phage types in S. Enteritidis. 67% of PT1 is associated with naladixic acid resistance. All naladixic acid resistant strains had reduced susceptibilities to ciprofloxacin (MIC’s  g/ml to 0.19  g/ml). No ciprofloxacin resistant isolates observed.

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. (2) Anderson ES, Ward LR, de Saxe MJ. Bacteriophage-typing designations of Salmonella Typhimurium. J Hyg 78: , 1977 (3) Swaminathan B., Barrett T.J., Hunter S., Tauxe R.V., and the CDC PulseNet Task Force. (2001). PulseNet: The Molecular Subtyping Network for Foodborne Bacterial Disease Surveillance, United States. Emerg. Infect. Diseases. 7, pp