1. Dangerous Dublin: The Virulent Salmonella Colette O’ Hare 1, Conor Burke 2, Suzanne Gray 3, Niall Delappe 1, Ger Doran 1, Grainne McHale 2, Dr. Gerard Solan 2 and Prof. Martin Cormican 3 1 Salmonella Reference Laboratory, Medical Microbiology, UCHG. 2 Medical Microbiology, Mayo General Hospital. 3 Medical Microbiology, UCHG ABSTRACT Salmonella Dublin is a particularly virulent serovar of Salmonella enterica. It is frequently recovered from cattle, but can be transferred to humans via meat and dairy products. In early 2003 Salmonella enterica was isolated from CSF and blood samples from a 1 month-old baby in a General Hospital. The isolates were sent to the Salmonella Reference Laboratory. Serotyping was performed by standard methods and susceptibility to 15 antimicrobial agents tested by the NCCLS disc diffusion method. These isolates and other Salmonella Dublin isolates were subcultured to ASAP agar (Aes Laboratoire Salmonella agar) and were typed by Pulsed Field Gel Electrophoresis (PFGE) using the Pulse-Net protocol. Isolates were examined for plasmids by the methods of Birhboim and Doly. Serotyping confirmed the isolates as Salmonella Dublin (9,12:g, p). Isolates were susceptible to all 15 antimicrobial agents tested. All Salmonella Dublin isolates failed to produce the expected pink/purple colonies on ASAP agar. On Pulsed Field Gel Electrophoresis (PFGE) the two isolates from the infant were indistinguishable from each other and indistinguishable from an isolate submitted from the Eastern region in 2001. A 80 kb plasmid, known to be associated with virulence in Salmonella Dublin was detected in most isolates. Salmonella Dublin was confirmed as the cause of blood stream infection and meningitis in the infant (isolate number 170/03). ASAP agar does not reliably detect Salmonella Dublin. PFGE allows discrimination of subtypes within the serovar Salmonella Dublin although the difference are relatively minor (1 to 2 band differences) in most cases. RESULTS ₪ Of the S. Dublin human isolates (n=34) 29% were isolated from non-fecal samples: blood (n=10), urine (n=1), CSF (n=1) and pleural fluid (n=1). ₪ 82% of non-human S. Dublin isolates were from bovine origin (incl milk/cheese) ₪ 35% of S. Dublin isolates were from children under 5 years of age (n=12) and 35% from adults over 60 years of age (n=12). Table 1 ₪ 12% (n=7) of isolates had resistance to one antibiotic. ₪ No C 8 esterase activity, as indicated by clear colony colour growth was obtained from 36 non-duplicate isolates of S. Dublin on ASAP agar ₪ 10/14 S. Dublin isolates showed presence of the 80kb serovar specific plasmid (ssp). Figure 1 ₪ Six banding patterns (A-A5) were observed (one to two band differences) for 28 human S. Dublin isolates using XbaI endonuclease. 78.5% were pattern A. Figure 2 CONCLUSIONS ₪ S. Dublin cause high percentage of invasive disease ₪ High numbers of S. Dublin isolated from patients at extremes of age ₪ Low levels of single antimicrobial resistance. ₪ No multiple antimicrobial resistance carraige ₪ Plasmid analysis poor discriminatory tool for genotyping ₪ PFGE good discrimination for genotyping MATERIALS AND METHODS ₪ 34 human and 22 non-human Salmonella Dublin isolates submitted from 2000-date ₪ Susceptibility testing against 15 antibiotics (Oxoid) using the NCCLS disc diffusion method (5) ₪ C8 esterase activity on ASAP agar (AES Laboratoire) determined by colony colour from growth aerobically at 37 o C (6) ₪ Plasmid isolation using the Birhboim and Doly method (7) ₪ PFGE using PULSENET protocol developed in CDC, Atlanta, USA INTRODUCTION Salmonella enterica is a major human pathogen. More than 2400 serovars are recognised (1). Human infection with most animal serovars is associated with acute self-limiting diarrhoea, however in the very young, the aged and other vulnerable individuals, life threatening invasive infection may occur. At present Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium are most common in Western Europe (2) and accounted for the majority of isolates in Ireland from 1996-2002 (3, 4). Salmonella Dublin is particularly associated with invasive disease and is frequently isolated from blood of infected patients. Its antigenic components include the Vi (virulence) antigen, which is also commonly found in Salmonella enterica serovar Typhi. From 2000-2003, 56 isolates of S. Dublin were received in the Reference Laboratory from humans and food/animals. This study was proposed to investigate both phenotypic and genotypic typing methods for S. Dublin REFERENCES (1) Popoff MY, Le Minor L. (1997) Antigenic formulae of the Salmonella serovars. 7e rev ed (2) EnterNet. (2002) Enter-net Quarterly Salmonella reports. (3) Cormican M, Butler C, Morris D, Corbett-Feeney G, Flynn J. (1998) Antibiotic resistance amongst Salmonella enterica species isolated in the Republic of Ireland. J. Antimicrob. Chemother. 42(1):116-118. (4) O’Hare C, Doran G, Delappe N, Morris D, Buckley V, Corbett-Feeney G, Cormican M. Antimicrobial resistance and phage types of human and non-human Salmonella enterica isolates in Ireland from 1998-2002. Submitted to CDPH (5) NCCLS. (2002) Performance standards for antimicrobial susceptibility tests, sixth edition: In: CDSC, NI (6) Gray S, Clancy J, O’ Hare C, Doran G, Cormican M. (2003) Failure to detect Salmonella enterica serovar Dublin on Aes Laboratoire Salmonella agar plate. J. Clin. Microbiol. 41 (8): 4003 (7) Birhboim H. C, Doly J. (1979) A rapid extraction procedure for screening recombinant DNA. Nucleic Acids Res. &: 1513-1523 ACKNOWLEDGMENTS We would like the thank Dr. Gabriel Fox, Mayo General Hospital for his case history Table 1. Figure 1. Plasmid analysis of S. Dublin isolates (lanes 2-5, 9-11). Control plasmid size markers (Lanes 6-8: 62Mdal, 72Mdal, 166Mdal). Supercoiled marker (lanes 1, 12) Figure 2. PFGE dendogram analysis of clinical S. Dublin isolates Contact Details Salmonella Reference Laboratory UCHG 091-544628 e.mail: colette.ohare@nuigalway.ie A A1 A2 A3 A4 A5