Salmonella Typhimurium DT104: to b or not to b Geraldine Doran, Niall DeLappe, Colette O Hare, Geraldine Corbett-Feeney, Martin G. Cormican. National Salmonella Reference Laboratory, University College Hospital, Galway Address: Room 320 Dept of Bacteriology Clinical Science Institute University College Hospital Galway Phone: Fax: ABSTRACT Salmonella enterica serovar Typhimurium is one of the most common enteric pathogens isolated from humans and animals in Europe and the US. The most frequently isolated phage type of this serovar is definitive type (DT)104. Phenotypic and genteic diversity among DT104 isolates is very limited as they are highly clonal. In Ireland and the UK, phage type DT104b is isolated with a similar frequency to DT104. DT104 and DT104b strains are closely related and these phage types are not readily distinguished from each other by Pulsed Field Gel Electrophoresis. In 2003 there was a small outbreak of Salmonella enterica serovar Typhimurium of phage type DT104b. A series of 15 isolates thought to be associated with the outbreak were submitted to the National Salmonella Reference Laboratory for typing however one of the collection of isolates was typed as DT104. This study describes the application of genotypic methods for clarification of the relationship of this isolate to the predominant outbreak strain. Antimicrobial susceptibility was performed according to the disk diffusion method the NCCLS. Pulsed field gel electrophoresis was carried out using the standardised protocol of Pulse-Net using Xba1 restriction enzyme. Plasmid analysis was performed using a modified method of Kado and Liu. All 15 isolates exhibited the same pattern of antimicrobial resistance. The DT104 isolate had an additional plasmid not present in the DT104b and on PFGE differed by a single band from the other fourteen isolates. Notwithstanding the strong epidemiological link between the DT104 isolate strain and the DT104b outbreak strain, molecular methods did not confirm this association. The DT104 isolated differed in two characteristics from the DT104b strain, (a) it had an additional plasmid and (b) was missing a band on PFGE electrophoresis. Genotypic methods can be helpful in clarifying relationships between isolates in a situation of suspected outbreak. INTRODUCTION  Salmonellosis is a major food-borne infection.  Salmonella Typhimurium is one of the most common serovars isolated from animals and humans in the US and Europe.  Salmonella Typhimurium and Salmonella Enteritidis account for most human isolates referred to the National Salmonella Reference Laboratory (NSRL).  S. Typhimurium phage types DT104 and DT104b accounted for 19% of human isolates submitted to the NSRL in  Most cases of salmonellosis are zoonotic (Wall et al., Villar et al.)  There is currently a pandemic clone of Salmonella Typhimurium DT104 The pandemic clone of DT104 has resistance to at least 5 antibiotics – ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline (ACSSuT). This resistance is primarily located on class 1 integrons on the chromosome (Ridley et al. 1998, Briggs et al. 1999). Resistance can be to up to nine antibiotics including trimethoprim and nalidixic acid. Reduced susceptibility to ciprofloxacin is also observed (Cormican et al. 1998, Ridley et al. 1998) Strains harbor a 62 MDa plasmid DT104 and DT104b are very closely related phage types and are often indistinguishable by Pulsed field gel electrophoresis (PFGE), plasmid analysis and antibiogram typing.  In 2003 a significant outbreak of Salmonella Typhimurium DT104b occured in Ireland.  15 isolates were sent to the NSRL – fourteenof phage type DT104b and one of DT104.  Phenotypic and genotypic methods were performed to establish the relationship between the strains. MATERIALS AND METHODS  Strains were confirmed as Salmonella enterica by API 20E (Biomeriux) and confirmed as S. Typhimurium according to the Kauffman and White serotyping scheme using slide agglutination with standard antisera (Murex Biotech Ltd., Dartford, England)  Antimicrobial susceptibility was performed according to the disk diffusion method of the NCCLS (NCCLS 2002)  The following antimicrobial agents were used: ampicillin (10µg), chloramphenicol (30µg), ciprofloxacin (5µg), kanamycin (30µg), nalidixic acid (30µg), sulphonamide(300µg), streptomycin (10µg), tetracycline (30µg), trimethoprim (5µg), cefotaxime (30µg), kanamycin (30µg), minocycline (30µg) and ceftazadime (30µg).  Phage typing was performed in accordance with the methods of the Health Protection Agency, Colindale, London, United Kingdom (Anderson et al. 1977).  Pulsed-field gel electropheresis was performed using the CHEF Mapper XA (Bio-Rad) system following the standardised protocol of Pulse-Net (Swaminathan et al. 2001) using XbaI restriction enzyme. Results were analysed using Bionumerics software. RESULTS  All 15 isolates were resistant to Ampicillin, Sulphonamide and Trimethoprim (ASuTm).  Phage typing14 DT104b 1 DT104  PFGE Analysis DT104 isolate missing a band observed in DT104b isolates (Fig. 1)  Plasmid Analysis DT104 isolate exhibited an additional plasmid to DT104b isolates (Fig. 2) CONCLUSIONS  Epidemiological and Phenotypic methods suggested a link between the DT104 isolate and DT104b outbreak strains Unusual antibiogram pattern in all isolates (ASuTm) DT104 and DT104b are closely related phage types Conversion between these phage types may be possible due to a loss or gain of a plasmid or phage  Two unrelated genetic differences did not support this link The DT104 isolate was missing a PFGE band The DT104 isolate had an additional plasmid  Genotypic methods were used to clarify the relationship between suspected outbreak isolates. Fig. 2 Plasmid Analysis 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13 Lane 1-3 and 9-13 : DT104b isolates Lane 8: DT104 isolate Lanes 4-6 Control Strains Lane 7: Molecular weight marker REFERENCES Anderson E. S., L. R. Ward, M. J. de Saxe, and J. D. de Sa Bacteriophage typing designations of Salmonella Typhimurium. J. Hyg 78: Briggs C. E. and P. M. Fratamico Molecular characterisation of an antibiotic resistance gene cluster of Salmonella Typhimurium DT 104. Antimicrobial Agents Chemother. 43: Cormican, M., C. Butler., D. Morris, G. Corbett-Feeney, and J. Flynn Antibiotic resistance amongst Salmonella enterica species isolated in the Republic of Ireland. J. Antimicrob. Chemother. 42: NCCLS Performance standards for antimicrobial susceptibility testing: Twelfth International Supplement M2-A7. Villanova, PA: NCCLS. Ridley A., and E. J. Threlfall Molecular epidemiology of antibiotic resistanve genes in multiresistant epidemic Salmonella Typhimurium DT104. Microb. Drug Res. 4: Swaminathan B., T.J. Barrett, S. Hunter, R.V. Tauxe and the CDC PulseNet Task Force. (2001). PulseNet: The molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg. Infect. Diseases. 7: Villar R.G., M.D. Macek, S. Simons et al Investigation of multidrug resistant Salmonella Typhimurium DT104 infections linked to rawmilk cheese in Washington State. JAMA 281: Wall P.G Transmission of multiresistant strains of Salmonella Typhimurium from cattle to man. Vet. Rec. 136: Fig. 1 Pulse-field Gel Electropheresis Lanes 1, 5, 10, 14 Control Strains Lanes 2-4 DT104 isolates not associated with outbreak Lane 6 DT104 isolate suspected to be part of outbreak Lanes 7-9 and DT104b isolates in outbreak Missing Band Extra plasmid Fig. 4 Typical phage typing plate