Emergence of new subclones of multiresistant Salmonella Typhimurium DT104 possibly associated with poultry meat

E. J. Threlfall, BSc, PhD, M. D. Hampton, BSc, MSc, H. Chart, BSc, PhD, K. L. Hopkins, BSc, PhD, L. R. Ward, BSc, Laboratory of Enteric Pathogens, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT G. Tebbutt, BSc, PhD, Public Health Laboratory, James Cook University Hospital, Marton Road, Middlesbrough TS4 3TA FROM 1990 to 2000, multiple antimicrobial drug resistance, that is, resistance to four or more drugs, increased dramatically in cases of human salmonellosis in England and Wales. An important factor in this was the epidemic spread of multiresistant Salmonella Typhimurium DT104 with chromosomally encoded resistance to ampicillin (A), chloramphenicol (C), streptomycin (S), sulphonamides (Su) and tetracyclines (T) (R-type ACSSuT) (Threlfall and others 1994). Strains of this phage type/R-type combination became widely distributed in bovine animals in the early 1990s and subsequently became common in poultry, pigs and sheep (Anon 1998). Human infection has been associated with the consumption of chicken, beef, pork sausages and meat paste (Wall and others 1994). The peak year for multiresistant DT104 infections in human beings was 1996, when 4006 isolates were identified (Threlfall 2000). Although isolations have subsequently declined, falling to 1168 in 2000 and 765 in 2002, multiresistant DT104 remains the most common drug-resistant phage type in human salmonellosis. Since 1992, there has been additional resistance to trimethoprim (Tm) (R-type ACSSuTTm) and decreased susceptibility to ciprofloxacin (CPL) (R-type ACSSuTCpL) (Threlfall and others 1997). In the majority of isolates of R-type ACSSuTTm, resistance to Tm has been encoded by a plasmid of 6-8 kb (R. A. Walker, unpublished observations), which also codes for resistance to Su (Threlfall and others 1996). Although some variants have been identified, the majority of multiresistant DT104 have been characterised by a distinctive XbaI-generated pulsed-field profile (PFP), designated Xtml (Ridley and Threlfall 1998, Lawson and others 2004), and by the possession of a single plasmid of approximately 90 kb (Threlfall and others 1994, 1996, Prager and others 1999, Lawson and others 2004). This plasmid, the S Typhimurium'serovar-specific' plasmid (ssP), is further characterised by possession of the 'salmonella plasmid virulence (spv) genes' responsible for the virulence of several common salmonella serotypes for BALB/C mice (Gulig and others 1993). This short communication reports the emergence of new subclones of multiresistant S Typhimurium DT104 possibly associated with poultry meat. In 2002, 44 per cent of DT104 isolates from cases ofhuman infection were of R-type ACSSuT and 25 per cent were of R-type ACSSuTTm (E. J. Threlfall, unpublished observations). Two substantive outbreaks, each involving over 40 cases, were identified. The first outbreak (outbreak 1) occurred in January 2002, and involved patients in north-west England. The outbreak strain (outbreak strain 1) was of R-type ACSSuTTm and was isolated from both human beings and cooked poultry meat samples (Anon 2002a). Sixty-four strains from patients and five from cooked poultry meat samples were examined. The second incident (outbreak 2) occurred between April and June 2002, and involved patients in north-east England (Anon 2002b). The outbreak strain (outbreak strain 2) was of R-type ACSSuT. This outbreak was associated with a sandwich bar. Isolations of multiresistant DT104 of R-type ACSSuT were also made from cooked chicken and the environment. Forty-one strains from patients, four isolates from foods and three environmental isolates were characterised. When studied by plasmid profile typing (Kado and Liu 1981), outbreak strain 1 (R-type ACSSuTTm) was characterised by three plasmids of 6-8, 3 0 and 1-5 kb. As with isolates of multiresistant DTl04 of R-type ACSSuTTm studied previously (Threlfall and others 1996), the 6-8 kb plasmid coded for resistance to Su and Tm. However, of particular note was that outbreak strain 1 did not possess the 90 kb S Typhimurium ssp, identified in all of over 3000 isolates of multiresistant DT104 previously studied in the UK (Threlfall and others 1996, Lawson and others 2004) and also in all isolates of multiresistant DT104 from human beings and food animals in Germany (Prager and others 1999) and the USA (Glynn and others 1998). When characterised by pulsed-field gel electrophoresis (Ridley and Threlfall 1998), the PFP of outbreak strain 1 was identical to Xtm 1, except that it lacked a fragment of approximately 90 kb. This fragment has been shown to correspond to the presence of S Typhimurium ssP (Ridley and Threlfall 1998). The PFP of outbreak strain 2 (R-type ACSSuT) was indistinguishable from that of outbreak strain 1. Outbreak strain 2 was characterised by two plasmids of 3 0 and 1.5 kb. As with outbreak strain 1, outbreak strain 2 did not possess the 90 kb S Typhimurium ssP. Subsequent studies have demonstrated that, when challenged with 103 viable bacteria by intraperitoneal injection (Chart and others 1991), neither of the two ssP-negative outbreak strains was pathogenic for BALB/C mice, in comparison with strains of multiresistant DT104 possessing the S Typhimurium ssP. The absence of the plasmid-encoded spv genes was further confirmed by PCR for the presence or absence of a 437 base pair internal fragment of the spvC gene (M. D. Hampton, unpublished observations), using a ssPpositive, spvC-positive strain of multiresistant DT104 as a control. Both outbreak strains were negative by PCR for this spvC gene fragment. The two outbreak strains of R-types ACSSuTTm and ACSSuT were thus identical in all respects other than the possession of a 6-8 kb SuTm plasmid by outbreak strain 1. The lack of the S Typhimurium ssp, coupled with the presence of plasmids of 3 0 and 1 5 kb in both strains, were crucial markers and suggest that both strains may have a common origin. The involvement of food of poultry origin in both outbreaks suggests that the reservoir of infection might have been poultry. However, the possible contribution of either imported poultry meat, which is an important source for the catering industry, or 'added-value' poultry meat products has not been ascertained. Further investigations are required to establish the occurrence ofthe two strains in poultry, poultry products and human beings in the UK and elsewhere.