Isolates of S. Typhimurium DT 12 and DT 120 from human, animal, food, and environmental sources received by the Laboratory of Enteric Pathogens from 1991 to 2000 were examined. For each phage type, 40 strains were selected, consisting of 15 antibiotic-sensitive strains, 12 strains of R-type ACSSuT, and 13 strains with non-ACSSuT R-types. Control strains of S. Typhimurium DT 104, both sensitive and MR, were also included.

All strains were characterized by phage type, in accordance with the scheme of Anderson et al. (5); antimicrobial susceptibility patterns were determined by using the breakpoint method (6), so that final concentrations (mg/mL^-1) were ampicillin, 8; chloramphenicol, 8; furazolidone (Fu), 8; gentamicin (G), 4; kanamycin (K), 8; neomycin (Ne), 8; streptomycin, 16; sulfonamides, 64; tetracyclines, 8; trimethoprim, 2; and ciprofloxacin, 0.125.

The presence of the 13 kbp ACSSuT resistance gene cluster was determined by a long-polymerase chain reaction (PCR) method developed by Walker et al. (7) that amplified the 10- kbp region between the aadA2 (streptomycin resistance) and bla _CARB-2 (ampicillin resistance) genes on the sequence of the antibiotic resistance gene cluster from S. Typhimurium DT 104 isolate H3380 (8). All strains of MR DT 104, DT 12, and DT 120 of R-type ACSSuT produced 10-kbp PCR amplicons; strains with other resistance patterns were negative.

The strains were examined for the presence of class 1 integrons by using a specific PCR assay (9). All strains of MR DT 104, DT 12, and DT 120 of R-type ACSSuT produced bands of 1.0 kbp and 1.2 kbp corresponding to the two integrons reported within the resistance gene cassettes (8,10). This corroborated the long-PCR result and demonstrated that there was no apparent variation in the arrangement of the genes within the ACSSuT resistance cassettes. Sensitive and non-ACSSuT-resistant strains were either negative by the class 1 integron PCR or produced bands other than 1.0 kbp and 1.2 kbp.

Pulsed-field gel electrophoresis (PFGE) analysis was performed by using the restriction enzyme XbaI according to the method of Powell et al. (11). Analysis showed that all sensitive and non-ACSSuT-resistant strains of DT 12 and DT 120 examined formed distinct groups of phage type-related PFGE profiles that clustered when analyzed by the nearest neighbor algorithm (12) (Figure 2). Nine distinct profiles (z1 to z9) were obtained from non-ACSSuT-resistant DT 12 and five different profiles (A to E) from the non-ACSSuT-resistant DT 120. Importantly, the ACSSuT-resistant strains of both DT 12 and DT 120 produced similar PFGE patterns that were distinct from those obtained by non-ACSSuT-resistant strains of the same phage type. However, these patterns were identical to those commonly observed in both sensitive and MR strains of DT 104 (profiles xtm1, xtm9, and xtm12). In contrast, the PFGE profiles of sensitive and ACSSuT-resistant strains of DT 104 typically differ by no more than a few bands. The PFGE profile of the ACSSuT-resistant DT 12 strains were all identical to that produced by most (80%- 90%) DT 104 isolates, termed profile xtm1 (10). The ACSSuT-resistant strains of DT 120 were mainly (8 of 12 strains) profile xtm1; the remainder were profiles xtm12 (3 of 12) and xtm9 (1 of 12). PFGE profiles xtm9 and xtm12 are less common variants of the basic xtm 1 pattern in DT 104. These variants occur in <5% of isolates and differ from the predominant pattern by two bands and one band, respectively (A. J. Lawson, unpub. data) (Figure 2).

Strains were analyzed for presence of plasmids by standard methods (13). All DT 104 and DT 12 strains contained a 90-kbp “serotype-specific” plasmid commonly found in S. Typhimurium (14). However, in DT 120 only those strains of R-type ACSSuT possessed this plasmid. Purified 90-kbp plasmid extracts from R-type ACSSuT strains of DT12, DT120, and DT104 were negative for integron-associated resistance gene cassettes by PCR (as described above), confirming the chromosomal location of the antibiotic resistance genes.

Strains of S. Typhimurium DT 12 and DT 120 of R-type ACSSuT possess antibiotic resistance gene cassettes indistinguishable from those typically present in multiresistant strains of the internationally distributed zoonotic pathogen S. Typhimurium DT 104. Strains of MR DT 12 and MR DT 120 of R-type ACSSuT have PFGE profiles distinct from those of other strains of the same phage type. Furthermore, the PFGE profiles of ACSSuT-resistant MR DT 12 and MR DT 120 are identical to those previously reported in MR DT 104. Additionally, ACSSuT-resistant strains of MR DT 120 possess a 90-kbp plasmid absent in DT 120 strains of other R-types, but present in DT 12 and DT 104 strains.

These data suggest that the occurrence of R-type ACSSuT in S. Typhimurium DT 12 and DT 120 strains is probably due to changes in phage susceptibility in a small proportion of MR DT 104 strains rather than to horizontal transfer of a resistance gene cassette. The Table shows selected phage reactions used in the S. Typhimurium phage typing scheme (5) and illustrates the similarity between the phage susceptibility profile of DT 12 and DT 120 and that of DT 104, especially the weaker reacting 104L profile. The causes of changes in phage susceptibility are complex and difficult to trace but are thought to result from the acquisition of new phages or plasmids and to changes in bacterial cell surface phage-receptors.

With regard to the international distribution of these strains, the ACSSuT-resistant DT 12 strains were all PFGE profile xtm1, and all originated from patients in England and Wales with no history of foreign travel. In contrast, the ACSSuT-resistant DT 120 strains consisted of a mixture of xtm1, xtm9, and xtm12 PFGE profiles and included cases from South Africa (xtm1), Sweden (xtm12), Northern Ireland (xtm9), and an isolate from a patient with a history of travel to the West Indies (xtm1). Continued surveillance coupled with molecular typing should be maintained to follow the spread of these new MR DT 104 variants in animals and humans.