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Sir, Multidrug-resistant Salmonella enterica serovar Typhimurium DT104 (where DT stands for definitive type) emerged during the 1980s as a cause of many outbreaks in humans and animals, most isolates exhibiting resistance to a core group of antimicrobials including ampicillin/amoxicillin, chloramphenicol/ florfenicol, streptomycin/spectinomycin, sulphonamides and tetracyclines (ACSSuT phenotype). The genes responsible were shown to be located on a 13 kb multidrug resistance region (MDR region) that constitutes a complex class 1 integron. This cluster, recently named In104, belongs to the integron In4 group and is located in a 43 kb genomic island named Salmonella genomic island 1 (SGI1). Several variants of SGI1 have also been described over time in a wide variety of S. enterica serovars and in Proteus mirabilis. In this study, we examined an S. enterica serovar Typhimurium strain 72-21880-11 isolated from the faeces of a healthy cow in Denmark in 2000, which displayed resistance to chloramphenicol/ florfenicol, streptomycin/spectinomycin and tetracycline. To assess whether this strain harbours SGI1, PCRs were performed using primers corresponding to the left and right junctions of SGI1 in the Salmonella chromosome as described previously. PCR results were positive for the left junction with the chromosomal thdF gene and for the right junction with the int2 gene of the retron sequence specifically found downstream of SGI1 in the serovar Typhimurium, indicating that this strain contained SGI1 at the same location as in other isolates. To confirm the presence of the entire SGI1, PCR mapping of the 5′ region of SGI1 (the first 10 kb) was performed as described previously. All of the PCR results were positive. The presence of the remaining non-MDR region of SGI1 was assessed by Southern blot hybridization of XbaI-digested genomic DNA with the p1-9 probe as previously described. The Southern blot profile of the serovar strain 72-21880-11 was similar to that obtained for the control strain of serovar Typhimurium DT104 harbouring SGI1 with two XbaI fragments of 4 and 9 kb (data not shown). To confirm the location of the MDR region within SGI1, PCRs were performed using primers S026-FW/int-RV and DB-T1/ MDR-B corresponding to the boundaries of the integron with the SGI1 backbone. The two PCRs gave positive results, indicating that the MDR region was at the normal position within the SGI1 backbone, i.e. between the res gene and the open reading frame S044. The unusual resistance pattern of the strain 72-21880-11 suggests that genetic variations occurred within the In104 complex integron, which were assessed by PCR mapping of the antibiotic resistance gene cluster as described previously (Figure 1a). All PCR products were obtained except the one corresponding to fragment E specific for the blaPSE-1 gene cassette. This result was confirmed by the cassette-array PCR for which only one fragment of 1 kb was obtained, corresponding to the aadA2 gene cassette. Additional PCRs were also carried out, indicating that the variant region is located downstream of orf2 (data not shown). The region between groEL/int1 and IS6100 was then amplified by PCR and the 1.2 kb product sequenced by Beckman Coulter Genomics (Takeley, UK). The nucleotide sequence obtained (deposited in GenBank under accession number GU830872) allowed us to resolve the genetic organization of the variant antibiotic resistance gene cluster, which consisted of the groEL/int1 gene, followed by only 403 bp of the blaPSE-1 gene and by the IS6100 element (Figure 1a). To confirm this genetic organization, we performed a Southern blot hybridization of genomic DNA cut either by HindIII or by XbaI and XhoI, and using as a probe the XbaI fragment of recombinant plasmid pSTF3, comprising nearly the entire In104 complex class 1 integron. Southern blot profiles of the studied strain were clearly distinct from those of the control strain harbouring the classic SGI1 (Figure 1b). The sizes of the bands correlated with the genetic organization characterized previously by PCRs and sequencing. The cluster described in this study constitutes a new SGI1 variant and we propose to name it SGI1-T according to the nomenclature. The genetic structure described here could be explained by an intramolecular transposition of the IS6100 element leading to the insertion of a second IS6100 copy within blaPSE-1 Subsequently, a single cross-over event between the two IS6100 elements in the same orientation would have led to the deletion of the central region including a part of blaPSE-1, qacED1sul1, orf5 and orf6. IS6100 has been described to transpose randomly by a replicative mode. The absence of direct repeats, as a result of the transposition event, is simply due to the intramolecular homologous recombination between the two IS elements, each with different direct repeat sequences. In S. enterica Typhimurium DT104, IS6100-mediated genetic rearrangements within SGI1 described previously led to the formation of the SGI1-E variant and to the deletion of the retron sequence and neighbouring genes. Thus, IS6100 seems to give an additional recombination potential to the In104 integron, which may lead, in the future, to novel genetic rearrangements.

IS6100-mediated genetic rearrangement within the complex class 1 integron In104 of the Salmonella genomic island 1