Evidence for a Chromosomally Located Third Integron in Salmonella enterica Serovar Typhimurium DT104b

Abstract

Salmonella enterica serovar Typhimurium isolates of phage types DT104 and DT104b are frequently associated with multiple antimicrobial resistance. We describe the characterization of a class 1 integron containing dfrA1 and aadA1, genes from two Salmonella serovar Typhimurium DT104b isolates. Genetic mapping located the integron to the bacterial chromosome in each case.

Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) and the closely related DT104b are recognized as international zoonotic pathogens (7). These phage types are often associated with simultaneous resistance to five antimicrobial agents, ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT resistance profile [R type]). The corresponding determinants for multiple drug resistance (MDR) are clustered in part of a 43-kbp Salmonella genomic island 1 (SGI1) on the bacterial chromosome (1). The development of the island probably occurred (2, 13) as a result of several recombination events between plasmid DNA and other mobile genetic elements including class 1 integrons (3, 10, 12). In addition to the MDR phenotype of DT104 and DT104b (4), isolates containing (unintegrated) plasmids conferring extended resistance to other antimicrobials including kanamycin and trimethoprim have been isolated (M. Daly and S. Fanning, unpublished data).

Involvement of mobile genetic elements facilitates the horizontal transmission of drug resistance markers among diverse gram-negative isolates (5). In this note, we report the detection of a third class 1 integron containing two gene cassettes in DT104b isolates of bovine origin. Genetic mapping was undertaken to establish the location of these resistance determinants.

Salmonella serovar Typhimurium isolates of phage type DT104b investigated in this study were obtained from the Cork Regional Veterinary Laboratory as part of ongoing research work. Both isolates were obtained from bovines and were originally cultured from feces (T180) and body fluid (T181) in May 2001.

All molecular methods used in this study were described previously (4-6, 8, 10).

Three amplicons were detected in the two Salmonella serovar Typhimurium DT104b study isolates, T180 and T181 (ACSSuT plus trimethoprim resistance [Tm] [ACSSuTTm]; Fig. 1, lanes 1 and 2). Two of these amplicons, of 1.0 and 1.2 kbp (encoding resistance to aminoglycoside and β-lactam antimicrobials, respectively), were detected previously in MDR DT104- and DT104-related isolates (5). The third amplicon, of 1.6 kbp, was fully characterized in this study and found to contain two open reading frames (ORFs), dfrA1 (474 bp) and aadA1 (792 bp), fused in a classical “head-to-tail” arrangement. The 59-base element recombination site was also located on the distal side of each ORF (Fig. 1). The former amplicons alone were present in the Salmonella serovar Typhimurium DT104b isolate (ACSSuT), as shown in lane 3 (Fig. 1).

FIG. 1.

Amplified fragments from the cassette region of class 1 integrons in Salmonella serovar Typhimurium DT104b. The corresponding R types are shown below the figure. Lane 1, CIT-T180; lane 2, CIT-T181; lane 3, CIT-T176. A schematic illustration of the 1.6-kbp amplicon is included, showing the two gene cassettes dfrA1 (474-bp; grey bar) and aadA1 (792 bp; open bar) in a head-to-tail arrangement, along with their 59-base elements (stippled box), located at the 3′ end of each ORF. The corresponding ORFs associated with the remaining amplicons (including the bla-_PSE shaded arrow) are also shown.

XbaI pulsed-field profiles of Salmonella serovar Typhimurium DT104b isolates T180 and T181 were obtained and compared against each other and unrelated DT104b ACSSuT and non-ACSSuT isolates. These profiles were indistinguishable both from each other (Fig. 2a, lanes 3 and 4) and from previously characterized MDR DT104 isolates (data not shown). Probing with the aadA1 gene identified the 10-kbp XbaI macrorestriction DNA fragment corresponding to most of the original SGI1 resistance island (1, 2) (Fig. 2a, lanes 5, 7, and 8). This resistance island occurs in all SGI1 MDR DT104 and DT104b isolates of R type ACSSuT. In contrast, three additional signals were detected for the Salmonella serovar Typhimurium DT104b isolates, T180 and T181 (Fig. 2a, lanes 7 and 8). Two of these signals appeared to be located as a doublet migrating close to the 97-kbp molecular size marker (Fig. 2a). The third signal was located at approximately 270 kbp (Fig. 2a, lanes 7 and 8). All three DNA fragments were similarly detected in the isolates T180 and T181 with the dfrA1 gene probe (data not shown). Notably, this probe did not hybridize to the 10-kbp XbaI DNA fragment of SGI1 nor to the Salmonella serovar Typhimurium DT104b isolate CIT-T179 (Fig. 2a, lane 6). In this case the dfrA1 marker was located on a plasmid (unpublished data). Furthermore, large and small plasmids isolated from T180 and T181 were similarly analyzed with dfrA1 and aadA1 gene probes. No corresponding signals were detected with either probe (data not shown), suggesting that these markers were not associated with plasmids in this instance.

FIG. 2.

Macrorestriction analysis by PFGE and Southern blot hybridization with aadA1 and dfrA1 gene probes. The lanes marked M contain digoxigenin-labeled DNA molecular size marker grade II (Roche Diagnostics, Lewes, East Sussex, United Kingdom). Unlabeled midrange PFG Markers (New England BioLabs, Hertfordshire, United Kingdom) in lane M* were included for fragment sizing before Southern transfer. (a) PFGE patterns of XbaI-digested DNA fragments of Salmonella serovar Typhimurium DT104b isolates are shown in the left panel, and the corresponding probe signals detected after hybridization with the aadA1 gene probe are in the right panel. Lanes 1 and 5, CIT-T178; lanes 2 and 6, CIT-T179; lanes 3 and 7, CIT-T180; lanes 4 and 8, CIT-T181. (b) Southern blot of CeuI-macrorestricted DNA fragments probed with aadA1 and dfrA1 gene probes. Hybridization signals detected by using the aadA1 gene probe are shown in the left panel, and similarly, the hybridization results obtained by using the dfrA1 gene probe are shown in the center panel, with corresponding PFGE profiles in the right panel. Lanes (phage types are given in parentheses): 1, 6, and 11, CIT-V37 (PTU302); 2, 7, and 12, CIT-F44 (DT104); 3, 8, and 13, CIT-T180 (DT104b); 4, 9, and 14, CIT-T181 (DT104b); 5, 10, and 15, CIT-HW264 (DT104) R types are given below the blot. TTm, tetracycline and trimethoprim resistance; ASu, resistance to ampicillin and sulfonamides. See the text for explanation of other R types.

To establish if these unique XbaI DNA fragments in T180 and T181 were located in close proximity to the original SGI1 resistance island, a second restriction enzyme, CeuI, was used to generate a pulsed-field gel electrophoresis (PFGE) profile that was probed as before. CeuI is a rare-cutting enzyme which produces very large restriction fragments. Similar to the XbaI profiles, the CeuI profiles were indistinguishable (Fig. 2b, lanes 11 through 15). Probing with aadA1 located SGI1 on a CeuI DNA fragment of 582 kbp (Fig. 2b, lanes 1, 3, 4, and 5) in Salmonella serovar Typhimurium DT104 and related isolates of R type ACSSuT. An additional second signal was also detected on a 242.5-kbp CeuI DNA macrofragment, identifying the location of the second aadA1 marker in the Salmonella serovar Typhimurium DT104b isolates T180 and T181 (Fig. 2b, lanes 3 and 4). The dfrA1 probe also hybridized to the latter fragment in T180 and T181 alone (Fig. 2b, lanes 8 and 9), uniquely locating the third integron to this new chromosomal locus. In contrast, this was not the case for CIT-V37 (R-type ACSSuTTm) (Fig. 2b, lane 1), since the Tm marker in this case was located on a plasmid (data not shown).

The SGI1 MDR gene cluster of Salmonella serovar Typhimurium DT104 isolates is well characterized (1, 2). In this study we described a class 1 integron containing the resistance genes dfrA1 and aadA1. Similar integrons were previously identified in Salmonella enterica serovars Panama, Ohio, and Brandenburg, Escherichia coli, and Klebsiella spp., among others (11, 15), and also more recently in Salmonella enterica serovar Bredeney and Salmonella serovar Typhimurium DT193 (Daly et al., unpublished data). Probing with the aadA1 gene probe identified the original SGI1 resistance island, together with additional signals at approximately 97 and 270 kbp in the Salmonella serovar Typhimurium DT104b isolates T180 and T181. Similarly, the dfrA1 gene probe detected the latter XbaI DNA fragments, but importantly it did not hybridize to the original SGI1 resistance island, suggesting that the dfrA1 resistance marker only maps to this new chromosomal locus. Furthermore, long-range PCR experiments (data not shown) demonstrated that the dfrA1-aadA1-containing integron did not disrupt the gene order of SGI1. Based on our observations, it seems reasonable to suggest that unlike the SGI1-derived variants described previously (1), our data identified an independent recombination event outside the original MDR cluster in Salmonella serovar Typhimurium DT104b. Although our findings did not confirm the exact chromosomal location of the third integron, neither do they exclude the possibility that all of these resistance genes identified are in close proximity. These data may at least in part signal an important step in the development of MDR chromosomal clusters (9, 14).