A total of 1,920 S. enterica isolates were investigated; 1,743 isolates were collected by laboratories in Argentina, Australia, Belgium, Canada, Denmark, Germany, Italy, the Philippines, South Africa, Spain, Taiwan, Uganda and the United States during September 2001–August 2002 (Table 1). These isolates were collected as part of a separate study that attempted to identify a genetically and geographically diverse group of S. enterica isolates with reduced susceptibilities to fluoroquinolones. The isolates were not selected but rather were collected consecutively, without regard to their antimicrobial drug susceptibility. In addition, 179 isolates were collected by the Allegheny County Department of Health in Pennsylvania during 2002–2003 as part of routine surveillance. Serotyping was performed by the collecting laboratories, except for isolates from Taiwan, which were serotyped by the Pennsylvania Department of Health.

The ACSSuT resistance phenotype has become increasingly prevalent in S. enterica, and that phenotype has been commonly associated with class 1 integron carriage in this species. For these reasons, we selected a subset of isolates from the collection that exhibited the ACSSuT resistance phenotype for further investigation. Isolates selected for integron investigation were confirmed to be S. enterica by PCR with primers specific for the invA region of the inv locus (16) (Table 2).

Antimicrobial drug resistance was determined by using the disc diffusion method on Mueller–Hinton agar (Becton, Dickinson and Co., Sparks, MD, USA), according to the manufacturer’s directions. Susceptibility to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline was determined according to the manufacturer’s breakpoints.

Genomic DNA from S. enterica isolates was prepared using the DNeasy Tissue Kit (QIAGEN, Valencia, CA, USA), according to the manufacturer’s directions. Class 1 integron carriage was determined by PCR using primers specific to the intI region of the integrase gene. Isolates positive for integrase were further characterized by PCR using primers specific for the 5′ and 3′ conserved segments (CS) of the integron structure. PCR was performed in a 50-µL volume consisting of 1× PCR buffer II (Applied Biosystems, Foster City, CA, USA), 1.5 mmol/L MgCl₂, 0.1 mmol/L dNTPs, 0.33 µM forward and reverse primers, and 1.66 units Amplitaq Gold polymerase (Applied Biosystems). PCR conditions were an initial denaturation at 95°C for 5 min; 35 cycles at 95°C for 1 min, 58°C for 1 min, and 72°C for 5 min plus 5 s each cycle (5 s longer in each subsequent cycle than in the previous cycle); and a final extension at 72°C for 7 min.

Long-range PCR was performed to detect integrons >2.0 kb by using the Gene Amp HiFidelity Kit (Applied Biosystems), according to the manufacturer’s directions. Long-range PCR conditions were an initial denaturation at 94°C for 2 min; 10 cycles at 94°C for 15 s, 58°C for 30 s, and 68°C for 4 min, followed by 20 cycles at 94°C for 15 s, 58°C for 30 s, and 68°C for 4 min plus 5 s each cycle (5 s longer in each subsequent cycle than in the previous cycle); and a final extension at 72°C for 7 min. PCR products were subjected to electrophoresis on 1% agarose gels, stained with ethidium bromide, and visualized using UV illumination on a Gel Doc 2000 documentation system (Bio-Rad, Hercules, CA, USA). For isolates that amplified multiple integrons, PCR products were separated by gel electrophoresis and purified by using either the Qiaquick Gel Extraction Kit (QIAGEN) or the Quantum Prep Freeze ’N Squeeze DNA Gel Extraction Spin Column Kit (Bio-Rad). Some PCR products were cloned before sequencing by using the Topo TA Cloning Kit for Sequencing (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s directions.

Isolates in this study were investigated for the presence of SGI1 and variant SGI1s by PCR using published primers specific for the right (104-RJ, 104-D) and left (U7-L12, LJ-R1) junctions of the chromosomal insertion site (19). Isolates were considered positive for the left or right junctions of the SGI1 if they generated PCR product of the appropriate size with primers specific for that junction. By this method, isolates with the SGI1 would be positive for the left junction but not for the right junction because of the presence of a retronphage between the 104-RJ and 104-D primer sites. Isolates with variant SGI1s would be positive for the left and right junctions.

Class 1 integron gene cassettes were identified by PCR and sequence analysis by using the 5′ and 3′ CS primers. Sequencing was performed with the BigDye terminator 3.1 kit (Applied Biosystems) according to the manufacturer’s instructions. Capillary sequence analysis was performed on a 3730 DNA sequence analyzer (Applied Biosystems). Sequences were analyzed and additional primers were designed by using the Lasergene 7.0.0 software package (DNAStar, Madison, WI, USA). Gene cassette homology searches were performed by using BLAST analysis (www.ncbi.nlm.nih.gov/BLAST) (20).

Ninety-one of the 121 integrons found in this study were sequenced in their entirety. In some cases, when integrons were identified that were of the same size as those previously sequenced, their gene cassettes were identified by PCR using primer pairs designed in this study (Table 2).

Genetic relatedness of isolates was assessed using multilocus sequence typing (MLST). MLST uses sequences ≈500 bp in length from 7 housekeeping genes to define a sequence type (ST). Isolates with the same alleles at all 7 loci are considered to be genetically indistinguishable by MLST and therefore define an ST. Isolates with the same alleles at 6 loci are considered to be closely related genetically.

MLST was performed using the 7-locus scheme described on the Salmonella MLST database (http://web.mpiib-berlin.mpg.de/mlst) (21–24). Visualization of PCR products and sequencing of gene fragments was accomplished as described above for integron gene cassettes. Sequences were analyzed using Bionumerics software V 5.10 (Applied Maths, Austin, TX, USA). Alleles and STs were assigned by the Salmonella MLST database. All isolates in this study and their associated sequence types have been deposited in the Salmonella MLST database.

Horizontal gene transfer was defined as S. enterica isolates belonging to different STs (STs that differ at >1 locus) but bearing the same integron. Clonal expansion was defined as S. enterica isolates bearing the same integron and belonging to the same ST or STs differing at only 1 locus but occurring in >1 location. Because isolates in this study were collected consecutively over a limited time in each location, and because source was the only epidemiologic information available, we could not determine whether genetically related isolates bearing the same integron in a given location were part of an outbreak or whether the isolates reflected clonal expansion beyond an outbreak. Therefore, S. enterica isolates collected from 1 location that belonged to the same ST and harbored identical integron structures were considered to be 1 isolate for classification as either horizontal gene transfer or clonal expansion.

Of the 1,920 isolates initially screened by antimicrobial drug susceptibility testing, 104 (4.9%) exhibited the ACSSuT resistance phenotype. The proportion of ACSSuT-resistant isolates ranged from 0% in Australia, Argentina, Belgium, and Canada to 19% in Taiwan and South Africa (Table 3).

Of the 104 nontyphoidal S. enterica isolates with the ACSSuT resistance phenotype, 90 (86.5%) were positive for the integrase gene and amplified gene cassette regions by PCR with primers for class 1 integron 5′ and 3′ CS regions. Most cassette-positive isolates contained either 1 (n = 61, 68%) or 2 (n = 26, 29%) integrons (Appendix Table). Three isolates contained >3 integrons. Although 16 different integrons were found in this collection, 19 distinct integron profiles could be identified because of multiple integrons in some isolates (Appendix Table). Six integrons contained only 1 gene cassette: aacA4, aadA2, aadB, blaPSE1, dfrA7, or dfrA15. Seven integrons contained 2 or 3 gene cassettes (listed in order of cassette occurrence in the individual integron): aadB/catB3, aac3A-Id/aadA7, blaOXA30/aadA1, dfrA12/orfF/aadA2, dfrA1/orfC, dfrA1/aadA1, and tnpA/dfrA7. A 4.0 kb integron containing the cassettes arr2/cmlA5/blaOXA10/aadA1was found alone or in combination with an integron containing the single gene cassette aacA4. Two other unique large integrons were found in this collection: a 5.8-kb integron with the gene cassettes qacH/dfrA17/ereA/aadA2/cmlA/aadA1 and a 6.0-kb integron with cassettes aac(6')-IIc/ereA2/IS1247/aac3/arr/ereA2.

A total of 121 class 1 integrons were identified. Of these, 91 were fully sequenced (Appendix Table). With 2 exceptions, genetic drift in gene cassette sequences was not observed. One integron containing the single cassette aadA2 showed 1 base difference from other aadA2 gene cassettes found in this study (a T→C transition at position 39 of the gene cassette, resulting in a synonymous change). One blaOXA30 gene cassette exhibited a point mutation (A→G) at position 31, also a synonymous change. All other gene cassettes within the study showed 100% nucleotide identity to GenBank reported cassettes, except for the dfrA17 gene cassette identified in the unique 5.8-kb integron in the Stanley isolates from Taiwan (Appendix Table). This cassette showed 91% sequence identity to a gene cassette found in uncultured bacteria (GenBank accession no. FM179325) and in E. coli (GenBank accession no. EU687490).

Most gene cassettes found in this study confer resistance to the aminoglycosides (aadA1, aadA2, aadA7, aadB, aacA4, aac, aac3A-Id, aac(6')-IIc), trimethoprim (dfrA1, dfrA7, dfrA12, dfrA15, dfrA17), and β-lactams (blaPSE1, blaOXA10, blaOXA30). Other resistance cassettes included those coding for chloramphenicol resistance (cmlA, cmlA5, catB3), erythromycin resistance (ereA2), rifampin resistance (arr2), and resistance to quaternary ammonium compounds (qacH). Because resistance to the aminoglycoside streptomycin and the β-lactam ampicillin were selection criteria for isolates in this study, the predominance of cassettes encoding resistance to those antimicrobial drugs is not unexpected.

Phenotypic resistance to chloramphenicol was also a selection criterion, but only 3 integrons contained gene cassettes for this resistance. None of the integrons identified in this study carried genes coding for tetracycline resistance, although phenotypic resistance to this antimicrobial drug was also an inclusion criterion. Because the SGI1 contains genes for chloramphenicol and tetracycline resistance, which are not located within integrons, tetracycline- and chloramphenicol-resistant isolates that are SGI1 positive most likely contain these genes. Alternatively, resistance to chloramphenicol and tetracycline may be encoded elsewhere on the chromosome or on structures other than integrons in isolates that are not positive for the SGI1.

The SGI1 was identified in 17 isolates (19%) (Appendix Table). Twelve of the SGI1-positive isolates were serovar Typhimurium, belonged to ST 19, and showed the integron pattern (blaPSE1, 1.0 kb and aadA2, 1.2 kb) characteristic of serotype Typhimurium phage type DT 104. An additional Typhimurium ST 19 isolate containing 4 integrons, including blaPSE1 and aadA2, was also positive for SGI1. Four serotype Albany isolates ST 292 were positive for a variant SGI1, which includes the integrons dfrA1/orfC and blaPSE1 (15). This result indicates chromosomal location of these integrons in these isolates.

The 90 integron-containing S. enterica isolates represented 17 different STs. Thirty-three (37%) Typhimurium isolates belonged to ST 19 or to STs that differ from 19 at only 1 locus. These isolates contained 10 different class 1 integrons, which combined to create 12 integron profiles (Appendix Table). Eleven (12%) isolates of serovar Isangi belonged to ST 216 or to STs that differ from 216 at 1 or 2 loci. These isolates contained 3 integrons and 4 integron profiles. The remaining isolates represented diverse STs, which differed from each other at 6 or 7 of the MLST loci.

This study identified 5 class 1 integrons that were distributed across different genetic lineages, supporting the role of horizontal gene transfer in the dissemination of antimicrobial drug resistance in nontyphoidal S. enterica. The class 1 integron dfrA12/orfF/aadA2, which confers resistance to trimethoprim and streptomycin/spectinomycin, was identified in S. enterica from 5 different serotypes belonging to 5 STs (Figure, panel A). This integron was geographically widespread, being found in isolates from Europe, the United States, Taiwan, the Philippines, and South Africa. The integron containing the single trimethoprim-encoding dfrA7 cassette was present in isolates from 2 serotypes, 2 STs and 3 different areas (Figure, panel B). The integron dfrA1/aadA1 was found in isolates from 3 serotypes, 2 STs, and 2 areas (Figure, panel C).

Isolates of ST 216, 335, 336, and 337 (serovar Isangi) and ST 19 (serovar Typhimurium) from South Africa contained an identical 4.0 kb integron not previously reported in S. enterica, with the cassettes arr2/cmlA5/blaOXA10/aadA1 (Figure, panel D). In some serovar Typhimurium ST 19 isolates, this integron was found in combination with the aacA4 integron. The presence of a unique integron in genetically unrelated isolates from the same area indicates that this integron may have been horizontally transferred.

An integron containing the single gene cassette aacA4 was found alone in isolates of serovar Isangi with ST 335 from Uganda and South Africa. This integron was also found in serovar Typhimurium isolates with ST 19, both alone and in combination with the integron arr2/cmlA5/blaOXA10/aadA1. These isolates belong to different genetic lineages, in that they differ at all 7 MLST loci. The occurrence of the same integron in these genetically unrelated isolates further supports a role for horizontal gene transfer in the dissemination of antimicrobial drug resistance in nontyphoidal S. enterica.

The serotype Typhimurium phage type DT104 integron pattern, with 2 class 1 integrons of sizes 1.0 and 1.2 kb and bearing the gene cassettes blaPSE1 and aadA2, was found in 12 (13%) serovar Typhimurium isolates of ST 19 (Table 4) and in 1 additional serovar Typhimurium ST 19 isolate, which also contained 2 other integrons. This result is consistent with the hypothesis that a common ancestor has undergone clonal expansion in a number of areas.

Serovar Typhimurium isolates of ST 19 from Taiwan and ST 328 from the Philippines contained 2 class 1 integrons with the cassettes aadB/catB3 and blaOXA30/aadA1. The integron blaOXA30/aadA1 was also found alone in an isolate of serovar Typhimurium ST 328 from Taiwan and in combination with an integron containing the cassette aadB in an isolate of serovar Typhimurium ST 313 from South Africa. ST 313 and ST 328 are closely related to ST 19, differing from it at only 1 locus (ST 313 differs from ST 19 at the MLST locus sucA, ST 328 and ST 19 differ at MLST locus aroC; see Appendix Table). Therefore, these isolates are all closely related, and their integrons may represent an instance of clonal expansion.

Although the class 1 integron dfrA12/orfF/aadA2 appears to have been circulated through horizontal gene transfer, this integron has also spread through clonal expansion of nontyphoidal S. enterica. This integron appeared in isolates of serovar Schwarzengrund ST 96 in Taiwan and Denmark and in serovar Typhimurium isolates of ST 19 in the United States and South Africa. Similarly, integrons with the single gene cassettes aacA4 and dfrA7 also exhibited both horizontal gene transfer and clonal expansion. The aacA4 integron was found in serovar Isangi isolates of ST 335 in Uganda and South Africa. The dfrA7 integron was found in serovar Enteritidis isolates of ST 11, also in Uganda and South Africa.