The S. enterica serovar Albany strain 7205.00 used in this study was isolated from a food fish from Thailand. This strain and control strains S. Typhimurium DT 104 BN9181 (8,13,14), S. Agona 959SA97 (8,13,14), S. Paratyphi B 44 (14), and Escherichia coli strain TOP10 (Invitrogen SARL, Cergy-Pontoise, France) used in cloning experiments were grown at 37°C in brain heart infusion broth or agar plates. The strains were tested for their antibiotic susceptibility by the disc-diffusion assay on Mueller-Hinton plates. Susceptibility was tested by using discs containing the following antibiotics: Ap (10 µg), Cm (30 µg), Ff (30 µg), Sm (10 IU), Sp (100 µg), Su (200 µg), Tc (30 IU), and Tm (5 µg). All antibiotic disks except for Ff were purchased from Bio-Rad (Marnes-la-Coquette, France). Ff disks and the drug itself were obtained from Schering-Plough Animal Health (Kenilworth, NJ). MICs of Ff and Cm were determined by using the standard agar doubling dilution method. MIC breakpoints for Cm and Ff were defined by the Comité de l’Antibiogramme de la Société Française de Microbiologie (CASFM) or by the manufacturer (i.e., susceptible [MIC <8 µg/mL], intermediate [MIC = 16 µg/mL], or resistant [MIC >32 µg/mL]).

Detection of SGI1 and its location were performed by using primers corresponding to left and right (with or without retron) junctions in the chromosome as described (Table; Figure 1) (6,7,14). Polymerase chain reaction (PCR) mapping of the typical antibiotic resistance genes and integrons associated with SGI1 was performed by using conditions and primers as described (Table; Figure 1) (13–15). The antibiotic resistance gene organization was also assessed by Southern blots of genomic DNA cut by either XbaI, XhoI, HindIII or EcoRI by using as a probe the XbaI insert from recombinant plasmid pSTF3, comprising nearly the entire DT104 antibiotic resistance gene cluster, as described (13,14). Presence of SGI1 regions outside the antibiotic resistance gene cluster was assessed by Southern blot of genomic DNA cut by XbaI by using probe p1-9 as described (6,14). This probe contains a 2-kb EcoRI insert corresponding to a central region of SGI1, comprising parts of S023 and S024 open reading frames (ORFs), which code for putative helicase and exonuclease proteins (6).

PCR amplification of the first integron was also performed by using primer int1 of PCR A and primer F3 of PCR B (Figure 1). Cloning of this PCR product in plasmid pCR2.1-TOPO was performed by using the TOPO TA cloning kit (Invitrogen). We used Genome Express (Meylan, France) for nucleotide sequencing of the insert.

Genomic DNA, contained in agarose plugs, was digested with BlnI or XbaI and fragments separated by pulsed-field gel electrophoresis (PFGE) performed by using the CHEF-DR III system (Bio-Rad, Hemel Hempstead, U.K.) at 6 V/cm for 24 h with pulse times of 10–30 sec. The nucleotide sequence of the S. Albany strain 7205.00 integron fragment harboring the Tm resistance gene has been deposited in GenBank under accession number AY146989.

SGI1 is the first genomic island containing an antibiotic resistance gene cluster identified in S. enterica; its acquisition in S. Typhimurium phage type DT104 was possibly an important trait in the worldwide epidemic of the resulting multidrug-resistant clone causing disease in animals as well as in humans. SGI1 has been further identified in other S. enterica serovars, such as in serovar Agona strains isolated from poultry in Belgium and in a serovar Paratyphi B strain isolated from a tropical fish in Singapore (6,13,14). The serovar Albany fish isolate from Thailand in this study represents the fourth S. enterica serovar in which SGI1 has been identified. The identification of SGI1 in several S. enterica serovars, shown by PFGE to be genetically distinct, suggests horizontal transfer of this region. That SGI1 has the same chromosomal location in the different serovars suggests that its insertion occured through site-specific recombination. Genes such as the tandemly arranged int and xis genes found adjacent to the DR-L of SGI1 may play a role in this recombination event because they encode a putative integrase and excisionase, respectively (6). Sequence analysis of the left and right junctions of SGI1 to the Salmonella chromosome provides additional clues about these recombination events in the different serovars. The right junction 18-bp DR-R direct repeat sequence found at the 3´ end of SGI1 appears to be a duplication of the last 18 bp of the thdf gene found upstream of SGI1. The left junction SGI1 18-bp DR-L direct repeat sequence is slightly different from the 3´-end of thdf in sensitive S. enterica serovars lacking SGI1 (Figure 2) but identical in all serovars carrying SGI1. This finding suggests that the origin of this sequence may be from the donor DNA. When SGI1 insertion takes place in a sensitive S. enterica serovar, the last 18 bp of its thdf gene would be duplicated and found at the 3´ end of SGI1 and replaced by the 18 bp of the donor DNA at the 5´ end of SGI1. In other words, the last 18 bp of thdf in sensitive S. enterica serovars may constitute a hotspot for homologous recombination with an 18-bp similar sequence of the SGI1 donor DNA. These DR-R and DR-L minor sequence differences also support the hypothesis that the SGI1 insertions in serovar Typhimurium DT104 and other serovars were separate events and not a result of genetic exchange between serovar Typhimurium DT104 and the other serovars (6). Yet, the origin of SGI1 remains to be determined.

A similar situation has been reported for an approximately 100-kb genomic island in V. cholerae called SXT conjugative, self-transmissible, integrating (constin) element. It carries multiple antibiotic resistance genes, including floR as in SGI1 (18,19). Integration of the element has been experimentally shown to occur through site-specific recombination in a 17-bp sequence found in the circular form of the SXT element and a similar 17-bp sequence of prfC of the V. cholerae and E. coli chromosomes (20). Chromosomal integration and excision of the SXT element required an element-encoded int gene that is found as first gene of the SXT element, as is the int gene of SGI1 (18,20). SGI1 may also exist in an intermediate circular form, but this remains to be demonstrated.

The antibiotic resistance gene cluster of SGI1 contains mainly five antibiotic resistance genes (6–15). Variant SGI1 antibiotic resistance gene clusters have been recently reported in serovars Typhimurium DT104 and Agona containing part of the antibiotic resistance genes or an additional resistance gene (i.e., dfrA10 coding for Tm resistance) (12,15). These variant antibiotic resistance gene clusters were probably generated by recombinational events such as deletions and insertions. The serovar Albany strain of our study represents the first SGI1 example in which gene replacement took place in one of the integron structures. The dfrA1 and ORF gene cassettes found instead of aadA2 may have been introduced by homologous recombination with a class 1 integron containing the same array of gene cassettes from another bacterium (21). Another possibility is the exchange between aadA2 and the two gene cassettes, which would imply excision, mediated by the integron-encoded integrase, of aadA2 and its replacement by the other gene cassettes (22). The array of gene cassettes found in the integron of the serovar Albany strain were the same as those recently reported in integrons of V. cholerae isolated in Thailand and India (16,17). Considering the origin of the serovar Albany strain (i.e., fish exported from Thailand), a possible explanation could be the exchange of antibiotic resistance gene cassettes between epidemic multidrug-resistant V. cholerae strains and Salmonella strains from Thailand. Moreover, during the choleralike epidemic among the Khmers in 1982, children and pregnant women were reportedly treated with trimethoprim-sulfamethoxazole (16,23). A high percentage of Khmer outbreak V. cholerae strains showed resistance to trimethoprim-sulfamethoxazole; the strains that acquired the dfrA1 gene cassette likely became predominant through selective pressure (16,24).

The multidrug-resistant V. cholerae epidemics in humans in Asia might be largely responsible for spread of antibiotic resistance genes. Recent reports describe that human colonization by V. cholerae creates a hyperinfectious bacterial state, which is perpetuated even after purging into natural aquatic reservoirs and may contribute to epidemic spread of cholera (25). These aquatic reservoirs may be an ecologic niche where antibiotic resistance gene exchange takes place between different enterobacterial pathogens. In various seawater places around Hong Kong where untreated sewage is discharged, several enterobacterial pathogens were simultaneously detected such as Salmonella and V. cholerae (26).

As shown in the present study, gene replacement in the integron structures is another way to contribute to variability of the antibiotic resistance gene cluster of SGI1. SGI1 may thus serve as a vehicle of various antibiotic resistance genes in different S. enterica serovars, a situation somewhat similar to that reported for SXT constins and integrons of multidrug-resistant V. cholerae strains (16–20).