Salmonella enterica Serotype Typhimurium DT 104 Antibiotic Resistance Genomic Island I in Serotype Paratyphi B

We have identified Salmonella genomic island I (SGI1) in an isolate of Salmonella enterica serotype Paratyphi B. This antibiotic-resistance gene cluster, which confers multidrug resistance, has been previously identified in S. enterica serotype Typhimurium phage types DT 104 and DT 120 and in S. enterica serotype Agona.

and a truncated sulI resistance gene. The second contains the beta-lactamase gene bla PSE-1 and a complete sulI gene. Flanked by these two integron structures are the floR gene (6), also called floSt (11) or cmlA-like (9), which confers crossresistance to chloramphenicol and florfenicol, and the tetracycline-resistance genes tetR and tet (G). Florfenicol resistance and detection of the floR gene by polymerase chain reaction (PCR)-based methods have been proposed as a means for rapidly identifying multidrug-resistant S. Typhimurium DT 104 strains (11), since phage typing is available only in specialized laboratories. However, recently SGI1 has been reported in another phage type of serotype Typhimurium and in serotype Agona, suggesting horizontal transfer of SGI1 (7,10,12). In serotype Agona, SGI1 has the same chromosomal location as in DT 104 strains, except that it lacks the retron sequence found downstream of SGI1; thus it is located between the thdf gene and the yidY gene of the chromosome (7).
Recently, Nastasi and Mammina reported the presence of the floR and tet(G) genes detected by PCR in an S. enterica serotype Paratyphi B strain of biovar Java isolated from a tropical fish in Singapore (13). We examined this isolate to determine the presence of SGI1.

The Study
The antibiotic-resistance phenotype of the serotype Paratyphi B strain was assessed by the disk-diffusion assay. All antibiotic disks except for florfenicol were purchased from Bio-Rad (Marnes-La-Coquette, France). Florfenicol disks and the drug itself were obtained from Schering-Plough Animal Health (Kenilworth, NJ). The serotype Paratyphi B strain showed a multidrug-resistance profile typical of serotype Typhimurium DT 104 or serotype Agona strains carrying SGI1, i.e., resistance to ampicillin, chloramphenicol and florfenicol, streptomycin and spectinomycin, sulfonamides, and tetracyclines. Moreover, this strain showed the same resistance level to florfenicol as serotypes Typhimurium DT 104 and Agona, i.e., a florfenicol MIC 32 µg/mL (12). The strain was susceptible to trimethoprim and the quinolones nalidixic acid, enrofloxacin, and ciprofloxacin.
No plasmids were detected in the serotype Paratyphi B strain, suggesting that all antibiotic-resistance genes were chromosomally located. PCR mapping of the typical antibiotic resistance genes and integrons associated with SGI1 was performed as described ( Figure 1) (12). PCR amplifications yielded products from genomic DNA extracted from the serotype Paratyphi B strain of the size expected from DNA of serotype Typhimurium DT 104 control strain BN9181 (data not shown) (6,12). Partial nucleotide sequencing of the floR gene showed 100% identity with that of serotype Typhimurium DT 104. Thus, these PCR mapping results indicated that the serotype Paratyphi B strain contains the entire antibiotic-resistance gene cluster of serotype Typhimurium DT 104.
The conservation of the antibiotic-resistance genes' organization was further assessed by Southern blot of HindIIIor XhoI-digested genomic DNA, with the 12-kb XbaI insert as *Institut National de la Recherche Agronomique, Nouzilly, France; †Health Canada, Winnipeg, Manitoba, Canada; ‡University of Palermo, Palermo, Italy; and §University of Florence, Florence, Italy M DISPATCHES probe (see XbaI fragment, Figure 1) from recombinant plasmid pSTF3 containing the DT 104 antibiotic-resistance gene cluster as described (6,12). The Southern blot profiles of the serotype Paratyphi B strain were identical to those of the DT 104 control strain BN9181 and serotype Agona strain 959SA97 (Figure 2), confirming that the organization of the antibioticresistance genes in the serotype Paratyphi B strain is the same as in serotype Typhimurium DT 104 or serotype Agona containing SGI1 (7,12).
To assess the presence of the entire SGI1 and its location in the chromosome of the serotype Paratyphi B strain, PCR was performed by using primers corresponding to the left and right junctions of SGI1 in the Salmonella chromosome. PCR took into account the presence or absence of the int2-retron sequence, which is located downstream of SGI1 in serotype Typhimurium DT 104 but not in serotype Agona ( Figure 1) (7,8). PCR was positive for the left junction of SGI1, as for serotype Typhimurium DT 104 or serotype Agona. If a sequence of the int2 gene of the retron was used as reverse primer, PCR was negative for the right junction of SGI1, but it was positive if the sequence of the yidY gene was used. The PCR products showed the expected sizes of approximately 500 bp for both the left junction and right junction PCR without the retron sequence in Figure 1. These data indicate that the serotype Paratyphi B strain contains SGI1 at the same chromosomal location as in serotype Typhimurium DT 104 or serotype Agona, that is, between the thdf and yidY genes, but lacks the retron sequence found in DT 104 strains and other serotype Typhimurium strains (7,8). The presence of entire SGI1 was also confirmed by Southern blot of XbaI-digested genomic DNA with the p1-9 probe containing a 2-kb EcoRI insert. This corresponds to a central region of SGI1, comprising parts of the S023 and S024 open reading frames, which code for putative helicase and exonuclease proteins, upstream of the antibiotic-resistance gene cluster (7). This probe revealed XbaI fragments of the 4-and 9-kb sizes expected in the serotype Paratyphi B strain and the control serotype Typhimurium DT 104 and serotype Agona strains ( Figure 2B).
Macrorestriction analysis by pulsed-field gel electrophoresis of the serotype Paratyphi B strain DNA cut by XbaI showed that it is genetically distinct from both serotypes Typhimurium DT 104 and Agona (Figure 3), further indicating at the molecular level that the occurrence of SGI1 in the serotype Paratyphi B strain probably results from horizontal transfer and not seroconversion of known S. enterica serotypes containing SGI1.

Conclusions
We have identified SGI1 in a S. Paratyphi B strain. These data, in conjunction with the identification of SGI1 in S. Agona and S. Typhimurium strains, suggest horizontal transfer of this region (7,10,12). That SGI1 has the same chromosomal location in S. Typhimurium, S. Agona, and S. Paratyphi B suggests that its insertion occurred through a homologous recombination event, perhaps through phage transduction (14). This hypothesis is experimentally supported by the fact that resistance genes of serotype Typhimurium DT 104 can be efficiently transduced by P22-like phage ES18 and phage PDT17, which is released by all DT 104 isolates analyzed (14). However, the question remains why the retron sequence downstream of SGI1 in serotype Typhimurium DT 104 and DT 120 strains (7) is not present downstream of SGI1 in other serotypes. A possible explanation could be that in the horizontal transfers described here, the SGI1 donor strains are not serotype Typhimurium strains. Once SGI1 has been acquired, it may become stable in the chromosome, as in vitro excision experiments failed to demonstrate its loss in a DT 104 strain in the absence of antibiotic selective pressure (8). This factor may contribute to evolution of S. enterica pathogens, similar to the acquisition of pathogenicity islands (15,16). In addition, the use of antimicrobial agents in agriculture might be of importance for the selection and spread of SGI1 among S. enterica serovars (17). The export of contaminated food or animals contributes to the spread of such multidrugresistant serovars and may be responsible for worldwide epidemics such as that caused by serovar Typhimurium phage type DT104. For example, florfenicol is a veterinary antimicrobial agent that has been used in aquaculture in Asia since the early 1980s and may have contributed to selection and horizontal transfer of SGI1 to phage type DT104 (7). Serovar Paratyphi B is the most frequently isolated serovar from imported seafood samples from Asian countries (18).
Acquisition of SGI1 may have been a key factor contributing to the DT 104 worldwide epidemic, perhaps not only through selection by agricultural use of antimicrobial agents (17) but also by possible virulence properties of SGI1 (10). Therefore, further surveillance is warranted for the emergence of horizontal transfer of SGI1 to S. enterica serotypes of public health importance.