Characterization of Variant Salmonella Genomic Island 1 Multidrug Resistance Regions from Serovars Typhimurium DT104 and Agona

Strains of multidrug-resistant Salmonella enterica serovar TyphimuriumDT104 (DT104) and S. enterica serovar Agona (Agona) have beenfound to harbor Salmonella genomic island 1 (SGI1), a 43-kbgenomic region that contains many of the drug resistance genes.Such strains are resistant to ampicillin (pse-1), chloramphenicol/florfenicol(floR), streptomycin/spectinomycin (aadA2), sulfonamides (sul1),and tetracycline [tet(G)] (commonly called the ACSSuT phenotype).All five resistance genes are found in a 13-kb multidrug resistance(MDR) region consisting of an unusual class I integron structurerelated to In4. We examined DT104 and Agona strains that exhibitedother resistance phenotypes to determine if the resistance geneswere associated with variant SGI1 MDR regions. All strains werefound to harbor variant SGI1-like elements by using a combinationof Southern hybridization, PCR mapping, and sequencing. VariantSGI1-like elements were found with MDR regions consisting of(i) an integron consisting of the SGI1 MDR region with the additionof a region containing a putative transposase gene (orf513)and dfrA10 located between duplicated qacE ${Delta}$ 1/sulI genes (SGI1-A;ACSSuTTm); (ii) an integron with either an aadA2 (SSu) or apse-1 (ASu) cassette (SGI1-C and SGI1-B, respectively); (iii)an integron consisting of the SGI1-C MDR region plus an orf513/dfrA10region as in SGI1-A (SGI1-D; ASSuTm; ampicillin resistance dueto a TEM ß-lactamase); and (iv) an integron relatedto that in SGI1 but which contains a 10-kb inversion betweentwo copies of IS6100, one which is inserted in floR (SGI1-E;ASSuT). We hypothesize that the MDR of SGI1 is subject to recombinationalevents that lead to the various resistance phenotypes in theSalmonella strains in which it is found.

INTRODUCTION

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Introduction

In the 1990s, the prevalence of multidrug-resistant (MDR) Salmonellaenterica serovar Typhimurium phage type DT104 (hereafter calledDT104) has increased dramatically in the United Kingdom (45,46), the United States (18, 22), and Canada (29). Many countrieshave also documented outbreaks associated with MDR DT104 inpoultry, beef, cheese, and swine (11, 13, 19, 26, 48). Althoughcase-control studies have suggested that MDR DT104 is more virulentthan sensitive strains of DT104 or other Salmonella serotypes(15, 49), another report suggested that the percentage of bacteremiawas no higher than with other Salmonella serotypes (42).

Nonetheless, encouraging findings have come from the UnitedKingdom which have documented a 22% decrease in 1999 from 1996levels of DT104 displaying the multidrug resistance phenotype(43). MDR DT104 strains have been described as resistant toa core group of antimicrobial agents, including resistance toampicillin, chloramphenicol/florfenicol, spectinomycin/streptomycin,sulfonamides, and tetracycline (commonly abbreviated ACSSuT).In addition to ACSSuT strains, others have been identified whichare resistant to fluoroquinolones, trimethoprim, and aminoglycosides(12, 17, 29, 44). The majority of ACSSuT DT104 isolates havea similar MDR region in their genomes comprised of the floRand tet(G) genes bracketed by the pse-1 and aadA2 gene cassettesof two class 1 integrons clustered in a 13-kb region (2, 6,28, 36, 38).

A number of Salmonella enterica serovar Agona (hereafter referredto as Agona) strains harboring the same antimicrobial resistanceregion have been characterized, suggesting horizontal gene transferof this region (10). The element containing the MDR region,Salmonella genomic island 1 (SGI1), has been cloned from thegenome of a Canadian isolate and comprises a 43-kb region betweenthdF and a novel retron sequence (5). Recently, the entire SGI1element has been sequenced and, in addition to the MDR region,contains at least 25 or more open reading frames (ORFs), includingan integrase gene and an excisionase gene, some of which showedsimilarity to genes commonly found on conjugative plasmids (4).

Among the members of the family Enterobacteriaceae and pseudomonads,transfer of antibiotic resistance genes is due largely to broad-host-rangeplasmids that carry transposons (14). Many of the resistancegenes in the transposons have been found to be mobile gene cassettescarried as part of integrons (16). Four classes of integronshave been described based on the similarities between theirintegrases, but the majority described belong to class 1, andthis type has been found in multiple Enterobacteriaceae, includingEscherichia coli, Citrobacter spp., Klebsiella spp., Enterobacterspp., Proteus spp., and Salmonella spp. (14, 20, 27, 40, 50).

Class 1 integrons contain a 5' conserved segment (5'-CS) whichconsists of the intI1 gene, encoding the site-specific integrase,and the associated attI1 site, the primary site of recombination,and the 3' conserved segment (3'-CS) of variable length butgenerally consisting of qacE ${Delta}$ 1, encoding low-level resistanceto some antiseptics; the sul1 gene, encoding sulfonamide resistance;and orf5, a gene of unknown function (16). The gene cassettes,of which over 60 have been described (16, 35), consist of thecoding region and the downstream 59-base element (59-be), whichis responsible for recognition and mobilization of cassettes.The IntI1-catalyzed recombination between the attI1 and 59-besites is the main reaction responsible for inserting gene cassettesinto the integron (33).

Transposon Tn402 is a mobile class 1 integron that containsthe 5'-CS and a transposition module consisting of four genes(tniA, -B, -Q, and -R) required for transposition (7). In addition,Tn402 is bound by inverted repeats of 25 bp, IRi at the integraseend and IRt at the tni end. Several class 1 integrons appearto have originated from a Tn402-like ancestor by incorporationof the common part of the 3'-CS, including qacE ${Delta}$ 1, sul1, andorf5. Most of these integrons, though still bound by IRi andIRt, have lost part or all of the tni module and are deemeddefective transposon derivatives (7).

Some of these integrons have been analyzed and shown to havean identical 5'-CS and 2-kb of the 3'-CS, after which this regiondiverges (21). One group, the In5 type, have lost parts of the3'-CS and tni module, likely due to IS1326-mediated and/or IS1353-mediateddeletions (7, 32). Another group, the In4 type, have a 3'-CSthat includes a copy of IS6100 but no transposition genes (34).In In4 itself, the IS6100 element is flanked by short segmentsfrom the IRt end of Tn402, the outer one being 152 bp and theinner one 123 bp, in inverse orientation to one another. Inother members of this group, only one or neither of the IRtends may be present, and the IS6100 element does not containthe partial copy (34). Thus, the SGI1 MDR region has a structuresimilar to that of an In4 integron (4, 5).

Since a number of DT104 strains have been identified which exhibitdifferent resistance phenotypes, e.g., SSu, ASu, and ASSuT (3,12, 36), we have examined a number of Typhimurium and Agonastrains for the presence of variant SGI1s. Furthermore, theMDR regions were characterized to determine their genetic structure,and a nomenclature system is suggested for SGI1 variants.

MATERIALS AND METHODS

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Materials and Methods

Bacteria and media. The Salmonella strains used in this study are listed in Table1. All strains were grown at 37°C in brain heart infusionbroth or Luria-Bertani (LB) medium. Stock cultures were storedat -70°C in Microbank vials (Pro-Lab Diagnostics, RichmondHill, Ontario, Canada).

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TABLE 1. Strains used in this study and Southern hybridization data with various probes

Antimicrobial susceptibility testing. Isolates were tested for susceptibility to various antimicrobialagents on Mueller-Hinton agar by the disk diffusion method aspreviously described (28). Disks containing: ampicillin at 10µg, chloramphenicol at 30 µg, florfenicol at 30µg, spectinomycin at 100 µg, streptomycin at 10IU, sulfonamides at 200 µg, and tetracyclines at 30 IUwere purchased from Sanofi Diagnostics Pasteur (Marnes-la-Coquette,France), except for disks with florfenicol, which were fromSchering-Plough Santé Animale (Segré, France).

DNA methodology. Genomic DNA was isolated as previously described (5). Primersused in the study are listed in Table 2. Standard PCRs and longPCR were carried out as previously described (4). Southern blottingwas carried out by standard methods (37) with probes labeledand detected by ECL kits using the manufacturer's instructions(Amersham Pharmacia Biotech). Probes were made by PCR usingthe primer pairs listed in Table 2.

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TABLE 2. Primers used in this study

Computer-aided analysis and annotation. Homology searches were carried out using the Blast suite ofprograms (1), and open reading frames (ORFs) were detected withORFinder via the World Wide Web interface of the National Centerfor Biotechnology Information (http://www.ncbi.nlm.nih).

Nucleotide sequence accession number. The complete nucleotide sequence of the region harboring thetrimethoprim resistance determinant has been deposited in theGenBank database under accession number AY049746.

RESULTS

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Results

DT104 and Agona strains isolated in Scotland and Belgium displayingthe ACSSuT, ACSSuTTm, ASu, SSu, ASSuTm, and ASSuT phenotypeswere characterized at the genetic level in this study. The strainswere shown to possess the SGI1 or a variation thereof by usinga combination of Southern blotting and PCR (Table 1). All strainsstudied displayed a PCR product using primers U7-L12 and LJ-R1(left junction) and 104-RJ and C9-L2 or 104-RJ and 104-D primers(right junction), indicating the presence of the left and rightSGI1 junction regions (Fig. 1 ). This result demonstrated thatthe SGI1 was located between thdF and a novel cryptic retronphagein all of the DT104 variants studied and located between thdFand yidY in the Agona strains, in which the retronphage is absent(5). In addition, all strains displayed an 8.9-kb and a 4.1-kbfragment when Southern blots of XbaI-digested genomic DNA wereprobed with p1-9, a probe spanning ORFs S023 to S024, suggestingthe majority of the SGI1 is present (Fig. 1; Table 1) (4).

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FIG. 1. Genetic organization of the MDR regions of the various strains in this study based on Southern hybridization data, PCR mapping, and sequence analysis. A schematic of SGI1 is shown at the top, with the approximate locations of the primers used to detect the left and right junction regions indicated. The cryptic retronphage region is absent in Agona strains. Direction of transcription of genes is indicated by arrows. Locations of PCR products used as probes are indicated (thin black bars), as are the locations of some primers used in mapping. IRi and and IRt are 25-bp imperfect inverted repeats defining the ends of class 1 integrons. Regions sequenced are indicated under the various MDR regions by a thick black bar. The nucleotide sequence of the insertion point of the IS6100 element in the floR gene of S/960081 is shown; the 8-bp region duplicated upon insertion is boxed (coordinates are for the floR gene only). IR-L and IR-R refer to the inverted repeats that define one end of IS6100. The sequence of the orf513/dfrA10 region from Agona 1169SA97 has been assigned accession no. AY049746.

Thus, Agona 959SA97, previously shown to harbor the same MDRregion as in ACSSuT DT104 (4), contains SGI1 (Fig. 1) (10).In the other strains studied, differences in SGI1 structurewere revealed with Southern hybridization analysis of XbaI-digestedgenomic DNA using probes directed against genes found in theMDR region (Table 1). These variable MDR regions will be discussedin detail below.

MDR region coding for ACSSuTTm. Agona 1169SA97 probed with aadA2, floR, or tet(G) showed thesame sizes of hybridizing bands as for Agona 959SA97; however,when probed with pse-1, qac/sul1, or IS6100/S044, an 8.8-kbfragment hybridized with the probe, which was approximately4.5 kb larger than the expected 4.3-kb XbaI fragment (Table1). PCR of this region with primers pse-L and MDR-B produceda major band of 8.9 kb and a minor band of 4.4 kb (data notshown). Sequence analysis of the purified 8.9-kb product revealeda 2,868-bp region inserted between copies of the qacE ${Delta}$ 1 and sul1genes that contains two other genes, dfrA10, encoding resistanceto trimethoprim, and orf513, a putative transposase (Fig. 1).This structure is identical to one found in In7 (31).

A 2,154-bp segment of this region, encompassing orf513, is alsofound upstream of the cat gene in In6 (31, 42), and the ampCand ampR genes in the integron in pSAL (47). The Orf513 proteinis 65% identical to the OrfA protein, whose gene and a truncatedversion of it surround a floR gene from a plasmid found in E.coli isolated from cattle (9), and 55% identical to the Orf2protein found in the SGI1 MDR region (4). An alignment of theseproteins is shown in Fig. 2. All these proteins have been annotatedas putative transposases or transposase-like, presumably dueto homology to known transposases. Interestingly, all membersof this group of proteins are found associated with resistancegenes.

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FIG. 2. Alignment of Orf513, OrfA, and Orf2 proteins. Identical residues in two or three of the proteins are boxed. Coordinates are at the right end of each line.

The size of a PCR product using the pse-L/MDR-B primer set is4.4 kb with DNA from DT104 96-5227, the same size as a minorband amplified when using 1169SA97 DNA. Restriction enzyme analysisof these two amplicons revealed identical patterns (data notshown). Thus, it appears that a small subpopulation of Agona1169SA97 cells may exist in which the orf513/dfrA10 region isdeleted. We propose to name the genomic island found in Agona1169SA97 SGI1-A.

MDR regions coding for ASu, SSu, and ASSuTm. DT104 strain S/960725, which was resistant to ampicillin andsulfonamides (ASu), was shown to contain SGI1 sequences upstreamof the MDR region by PCR and Southern hybridization (Table 1).Southern blots of XbaI-digested S/960725 chromosomal DNA probedwith either aadA2, floR, or tet(G) did not produce a hybridizationsignal, suggesting that these genes were absent in this strain.The hybridization signals generated when qac/sul1 or pse-1 (containsa XbaI site) were used to probe the blot suggested a singleintegron was present in this MDR region (Table 1). Indeed, PCRand sequence analysis of this region in DT104 S/960725 revealedthat a single complete pse-1-containing integron was presentbetween the res and IS6100 sequences (Fig. 1). We propose toname the genomic island found in DT104 S/960725 SGI1-B.

Two strains studied, Agona 0047SA97 and DT104 S/954435, displayedresistance to only two antimicrobials tested, streptomycin andsulfonamides (SSu). Southern blots of XbaI-digested genomicDNA did not reveal any sequences homologous to floR, tet(G),or pse-1, suggesting these sequences were absent in the strains(Table 1). Further probing of DT104 S/954435 and Agona 0047SA97with aadA2, qac/sul1, or IS6100/S044 showed hybridization productsof 6.6 kb, suggesting only one copy of qac/sul1 was presentand it was localized to the same XbaI fragment that containsthe aadA2 gene (Table 1). These results suggested a single integronmay be present in the MDR region of these strains, and PCR andsequence analysis of this region in DT104 S/954435 confirmedthat a single complete aadA2-containing integron was presentbetween res and IS6100 (Fig. 1). We propose to name the genomicisland found in DT104 S/954435 and Agona 0047SA97 SGI1-C.

One strain, Agona 953SA98, was resistant to ampicillin, streptomycin,sulfonamides, and trimethoprim (ASSuTm). PCR analysis showedsequences from an aadA2-containing integron were present, butreactions with primers from pse-1 were negative. However, PCRanalysis with TEM-specific primers were positive, revealingthat ampicillin resistance in this strain is due to a TEM-1-relatedß-lactamase (data not shown). Further PCR and sequenceanalysis showed that the trimethoprim resistance in Agona 953SA98was due the presence of the same orf513/dfrA10 region as foundin Agona 1169SA97 (Fig. 1). We propose to name the genomic islandfound in Agona 953SA98 SGI1-D.

MDR region coding for ASSuT. Strain DT104 S/960081 displayed a resistance phenotype of ASSuT,and initial PCR analysis of the MDR region suggested all ofthe genes were present and in the same order as in ACSSuT strains,including the floR gene. However, PCR using the StCM-L/StCM-Rprimer pair did not produce a product, suggesting that eithera mutated primer binding site was present in floR or a deletionexisted within floR that encompassed a primer-binding site.Southern hybridization results, however, suggested that eitherIS6100 or S044 was duplicated, as this probe hybridized with7.9-kb and 9.1-kb XbaI fragments, as opposed to a single 4.3-kbfragment as in the ACSSuT strains (Table 1). The floR probehybridized to the same two fragments, while the qac/sul1 probehybridized only to the 9.1-kb fragment, suggesting a possibleinversion had taken place within the MDR region which may haveinvolved a portion of the floR gene and the IS6100-S044 region.

To test this hypothesis, PCRs using primer pair MDR-7/DB-T1and primer pair F6/MDR-B were attempted, as both reactions werepostulated to produce amplicons <2.0 kb in size if the inversiondescribed above had occurred (Fig. 1). The MDR-7/DB-T1 reactionproduced a 1.6-kb fragment, and the F6/MDR-B reaction produceda 1.9-kb fragment, both of which were sequenced. Analysis revealedthat a second IS6100 element had interrupted floR, and an inversionof the region between the two IS6100 elements had taken place(Fig. 1 and Fig. 3). We propose to name the MDR region foundin DT104 S/960081 SGI1-E.

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FIG. 3. Schematic diagram of the generation of variant SGI1 MDR regions from SGI1. Mechanisms are discussed in the text.

DISCUSSION

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Discussion

Variant MDRs are In4-type integrons. Original analysis of the DT104 SGI1 MDR region revealed an IS6100element in a position identical to that in In4 except for theabsence of the partial IS6100 copy (4, 5) (Fig. 1). Furtheranalysis revealed the presence of IRi (positions 26637 to 26661of the sequence with accession no. AF261825) between the resand intI1 genes of the aadA2 integron (Fig. 1). Thus, the entireMDR region of SGI1 (positions 26637 to 41698 of the sequencewith accession no. AF261825) can be considered a single integronbelonging to the In4 group (34). Additionally, this region isflanked by 5-bp direct repeats, ACTTG, strongly suggesting thatit has been integrated by a transposition event. Hence, theMDR regions of the variant SGI1s described here can be describedas In4-type integrons (34).

Generation of variant MDRs. SGI1 (ACSSuT) has been found in DT104, DT120, and Agona serovarsof S. enterica (4, 5, 10). In addition, we have detected SGI1in a strain of S. enterica serovar Paratyphi B isolated froma tropical fish (25). In every case, SGI1 is located at theend of the thdF gene, suggesting site-specific insertion possiblyinvolving the product of the int gene found at one end of thegenomic island (Fig. 1) (4, 5). Thus, it seems likely that whateverthe donor strain, SGI1 exists on a transferable element andis acquired by other serovars via horizontal transfer. As thecryptic retronphage downstream of SGI1 has been found only inTyphimurium strains, it seems unlikely that transfer is mediatedby phage transduction, though the resistance genes have beenshown to be transduced (39).

However SGI1 is acquired, the following questions arise: whatis the origin of the SGI1 MDR region and how are the variantsgenerated? It seems reasonable to assume that the SGI1 MDR regionevolved from insertions into an MDR region consisting of a singleclass 1 integron, i.e., SGI1-B (ASu) or SGI1-C (SSu). One scenariois that either SGI1-B or SGI1-C acquired another integron thatincluded the floR/tet(G)/orf1/orf2/groEL region (flo/tet) aspart of its 3'-CS, or acquired a second integron first, withsubsequent integration of the flo/tet region between the twointegrons. In either scenario, truncation of the sul1 and intI1genes, as found in SGI1, would most likely have occurred duringintegration of the flo/tet region into the integron. The originof the flo/tet region itself is unknown, and this combinationof genes exists only in SGI1, though segments similar to partsof this region are found in various plasmids.

Homologs of floR (>95% identity) and the upstream sequenceincluding the 99-bp direct repeat in SGI1 that brackets thefloR gene (4, 6) have been found in plasmids from Klebsiellapneumoniae, E. coli, and Photobacterium damselae (8, 23). Inthe P. damselae and E. coli plasmids, the floR genes are associatedwith a putative transposase gene (orfA) whose product has homologyto the product of orf513 (Fig. 2) and to a lesser extent tothe product of orf2 of SGI1 (see above and Fig. 2). In the K.pneumoniae plasmid R55, the floR gene is followed by a genewhose product has 67% identity to the LysR-like Orf1 proteinfrom the MDR region of SGI1 and a gene whose product has a C-terminalend identical to that of OrfA (8). Thus, all floR variants characterizedto date are associated with putative transposase genes. As well,a 361-bp region highly similar to the region upstream of floRincluding the direct repeat sequence is found just upstreamof a tetR/tet(G) region from Pseudomonas plasmid pPSTG1 (41).Thus, it can be speculated that the direct repeat sequence mayplay a role in the acquisition and/or mobility of genes in elementswhere it is found.

However the SGI1 MDR region originated, various mechanisms couldaccount for the generation of the variant SGI-like elements.It may be that each type of MDR region has been assembled onanother element (i.e., a resident plasmid) and then integratedinto the genomic island in an insertional hotspot between theres gene and S044 in a strain already carrying the SGI1 progenitorin its genome. A similar explanation is that each type of SGI1-likeelement could have been assembled outside of the genome andthen acquired by site-specific integration into the end of thethdF gene in a previously antibiotic-sensitive strain. However,the generation of the variant MDR regions from an SGI MDR regioncan most easily be postulated to have occurred by recombinationbetween homologous regions (Fig. 3).

The structure of SGI1-A can be explained by the insertion ofthe orf513/dfrA10 region via homologous recombination betweenthe qacE ${Delta}$ 1 and sul1 regions, as has been suggested for In7 (42).This hypothesis is partially supported by the findings thatdeletion of the orf513/dfrA10 region from SGI1-A can be detectedby PCR, indicating this region may not be stable and lost withoutselective pressure. The simplest MDR regions are those containingsingle intact class 1 integrons, as was found in SGI1-B andSGI1-C (Fig. 1). It is important to note that these integronsare different from the two which exist in the SGI1 MDR regionin that intact intI1 and sul1 genes are found in SGI1-B andSGI-C (Fig. 1) (4, 5). The generation of both single intactintegrons in SGI1-B and SGI1-C from SGI can be explained bya single crossover between the copies of the intI1 sequencesor qacE ${Delta}$ 1/sul1 sequences, respectively, with concomitant lossof the intervening DNA (Fig. 3). Similarly, SGI1-C and SGI1-Dcould be generated from SGI1-A by crossover between the differentqacE ${Delta}$ 1/sul1 copies (Fig. 3).

A single insertional event of the orf513/dfrA10 element intoSGI1-C would give rise to SGI1-D (Fig. 3). The origin of theorf513/dfrA10 region is unknown, though the putative transposaseOrf513 may be involved in its mobility and in the acquisitionof different associated resistance genes (i.e., as in In6, In7,and pSAL). The SGI1-E variant likely arose from SGI1 after intramoleculartransposition of IS6100 occurred (Fig. 3). The second copy insertedinto floR in the opposite orientation to that of the other IS6100element, generating small direct repeats of the host targetsequence (shown in Fig. 1), as has been observed with the insertionof many insertion sequence elements (24). Subsequently, a singlecrossover event between the two IS6100 elements led to the inversionof the intervening region and the observed SGI1-E MDR region.Support for this hypothesis is provided for by the fact thateach copy of IS6100 in the SGI1-E MDR region is associated withonly one of the IRt sequences that are found surrounding thesingle IS6100 element in SGI1, the 123-bp region is found adjacentto the 5' floR region, and the 152-bp region is found adjacentto the 3' floR region (Fig. 1 and 3). A reverse scenario toproduce the MDR region of SGI1, though possible, is unlikelyto have occurred, as regeneration of an intact floR would necessitatedeletion of one of the short direct repeats, and a copy of IS6100would also have to have been deleted.

Conclusions. This report describes the characterization of the MDR regionsfrom a number of different DT104 and Agona strains which displayantimicrobial resistance patterns other than the classical ACSSuTphenotype often described in strains involved in outbreaks.It will be interesting to see if strains containing other variantSGI1 elements coding for other resistance phenotypes can beisolated, e.g., ASSu, ASuTm, ACSu, CSSu, ACSuTm, and CSSuTm.Also, do MDR regions exist without at least one integron, i.e.,CT, CTTm, C, T, CTm, or TTm? Knowing the relationship of thedrug resistance phenotype to the type of SGI1 present may haveimportant epidemiological implications if variants are isolatedduring the course of an outbreak situation involving an MDRSalmonella serovar containing an SGI1-like element. Investigatorsshould also be aware that strains may exist with identical resistancephenotypes where a resistance may be due to different genes,such as ampicillin resistance due to the presence of a pse-1-containingMDR region in one strain but due to the presence of a TEM ß-lactamasein the other strain.

In this report, we have initiated a nomenclature system to identifyvariants of the SGI1 regions in Salmonella strains. We encourageinvestigators to register any additional variants with us inorder to maintain an ordered nomenclature system for SGI1 variants.Please contact the corresponding author with relevant informationregarding novel SGI1 variants.

ACKNOWLEDGMENTS

We thank S. Rankin (Scotland) and H. Imberechts (Belgium) forproviding isolates and Tim Du and Romeo Hizon for excellenttechnical assistance.

FOOTNOTES

* Corresponding author. Mailing address: Nosocomial Infections, National Microbiology Laboratory, Health Canada, 1015 Arlington St., Winnipeg, Manitoba, Canada R3E 3R2. Phone: (204) 789-2133. Fax: (204) 789-5020. E-mail: Michael_Mulvey{at}hc-sc.gc.ca.

REFERENCES

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