Salmonella Genomic Island 1 Multidrug Resistance Gene Clusters in Salmonella enterica Serovar Agona Isolated in Belgium in 1992 to 2002

Salmonella genomic island 1 (SGI1) harbors a multidrug resistance(MDR) gene cluster which is a complex class 1 integron. VariantSGI1 MDR gene clusters conferring different MDR profiles havealso been identified in several Salmonella enterica serovarsand classified as SGI1-A to -F. A retrospective study was undertakento characterize MDR regions from serovar Agona strains harboringSGI1 isolated from poultry in Belgium between 1992 and 2002.A total of 171 serovar Agona strains, displaying resistanceto at least one antibiotic, were studied for the presence ofSGI1. SGI1 was detected in 94 serovar Agona strains. The mostprevalent variant was SGI1-A (85%), which harbors within theSGI1 complex class 1 integron a common region (CR1) containingorf513, a putative transposase gene, adjacent to the dfrA10trimethoprim resistance gene. A new variant SGI1 named SGI1-Gwas identified in two strains. It consisted of the pse-1 genecassette, as in SGI1-B, but with additional insertion of theorf513/dfrA10 region structure. Seven strains displaying thetypical SGI1 MDR profile (Ap Cm Ff Sm Sp Su Tc) showed geneticvariation at the 3' end of SGI1. These strains harbored theinsertion of the CR1 containing orf513 as in SGI1-A, -D, and-G. However, downstream the right end of CR1, they presenteddifferent 7.4- to 8.5-kb deletions of the SGI1 3' end that extendedto the chromosomal genes yieE and yieF. These results suggesta possible role of CR1 in deletion formation, as has been reportedfor some insertion sequences. Pulsed-field gel electrophoresisanalysis showed that all the serovar Agona SGI1-carrying strainsbelonged to a single clone. Thus, SGI1 is largely encounteredin serovar Agona strains isolated from poultry in Belgium, themost prevalent variant being SGI1-A. SGI1 MDR region undergoesrecombinational events resulting in a diversity of MDR geneclusters.

INTRODUCTION

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Introduction

Multidrug-resistant (MDR) Salmonella enterica serovar Typhimuriumphage type DT104 emerged during the last decade as a globalhealth problem because of its involvement in diseases in animalsand humans (12, 18, 19, 27, 28, 31, 32). MDR DT104 strains arecommonly resistant to ampicillin (Ap^r), chloramphenicol (Cm^r)and florfenicol (Ff^r), streptomycin (Sm^r) and spectinomycin(Sp^r), sulfonamides (Su^r), and tetracyclines (Tc^r).

This MDR profile is conferred by an MDR gene cluster includedin a chromosomal genomic island called Salmonella genomic island1 (SGI1) (5, 6). The 43-kb SGI1 is located between the thdFand int2 genes of the chromosome of S. enterica serovar TyphimuriumDT104. The int2 gene is part of a retron sequence which hasbeen reported to date only in serovar Typhimurium strains (5,6). In other S. enterica serovars SGI1 is located between thdFand the yidY gene (4, 5, 15, 22). All of the antibiotic resistancegenes are located near the 3' end of SGI1 and are part of acomplex class 1 integron that belongs to the In4 group (4).Class 1 integrons contain a 5'-conserved segment (5'-CS) whichconsists of the intI1 gene encoding the site-specific integraseand the associated attI1 site, the primary site of recombination,and the 3'-CS of variable length but generally consisting ofqacE ${Delta}$ 1, which encodes low-level resistance to some antiseptics;the sul1 gene, which encodes sulfonamide resistance; and orf5,a gene of unknown function (16). One or more gene cassettesconsisting of the coding region(s) and the downstream 59-baseelement (59-be), which is responsible for recognition and mobilizationof cassettes, are found between the 5'-CS and 3'-CS (16). TransposonTn402 is a mobile class 1 integron that contains the 5'-CS anda transposition module (tni region) consisting of four genesrequired for transposition (8). In addition, Tn402 is boundby inverted repeats (IRs) of 25 bp, IRi at the integrase endand IRt at the tni end. Several class 1 integrons appear tohave originated from a Tn402-like ancestor by incorporationof the common part of the 3'-CS including qacE ${Delta}$ 1, sul1, and orf5.Most of these integrons, though still bound by IRi and IRt,have lost part or all of the tni module and are deemed defectivetransposon derivatives (8). The In4 group have a 3'-CS thatincludes a copy of IS6100 but no transposition genes, and mostmembers are bound by IRi and IRt (24, 26). The MDR gene clusterof SGI1 is bound by IRi and IRt and thus can be considered acomplex In4-type integron (4). Further, the MDR region is surroundedby 5-bp direct repeats, strongly suggesting it was integratedby a transposition event (4, 24, 26). Interestingly, in SGI1there is a duplication of the 5'-CS, each one followed by agene cassette. The first cassette carries the aadA2 gene, whichconfers resistance to streptomycin and spectinomycin, and a3'-CS with a truncated sul1 (sul1 ${Delta}$ ) gene. The second cassettecontains the ß-lactamase gene pse-1, which confersresistance to ampicillin and a 3'-CS with a complete sul1 geneconferring resistance to sulfonamides. Flanked by the two cassettesare the floR gene, which confers cross-resistance to chloramphenicoland florfenicol, and the tetracycline resistance genes tetRand tet(G) (1, 3, 7). Variant MDR gene clusters of SGI1 haverecently been described in serovars Typhimurium DT104, Agona,and Albany (4, 9, 15). In some, there is only one 5'-CS and,hence, a single gene cassette, either aadA2 (SGI1-C) or pse-1(SGI1-B), and floR and tet(G) are not present. In two othervariants, SGI-A and SGI1-D, the 3'-CS, designated 3'-CS1, isfollowed by a 2,154-bp common region (CR) originally describedin In6 and In7 (23, 30). Adjacent to the CR is a unique regionthat includes dfrA10 (trimethoprim resistance) as in In7 (23).Following the unique region is a second partial copy of the3'-CS, designated 3'-CS2. The CR itself contains a gene, designatedorf513, whose putative product, Orf513, due to its weak similarityto the transposase of IS801, is thought to be a putative transposase(4, 25). The CR described above, designated CR1, has been describedin other class 1 integrons always in association with antibioticresistance genes (25). Orf513 exhibits 66% identity to OrfAand 55% identity to Orf2, two proteins whose genes, like orf513in CR1, are found adjacent to antibiotic resistance genes (4,10, 25). The orfA gene was originally found adjacent to a floRgene in a plasmid isolated from E. coli in cattle (10), andorf2 is found adjacent to tet(G) in SGI1 and some variants (4,5, 11). An examination of the downstream sequences revealeda 27-bp conserved region that defines the right-hand boundaryof related CRs (25). Thus, each CR, CR1, CR2 (orfA), and CR3(orf2) encodes a putative transposase which is postulated tointeract with the 27-bp boundary sequence and mobilize antibioticresistance genes (25).

Recently, SGI1 has also been identified in other serovar Typhimuriumphage types, i.e., DT120, and in other S. enterica serovars,i.e., Agona, Paratyphi B, and Albany, indicating the horizontaltransfer potential of SGI1 (4, 5, 11, 15, 22). SGI1-carryingMDR serovar Agona, Paratyphi B, and Albany strains were isolatedfrom different animal species such as poultry in Belgium, tropicalfish from Singapore, and food fish imported from Thailand toFrance, respectively (4, 5, 11, 15, 22).

Since 1992 an increasing number of MDR serovar Agona strainsexhibiting different antibiotic resistance profiles have beenisolated from poultry in Belgium. Some recent isolates werestudied and reported to carry SGI1 or SGI1 variants (4, 5).In the present work, we conducted a retrospective study to identifyand characterize SGI1 in all serovar Agona strains that wereresistant to at least one antibiotic and were isolated frompoultry in Belgium from 1992 to 2002. The aim of this studywas thus to determine what was the most prevalent SGI1 variant,to identify possible new SGI1 variants, and to determine atthe epidemiological level whether these strains were clonalor not.

MATERIALS AND METHODS

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Materials and Methods

Bacteria, media, and antibiotic susceptibility testing. One hundred and seventy-one serovar Agona strains used in thisstudy, displaying resistance to at least one antibiotic, wereisolated from poultry in Belgium between 1992 and 2002. Thesestrains and control strains serovar Typhimurium DT104 BN9181and serovar Paratyphi B 44 and E. coli strain TOP10 (Invitrogen)used in cloning experiments were grown at 37°C in brainheart infusion broth or agar plates. The strains were testedfor their antibiotic susceptibility by the disk-diffusion assayon Mueller-Hinton plates. Susceptibility was tested using diskscontaining the following antibiotics: ampicillin (10 µg),chloramphenicol (30 µg), florfenicol (30 µg), streptomycin(10 IU), spectinomycin (100 µg), sulfonamides (200 µg),tetracyclines (30 IU), and trimethoprim (Tm^s) (5 µg).Susceptibilities to cephalothin (Cf^s) (30 µg), ceftriaxone(Cro^s) (30 µg) and ceftiofur (Xnl^s) (30 µg) werealso tested. All antibiotic disks except for florfenicol werepurchased from Bio-Rad (Marnes-la-Coquette, France). Florfenicoldisks and the drug itself were obtained from Schering-PloughAnimal Health (Kenilworth, N.J.). The MICs of florfenicol andchloramphenicol were determined by the standard agar doublingdilution method (21). The MIC breakpoints for chloramphenicoland florfenicol were defined by the Comité de l'Antibiogrammede la Société Française de Microbiologie(21) or by the manufacturer, i.e., susceptible (MIC $≤$ 8 µg/ml),intermediate (MIC = 16 µg/ml), or resistant (MIC $≥$ 32 µg/ml).

PCR mapping, cloning, and sequencing. Detection of SGI1 and its location were performed using primerscorresponding to left and right (with or without retron) junctionsin the chromosome as described previously (Table 1; Fig. 1)(4, 15, 22). PCR mapping of the typical antibiotic resistancegenes associated with SGI1 was performed using conditions andprimers described previously (11, 15, 22) (Table 1) (Fig. 1).

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TABLE 1. Primers used for PCR in this study

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FIG. 1. Genetic organization of the MDR gene clusters of SGI1, SGI1-A variant, and new SGI1-G variant. Black and grey arrows correspond to antibiotic resistance genes and chromosomal genes flanking SGI1, respectively. DR-L and DR-R are the left and right direct repeats, respectively, bracketing SGI1. IRi and IRt are 25-bp imperfect IRs defining the left and right ends of complex class 1 integrons. Vertical thick black bars indicate homology regions between SGI1 variants. PCRs used to assess the genetic organization of the antibiotic resistance genes (PCRs floR, A, B, C, D, E, F, G, H, and I) and the SGI1 junctions to the chromosome (PCRs LJ and RJ for left and right junctions, respectively) are indicated. Abbreviation for restriction site: X, XbaI.

To assess a new variant SGI1 MDR gene cluster identified inthis study, PCR amplification of a region of the complex class1 integron was also performed using primer Fresol and primerRsul of PCR I (Fig. 1). Cloning of this PCR product in plasmidpCR2.1-TOPO was performed using the TOPO TA cloning kit (Invitrogen,Cergy Pontoise, France). Nucleotide sequencing of the insertwas achieved by Genome Express (Meylan, France). Sequence analysiswas done by using BLAST (available from http://www3.ncbi.nlm.nih.gov/BLAST/).

A set of PCRs was performed to determine the extent of the deletionsat the 3' end of SGI1 in the serovar Agona strains with negativeresults for the right junction of SGI1. PCR products correspondingto the new right junctions were sequenced.

PFGE. Strains positive for SGI1 were investigated by pulsed-fieldgel electrophoresis (PFGE). Preparation of genomic DNA was performedas previously described (11, 15, 22). Genomic DNA, containedin agarose plugs, was digested with BlnI (BioWhttaker, Emerainville,France) or XbaI (Qbiogene, Illkrich, France). Electrophoresiswas performed using the CHEF-DR III system (Bio-Rad, Hemel Hempstead,United Kingdom) at 14°C in 0.5x Tris-borate-EDTA runningbuffer. Electrophoresis conditions were as follows: 0.6 V/cmfor 18 h with pulse times of 2.16 to 54.17 s.

Nucleotide sequence accession numbers. The complete nucleotide sequences of regions resulting fromthe different SGI1 deletions have been deposited in the GenBankdatabase under accession numbers AY434091, AY434092, and AY434093.

RESULTS

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Results

Detection of SGI1 in MDR serovar Agona isolated in Belgium in 1992 to 2002. One hundred and seventy-one serovar Agona strains resistantto at least one antibiotic and isolated between 1992 and 2002were analyzed for the presence of SGI1. The SGI1 detection andlocation based on junction PCRs were realized by using primerscorresponding to the left and right junctions of SGI1 in theSalmonella chromosome (Fig. 1). We also used PCR to detect thepresence or absence of the retron sequence, which is locateddownstream of SGI1 in serovar Typhimurium DT104 strains butabsent to date in other S. enterica serovars (4, 5, 15, 22).SGI1 was thus detected in 87 serovar Agona strains between thethdF and the yidY genes of the chromosome (Table 2). All strainslacked the retron sequence found downstream of SGI1 in serovarTyphimurium DT104. For seven MDR serovar Agona strains, PCRwas positive for the left junction of SGI1 with thdF but was,however, negative for the right junction with yidY (data notshown) (Table 2). Thus, these data indicate that 55% of MDRserovar Agona strains tested harbor SGI1 at the same chromosomallocation, i.e., between the thdF and yidY genes, as previouslyfound (5). The earliest MDR serovar Agona strain harboring SGI1was isolated in 1993 (Table 2). Among 19 serovar Agona strainstested from 1992, none contained SGI1. Seven serovar Agona strainsisolated between 2000 and 2002 that were negative for the rightjunction but positive for the left junction PCR appeared toharbor SGI1 with some genetic variation at its 3' end.

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TABLE 2. Characteristics of the 94 serovar Agona SGI1-carrying strains tested in this study

MDR profiles. SGI1-positive serovar Agona strains showed five different MDRprofiles (Table 2). They consisted of the following: Ap Cm FfSm Sp Su Tc Tm (81 strains), Ap Cm Ff Sm Sp Su Tc (9 strains),Sm Sp Su (2 strains), Ap Su Tm (2 strains), and Ap Cf Cm CroFf Sm Sp Su Tc Tm Xnl (1 strain). The serovar Agona strainsresistant to florfenicol showed the same level of resistanceto florfenicol as previously reported for serovars TyphimuriumDT104, Paratyphi B, and Albany harboring SGI1 (MIC of florfenicol,64 µg/ml) (Table 2) (11, 15, 22).

MDR gene clusters. PCR mapping of the typical antibiotic resistance genes associatedwith SGI1-A is schematized in Fig. 1. PCRs A to I and floR wereperformed on genomic DNAs extracted from SGI1-positive serovarAgona strains. Eighty-one strains displaying the MDR profileAp Cm Ff Sm Sp Su Tc Tm yielded fragments A to I and floR ofthe sizes expected from the control serovar Agona strain 1169SA97harboring SGI1-A described previously (data not shown) (Table2) (4). Thus, these PCR mapping results indicated most serovarAgona strains harbor the variant MDR gene cluster of SGI1-A.

Two serovar Agona strains (1233SA96 and 959SA97) isolated in1996 and 1997 showed the typical MDR profile of serovar TyphimuriumDT104 harboring SGI1 (Ap Cm Ff Sm Sp Su Tc) with lack of resistanceto trimethoprim (Table 2). PCRs A to E and partial floR resultswere positive and yielded fragments of expected sizes as forserovar Typhimurium DT104 control strain BN9181 (data not shown).However, PCR fragments F to I specific for the orf513/dfrA10region were not obtained. The absence of this region was furtherassessed by PCR with a forward primer of fragment F and a reverseprimer of fragment I. This PCR result was positive and yieldeda 4.4-kb fragment as with DNA from serovar Typhimurium DT104control strain BN9181 (data not shown). These results indicatedthat these two serovar Agona strains harbor SGI1 with the classicalMDR gene cluster described for serovar Typhimurium DT104 (1,3, 5, 6, 7).

Two others serovar Agona strains (47SA97 and 278SA00) isolatedin 1997 and 2000 were only resistant to streptomycin or spectinomycinand sulfonamides (Table 2). PCR mapping results of these strainswere only positive for fragment A specific for the aadA2 genecassette. This SGI1 variant containing a single aadA2 gene cassettewas previously characterized for strain 47SA97 and was namedSGI1-C (4). Thus, the other serovar Agona strain isolated in2000 was also shown to harbor SGI1-C (4).

New variant SGI1 MDR gene cluster. Two SGI1-positive serovar Agona strains (712SA96 and 169SA00)isolated in 1996 and 2000 displayed the MDR profile Ap Su Tm,which did not correspond to any variant SGI1 MDR gene clusterpreviously described (Table 2) (4, 9). All PCRs were negativeexcept PCRs F to I (data not shown) (Fig. 1). Thus, PCR mappingof the antibiotic resistance genes showed the presence of thepse-1 gene cassette and the orf513 and dfrA10 genes. To furtherassess the pse-1 gene cassette region in this complex integron,an additional PCR amplification using primer Fresol and primerRsul of PCR H was performed (Table 1, Fig. 1). A fragment ofapproximately 3.7 kb was obtained, and its nucleotide sequencewas determined. Sequence analysis showed 100% identity withthe nucleotide sequences of the tnpR gene and the pse-1 genecassette from the serovar Typhimurium DT104 SGI1 MDR gene cluster(5). This MDR gene cluster thus shows the same genetic organizationas in the SGI1-B variant with a complete integrase gene intI1,the pse-1 gene cassette and the 3'-CS of the integron (4). However,it constitutes a new SGI1 variant by the additional insertionof the orf513/dfrA10 region (Fig. 1); we propose the name SGI1-Gfor this new SGI1 variant, according to the previously proposednomenclature (4, 15).

SGI1 3'-end deletions. Seven serovar Agona strains (1690SA00, 1691SA00, 507SA01, 519SA01,773SA01, 781SA01, and 1254SA02) isolated between 2000 and 2002were negative for the right junction PCR of SGI1 with the yidYgene of the chromosome (Table 2). These strains showed the typicalMDR profile Ap Cm Ff Sm Sp Su Tc from serovar Typhimurium DT104harboring SGI1. PCRs A to E and floR were positive, whereasPCRs F to I were negative (data not shown) (Fig. 1). A set offurther PCR mappings was done to try to determine the geneticvariation, possibly involving deletions, at the SGI1 3' endof these strains. PCR mapping of the SGI1 3' end showed foreach strain the presence of the complete pse-1 gene cassetteand a specific orf513 fragment of the expected size (data notshown). PCR amplifications corresponding to dfrA10, qacE ${Delta}$ 1, sul1,orf5, orf6, IS6100, and S044 were not obtained (Fig. 2). Thus,these data indicated deletions of the respective genes downstreamof orf513. Further PCR mapping of the chromosome downstreamof orf513 showed that in these seven strains, the yidY, yidZ,and yieE genes were absent. However, the yieF gene was detectedin all strains except in two (1690SA00 and 1691SA00) isolatedin 2000 (data not shown). Genes downstream of yieF were detectedin all strains. A small insertion of about 500 bp between theyieF and yieG genes was found in all strains which was not presentin the genome sequence of serovar Typhimurium LT2 (GenBank accessionnumber NC_003197) (Fig. 2). Thus, PCR mapping results indicatedthat three different deletions had occurred in these strainsindicated as SGI1-A ${Delta}$ 1R, SGI1-A ${Delta}$ 2R, and SGI1-A ${Delta}$ 3R in Table 1. APCR product of about 3.5 kb between the end of the sul1 genedownstream of the pse-1 gene cassette to the yieG gene of thechromosome was amplified and sequenced for strain 1690SA00 (GenBankaccession number AY434092). A PCR product of shorter size endingin the insertion between the yieF and yieG genes of the chromosomewas amplified and sequenced for strain 507SA01 (Fig. 2) (GenBankaccession number AY434091). A PCR product of about 2.8 kb wasobtained for strain 1254SA02 that corresponds to a fragmentbetween the beginning of the CR1 to the yieE gene of the chromosomeand was also sequenced (GenBank accession number AY434093).Sequence analysis showed that for all strains SGI1 deletionsthat extended to chromosomal genes started downstream of theCR1 of 2,154 bp that includes orf513 (25). For the sequencedstrain 1254SA02, the deletion extended from the end of the CR1to bp 64 of the yieE gene of the chromosome, which representsan approximately 7.4-kb deleted fragment from SGI1-A and thechromosome (Fig. 2). For the sequenced strain 507SA01, the deletionended at bp 362 of the yieE gene of the chromosome, which representsa 7.7-kb deletion. For strain 1690SA00, the deletion was approximately8.5 kb long to bp 408 of the yieF gene of the chromosome. Thus,we named these SGI1 deletion variants SGI1-A ${Delta}$ 1R (strain 1254SA02),SGI1-A ${Delta}$ 2R (strains 507SA01, 519SA01, 773SA01, and 781SA01), andSGI1-A ${Delta}$ 3R (strains 1690SA00 and 1691SA00) (Table 2; Fig. 2).Sequence analysis of the insertion of about 500 bp between yieFand yieG genes in these seven serovar Agona strains, relativeto the genome of serovar Typhimurium LT2 (GenBank accessionnumber NC_003197), revealed the presence of an open readingframe of 426 bp named orfX, with absence of homology to anyknown proteins (Fig. 2). Sequence alignment of CR1 from strains1690SA00 and 507SA01 with the previously sequenced CR1 fromSGI1-D of serovar Agona strain 953SA98 (GenBank accession numberAY049746) showed two and six nucleotide substitutions, respectively.The nucleotide sequence of the CR1 from strain 1254SA02 was100% identical to that of the CR1 from SGI1-D. The deduced aminoacid sequence of orf513 from strain 1690SA00 was 100% identicalto that of strain 953SA98 harboring SGI1-D (4). For strain 507SA01,all six nucleotide substitutions in orf513 led to amino acidchanges in the deduced protein. The Orf513 protein from strain507SA01 was thus 98.8% identical to that from the others.

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FIG. 2. Schematic view of the 3' end of SGI1-A ${Delta}$ Rs characterized in this study. SGI1-A 3' end is shown at the top. Black and grey arrows correspond to antibiotic resistance genes and chromosomal genes found downstream of SGI1, respectively. Right-hand boundary sequences are indicated under each SGI1-A ${Delta}$ R. Uppercase letters represent the CR1 sequences. Nucleotides in boldface letters indicate the 27-bp potential recognition site of Orf513. Nucleotides in lowercase letters on a grey background represent chromosomal gene sequences found downstream of SGI1. The extents of deletions are indicated by arrows. Locations of sequenced regions are indicated by thick black bars, and locations of the CR1 sequences are indicated by dotted lines.

Macrorestriction analysis by PFGE. Macrorestriction analysis by PFGE of the SGI1 carrying serovarAgona strains DNAs cut by XbaI showed that all strains had identicalXbaI PFGE patterns (Table 2; Fig. 3). Four BlnI PFGE patterns(A1 to A4) were obtained and corresponded to only one band differenceeach in the bottom of the gel. These different BlnI patternscorrelated well with the type of SGI1 variant; i.e., patternA1 correlated well with SGI1-A, pattern A2 correlated well withSGI1-G and SGI1-A ${Delta}$ Rs, pattern A3 correlated well with SGI1 andSGI1-A ${Delta}$ Rs, and pattern A4 correlated well with SGI1-C. Thus,these results indicate that all serovar Agona SGI1-carryingstrains from this study are clonally related.

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FIG. 3. Macrorestriction analysis by PFGE of genomic DNAs cut by XbaI or BlnI of serovar Agona strains harboring SGI1-A (lanes 1), SGI1 (lanes 2), SGI1-G (lanes 3), and SGI1-C (lanes 4).

DISCUSSION

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Discussion

SGI1 is the first genomic island containing an MDR gene clusteridentified in S. enterica. Among MDR serovar Typhimurium DT104strains, most of them show the Ap Cm Ff Sm Sp Su Tc resistanceprofile and harbor the classical MDR gene cluster from SGI1.Acquisition of SGI1 may have been an important trait in theworldwide epidemic of the MDR serovar Typhimurium DT104 clonecausing disease in animals as well as in humans. SGI1 has beenfurther identified in other S. enterica serovars, such as serovarAgona strains isolated from poultry in Belgium and serovarsParatyphi B and Albany strains isolated from fish in Asia (4,5, 11, 15, 22). The identification of SGI1 at the same chromosomallocation in several S. enterica serovars indicates horizontaltransfer of this island and a SGI1 site-specific insertion inthe chromosome. SGI1-carrying MDR serovar Agona strains isolatedfrom poultry in Belgium between 1992 and 2002 represent 55%of serovar Agona strains tested (94 SGI1-positive serovar Agonastrains). It appears from the present study that the emergenceof SGI1 in serovar Agona strains from poultry in Belgium startedin 1993. The most prevalent SGI1 variant identified in thesestrains was SGI1-A, which accounts for 85% of the total SGI1-positivestrains. This SGI1-A variant, which confers additional Tm^r,relative to that of SGI1 mostly encountered in MDR serovar TyphimuriumDT104 strains, carries in its MDR gene cluster the additionalstructure orf513/dfrA10 downstream of the pse-1 gene cassette3'-CS1 (4). A new SGI1 variant identified in this study, namedSGI1-G, consists of the complex integron structure that includesonly the ß-lactamase pse-1 gene cassette associatedwith the orf513/dfrA10 region downstream of the 3'-CS1. Thegeneration of this variant SGI1-G could be explained by theinsertion of the orf513/dfrA10 structure via homologous recombinationbetween the duplicated qacE ${Delta}$ 1/sul1 genes of this variant (Fig.4) (4, 25). This hypothesis is supported by a recent study ofPartridge et al. (25) who showed that an artificially excisedcircular molecule which consists of the orf513/dfrA10 regionfrom In34 could recombine with the 3'-CS in another class 1integron. Another explanation could be that SGI1-G originatedfrom SGI1-A by a single crossover between the copies of intI1with loss of the aadA2, qacE ${Delta}$ 1, sul1, floR, tetR, tet(G), orf1,and orf2 region (Fig. 4).

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FIG. 4. Schematic diagram of the generation of variant SGI1-G from SGI1-A or SGI1-B.

Over the past 10 years, a number of complex class 1 integronshave been described as containing CR1 that includes orf513 (2,4, 13, 25, 29, 30, 35, 36). These complex integrons containthe 5'-CS and two 3'-CSs designated 3'-CS1 and 3'-CS2. 5'-CS(intI1 gene, attI) and 3'-CS1 (qacE ${Delta}$ 1 and sul1 genes) constitutea typical class 1 integron with one or more gene cassettes containinga typical 59-be. Following the 3'-CS1 is the CR1 of 2,154 bpthat contains orf513 previously known as orf341 (34, 35). Downstreamof the CR1 is a unique region that includes antibiotic resistancegenes such as catA2, dfrA10, dfrA19, bla_DHA-1, bla_CTX-M-9, bla_CTX-M-2,bla_CMY-9, and qnr, which are not present as gene cassettes asthey are in integrons (2, 4, 13, 25, 29, 30, 34, 35, 36). Theseresistance genes are indeed not followed by the 59-be presentin class 1 integrons. Downstream of the unique region is the3'-CS2 of a typical class 1 integron (qacE ${Delta}$ 1 and sul1 genes).SGI1-A, -D, and -G variant MDR gene clusters present the sameorganization as complex In4-like integrons (4). As seen in thepresent study, in contrast to SGI1-carrying serovar TyphimuriumDT104 strains, the CR1 that includes orf513 is dominantly prevalentin SGI1-carrying Agona strains (SGI1-A, -D, -G, -A ${Delta}$ 1R, -A ${Delta}$ 2R,and -A ${Delta}$ 3R). The role of the putative transposase-recombinaseOrf513 has not yet been experimentally determined (4, 10, 25).In the early 1990s, it was speculated that antibiotic resistancegenes could have been inserted as gene cassettes, using a 59-be,with the subsequent loss of this element (13, 23, 25, 30). Nowadays,with the increasing number of complex integrons described, itappears that Orf513 may recognize a 27-bp segment well conservedat the 3' end of the CR1 where Orf513 will thus catalyze recombinationalevents to incorporate antibiotic resistance genes adjacent tothis recognition site (2, 13, 25, 29).

Three different deletions at the 3' end of SGI1 downstream ofthe CR1 are described in the present study (i.e., SGI1-A ${Delta}$ 1R,-A ${Delta}$ 2R, -A ${Delta}$ 3R). These deletions of different sizes (7.4, 7.7, and8.5 kb) start at the precise right-hand end of the CR1, downstreamfrom the 27-bp putative recognition site of Orf513, and extendfurther downstream of SGI1 to proximate chromosomal genes. Mostlikely these deletions originated from SGI1-A according to thehigh prevalence of SGI1-A. The insertion of the CR1 from theorf513/dfrA10 region downstream from the pse-1 gene cassettewhen recombined with the SGI1 3' end could have led to concomitantdeletion of downstream chromosomal genes in some strains. Recombinationalevents at the 27-bp segment involving the putative transposaseOrf513 may thus have resulted in deletion of the SGI1-A 3' endand part of the adjacent chromosomal region. Considering thesimilarity of Orf513 to the transposase of IS801 (4, 10, 25),our results suggest a possible role of the CR1 in generatingdeletions, as has been reported for some insertion sequences(ISs), and also raise the question of mobility of the CR1. Indeed,IS elements may mediate deletion events and remove DNA fromone or the other side of the elements, as seen with deletionsfound downstream of the CR1 described in this study (17, 20,33). However, in the case of IS, deletion formation appearsto be directly related to insertion and transposition events(17). The most studied are IS1-mediated deletions that are generatedby intramolecular tranposition (17, 20, 33). Another possibleexplanation in the case of the CR1 that includes orf513 is thata copy of the CR1 moved and inserted in the yieE or yieF genesin strains carrying SGI1-A. Subsequently, a single crossoverevent between the two CR1 led to the deletion of the internalregion. Thus, further studies concerning mobility of the CR1and recombination events mediated by it, are of interest.

Macrorestrction PFGE analysis showed that all serovar Agonastrains carrying SGI1 and variants were clonally related. Accordingto published data, it is likely that horizontal transfer ofSGI1 is not a frequent event and that, as in the case of MDRserovar Typhimurium DT104 strains, S. enterica serovars acquiringSGI1 spread clonally in animals and humans (12, 18, 19, 27,28, 31, 32). Only recently, we have reported the first humancase of infection with a serovar Agona strain harboring thevariant SGI1-A (14). The patient was probably infected by oneof the clonal SGI1-carrying serovar Agona strains from poultry,on the basis of the fact that XbaI and BlnI PFGE patterns ofthis human strain were identical to those of poultry strains.

In conclusion, SGI1 is largely encountered in clonally relatedserovar Agona strains isolated from poultry in Belgium, withthe most prevalent variant being SGI1-A. SGI1 MDR gene clustersare subjected to recombinational events, which results in agreat diversity of MDR gene clusters, such as the new one, SGI1-G,described in this study. The deletions found at the SGI1 3'end that extend to proximate chromosomal genes also raise newquestions about possible recombinational events mediated bythe CR1 as well as CR1’s mobility.

ACKNOWLEDGMENTS

We thank J. Borlang, C. Mouline, and V. Verberen for experttechnical assistance.

FOOTNOTES

* Corresponding author. Mailing address: Unité BioAgresseurs, Santé et Environnement, Institut National de la Recherche Agronomique, 37380 Nouzilly, France. Phone: (33) 2 47427750. Fax: (33) 2 47427774. E-mail: cloeckae{at}tours.inra.fr.

REFERENCES

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