IS21-558 Insertion Sequences Are Involved in the Mobility of the Multiresistance Gene cfr

Abstract

During a study of florfenicol-resistant porcine staphylococci from Denmark, the genes cfr and fexA were detected in the chromosomal DNA or on plasmids of Staphylococcus hyicus, Staphylococcus warneri, and Staphylococcus simulans. A novel variant of the phenicol resistance transposon Tn558 was detected on the ca. 43-kb plasmid pSCFS6 in S. warneri and S. simulans isolates. Sequence analysis of a 22,010-bp segment revealed that the new Tn558 variant harbored an additional resistance gene region integrated into the tnpC reading frame. This resistance gene region consisted of the clindamycin exporter gene lsa(B) and the gene cfr for combined resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics bracketed by IS21-558 insertion sequences orientated in the same direction. A 6-bp target site duplication was detected at the integration site within tnpC. Transpositionally active forms of the IS21-558 element, known as minicircles, were detected by PCR and suggest that this insertion sequence is involved in the mobility of the multiresistance gene cfr. Based on the knowledge of the transposition pathways of IS21-like insertion sequences and the sequence features detected, the resistance gene region of plasmid pSCFS6 is believed to have developed via IS21-558-mediated cointegrate formation. The data obtained in this study identified the multiresistance gene cfr not only in three novel host species but also in a novel genetic context whose further analysis suggested that insertion sequences of the type IS21-558 are likely to be involved in the dissemination of cfr.

The gene cfr was initially discovered in a bovine Staphylococcus sciuri isolate during a florfenicol surveillance study of staphylococci from animals (19). In 2005, it was shown that the Cfr protein represents a methyltransferase which modifies the 23S rRNA (6). Detailed footprint analysis and mass spectrometric studies showed that the Cfr enzyme adds an additional methyl group at position A2503 (6). This Cfr-mediated modification affected not only the binding of phenicols but also that of members of another four different drug classes to the bacterial ribosome. The corresponding multidrug resistance phenotype was characterized by elevated MICs to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A (PhLOPS_A) antibiotics, all of which are important antimicrobial agents for the treatment of bacterial infections in humans and/or animals (7). The PhLOPS_A phenotype was expressed not only in Staphylococcus aureus but also in Escherichia coli carrying the gene cfr (7).

So far, the gene cfr has been detected in six staphylococcal isolates of animal origin, located either on the 17.1-kb plasmid type pSCFS1 or on the 35.7-kb plasmid type pSCFS3 (4). Recently, cfr has also been detected in a methicillin-resistant S. aureus isolate of human origin (8a). In the completely sequenced plasmid pSCFS1, the gene cfr was located in close proximity to other antibiotic resistance genes, such as lsa(B), erm(33), and spc, which mediated low-level clindamycin resistance; inducible resistance to macrolides, lincosamides, and streptogramin B antibiotics; or spectinomycin resistance, respectively (5). When the cfr gene is located on this plasmid type, coselection of the gene may occur under selective pressure imposed by the use of macrolides or spectinomycin. In the second cfr-carrying plasmid type, pSCFS3, the regions up- and downstream of the gene cfr differ distinctly from the situation in plasmid pSCFS1. The cfr gene was found to be integrated into a partially deleted variant of transposon Tn558 in plasmid pSCFS3 (4). This transposon carries the gene fexA, which codes for a phenicol-specific efflux protein of the major facilitator superfamily (2, 3). In this Tn558 variant of plasmid pSCFS3, two of the three transposase genes, tnpA and tnpB, assumed to be involved in the mobility of Tn558 were truncated. The 3′ end of tnpA and the 5′ end of tnpB were replaced by a 4,674-bp segment consisting of the resistance gene cfr and a novel IS21-like insertion sequence, designated IS21-558 (4). This novel insertion sequence contained two overlapping reading frames, istAS and istBS, that coded for 445-amino-acid (aa) and 250-aa proteins, respectively. Although the novel insertion sequence was located close to cfr, its involvement in the mobility of the gene cfr remained to be clarified.

In the present study, we identified a novel plasmid-borne variant of Tn558 with a large inserted segment. This segment harbored the genes cfr and lsa(B) bracketed by copies of the insertion sequence IS21-558. Based on the knowledge of the mode of transfer of IS21-like insertion sequences and further analysis, a model for the mobility of cfr involving the IS21-558 sequences was developed.

MATERIALS AND METHODS

Bacterial isolates and antimicrobial susceptibility testing.

In 2005, a total of 23 florfenicol-resistant staphylococci were collected from two pig farms in Denmark. Both farms, owned by the same farmer, had a known history of florfenicol pretreatment of the animals and additional usage of other classes of antimicrobials to treat and/or prevent respiratory tract infections in 2004 and 2005. The drugs used at these farms included penicillin, amoxicillin, florfenicol, gentamicin, lincomycin, spectinomycin, tetracycline, tiamulin, tylosin, and trimethoprim plus sulfonamides. The staphylococcal isolates were cultivated overnight at 37°C on blood agar (Oxoid, Wesel, Germany) supplemented with 5% (vol/vol) sheep blood. The species identification was confirmed biochemically using the ID32 Staph system (bioMérieux, Marcy l'Etoile, France). Antimicrobial susceptibility testing was conducted by agar disk diffusion with disks containing ampicillin (10 μg), chloramphenicol (30 μg), clindamycin (2 μg), erythromycin (15 μg), florfenicol (30 μg), gentamicin (10 μg), kanamycin (30 μg), streptomycin (10 μg), spectinomycin (100 μg), sulfamethoxazole-trimethoprim (23.75/1.25 μg, respectively), or tetracycline (30 μg). MICs were determined for chloramphenicol, florfenicol, and clindamycin by the broth macrodilution method (range of concentrations tested, 2 to 256 μg/ml). Staphylococcus aureus strains ATCC 29213 and ATCC 25923 served as reference strains for quality control purposes. All susceptibility tests were performed and evaluated according to CLSI guidelines M31-A2 and M31-S1 (12, 13).

Macrorestriction analysis.

Since the bacterial strains were collected from only two neighboring pig farms, macrorestriction analysis was performed to investigate the clonality of the isolates. DNA preparation and restriction analysis were conducted as described previously (20). The SmaI (Roche Diagnostics, Mannheim, Germany) fragments of staphylococcal whole-cell DNA were separated in a CHEF DR III system (Bio-Rad) at 15 V/cm with 0.5× Tris-borate-EDTA buffer as running buffer. The pulse time was increased from 7 to 12 s for 12 h and from 20 to 65 s for another 13 h.

Isolation of DNA and transformation experiments.

The preparation of whole-cell DNA followed a previously described protocol (4). Plasmids were prepared according to a modification of the alkaline lysis procedure (4). For the determination of plasmid sizes, the sum of fragment sizes obtained after digestion of the plasmids with either BglII or EcoRI (Roche Diagnostics, Mannheim, Germany) was calculated. The use of the restriction endonucleases followed the manufacturer's recommendations. Comparisons of plasmids were performed on the basis of their fragment patterns. Transformation experiments into protoplasts of the recipient strain S. aureus RN4220 were used to identify phenicol resistance plasmids (18). Transformants were picked after 48 to 72 h on regeneration plates supplemented with either 10 μg/ml florfenicol or 10 μg/ml chloramphenicol.

PCR assays and hybridization experiments.

The presence of the resistance genes cfr and fexA as well as of parts of the transposon Tn558 was confirmed by PCR assays and Southern blot hybridization experiments using previously described primers and probes (4). To detect the IS21-558 element and the associated reading frames istAS and istBS, the following PCR primers were used: istAS-fw (5′-GAGATTGTGGAAAGGCTCAAGG-3′), istAS-rv (5′-TGTTCAAGCACTCCTTCATCGAC-3′), istBS-fw (5′-GAATTACGTCTTCCTGGCATCC-3′), and istBS-rv (5′-GTAAGATGGGCCGATCATATTCA-3′). A combination of istAS-fw and istBS-rv was used to detect almost the complete IS21-558. The expected amplicon sizes were 1,322 bp (istAS), 672 bp (istBS), and 2,029 bp (almost complete IS21-558); the annealing temperature of 58°C was used for all three PCR assays.

Transposition activities of IS21 insertion sequences and related elements are reported to occur via different transposition pathways. A single copy of IS21 is thought to generate simple transpositions via a cut-and-paste mechanism (1), and since this specific mechanism involves a circularization of IS21, inverse PCR assays were performed to detect this minicircle formation. For this, a combination of the primers istBS-fw and istAS-rv and the Phusion polymerase (New England Biolabs, Frankfurt, Germany) were used. The PCR conditions were the same as those for the istAS amplification protocol. To determine whether tandem repeats of IS21-558 are present, plasmid and chromosomal DNA were digested with either EcoRV or BglII (the single restriction sites for these enzymes in IS21-558 are located within istAS) and hybridized with the istBS amplicon or digested with HindIII (the single restriction site for this enzyme in IS21-558 is located within istBS) and hybridized with the istAS amplicon.

Cloning and sequencing of plasmid DNA.

The BglII fragments of plasmid DNA obtained from the transformants were cloned into the BamHI site of vector pBluescript II SK(+), and recombinant plasmids were introduced into E. coli JM109. In total, a segment of 22,010 bp, including the resistance gene regions and their up- and downstream flanking parts, was sequenced on both strands by primer walking (MWG-Biotech, Martinsried, Germany). Cloning of PCR amplicons of the minicircles into vector pCR-Blunt II-TOPO and transformation into E. coli TOP10 followed the manufacturer's instructions (Invitrogen, Groningen, The Netherlands). The minicircle amplicons were sequenced using standard primers (MWG-Biotech).

Nucleotide sequence accession number.

The 22,010-bp sequence of the novel Tn558 variant of pSCFS6 including the up- and downstream flanking regions has been deposited in the EMBL database under accession number AM408573.

RESULTS AND DISCUSSION

Species identification, antimicrobial resistance, and clonality of the isolates.

Among the 23 staphylococci included in this study, 14 corresponded in their biochemical characteristics to the species Staphylococcus hyicus, 8 were identified as Staphylococcus warneri, and a single isolate was classified as S. simulans. Macrorestriction analysis of the 14 S. hyicus isolates detected only slight differences in the SmaI patterns of two isolates, while the fragment patterns of the remaining S. hyicus isolates were indistinguishable from each other (Fig. 1). A similar situation was seen with the S. warneri isolates, among which only two very similar SmaI macrorestriction patterns were identified. As expected, the S. simulans isolate showed a unique SmaI restriction pattern. Therefore, all the S. hyicus as well as the S. warneri isolates were considered closely related, a finding that was expected with regard to the origin of these strains from only two neighboring farms. Based on the clonality of the isolates, two S. hyicus and two S. warneri isolates with slight differences in their macrorestriction profiles and the single S. simulans isolate were chosen for further analysis.

FIG. 1.

SmaI macrorestriction patterns of 10 S. hyicus isolates (lanes 1 to 10). Lanes M contain the SmaI pattern of S. aureus 8325 with the fragment sizes given in kilobases on the right-hand side.

Agar disk diffusion (12, 13) revealed that all selected isolates were resistant to florfenicol, chloramphenicol, clindamycin, erythromycin, streptomycin, and tetracyclines. In addition, both S. hyicus isolates were also resistant to gentamicin and kanamycin and the two S. warneri isolates showed no zone of growth inhibition around the spectinomycin disk. The MICs for chloramphenicol and florfenicol varied between 128 and >256 μg/ml; those for clindamycin were >256 μg/ml for all five isolates.

Localization of the resistance genes cfr and fexA.

Protoplast transformation experiments using plasmid DNA obtained from the two S. warneri isolates as well as from the S. simulans isolate identified a ca. 43-kb plasmid to confer phenicol and clindamycin resistance on the recipient strain S. aureus RN4220. Comparisons of the restriction patterns of the 43-kb plasmids from transformants originating from either S. warneri or S. simulans did not detect any differences (data not shown), suggesting that a single plasmid type was present among these porcine staphylococci. This plasmid type was designated pSCFS6. The MICs for the RN4220::pSCFS6 transformants as determined by the macrodilution broth method (12) were 256 μg/ml for florfenicol as well as 256 μg/ml for chloramphenicol and increased to >256 μg/ml after preincubation in the presence of 0.5 μg of the respective drug. The clindamycin MICs for all transformants were >256 μg/ml.

PCR assays as well as hybridization experiments confirmed the localization of the phenicol exporter gene fexA as well as the multidrug resistance gene cfr on plasmid type pSCFS6. When pSCFS6 plasmid DNA was used as template, PCR amplicons for the transposase genes tnpA and tnpB, but not for tnpC or the circular form of transposon Tn558, were obtained. Furthermore, PCR assays and Southern blot hybridization identified the open reading frames istAS and istBS as well as two almost complete IS21-558 sequences on plasmid pSCFS6. When the primer combination istAS-fw and istBS-rv was used, only the expected amplicon size of 2,029 bp was obtained and no larger amplification products were identified which might give a hint towards a tandem formation of IS21-558 elements.

In contrast, repeated negative results for protoplast transformation experiments as well as hybridization of plasmid profiles but positive hybridization results when whole-cell DNA was used as template suggested that the genes fexA and cfr were located in the chromosomal DNA of the S. hyicus isolates. The istAS and istBS reading frames and the almost complete IS21-558 sequence were also identified in the chromosomal DNA of these isolates. While previous studies identified the gene cfr exclusively on plasmids (4, 5, 19), this is the first report of a chromosomal location of cfr.

Sequence analysis of the cfr-IS21-558 resistance gene area on plasmid pSCFS6.

Since only a single localization of the novel IS21-558 element has been described so far (4), the resistance gene area of plasmid pSCFS6 was cloned and sequenced. Analysis of this sequenced part of 22,010 bp led to the detection of a novel variant of transposon Tn558. This variant comprised in total 16,253 bp and consisted of a Tn558-homologous part of 6,659 bp that was interrupted by the insertion of a 9,594-bp region. The Tn558-homologous part included the complete transposase genes tnpA and tnpB as well as the phenicol exporter gene fexA and orf138. While orf138 corresponded exactly to the respective open reading frame of Tn558 (accession no. AJ715531), single bp exchanges were identified in tnpA (4 bp), tnpB (2 bp), and fexA (1 bp) or in the noncoding regions. Compared to all so-far-known Tn558 sequences, the novel variant exhibited the typical 6-bp core sequence 5′-GATGTA-3′ at the left-end junction but differed slightly in the right-end junction (5′-GATCCG-3′). Serial transposition experiments with Tn554 (9), the best-studied transposon of the family to which Tn558 belongs, revealed that the junctions varied with respect to the previous target sites (10). The observed altered core sequence at the right-end junction in plasmid pSCFS6 is therefore likely to have developed during transposition from a former target site. Analysis of the attachment site of the Tn558 variant on plasmid pSCFS6 identified a sequence similar to att554 (11) and att558 on pSCFS3 (3). Circular intermediates of Tn558, previously described as a hint towards a functional activity of this transposon (3), were not detectable for the Tn558 variant of plasmid pSCFS6. The transposase gene tnpC of the novel Tn558 variant was interrupted by the insertion of a 9,594-bp region, with 270 bp of tnpC located upstream and 102 bp downstream of this integrated segment (Fig. 2). The presumable target sequence for the inserted region, 5′-GACGTA-3′, was duplicated at the right- and left-end junctions of the novel region, forming 6-bp direct repeats (Fig. 2).

FIG. 2.

Organization of the novel variant of transposon Tn558 of plasmid pSCFS6 carrying an lsa(B)-cfr resistance gene region bracketed by IS21-558 elements. This Tn558 variant is shown in comparison to the original fexA-carrying transposon Tn558 located on plasmid pSCFS2 (AJ715531) and to the other so-far-known Tn558 variant detected on plasmid pSCFS3 (AM086211). The positions and orientations of the genes coding for transposition functions (tnpA, tnpB, and tnpC), antimicrobial resistance [fexA, resistance to florfenicol and chloramphenicol; cfr, resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics; lsa(B), reduced susceptibility to lincosamides], or unknown functions (orf138) are indicated by arrows with the direction of transcription shown by the arrowhead. A distance scale in kilobases is given below each map. The 6-bp direct repeats at the junctions of the IS21-558 elements in plasmid pSCFS6 are shown in boxes.

Analysis of the inserted 9,594-bp region revealed a structure consisting of two terminal IS21-558 insertion sequences present in the same orientation and a central part carrying the multidrug resistance gene cfr and the low-level clindamycin exporter gene lsa(B) (Fig. 2). Both IS21-558 elements were 2,452 bp in size, differed by only 4-bp exchanges from each other, and contained the two overlapping reading frames istAS and istBS, coding for proteins of 445 aa and 250 aa, respectively. Both IS21-558 elements of pSCFS6 exhibited 99% nucleotide sequence identity to the 6-bp-shorter IS21-558 of plasmid pSCFS3 (accession no. AM086211). Moreover, both pSCFS6-associated insertion sequences exhibited imperfect terminal inverted repeats of 56 or 57 bp ending with the nucleotides CA which are characteristic for members of the IS21 family. The multiple repeats, previously reported to be present near the termini in other members of the IS21 family (1, 8), could not be detected in these pSCFS6-associated IS21-558 elements.

A 4,690-bp region with striking homology to the cfr-lsa(B) resistance gene area of plasmid pSCFS1 (accession no. AJ579365) was located between the two IS21-558 elements (Fig. 2). While cfr was indistinguishable from the gene described on plasmid pSCFS1 and only two base pair exchanges were detected in the pSCFS1-homologous, noncoding regions, lsa(B) differed in 2 bp from lsa(B) of plasmid pSCFS1 and three single-base-pair insertions and one 14-bp insertion were detectable in the lsa(B) up- and downstream areas. A stretch of 94 bp without homology to any database entries was identified between these cfr and lsa(B) parts (Fig. 2).

Analysis of potential mobility pathways for cfr-IS21-558.

For the IS21 family of insertion sequences, a double function of the istA gene product as a transposase or a cointegrase was described, depending on the use of alternative start codons that may enlarge the IstA protein by 3 to 11 aa (1, 17). For the pSCFS6-associated IS21-558 elements, such an alternative start codon, GTG, was also detected 12 bp upstream of the ATG start codon. While the enlarged IstA protein has been reported to promote mainly simple transposition events, the shorter IstA protein primarily promotes replicon fusions (17). The simple transposition event is thought to occur via a “cut-and-paste” mechanism of a single IS21 copy and involves circularization of the IS21 element (Fig. 3a), while the replicon fusion occurs preferentially between two IS21 elements arranged in a tandem formation and a second target replicon (Fig. 3b) (1, 8, 14, 15). All these processes are assisted by the IstB helper protein (16) and enable the translocation of DNA fragments from a donor site to a target site. As a result of this replicon fusion pathway, a cointegrate is formed which carries two IS21 elements located in the same orientation at the former replicon junctions (1, 14). Such a cointegrate, flanked by two IS21-558 elements, was detected on plasmid pSCFS6. Although a tandem formation of IS21 elements normally precedes the cointegrate formation (1, 15), IS21-558 tandems could not be detected by sequence analysis, PCR assays, or hybridization experiments in any of the strains investigated in this study. Nevertheless, this specific transposition or “replicon fusion” process (17) seems to be the most likely way by which the insertion of the resistance genes cfr and lsa(B) into Tn558 has occurred. This assumption is supported not only by the characteristic arrangement of the IS21-558 insertion sequences but also by the 6-bp direct repeats at the junctions of the inserted element. Concerning their size, these duplications are in the typical range (4 to 7 bp) of target site duplications described elsewhere for IS21-like elements (1). PCR assays and subsequent sequence analysis were conducted to detect the circular intermediates promoted by the IstAS transposase. Amplicons were obtained from both S. hyicus isolates, but neither from the S. warneri or S. simulans isolates nor from pSCFS6-carrying S. aureus RN4220 transformants. Sequence analysis of the amplicons confirmed the linkage between the 3′ and 5′ ends of IS21-558, as expected from a circularization of the element, and both ends were separated by two additional base pairs, 5′-CA-3′. Since the results of hybridization experiments did not confirm the presence of an IS21-558 tandem formation, the amplicons obtained are considered indicative for the presence of minicircles in S. hyicus and thus suggest a functional activity of the IS21-558 elements.

FIG. 3.

Schematic presentation of the two transposition pathways of IS21-like elements, simple transposition and cointegrate formation (replicon fusion), according to reference 1. (a) Simple transposition is believed to involve minicircle formation of IS21 elements prior to integration into a new recipient replicon. Tandem formation may arise from transposition of an IS21-like element next to the terminus of an IS21-like element already present on a replicon or by recombination between an IS21-like element located on a replicon and an IS21-like minicircle (1). (b) Cointegrate formation involves an IS21-mediated fusion of two replicons and generates typical target site duplications. The 6-bp target duplication in plasmid pSCFS6, GACGTA (which is part of the tnpC coding sequence), flanking the inserted segment (replicon 2), is shown in boxes.

In contrast to the simple transposition pathway, IS21-mediated transposition events resulting in cointegrate formation are occasionally followed by recombination-derived deletions which may affect one insertion sequence element. In such cases, flanking DNA can be deleted concomitantly (1). Recombination processes might explain the presence of a single IS21-558 element and the cfr gene on plasmid pSCFS3 (4) (Fig. 2).

In conclusion, the data described in this study show the presence of the multidrug resistance gene cfr not only in several new staphylococcal host species but also in a new genetic context. Its identification in pathogenic porcine staphylococci, including S. hyicus, underlines a wider distribution than originally thought (4). Moreover, the detection of the entire cointegrate structure on plasmid pSCFS6 might explain a potential IS21-558-mediated transfer of the resistance genes cfr and lsa(B) and bears the danger of a further dissemination of the multidrug resistance gene cfr.