Characterization of New Staphylococcal Cassette Chromosome mec (SCCmec) and Topoisomerase Genes in Fluoroquinolone- and Methicillin-Resistant Staphylococcus pseudintermedius

Strain identification and antibiotic resistance profile.

The isolated strains (n = 15) were cultured on tryptone soy agar plates containing 5% sheep blood (Oxoid Ltd., Basingstoke, England) and were identified by catalase activity, Gram staining, and sequencing of the sodA gene as described previously (24, 26). All isolates harbored the leukocidin gene lukS (25) but not the Panton-Valentine leukocidin gene lukS-PV (28), as determined by PCR using the primers listed in Table 2. Antibiotic susceptibility was determined by broth microdilution using a custom Sensititre susceptibility plate, model NLV57 (Trek Diagnostics Systems, East Grinstead, England; MCS Diagnostics BV, Swalmen, The Netherlands), according to CLSI (formerly NCCLS) guidelines (4). Resistance genes were detected using a microarray (22) (Table 1). In addition to fluoroquinolone and oxacillin (methicillin) resistance, the isolates also displayed resistance to other antibiotics (Table 1).

Characterization of mutations in topoisomerase genes.

The mechanism of resistance to fluoroquinolones was investigated by sequence analysis of the topoisomerase II (gyrA and gyrB) and IV (grlA and grlB) genes, since mutations in these genes have been shown to confer resistance to fluoroquinolones on Staphylococcus aureus (8, 11, 13, 14, 18, 27, 30). Fragments of gyrA, gyrB, grlA, and grlB of type strains S. pseudintermedius CCUG49543^T (also known as LMG 22219^T [10]) (CCUG, Culture Collection, University of Göteborg, Göteborg, Sweden) and Staphylococcus intermedius DSM20373^T (also known as NCTC 11048^T) (DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were amplified by PCR and sequenced on an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA) using oligonucleotide primers (Table 2) designed from conserved regions found after alignment of DNA sequences of grlB/grlA and gyrB/gyrA from S. aureus (GenBank accession no. L25288 and BA000018), Staphylococcus epidermidis (GenBank accession no. CP000029 and AE015929), and Staphylococcus haemolyticus (GenBank accession no. AY341071, AY341072, AY341073, and AY341074) using MultAlin (7). The nucleotide sequences flanking the amplified fragments were determined by genome walking using the Universal Vectorette system according to the manufacturer's protocol (Sigma-Genosys, St. Louis, MO) and genomic DNA digested with Sau3A, EcoRI, HindIII, and ApoI. The nucleotide sequences of the grlB/grlA and gyrB/gyrA loci of S. pseudintermedius CCUG49543 share 89% and 94% identity, respectively, with those of S. intermedius DSM20373. The entire gyrB/gyrA and grlB/grlA loci of fluoroquinolone-resistant and fluoroquinolone-susceptible S. pseudintermedius strains were then amplified using the Expand Long Template PCR system (Roche Applied Science, Indianapolis, IN) (annealing at 54°C for 30 s and extension at 68°C for 5 min) with specific primer pairs gyrB-si-PF0-gyrA-si-RV and grlB-si-PF-grlA-si-R0 and were sequenced. Nucleotide sequence comparison revealed high levels of nucleotide polymorphism in the gyrase and topoisomerase IV genes. Most of the mutations are silent (Fig. 1). Mutations that cause amino acid substitutions in the topoisomerases were found in both resistant and susceptible strains and cannot, therefore, be responsible for resistance. However, other amino acid mutations were found only in fluoroquinolone-resistant strains (Table 3). Some of these amino acid changes occurred at the same positions as those reported for fluoroquinolone-resistant S. aureus, S. intermedius, and Staphylococcus schleiferi strains, including positions 251 (Ser84Leu) and 263 (Glu88Gly) on gyrA and positions 239 (Ser80Ile) and 250 (Asp84Asn) on grlA (8, 13-15, 18, 27, 30) (Table 3). S. pseudintermedius strains harboring at least one of these mutations showed decreased susceptibility to enrofloxacin (MICs, 4 to 8 μg/ml). Higher MICs (≥16 μg/ml) were observed when two additional mutations (Thr678Ala and Glu714Lys) were present in gyrA (Table 3). Additional amino acid substitutions were found in the topoisomerase genes, but their roles in fluoroquinolone resistance remain to be determined (Fig. 1).

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FIG. 1.

Consensus nucleotide mutations present in the topoisomerase IV and gyrase genes of 12 fluoroquinolone-resistant and 4 fluoroquinolone-susceptible strains. Silent mutations (not boxed) are indicated with thin arrows. Open boxes, mutations that cause amino acid substitutions in both fluoroquinolone-resistant and fluoroquinolone-susceptible strains; solid boxes, mutations that cause amino acid substitutions at the same positions as those described for fluoroquinolone-resistant S. aureus; shaded boxes, additional mutations that cause amino acid substitutions in fluoroquinolone-resistant S. pseudintermedius.

Characterization of two new SCCmec elements.

Methicillin resistance is mediated by the mecA gene, which is located on a large staphylococcal cassette chromosome mec (SCCmec) element. SCCmec elements are characterized by the completeness of the methicillin resistance regulon containing mecA, by the allotype of the recombinase genes ccrA and ccrB, and by the general genetic structure (2, 12, 20). SCCmec are delineated by two inverted repeats, IR-L and IR-R (Fig. 2). Six different SCCmec (I to VI) have been described to date for Staphylococcus (2, 12, 16, 20, 21). In the course of our study, SCCmec of S. pseudintermedius could not be classified using PCR methods previously developed to determine the SCCmec class (19, 31) and were therefore sequenced. First, the regions spanning orfX to mecI and mecI to ccrA of all methicillin-resistant S. pseudintermedius strains were amplified using the Expand Long Template PCR system (Roche Applied Science, Indianapolis, IN) with primers ccrA4-F1 and mecI-R (fragment A) and mecI-F and ORFX1r (9) (fragment B) (annealing at 50°C for 30 s; extension at 68°C for 15 min) (Table 2; Fig. 2). Restriction analysis of fragments A and B, digested with HindIII and PstI, respectively, revealed two types of SCCmec. The SCCmec of KM241 had a unique profile, whereas all the other profiles were identical to that of KM1381. The fragments were sequenced using a primer-walking strategy. To complete the entire nucleotide sequence of the cassette, the 5′ end of the SCCmec of KM1381 situated upstream of ccrA was amplified using primer ccrA-R and primer SccmecIR-F, which is specific to S. aureus SCCmec III IR-L (GenBank accession no. AB037671). The 5′-end sequence of the SCCmec of KM241 was determined using the Universal Vectorette system (Sigma-Genosys Co., St. Louis, MO). Sequence analysis of the two entire cassettes revealed two new SCCmec, SCCmec II-III in KM1381 and SCCmec VII in KM241 (Fig. 2). SCCmec II-III consists of a combination of S. aureus SCCmec III (accession no. AB037671) (100% nucleotide identity from IR-L to ORF12) and S. epidermidis SCCmec II (GenBank accession no. CP000029) (98.9% nucleotide identity from ORF13 to IR-R) (Fig. 2). SCCmec VII contains new recombinase genes, ccrA5 and ccrB5, classifying it as a new allotype, allotype 5. The amino acid sequences of ccrA5 and ccrB5 showed 75.6% identity overall to S. aureus CcrA, allotype 3 (GenBank accession no. BAA88754), and 92.3% identity overall to S. aureus CcrB, allotype 3 (GenBank accession no. BAA88755). The rest of the SCCmec VII downstream of the ccrA5 and ccrB5 loci until IS431 showed 99% nucleotide identity to S. aureus SCCmec III (GenBank accession no. AB037671) (Fig. 2) but differed from SCCmec III by a complete mecA regulon. Both SCCmec II-III and SCCmec VII are new cassettes, which belong to class A, allotype 3, and class A, allotype 5, respectively.

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FIG. 2.

Alignment of S. pseudintermedius SCCmec II-III and SCCmec VII of with S. aureus SCCmec III and S. epidermidis SCCmec II. Solid arrows represent the mecA regulon. Diagonally lined, vertically lined, and checked arrows represent the different cassette chromosome recombinases A and B. Horizontally lined arrows, transposases; open arrows, cadmium resistance genes; shaded arrows, hypothetical proteins. Cassettes are delimited by inverted repeats IR-L and IR-R (dashed lines).

Differentiation of strains by ST.

Sequence typing (ST) of five gene loci (the 16S rRNA gene, tuf, cpn60, pta, and agrD) and examination of the allelic variation of agrD (1) showed that all methicillin-resistant strains containing SCCmec II-III were clonally related. They belong to ST71, agr type III (Table 1), which is the predominant clonal group in North and Central Europe (1). They also contain the same mutations in the gyrB/gyrA and grlB/grlA genes (gyr/grl group 5), except for strain KM1395. S. pseudintermedius strain KM241 (SCCmec VII) belongs to a new group (ST73, agr IV). The methicillin- and fluoroquinolone-susceptible strains belong to the distinct ST group ST41, agr II, and to new ST groups ST74, agr II, and ST75, agr I (Table 1).

New SCCmec with new recombinase genes in S. pseudintermedius represent a new reservoir of the mecA gene for methicillin-sensitive Staphylococcus species. Until now, mainly SCCmec III has been detected in S. pseudintermedius (26). Additionally, sequence analysis of topoisomerase genes has allowed us to determine, for the first time for S. pseudintermedius, mutations that play a role in fluoroquinolone resistance.

The presence of multidrug-resistant Staphylococcus of animal origin is a further demonstration that the use of antibiotics in veterinary medicine selects for resistant strains. Guidelines regarding the use and choice of antibiotics should be followed in veterinary medicine to suppress the rapid, nationwide dissemination of multidrug-resistant S. pseudintermedius clones.

Nucleotide sequence accession numbers.

Nucleotide sequences were deposited in the EMBL/GenBank/DDBJ databases. SCCmec II-III and SCCmec VII were assigned accession no. AM904732 and AM904731. The gyrB/gyrA and grlB/grlA loci of S. pseudintermedius CCUG49543^T and KM1381 and of S. intermedius DSM20373^T were assigned accession no. AM262968 and AM262971, AM262969 and AM262972, and AM262967 and AM262970, respectively.