Characterization of a Novel Composite Staphylococcal Cassette Chromosome mec in Methicillin-Resistant Staphylococcus pseudintermedius from Thailand

Abstract

A novel staphylococcal cassette chromosome mec (SCCmec) composite island (SCCmec_AI16-SCCczr_AI16-CI) was identified in Staphylococcus pseudintermedius. Four integration site sequences for SCC subdivided the 60,734-bp island into 41,232-bp SCCmec_AI16, 19,400-bp SCCczr_AI16, and 102-bp SCC-like_AI16 elements. SCCmec_AI16 represents a new combination of ccrA1B3 genes with a class A mec complex. SCCczr_AI16 contains ccrA1B6 and genes related to restriction modification and heavy metal resistance. SCCmec_AI16-SCCczr_AI16-CI was found in methicillin-resistant S. pseudintermedius sequence type 112 (ST112) and ST111 isolated from dogs and veterinarians in Thailand.

TEXT

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) has been associated with clinical manifestations in companion animals and occasionally causes diseases in humans (1, 2). MRSP contains the staphylococcal cassette chromosome mec (SCCmec) element, which is defined by the presence of the mec gene that mediates β-lactam resistance and by the cassette chromosome recombinase (ccr) gene(s) responsible for site-specific integration/excision of the element (3). Composite SCC structures containing two ccr gene complexes likely resulting from multiple element integrations have been increasingly reported in Staphylococcus aureus and in coagulase-negative staphylococci where such structures frequently carry heavy metal resistance gene clusters (3–9). So far, only five SCCmec elements have been completely characterized in MRSP, including SCCmec II-III, SCCmec VII-241, SCCmec V, ΨSCCmec₅₇₃₉₅, and SCCmec IV, and are mainly associated with predominant clones of the following sequence types (ST): ST71, ST93, ST68, ST233, ST45, and ST261 (10–14). Recently, MRSP strains of ST112 and ST111 (a single locus variant of ST112) isolated from dogs and veterinarians in Thailand were found to contain an unusual combination of the class A mec gene complex with the type I ccr gene complex, suggesting a new SCCmec type (15). We therefore aimed to characterize this novel SCCmec element and to determine whether it is conserved within other MRSP isolates of the same clonal lineage.

Identification of the SCCmec_AI16-SCCczr_AI16 composite element (CI).

MRSP strains of ST111 and ST112 were obtained from a previous study and are listed in Table 1 (15). Phenotypic and genotypic characteristics, including those determined by antimicrobial susceptibility testing and antibiotic resistance gene detection, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST), were determined previously (15). In this study, the strains were additionally tested for mec-associated direct repeat unit (dru) and spa types (16, 17), for the presence of the novel SCCmec element, and for resistance to heavy metals. The genomic DNA of all strains was extracted using the peqGOLD bacterial DNA kit (Peqlab Biotechnologie GmBH, Jena, Germany), and that of strain AI16 was sequenced using Ion Torrent semiconductor (Life Technologies, Carlsbad, CA) and Illumina MiSeq (Illumina, San Diego, CA) technologies. Sequence reads from the Ion Torrent were assembled de novo using MIRA v3.4.1.1, generating 64 contigs with an N₅₀ (length weighted median) of 121,469 bp and a contig sum of 2,829,899 bp (mean read length, 265 bp; average coverage, >100-fold). The sequences were corrected by read mapping with MiSeq reads using Geneious version R8 (Biomatters, Auckland, New Zealand) (18), and unresolved discrepancies were verified by Sanger sequencing (ABI Prism 3100 genetic analyzer; Applied Biosystems, Foster City, CA). Nucleotide analysis searching for SCC-associated components (orfX gene, mec gene complexes, ccr gene complexes, and integration site sequence [ISS] for SCC [19]) using BLAST (http://blast.ncbi.nlm.nih.gov/) enabled the identification of a 60,734-bp SCC composite element within a 100,011-bp contig. Open reading frames (ORF) were defined using Prodigal software for locating genes in prokaryotes (34) and were annotated manually using BLASTn and BLASTp algorithms. The element was integrated at the 3′ end of the chromosomal orfX gene and contained a mec gene complex, two ccr gene complexes, and four ISSs. The ISSs contained direct repeats (DRs) that flanked the composite SCC element and that also divided it into three subunits, including SCCmec_AI16, SCCczr_AI16, and SCC-like_AI16 according to the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) (4) (Fig. 1). ISSs were also linked to imperfect inverted repeats (IRs) at all subunit boundaries, forming parts of the SCC attachment sites (att) (19) (Fig. 1).

TABLE 1.

Origin and molecular characteristics of the six methicillin-resistant Staphylococcus pseudintermedius strains used in this study

Strain	Source	Site	Sequence typea	PFGE resulta	SCCmecb	spa typec	dru typed	Antimicrobial resistance profile [resistance genes]e
AI16	Dog	Groin with crusty exudate	ST112	A	SCCmecAI16-SCCczrAI16-CI	t05	11y	OXA-PEN-TET-GEN-KAN-STR-ERY-iCLI-SMX-TMP [mecA, blaZ, tet(M), aac(6′)-Ie, ant(6′)-Ia, aph(2′)-Ia, aph(3′)-III, erm(B), sat4 and dfrG]
AJ1	Dog	Perineal carriage	ST112	A	SCCmecAI16-SCCczrAI16-CI with 1,868-bp insertion	t05	11cv	OXA-PEN-TET-GEN-KAN-STR-ERY-CLI-SMX-TMP-MUP [mecA, blaZ, tet(K), tet(M), aac(6′)-Ie, ant(6′)-Ia, aph(2′)-Ia, aph(3′)-III, erm(A), erm(B), sat4, dfrG and mupA]
AK5	Dog	Nasal carriage	ST112	A	SCCmecAI16-SCCczrAI16-CI	t05	11y	OXA-PEN-TET-GEN-KAN-STR-ERY-iCLI-SMX-TMP [mecA, blaZ, tet(K), tet(M), aac(6′)-Ie, ant(6′)-Ia, aph(2′)-Ia, aph(3′)-III, erm(B), sat4 and dfrG]
AM33	Dog	Perineal carriage	ST111	C	SCCmecAI16-SCCczrAI16-CI	Negative	11y	OXA-PEN-TET-GEN-KAN-STR-ERY-iCLI-SMX-TMP [mecA, blaZ, tet(K), aac(6′)-Ie, ant(6′)-Ia, aph(2′)-Ia, aph(3′)-III, erm(B), sat4 and dfrG]
VA26	Human	Nasal carriage	ST112	A	SCCmecAI16-SCCczrAI16-CI with 1,868-bp insertion	t06	11y	OXA-PEN-TET-GEN-KAN-STR-ERY-iCLI-SMX-TMP-CIP [mecA, blaZ, tet(M), aac(6′)-Ie, ant(6′)-Ia, aph(2′)-Ia, aph(3′)-III, erm(B), sat4 and dfrG]
VB16	Human	Nasal carriage	ST112	A	SCCmecAI16-SCCczrAI16-CI	t06	11y	OXA-PEN-TET-GEN-KAN-STR-ERY-CLI-SMX-TMP-MUP-CIP [mecA, blaZ, tet(M), aac(6′)-Ie, ant(6′)-Ia, aph(2′)-Ia, aph(3′)-III, erm(B), sat4, dfrG and mupA]

^aMLST and PFGE analyses were performed as a part of the previous study (15).

^bSCCmec_AI16-SCCczr_AI16-CI was identified in all strains by long-range PCR and restriction analysis. AJ1 and VA26 contained a 1,868-bp insertion.

^cThe spa typing resulted in two spa types, t05 (r01-r02-r03-r03-r03-r03-r06-r05) and t06 (r01-r02-r03-r03-r06-r05), and one negative strain with no spa amplification by PCR.

^dThe dru types presented dru repeats as follows: 11y, 5a-2d-4a-1b-2d-5b-3a-2g-3b-4e-3e; 11cv, 5a-2d-4a-1b-2d-6f-3a-2g-3b-4e-3e.

^eAntimicrobial resistance phenotypes and detection of resistance genes were determined in the previous study using broth dilution methods and microarray (15). Abbreviation of antimicrobials are as follows: OXA, oxacillin; PEN, penicillin; TET, tetracycline; GEN, gentamicin; KAN, kanamycin; STR, streptomycin; ERY, erythromycin; CLI, clindamycin; iCLI, inducible resistance to clindamycin; SMX, sulfamethoxazole; TMP, trimethoprim; MUP, mupirocin; CIP, ciprofloxacin. Antibiotic resistance genes and functions are as follows: mecA, penicillin-binding protein 2a; blaZ, β-lactamase; tet(K), tetracycline efflux protein; tet(M), ribosomal protective protein; aac(6′)-Ie, aminoglycoside acetyltransferase; ant(6′)-Ia, aminoglycoside nucleotidyltransferase; aph(2′)-Ia and aph(3′)-III, aminoglycoside phosphotransferases; erm(B) and erm(C), erythromycin resistance methylase; sat4, streptothricin acetyltransferase; dfrG, dihydrofolate reductase; mupA, isoleucyl-tRNA synthetase.

FIG 1.

Genetic organization of the novel composite SCCmec_AI16-SCCczr_AI16-CI element of Staphylococcus pseudintermedius strain AI16 and formation of circular intermediates. The single units of the SCCmec_AI16-SCCczr_AI16-CI are flanked by direct repeats (DR) containing the integration site sequence (ISS) (red) and by inverted repeats (IR) (harpoon arrows) forming part of the attachment sites (att). White arrows represent sequences encoding hypothetical proteins, and other genes are color coded as follows: orfX, gray; IS431, pale green; mec operon, sky blue; transposases of Tn554, light gray; cyclopentanol dehydrogenase, brick red; type I restriction-modification system, orange; and czr operon, brown. The ccrA1, ccrB3, and ccrB6 recombinase genes are shown in blue, green, and cyan, respectively. Small blue arrows indicate the primers used for PCR, identifying circularization and excision of the SCCmec_AI16-SCCczr_AI16-CI subunits (see Tables S2 and S3 in the supplemental material). The genetic map of SCCmec_AI16-SCCczr_AI16-CI was drawn using the Easyfig software (32).

SCCmec_AI16.

SCCmec_AI16 consisted of a classical SCCmec element, as it contained the mec gene as well as the ccr genes and was flanked by two DRs (DR1 and DR2). It was located downstream of orfX, had a size of 41,232 bp, and contained 44 ORFs. The class A mec gene complex was located directly downstream of orfX and was only separated by a small 108-bp noncoding joining region (Fig. 1). The second joining region of SCCmec_AI16 spanning the region between the mec gene complex and the ccr gene complex contained several genes coding for hypothetical protein, cadmium resistance (cadCAD), transposase B and C of transposon Tn554, and a putative cyclopentanol dehydrogenase (cpnA). The entire 5′ region of SCCmec_AI16, including the mec gene complex up to cpnA, was highly identical (99%) to the nucleotide sequence of SCCmec VII-241 (without cpnA) and to a hybrid SCCmec-mecC region of Staphylococcus sciuri GVGS2 (including cpnA) (10, 21). However, SCCmec_AI16 differed from those two elements by the presence of a different type of ccr gene complex and a different 3′ end-joining region that encoded various hypothetical proteins, some of them already identified in other SCCmec elements (see Table S1 in the supplemental material). The ccr complex of SCCmec_AI16 contained ccrA1 and ccrB3 genes and shared the closest identity to those of S. aureus JCSC6945 and S. sciuri MCS24 with 92% and 89% nucleotide identity, respectively (20, 22) (see Fig. S1 in the supplemental material). ccrA1B3 was assigned as a type 8 ccr complex, which was originally found in SCCmec XI together with a class E mec complex in S. aureus M10/0061 (23). However, the ccrA1 of SCCmec_AI16 shared only 81% nucleotide identity with the ccrA1 of SCCmec XI but shared up to 90% sequence similarity with the ccrA1 in SCCmec IX and X in livestock-associated methicillin-resistant S. aureus (LA-MRSA) in Thailand (20, 24). The combination of the type 8 ccr complex and the class A mec complex of SCCmec_AI16 has not been previously reported and represents a new SCCmec type in S. pseudintermedius.

SCCczr_AI16 and SCC-like_AI16.

The SCCczr_AI16 element contained recombinase genes but no mec gene. It was flanked by DR2 and DR3, arranged in tandem with the SCCmec_AI16, and followed by a 102-bp noncoding SCC-like_AI16 fragment demarcated by DR3 and DR4. SCCczr_AI16 was 19 kb in length, contained 18 ORFs (see Table S1 in the supplemental material), and consisted of three main segments displaying different functions. The first segment contained genes coding for proteins (HsdR, HsdM, and HsdS) associated with a type I restriction-modification system. HsdR and HsdM showed 99% and 98% amino acid (aa) identity with those encoded on SCCfusC, respectively (25), and were combined with a novel HsdS that shared 61% aa identity to the closest HsdS (see Table S1), indicating a new restriction specificity in this MRSP lineage (26). The second segment contained a type 7 ccr complex, with ccrA1 and ccrB6 sharing 99% and 89% identity to those of SCCmec X from LA-MRSA ST398 (JCSC6945) isolated from a human in Thailand (20) (see Fig. S1 in the supplemental material). This ccr complex was preceded by three ORFs that also displayed high similarity (96%, 100%, and 99%) to those found upstream of the ccr genes of SCCmec X. The third segment situated at the 3′ region of SCCczr_AI16 showed 99% DNA identity to the 3′ region of SCC_SH32 identified in Staphylococcus haemolyticus SH32 isolated from a Chinese patient (27) and to a fragment of the SCCmec V(5C2&5)c found in S. aureus (28) and S. haemolyticus (9). It carried six ORFs, including czrC, which has been shown to confer resistance to zinc and cadmium in S. aureus (29). While resistance to cadmium increased up to 4-fold in S. pseudintermedius strains containing cadA compared to that in strains which lack cadA or carry only czrC, we did not observe reduced susceptibility to zinc in any of the tested strains, including those containing czrC, using broth microdilution assays after 20-h and 48-h (data not shown) incubations at 37°C (30) (Table 2). The same MIC of zinc for strains with and without czrC indicates that this gene does not confer measurable zinc resistance in S. pseudintermedius. Additionally, decreased susceptibility to other heavy metals was not observed (Table 2). The presence of cadA and czrC genes was confirmed by PCR using the primers listed in Table S2 in the supplemental material.

TABLE 2.

Heavy metal susceptibility of Staphylococcus aureus and Staphylococcus pseudintermedius strains as determined using broth microdilution test

Strain	Metal resistance gene(s)	MIC (mM) of metal						Source
		CuSO4	CdCl2	ZnSO4	AsNaO2	Pb(CH3COO)2	HgCl2
S. aureus
RN4220		4	≤0.015	0.5	0.03	≤0.125	≤0.015	33
S. pseudintermedius
CCUG 49543 (=LMG 22219)		16	0.25	1	2	1	≤0.015	Culture Collection, University of Göteborg, Sweden
Methicillin-resistant S. pseudintermedius
E120		16	0.25	1	2	1	≤0.015	1
KM241	cadA	16	1	1	2	1	≤0.015	10
196511	czrC	16	0.25	1	2	1	≤0.015	D. Elad, Kimron Veterinary Institute, Bet Dagan, Israel
AI16	cadA, czrC	16	1	1	2	1	≤0.015	This study, 15

Excision and circularization of SCCmec_AI16-SCCczr_AI16-CI subunits.

Ccr recombinases recognize the att sites of SCC and catalyze DNA cleavage, strand exchange, and recombination to integrate an element or to release a nonreplicative circular element. Sequencing PCR products obtained using primer pairs reading inwards and outwards from the att sites identified circular forms containing one copy of ISS as a joining region as well as chromosomal segments remaining after element excision. Such circular forms were detected for the individual SCCmec_AI16 and SCCczr_AI16 and for the composites SCCmec_AI16-SCCczr_AI16 and SCCmec_AI16-SCCczr_AI16-CI (Fig. 1; see also Table S3 in the supplemental material). This indicated CcrAB activity in the strain AI16 and possible mobilization of the individual SCCmec_AI16-SCCczr_AI16-CI circular subunits. Notably, ISS3 was preferred over ISS4 for recombination, which may be caused by the presence of a divergent IR in the ISS4-containing att site (Fig. 1; see also Table S3).

Presence of SCCmec_AI16-SCCczr_AI16-CI in additional MRSP isolates.

Long-range PCR amplification followed by restriction analysis (see Tables S2 and S4 in the supplemental material) confirmed the overall structure of SCCmec_AI16-SCCczr_AI16-CI in MRSP strain AI16 and allowed detection of similar composite elements in five additional clonally related MRSP strains of ST111 and ST112 isolated from dogs and humans (Table 1). Two strains, AJ1 (ST112) and VA26 (ST112), showed a minor variation in the restriction analysis caused by a 1,868-bp insertion between czrC and ISS3 as confirmed by primer walking and Sanger sequencing. In addition, the SCCmec element of AJ1 displayed an alternative hypervariable repeat region (dru 11cv) that was different from that of the other strains (dru 11y), suggesting different evolutionary processes within SCC of the same clonal lineage.

Characterization of the novel SCCmec_AI16-SCCczr_AI16-CI element in S. pseudintermedius revealed regions with homology to other SCCmec elements. The 5′ region of SCCmec_AI16 obviously shared a common ancestral origin with SCCmec elements found in S. pseudintermedius KM241 and in S. sciuri GVGS2 but displayed a substituted 3′ region resulting in a new combination of the class A mec complex with ccrA1B3 genes. The presence of SCCczr_AI16 harboring a second ccrAB complex illustrates the challenge for PCR-based SCCmec typing. Furthermore, the location of two related ccrAB gene complexes aligned in proximity offer a source for homologous recombination-mediated deletion, a mechanism observed previously with ccrC sequences (28, 31). The presence of this composite element in other strains of the same lineage rather supported a spread of this cassette with a clone as opposed to an autonomous transfer between strains. However, the ability of the SCCmec_AI16-SCCczr_AI16-CI subunits to circularize independently may play a role in the mobilization and in the further evolutionary diversification of this composite SCCmec structure.

Nucleotide sequence accession numbers.

The nucleotide sequences of the SCCmec_AI16-SCCczr_AI16-CI of S. pseudintermedius AI16 and the 1,868-bp insertion in S. pseudintermedius VA26 have been deposited in GenBank under accession numbers LN864705 and LN874217, respectively.