Successful multiresistant community-associated methicillin-resistant Staphylococcus aureus lineage from Taipei, Taiwan, that carries either the novel Staphylococcal chromosome cassette mec (SCCmec) type VT or SCCmec type IV

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) isolates carry the methicillin resistance gene (mecA) on a horizontally transferred genetic element called the staphylococcal chromosome cassette mec (SCCmec). Community-acquired MRSA (CAMRSA) isolates usually carry SCCmec type IV. We previously reported that 76% of 17 CAMRSA isolates (multilocus sequence type 59) obtained from pediatric patients with skin and soft tissue infections (SSTI) from Taipei did not carry SCCmec types I to IV. We used DNA sequence analysis to determine that the element harbored by these nontypeable isolates is a novel subtype of SCCmec V called SCCmec V(T.) It contains a ccrC recombinase gene variant (ccrC2) and mec complex C2. One SSTI isolate contained molecular features of SCCmec IV but also contained ccrC2 (a feature of SCCmec V(T)), suggesting that it may harbor a composite SCCmec element. The genes lukS-PV and lukF-PV encoding the Panton-Valentine leukocidin (PVL) were present in all CAMRSA SSTI isolates whether they contained SCCmec type IV or V(T). SCCmec V(T) was also present in 5 of 34 (14.7%) CAMRSA colonization isolates collected from healthy children from Taipei who lacked MRSA risk factors. Four (80%) of the these isolates contained lukS-PV and lukF-PV, as did 1 of 27 (3.7%) SCCmec IV-containing colonization isolates. A total of 63% (10 of 16) of the SSTI isolates and 61.7% (21 of 34) of the colonization isolates tested were resistant to at least four classes of non-beta-lactam antimicrobials. SCCmec V(T) is a novel SCCmec variant that is found in multiply resistant CAMRSA strains with sequence type 59 in Taipei in association with the PVL leukotoxin genes.

FIG. 1.

Multiple resistance to non-β-lactam antimicrobials among SSTI and colonization isolates. The percentages of isolates with resistance to ≤2, 3, and ≥4 non-β-lactam antimicrobials in SSTI and colonization CAMRSA from Taipei are shown. The antimicrobials tested are listed in Table 2.

FIG. 2.

Southern blotting analysis of ST 59 CAMRSA isolates containing either SCCmec V_T or SCCmec IV. (A) Map illustrating the hybridization probes (black bars above and below the map), mecA and orfX. (B) Pulsed-field gel containing SmaI-digested DNA from ST 59 isolates containing SCCmec IV (lane 5, strain TSGH 5) or SCCmec V_T. Lanes 1, 2, 3, 4, 7, 8, and 9 represent strains TSGH 1, 2, 3, 4, 7, 8, and 9, respectively. Lane 6 contains DNA from strain TSGH 6, an MSSA isolate with ST 59. Lane 39 contains DNA from an MSSA healthcare-associated strain TSGH 39 that has a different ST. (C) Southern blot of the gel shown in panel B hybridized with the mecA probe. (D) Southern blot produced after the blot in panel C was stripped and rehybridized with a probe specific for orfX, the ORF adjacent to the right junction of SCCmec that contains the integration site. The faint bands appearing toward the bottom of the membrane are mecA-specific bands remaining after incomplete stripping of the membrane. Lane S contains DNA from a pulsed-field gel marker from Sigma. Lane Se contains DNA from Staphylococcus epidermidis.

FIG. 3.

Comparisons of ccrC genes. (A) ORF architecture of the ccrC homologues from SCCmec V_T (strain TSGH 17), SCCmec V (strain WIS), SCCmec III (strain 85/2082), SCCmec III (strain 85/3907), and SCCcap1 (strain M). (B) Sequence alignment tree built from a CLUSTAL W alignment between the 1,677-bp ccrC ORF from strain TSGH 17 and the corresponding ∼1,680-bp region encompassing the ccrC ORFs from SCCmec types III, V, and SCCcap1 (same sequences as those used in panel A) using the neighbor-joining method of Saitou and Nei (35) using AlignX program in the Vector NTI software suite. Calculated distance based on distances between all pairs of sequence values are shown in parenthesis following the molecule name. (C) Sequence alignment of the 3′ region of ccrC between strains TSGH 17 and WIS and the consensus sequence for the corresponding region from 18 other SCCmec V_T isolates (starting at position 1249 of TSGH 17 ccrC to the end of the ORF). The accession numbers used in the above alignments were: SCCmec V (strain WIS, accession no. AB121219, nucleotides 16132 to 17813), SCCmec III (strain 85/2082, accession no. AB037671, nucleotides 7232 to 8913, reverse complement), SCCmec III (strain 85/3907, accession no. AB047089, nucleotides 7237 to 8917, reverse complement), and SCCcap1 (strain M, accession no. U10927, nucleotides 6864 to 8546). A “^” symbol beneath the aligned sequences indicates a nucleotide difference between SCCmecV_T and SCCmecV. The alignment was performed using default settings with public domain software (http://align.genome.jp) and the Fast/Approximate algorithm. (D) Unrooted dendrogram calculated from a CLUSTAL W multiple sequence alignment between ccrA1, ccrA2, ccrA3, ccrB1, ccrB2, ccrB3, and ccrC1 (from SCCmecV); ccrC2 (from SCCmec V_T); ccrC3 from SCCmec III; and ccrC4 from SCCcap1.

FIG. 3.

Comparisons of ccrC genes. (A) ORF architecture of the ccrC homologues from SCCmec V_T (strain TSGH 17), SCCmec V (strain WIS), SCCmec III (strain 85/2082), SCCmec III (strain 85/3907), and SCCcap1 (strain M). (B) Sequence alignment tree built from a CLUSTAL W alignment between the 1,677-bp ccrC ORF from strain TSGH 17 and the corresponding ∼1,680-bp region encompassing the ccrC ORFs from SCCmec types III, V, and SCCcap1 (same sequences as those used in panel A) using the neighbor-joining method of Saitou and Nei (35) using AlignX program in the Vector NTI software suite. Calculated distance based on distances between all pairs of sequence values are shown in parenthesis following the molecule name. (C) Sequence alignment of the 3′ region of ccrC between strains TSGH 17 and WIS and the consensus sequence for the corresponding region from 18 other SCCmec V_T isolates (starting at position 1249 of TSGH 17 ccrC to the end of the ORF). The accession numbers used in the above alignments were: SCCmec V (strain WIS, accession no. AB121219, nucleotides 16132 to 17813), SCCmec III (strain 85/2082, accession no. AB037671, nucleotides 7232 to 8913, reverse complement), SCCmec III (strain 85/3907, accession no. AB047089, nucleotides 7237 to 8917, reverse complement), and SCCcap1 (strain M, accession no. U10927, nucleotides 6864 to 8546). A “^” symbol beneath the aligned sequences indicates a nucleotide difference between SCCmecV_T and SCCmecV. The alignment was performed using default settings with public domain software (http://align.genome.jp) and the Fast/Approximate algorithm. (D) Unrooted dendrogram calculated from a CLUSTAL W multiple sequence alignment between ccrA1, ccrA2, ccrA3, ccrB1, ccrB2, ccrB3, and ccrC1 (from SCCmecV); ccrC2 (from SCCmec V_T); ccrC3 from SCCmec III; and ccrC4 from SCCcap1.

FIG. 4.

Comparison of mec complexes in SCCmec V_T and SCCmec V. The map depicts the mec C2 complex (indicated by a dashed line beneath the diagram) of SCCmec V_T from strain TSGH 17 (top) and the mec C2 complex of SCCmec V from strain WIS (accession no. AB121219) (bottom). The SCCmec V_T signature premature stop codon forming a truncated transposase (tnp′) in IS431 in strain TSGH 17 is not present in SCCmec V. The horizontal bar between the two SCCmec elements depicts >95% nucleotide sequence identity. The vertical striped bar indicates the presence of an extra dru in the intergenic hypervariable region in SCCmec V_T in strain TSGH 17 compared to strain WIS.

FIG. 5.

PCR assay demonstrating the presence of both ccrAB type 2 and ccrC2 in strain TSGH 10. PCR products were resolved by agarose gel electrophoresis stained with ethidium bromide. Lanes contain PCR products produced with either the primer pair γF and CDS15-R to detect ccrC (ccrC) or the multiplex primers β2, α2, α3, and α4 for detecting ccrAB 1, 2, and 3. Lanes contain reactions from either SSTI isolates TSGH 17 (lane 17) or TSGH 10 (lane 10). DNA from control strains containing SCCmec I, II, III, and IV are in lanes labeled I (NCTC 10442/SCCmec I), II (strain N315, SCCmec II), III (strain 85/2082, SCCmec III), and IV (strain MW2, SCCmec IV) and contain ccrAB types 1, 2, 3, and 2, respectively. Lanes c, negative control lanes containing water instead of a DNA template.