Article Figures & Data
Figures
- Fig. 1.
Genetic organization of the mecA gene complex in staphylococci and the PCR primers for the detection of IS431L and IS1272 upstream of mecA. The structures of five classes of mec complexes found in MRSA and MRC-NS are shown. In all five classes, IS431R was present downstream of mecA. The class A meccomplex has the structuremecI-mecR1-mecA-IS431mec. In S. haemolyticus, the class A mec complex was accompanied by an upstream IS431 copy (IS431L). The class Bmec complex has the structure ψIS1272-ΔmecR1-mecA-IS431. Class C1 and C2 mec complexes have a deletion in the left side of the class A mec complex associated with an insertion of IS431. In class C1, IS431L is inserted in the same orientation as mecA, and in class C2, IS431Lis inserted in the opposite orientation relative to mecA. Class D has a deletion without any insertion sequence adjacent to the deletion point. Arrows above the genes indicate the direction of transcription. The arrowheads indicate the location and direction of primers (see Materials and Methods).
- Fig. 2.
Nucleotide sequencing analysis of meccomplexes and their surrounding regions. The arrows indicate the direction of transcription of genes. The region upstream of ΔmecR1 of S. caprae strain JA186 (class D) was compared with SCCmec of N315 (carrying the class Amec complex). Eight nucleotides flanking the IS431 copies are also shown to infer a possible relationship of individual IS copies. The single asterisk and the double asterisks indicate pairs of the same sequence. No conserved direct repeats indicating target duplication were found in the 8 bp flanking the IS431-mecA-IS431 structures (5).
- Fig. 3.
Detection of IS431L-mediated deletion in resistant mutants derived from S. haemolyticus SH621. Portions (3 μl) of the PCR mixture were applied to an agarose gel and subjected to electrophoresis. Lanes 1, λ-HindIII digest as a DNA molecular mass standard marker (the sizes were 23.1, 9.5, 6.6, 4.3, 2.3, and 2.2 kb from top to bottom); 2, SH621; 3 to 5, SH621-derived mutants with increased methicillin resistance. Mutants were selected with 1 (lane 3), 3 (lane 4), or 8 (lane 5) mg of methicillin per liter, respectively. (A) Primers iS-1 plus mA2 were used to detect the region between IS431L andmecA. (B) Primers mA4 plus iS-2 were used to detect the region between mecA and IS431R(IS431R). Note that there are varied band sizes only in lanes 3 and 4 in panel A.
- Fig. 4.
IS431L-mediated deletion of the class Amec complex. The structures of three subclasses of the class C1 deletion of the mec complex in the mutants are shown as follows: subclass C1a (IS431-ΔmecI-mecR1-mecA), subclass C1b (IS431-ΔmecR1 [retaining the MS domain]-mecA), and subclass C1c (IS431-ΔmecR1 [retaining only the 5′ end]-mecA). The flanking sequences of IS431L and inverted repeats (IR-L; underlined) are presented to show the deletion points in detail. The bold type indicates the names of the (mutant) strains and the sizes of the deletion. The arrows indicate the direction of transcription. A total of 15 mutants including all 10 mutants obtained by selection with 8 mg of methicillin per liter retained the class A mec complex. Others were classified as either one of the three class C subtypes. The asterisk in the mutant strain 33 shows that the position of IS431L was identical to that found in clinically isolated strain SH631.
- Fig. 5.
Population analysis of mutant strains SH621-37, SH621-34, and SH621-81 in comparison with the parent strains SH621 and N315. See Materials and Methods for the procedure of population analysis. Symbols: ⧫, SH621 (class A); ■, SH621-37 isolated with 3 mg of methicillin per liter (class C1); ●, SH621-34 isolated with 3 mg of methicillin per liter (class A); ▴, SH621-81 isolated with 8 mg of methicillin per liter (class A); ○, S. aureus N315 (class A).
Tables
- Table 1.
Synthetic oligonucleotides primers used for the analysis of mec complex structure
Primer designation Sequencea Localization Positions Reference(s) mA2 5′-AACGTTGTAACCACCCCAAGA-3′ mecA 1461–1441 11,28 mA4 5′-AGTGTATGATGAGCTATATGAGA-3′ mecA 1959–1981 28 mA5 5′-CGCTCAGAAATTTGTTGTGC-3′ mecA 212–180 28 mA6 5′-TATACCAAACCCGACAAC-3′ mecA 83–66 28 mA7 5′-ATCGTTGACGATAATAGCAATACA-3′ mecA 907–930 28 mA8 5′-ATGTTTGGATTATCTTTATCATAT-3′ mecA 1863–1886 28 iS-1 5′-ACATTAGATATTTGGTTGCGT-3′ IS431mec 566–596 3 iS-2 5′-TGAGGTTATTCAGATATTTCGATGT-3′ IS431mec 767–743 3 iS-3 5′-TCGGATGCTATCATTAAGCAT-3′ IS1272 1668–1688 2 iS-4 5′-ACAATCTGTATTCTCAGGTCGT-3′ IS1272 1722–1701 2 mI-1 5′-AATGGCGAAAAAGCACAACA-3′ mecI 1923–1942 10, 30 mI-2 5′-GACTTGATTGTTTCCTCTGTT-3′ mecI 2403–2283 10, 30 mcR2 5′-CGCTCAGAAATTTGTTGTGC-3′ mecR1 1954–1935 10, 30 mcR3 5′-ATCTCCACGTTAATTCCATT-3′ mecR1 357–376 10, 30 ↵a The location of each primer is shown in Fig. 1.
- Table 2.
Distribution of various classes of mec complex among C-NS species
mec complex classa No. (%) of isolates of: S. aureus (n = 38) S. haemolyticus (n = 38) S. epidermidis(n = 34) S. sciuri (n = 9) S. caprae (n = 5) S. hominis (n = 2) S. capitis (n = 2) S. warneri (n = 1) A 26 (68.4) 5 (13.2) 19 (55.9) 9 4 (80.0) 2 2 1 B 12 (31.6) 2 (5.3) 14 (41.2) 0 0 0 0 0 C1 0 1 (2.6) 0 0 0 0 0 0 C2 0 30 (78.9) 1 (2.9) 0 0 0 0 0 D 0 0 0 0 1 (20.0) 0 0 0 ↵a Corresponds to the meccomplex shown in Fig. 4.
- Table 3.
Correlation between methicillin MIC and the class ofmec complex carried by C-NS clinical strains
Species Total no. of strains tested meccomplex classa No. of strains for which the MIC (mg/liter) is: 2 4 8 16 32 64 ≧128 S. epidermidis 16 A 1 4 5 6 5 B 1 1 3 1 C2 1 S. haemolyticus 5 A 3 2 2 B 1 1 1 C1 1 30 C2 2 7 10 1 1 9 S. sciuri 7 A 2 4 1 S. caprae 4 A 4 1 D 1 S. hominis 2 A 2 S. capitis 2 A 2 S. warneri 1 A 1 ↵a Corresponds to the meccomplex shown in Fig. 4.
- Table 4.
Structure of mec complex of and methicillin MIC for mutants derived from S. haemolyticus strain SH621
Strain PBP2 productiona Selective methicillin concn (mg/liter)b Class of meccomplexc PCRdstructure of: MIC of methicillin (mg/liter) mecI mecR1 PB MS SH621 − A + + + 2 SH621-15 + 1 A + + + 8 SH621-16 + 1 A + + + 4 SH621-32 + 3 A + + + 512 SH621-34 + 3 A + + + <1,024 SH621-310 + 3 A + + + 512 SH621-81 + 8 A + + + <1,024 SH621-82 + 8 A + + + <1,024 SH621-83 + 8 A + + + 1,024 SH621-84 + 8 A + + + 1,024 SH621-85 + 8 A + + + <1,024 SH621-86 + 8 A + + + <1,024 SH621-87 + 8 A + + + 1,024 SH621-88 + 8 A + + + 1,024 SH621-89 + 8 A + + + <1,024 SH621-810 + 8 A + + + 512 SH621-17 + 1 C1a Δ + + 16 SH621-37 + 3 C1a Δ + + 16 SH621-38 + 3 C1a Δ + + 16 SH621-39 + 3 C1a Δ + + 16 SH621-13 + 1 C1b − Δ + 32 SH621-35 + 3 C1b − Δ + 64 SH621-31 + 3 C1b − Δ + 32 SH621-33 + 3 C1b − Δ + 16 SH621-36 + 3 C1b − Δ + 16 SH621-11 + 1 C1c − − Δ 16 SH621-12 + 1 C1c − − Δ 16 ↵a Judged by the rapid slide latex agglutination assay kit (24).
↵b The concentration of methicillin used for the selection of mutants.
↵c Corresponds to the mec complex shown in Fig. 4.
↵d + indicates that the size of the DNA fragment amplified by PCR was indistinguishable from that of SH621. Δ indicates that the amplified DNA fragment contained a deletion.
