Genetic organization of the chromosome region surrounding mecA in clinical staphylococcal strains: role of IS431-mediated mecI deletion in expression of resistance in mecA-carrying, low-level methicillin-resistant Staphylococcus haemolyticus

Abstract

We report on the structural diversity of mecA gene complexes carried by 38 methicillin-resistant Staphylococcus aureus and 91 methicillin-resistant coagulase-negative Staphylococcus strains of seven different species with a special reference to its correlation with phenotypic expression of methicillin resistance. The most prevalent and widely disseminated mec complex had the structure mecI-mecR1-mecA-IS431R (or IS431mec), designated the class A mecA gene complex. In contrast, in S. haemolyticus, mecA was bracketed by two copies of IS431, forming the structure IS431L-mecA-IS431R. Of the 38 S. haemolyticus strains, 5 had low-level methicillin resistance (MIC, 1 to 4 mg/liter) and characteristic heterogeneous methicillin resistance as judged by population analysis. In these five strains, IS431L was located to the left of an intact mecI gene, forming the structure IS431L-class A mecA-gene complex. In other S. haemolyticus strains, IS431L was associated with the deletion of mecI and mecR1, forming the structure IS431L-DeltamecR1-mecA-IS431mec, designated the class C mecA gene complex. Mutants with the class C mecA gene complex were obtained in vitro by selecting strain SH621, containing the IS431L-class A mecA gene complex with low concentrations of methicillin (1 and 3 mg/liter). The mutants had intermediate level of methicillin resistance (MIC, 16 to 64 mg/liter). The mecA gene transcription was shown to be derepressed in a representative mutant strain, SH621-37. Our study indicated that the mecI-encoded repressor function is responsible for the low-level methicillin resistance of some S. haemolyticus clinical strains and that the IS431-mediated mecI gene deletion causes the expression of methicillin resistance through the derepression of mecA gene transcription.

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 mec complex has the structure mecI-mecR1-mecA-IS431mec. In S. haemolyticus, the class A mec complex was accompanied by an upstream IS431 copy (IS431L). The class B mec 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, IS431L is 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 mec complexes 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 A mec 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 and mecA. (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 A mec 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).