Mechanisms of heteroresistance in methicillin-resistant Staphylococcus aureus.

Characteristic for methicillin-resistant (Mcr) staphylococci is the heterogeneous expression of the intrinsic methicillin resistance. The majority of the cells express resistance to low concentrations of methicillin, and a minority of the cells express resistance to much higher concentrations. We show here (i) that the presence of the mecA encoding region on plasmid pBBB79 was sufficient to render a methicillin-susceptible (Mcs) Staphylococcus aureus strain heteroresistant and (ii) that this Mcr strain segregated highly resistant subclones which retained the high-resistance phenotype under nonselective growth conditions. The Mcr strain with only mecA on plasmid pBBB79 thus behaved identically to a Mcr strain carrying the complete mec determinant integrated at its proper chromosomal site. (iii) Curing a such highly resistant subclone from plasmid pBBB79 yielded an Mcs strain that was as susceptible as the original Mcs parent strain. (iv) Comparisons were made between the original parent and the cured Mcs strain by backcrossing pBBB79 into them and looking at their progeny. Transductants derived from the formerly highly resistant cured strain became resistant to high concentrations of methicillin, whereas transductants derived from the original parent strain were resistant to lower concentrations of methicillin and showed the typical heterogeneous resistance. We deduced therefrom that the high-level resistance expressed by the minority of the population of Mcr S. aureus was due to a chromosomal mutation(s) (chr*) involving neither mecA nor the additional 30 kb of mec-associated DNA. Moreover, we could show that this postulated mutation chr* was not linked to the femAB operon, which is known to affect methicillin resistance levels.

Survey of the methicillin resistance-associated genes mecA, mecR1-mecI, and femA-femB in clinical isolates of methicillin-resistant Staphylococcus aureus.

The restriction site polymorphism of the chromosomal femAB region and the first appearance of the regulatory element mecR1-mecI associated with the methicillin resistance determinant (mec) were analyzed in 192 initially methicillin resistant (Mcr) Staphylococcus aureus clinical isolates collected between 1965 and 1990 in the Zurich area. Forty-three of the strains lost the resistance spontaneously. All isolates that were still Mcr hybridized with mecA, the gene for the low-affinity penicillin-binding protein PBP 2'. Mcr strains isolated before 1977 lacked sequences that hybridized with mecR1-mecI, a regulatory element controlling the expression of mecA; exceptions to this were one strain isolated in 1966 and one strain isolated in 1972. The size of the EcoRV fragment carrying femA, a chromosomally encoded factor involved in pentaglycine side chain formation of the peptidoglycan and essential for the expression of methicillin resistance, was conserved in all strains but one, which was susceptible to methicillin even though it carried a functional mecA gene. The methicillin susceptibility of this particular strain was presumably due to a spontaneous femA-like mutation. The 192 strains belonged to seven different EcoRV restriction fragment patterns recognizable with a 10.5-kb probe covering the femAB region. Some 93% of the 149 Mcr strains belonged to pattern A, and the remaining Mcr strains shared patterns A' and B. The 42 isolates which spontaneously lost their resistance upon storage and revival represented all seven different patterns. This strong conservation of femA suggests an important role for femA in cell wall metabolism and methicillin resistance.

Correlation between regulation of mecA transcription and expression of methicillin resistance in staphylococci.

Total RNA was used to study the effect of penicillinase plasmid pI524 and of mecR, the regulatory region located on the methicillin resistance determinant (mec), on the expression of mecA, the gene coding for the low-affinity penicillin-binding protein PBP2', in methicillin-resistant staphylococci. In the present report, we show that the regulation of methicillin resistance occurs primarily at the level of mecA transcription and that in the presence of intact plasmid pI524 or mecR, the gene undergoes negative control. The relative amount of mecA mRNA present during exponential growth in uninduced cultures matches the type of mecA regulation and decreases in the following order: constitutive greater than pI524 greater than mecR-dependent mecA expression. Induction of mecA by methicillin is faster in pI524- than in mecR-controlled strains. The overall mRNA half-life is similar for all strains analyzed. Our results indicate that methicillin resistance under mecR control in certain staphylococcal strains could escape detection by the standard disk diffusion test and broth microdilution test because of the very slow derepression of the mecA gene. This finding is of importance for the clinical detection of this type of methicillin resistance.

The Staphylococcus aureus mec determinant comprises an unusual cluster of direct repeats and codes for a gene product similar to the Escherichia coli sn-glycerophosphoryl diester phosphodiesterase.

The DNA sequence located between mecA, the gene that codes for penicillin-binding protein PBP2', and insertion sequence-like element IS431mec has been termed hypervariable because of its length polymorphism among different staphylococcal isolates. We sequenced and characterized the hypervariable region of the methicillin resistance determinant (mec) isolated from Staphylococcus aureus BB270. Within the 2,040-bp hypervariable region, we identified an unusual accumulation of long direct repeats. Analysis of the DNA sequence revealed a minimal direct repeat unit (dru) of 40 bp which was repeated 10 times within 500 bp. The dru sequences are responsible for the length polymorphism of mec. Moreover, we identified an open reading frame that codes for 145 amino acids (ORF145), whose deduced amino acid sequence showed 57% amino acid sequence similarity to the N terminus of the glycerophosphoryl diester phosphodiesterase (UgpQ) of Escherichia coli.

Evidence of a novel staphylococcal mec-encoded element (mecR) controlling expression of penicillin-binding protein 2'.

A region was identified on the methicillin resistance determinant (mec) isolated from Staphylococcus epidermidis and cloned into Staphylococcus carnosus which was responsible for a novel downregulation of the expression of methicillin resistance. The presence of this region reduced the overall expression of methicillin resistance and the synthesis of the mec-encoded penicillin-binding protein 2' (PBP 2') in S. carnosus. This region was located by Bal31 deletion mutagenesis upstream of the structural gene for PBP 2'. Deletions within this region resulted in higher levels of expression of methicillin resistance and increased levels of PBP 2' synthesis. We tentatively called this region mecR. Analysis of selected Mcr strains of Staphylococcus aureus and S. epidermidis by Southern hybridization suggested that the natural occurrence of two types of mec resistance determinants differ by the presence or absence of mecR-specific sequences.