A simple and reliable method using a polymerase chain reaction (PCR) was devised to identify methicillinresistant staphylococci. By using lysates of the strain to be tested as templates and 22-mer oligonucleotides as primers, a 533-bp region of mecA, the structural gene of a low-affinity penicillin-binding protein (PBP 2'), was amplified by PCR and detected by agarose gel electrophoresis. Results obtained by this method were compared with those obtained by broth microdilution MIC determination for 210 and 100 clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, respectively. Of 99 mecA-negative S. aureus isolates, 100% of the strains were methicillin susceptible and 98% of the strains were oxacillin susceptible. Three strains (3%) of 111 mecA-positive S. aureus isolates exhibited almost the same susceptibility to ,(-lactams as the mecA-negative ones and did not produce detectable amounts of PBP 2' despite the presence of the mecA gene. One of them yielded typically methicillin-resistant variants at a low frequency with concomitant recovery of PBP 2' production. The mecA gene was also found in coagulase-negative Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, and Staphylococcus caprae and conferred resistance on most of the bacteria. * Corresponding author. species were identified by Staphyogram (Terumo, Tokyo, Japan), an identification kit, and the coagulase test. S. aureus SR3626, SR3633, SR3636, SR3639, SR3665, SR3681, and SR3716 were used as mecA-positive control organisms (12), and S. aureus ATCC 25923, 209P JC-1, and Smith were used as mecA-negative control organisms.

A methicillin-susceptible, novobiocin-resistant strain of Staphylococcus aureus (RN2677; methicillin MIC, 0.8 jig/ml) was transformed with DNA prepared from highly and homogeneously methicillin-resistant S. aureus strains (methicillin MIC, .400 ,ug/ml) or from heterogeneous strains in which the majority of cells had a low level of resistance (methicillin MIC, 6.3 tug/ml). All methicillin-resistant transformants showed low and heterogeneous resistance (methicillin MIC, 3.1 pg/ml) irrespective of the resistance level of DNA donors. All transformants examined produced normal amounts of the low-affinity penicillin-binding protein (PBP) 2a, and methicillin resistance and the capacity to produce PBP 2a showed the same degree of genetic linkage to the novobiocin resistance marker with both homogeneous and heterogeneous DNA donors. Next, we isolated a methicillin-susceptible mutant from a highly and homogeneously resistant strain which had a TnS51 insertion near or within the PBP 2a gene and thus did not produce PBP 2a. With this mutant used as the recipient, genetic transformation of the methicillin resistance gene was repeated with DNA isolated either from highly and homogeneously resistant strains or from heterogeneous (low-resistance) strains. All transformants obtained expressed high and homogeneous resistance and produced PBP 2a irrespective of the resistance level of the DNA donors. Our findings suggest that (i) the methicillin resistance locus is identical to the structural gene for PBP 2a, (ii) although the ability to produce PBP 2a is essential for resistance, the MICs for the majority of cells are not related to the cellular concentration of PBP 2a, and (iii) high MICs and homogeneous expression of resistance require the products of other distinct genetic elements as well.

Tn918 mutagenesis of a high-level methicillin-resistant Staphylococcus aureus (methicillin MIC, 800 pug/ml) led to the isolation of a low-resistance mutant. The Tn918 insert was transferred back to the parent to produce strain SRM563 (methicillin MIC, 12.5 jig/ml), which showed heterogeneous resistance. Twenty-two clinical isolates of methicillin-resistant S. aureus were transformed with DNA of SRM563. In the transformants of most strains, instances of reduced resistance were observed with concomitant increases of autolysis rate induced by Triton X-100 and were generally more profound in high-resistance strains. Two transformants exhibited a decrease of the autolysis rate and little reduction of resistance. In the transformant of methicillin-susceptible strain RN2677, an increase of the autolysis rate and little reduction of resistance were observed. The production of low-affinity penicillin-binding protein (PBP2') did not significantly decrease in the mutants.Insertion of Tn.918 occurred within the 3'-terminal region of a novel gene designated llm, which was cloned and sequenced. RNA blot analysis demonstrated that the gene was transcribed. The encoded protein was composed of 351 amino acid residues with a molecular weight of 38,512 and was hydrophobic, suggesting its location on the membrane. The gene was detected by PCR in all S. aureus strains tested but not in the other 26 staphylococcal species. Comparison of the 3'-terminal sequences of the gene among several S. aureus strains showed that, whereas nucleotide substitutions occurred at the third position in seven of eight 3'-terminal codons, only C-terminal amino acid variation of glutamate or aspartate was observed.Methicillin-resistant strains of Staphylococcus aureus produced a low-affinity penicillin-binding protein (PBP), PBP2' or PBP2a, in addition to the ordinary PBPs (16, 39). PBP2' was considered to take over the function of peptidoglycan biosynthesis from ordinary PBPs in the presence of otherwise inhibitory concentrations of P-lactam antibiotics such as methicillin because of the low affinity of PBP2' for r-lactam antibiotics (11,32). While the production of PBP2' was known to be prerequisite for methicillin resistance, earlier reports indicated that the amount of PBP2' produced did not correspond to the resistance level; that is, strains with a high level of resistance did not necessarily produce a larger amount of PBP2' than strains with a low level of resistance (4, 17, 28). These observations were subsequently confirmed and extended by the finding that mecA, the structural gene for PBP2', was not the single factor determining the resistance level-another genetic determinant(s) was involved in the expression of the high-level methicillin resistance, thus differentiating high-level resistant strains from low-level ones (29). Furthermore, transposon insertional mutagenesis with Tn551 revealed that, for the expression of both low-level and high-level methicillin resistance, several other genetic determinants such as fem genes or auxiliary genes w...

From high-level methicillin-resistant Staphylococcus aureus SRM551, the low-level heterogeneously resistant mutant, SRM563, was isolated by transposon mutagenesis. The transposon insertion occurred in the 3 region of the llm gene in the mutant (H. Maki, T. Yamaguchi, and K. Murakami, J. Bacteriol. 176:4993-5000, 1994). Resistant revertants were generated from the mutant strain SRM563 on the plate containing methicillin at a concentration of 12.5 g/ml or more. In some revertants, the insertion sequence IS256 was observed to be transposed into one of five sites localized 88 to 212 bp upstream of the mutant llm at a frequency of 2.8 ؋ 10 ؊7in the bacterial population. The IS256 transposition created a new hybrid promoter in which the ؊35 region at the end of IS256 was properly arranged in relation to the ؊10-like sequence upstream of llm. The new promoters greatly enhanced the transcription of the mutant llm, as judged by blotting analysis of llm mRNA, with concomitant elevation of the methicillin resistance. Involvement of the insertion sequence in the heteroresistance characteristics of methicillin-resistant S. aureus was suggested.Various insertion sequences (ISs) are widely distributed among bacteria. They can be transposed to various loci in chromosomal DNA, which sometimes results not only in disruption of genes but also in activation of downstream genes (5). With some IS elements, such as IS10 in Escherichia coli, the activation is caused by a promoter located within the element and directed outward into the adjacent gene (3). On the other hand, some elements, such as IS1 (19) or IS2 (10), can activate the downstream genes via the formation of a hybrid promoter in which the Ϫ35 region at the end of the element is properly positioned close to the Ϫ10 region in the host DNA. Many IS elements have been shown to contain the Ϫ35-like sequence of the promoter in their terminals (5), although not all elements have been demonstrated to be involved in the formation of functional promoters. In Staphylococcus aureus, IS256 and IS257, which compose both ends of Tn4001 (14) and Tn4003 (15), respectively, belong to this category of IS elements. These elements appear to construct the hybrid promoter for the aminoglycoside resistance gene, aacA-aphD, in Tn4001 (23), and that for the trimethoprim resistance gene, dfrA, in Tn4003 (24), respectively. However, their transposition with concomitant activation of adjacent genes has not been observed.Previous studies have shown that S. aureus became methicillin resistant via the production of a low-affinity penicillinbinding protein, designated PBP2Ј or PBP2a, encoded by mecA of foreign origin (7, 30). The MIC of methicillin, however, varied from 3.1 g/ml for some strains to more than 800 g/ml for others; thus, in addition to mecA, other, unknown genetic factors seem to determine the resistance level (18, 25). We introduced transposon Tn918 with a tetracycline resistance marker into the chromosome of high-level methicillin-resistant S. aureus (MRSA) and isolated an insertional muta...