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Methicillin resistance of staphylococci is defined by production of penicillin-binding protein 2a (5, 15), encoded by the mecA gene, which is located on the bacterial chromosome (13). Expression of mecA is primarily controlled by regulator elements MecR1 and MecI, encoded by mecR1 and mecI, which are located upstream of mecA (6). Although isolates of methicillin-resistant Staphylococcus aureus (MRSA) with intact mec regulatory genes show inducible resistance to beta-lactams, most recent isolates of MRSA are constitutively resistant to methicillin and have changes in the mec regulatory region (7, 8). Indeed, point mutations in the mecI or mecA operator region which are believed to have inactivated MecI were detected (7, 11, 14).

Another genomic variation, the deletion of mecI accompanied by a partial deletion of mecR1, has been reported (1, 10, 14). Archer and coworkers analyzed the MRSA strain COL, which lacks mecI and the 3' half of mecR1, and described the presence of the insertion sequence-like element IS1272 adjacent to the incomplete mecR1 gene (1). IS1272 contains two open reading frames (ORFs) and an inverted repeat (IR) comprising 16 bases at each terminus (IRR and IRL) (2). The truncated mecR1 gene (mecR1) adjacent to IS1272 has also been observed in Staphylococcus epidermidis and Staphylococcus haemolyticus; furthermore, this mecR1 deletion junction sequence was the same in S. aureus and S. epidermidis strains, as well as in an S. haemolyticus strain, Y176 (1, 2). These observations suggested that an IS1272-mediated mecR1 deletion event may have occurred in coagulase-negative staphylococci, with subsequent horizontal transfer of the rearranged region to S. aureus.

However, it is not clear whether there are other rearranged forms of mecR1 and IS1272 different from the one detected in strain COL or whether the COL-type mec DNA was transmitted from coagulase-negative staphylococci into a single clone or multiple clones of S. aureus. To investigate these points, a total of 176 clinical isolates of staphylococci from different patients admitted to the Sapporo Medical University Hospital, Sapporo, Japan, during the period from 1993 to 1998 (99 S. aureus isolates, 59 S. epidermidis isolates, and 18 S. haemolyticus isolates) were analyzed in this study. S. aureus isolates of various coagulase types were selected for the present study. In addition, we analyzed three Japanese MRSA strains, MR108, MR6, and JO18, possessing truncated mecR1 genes (14), which were provided by the Culture Collection Laboratory of the Institute of Medical Science at the University of Tokyo, Tokyo, Japan, and five type strains of S. haemolyticus (ATCC 15796, ATCC 29968, ATCC 29969, ATCC 29970, and ATCC 43253).

The presence of mecA, mecI, and the 5' and 3' halves of mecR1 was examined by PCR as described previously (9, 10). Based on the results, staphylococcal strains were classified into four groups: type 1 methicillin-resistant (MR) strains, which have both mecR1 and mecI (type 1 mec region); type 2 MR strains, which lack mecI and the 3' half of mecR1 (type 2 mec region); type 3 MR strains, lacking both mec regulator genes (type 3 mec region); and methicillin-susceptible (MS) strains possessing neither the mecA nor mec regulator gene.

Detection of IS1272 was performed by PCR with two primer pairs that amplify different portions of IS1272 ORFs. IS1272 was detected in a total of 52 MR and MS isolates of the three staphylococcal species (Table 1). In S. aureus, IS1272 was found in all 10 type 2 MRSA and four MS S. aureus (MSSA) isolates, while it was not detected in isolates of other mec region types. Similarly, in S. epidermidis, all of the type 2 and some of the MS isolates possessed IS1272. However, some isolates of type 1 and type 3 MR S. epidermidis were also IS1272 positive. IS1272 was detected in all of the MR and MS S. haemolyticus isolates, regardless of the mec region type, except for a type 1 isolate (SH339) and an MS strain (ATCC 29968).

S. aureus isolates were genetically classified by protein A typing, which measures the number of 24-bp repeating units in the Xr region of the protein A gene, as described previously (4, 12). As shown in Table 2, IS1272 was found in coagulase type III, IV, and VII MRSA with different protein A types (repeat no. 5, 8, 9, 10, 11, and 12). The three MRSA strains not from the Sapporo Medical University HospitalMR108, MR6, and JO18possessed IS1272 and showed identical coagulase (IV) and protein A (repeat no. 9) types. IS1272 was detected in coagulase type IV, V, and VII MSSA isolates with different protein A types. In contrast, no coagulase type II MRSA and MSSA possessed this genetic element.

S. epidermidis and S. haemolyticus were typed by arbitrarily primed PCR fingerprinting with ERIC2 primer (16) and M13 reverse primer (3) as described previously (16). As a result, S. epidermidis and S. haemolyticus carrying IS1272 were differentiated into seven and two genetic types, respectively (data not shown).

The presence of the junction sequence between mecR1 and IS1272 was examined for all of the type 2 isolates by PCR with primer pair mecRA1 (10) and mDA2 (5'-GATGTCTGTCGAGGACTC-3'), which are complementary to the sequences in mecR1 and IS1272, respectively. A PCR product of 1,287 bp, which was expected from the sequence of S. aureus COL (GenBank accession no. L14017), was obtained for 22 isolates, although no PCR products were generated for the two MR S. haemolyticus isolates with a type 2 mec region. However, a PCR product which was approximately 1,400 bp longer than those from other isolates was generated for one S. epidermidis strain (SH194).

The nucleotide sequence between the 3' end of mecR1 and the IRL of IS1272 was determined directly from these PCR products by the dideoxynucleotide chain termination method (12). The 3' ends of the mecR1 genes of the 23 type 2 isolates were identical to those of S. aureus COL and S. haemolyticus Y176 (1, 2) (GenBank accession no. L14017 and U35635, respectively), containing a 9-base divergent sequence not found in mecR1 (Fig. 1a). The truncated mecR1 had an ORF of 984 bp and was suggested to encode an incomplete MecR1 product comprising 328 amino acids. The mecR1-IS1272 junction sequences of all isolates except for SH194 consisted of 248 bases, a length almost identical to those of strains COL and Y176. However, compared with published sequences of these strains, two additional nucleotides, T and A, were identified in all staphylococcal genomes examined (Fig. 1a). Furthermore, a single nucleotide was substituted in the junction sequence of S. aureus SH475 (Fig. 1a).

View larger version (43K): [in this window] [in a new window]   FIG. 1.   (a) Nucleotide sequences of the 3'-end portion of mecR1, the mecR1-IS1272 junction sequence (numbered on the sequence from 1 to 248), and the IRL side of IS1272 determined for staphylococci in the present study. Termination codons for mecR1 and ORF2 of IS1272 are shown in boldface type and are followed by vertical lines. Dotted underlining indicates divergent terminal sequence of mecR1, while solid underlining shows the IRLs. Lines above the nucleotide sequences TT and AAAAAA, under solid inverted triangles, denote the regions where an additional T and A were identified, respectively, compared with those of strains COL and Y176 (1, 2). A single nucleotide in the junction sequence which was substituted in S. aureus SH475 is boxed. (b) In S. epidermidis SH194, an additional gene sequence (approximately 1,400 bp) was found in the IRL-side terminal portion of the junction sequence. Lowercase letters indicate partial nucleotide sequences at both ends of the inserted sequence, which initiates with adenine (*1) after the 247th nucleotide of the junction sequence (a) and terminates at the guanine (*2) close to the IRL of IS1272 (b).

In S. epidermidis SH194, the truncated portion of mecR1, the mecR1-side 247-base junction sequence (including two additional bases), and the IRL of IS1272 were identical to those of most other isolates. However, an insertion of additional DNA was found in the junction sequence at the site close to the IRL. Partial sequences of both sides of the additional DNA are shown in Fig. 1.

In the present study, IS1272 was found in S. aureus, S. epidermidis, and S. haemolyticus strains with the type 2 mec region, together with almost identical mecR1-IS1272 junction sequences. This finding supports the hypothesis that a mec element with mecR1 and IS1272 has been transmitted horizontally among staphylococci. However, IS1272 was also detected in type 1 and type 3 staphylococci and in MSSA in the present study. Hence, IS1272 may have been disseminated among staphylococci irrespective of the presence of mec DNA, although this genetic element appears to have been originally resident in S. haemolyticus (2).

Molecular epidemiologic typing of S. aureus indicated that truncated mecR1 and IS1272 are distributed in S. aureus strains of different coagulase and protein A types and also in S. epidermidis and S. haemolyticus strains with some distinct genetic types. These findings suggested that the mec element containing mecR1 and IS1272 might have been transmitted to multiple clones of staphylococci.

It is notable that an additional large DNA sequence is inserted in the IRL-side terminal portion of the mecR1-IS1272 junction sequence in S. epidermidis SH194, which exhibited a novel rearrangement of mecR1 with IS1272. Furthermore, the mecR1-IS1272 junction sequence was not detected in two isolates of type 2 MR S. haemolyticus, suggesting the presence of an unknown form of deletion junction sequence in mec regulator genes. Thus, mec regulator genes may contain various genomic changes, which seem to have occurred multiple times in coagulase-negative staphylococci.