ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1990, 0066-4804/90/040600-05$02.00/0 Copyright © 1990, American Society for Microbiology

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Role of Penicillinase Plasmids in the Stability of the mecA Gene in Methicillin-Resistant Staphylococcus aureus KEIICHI HIRAMATSU,* EIKO SUZUKI, HIROMI TAKAYAMA, YUKO KATAYAMA, AND TAKESHI YOKOTA Department of Microbiology, Faculty of Medicine, Juntendo University, Hongo 2-1-1, Bunklyo-ku, Tokyo, Japan 113 Received 18 July 1989/Accepted 27 December 1989

The stability of methicillin resistance (Mcr) in three independent clinical isolates, MR108, MR6, and MR61, of methiciflin-resistant Staphylococcus aureus (MRSA) was studied. The Mcr phenotype was stably maintained in the progeny of all three MRSA clones carrying penicillinase plasmids. However, when the clones were tested after elimination of the plasmids, methicillin-susceptible (Mc') subclones appeared at various frequencies. Seven Mcs subclones were classified into two groups based on their stabilities. Five Mcs subclones, which were derived from homogeneous strains MR108 and MR61, were stably susceptible. They lost penicillin-binding protein 2' production, and moreover, the mecA gene was deleted in four of five subclones. Two subclones were derived from heterogeneous strain MR6. They were very unstable, and more than half of their progeny were Mcr revertants. However, the remainder were stably Mcs and had lost penicillin-binding protein 2' and the mecA gene. We propose that penicillinase plasmids, which are present in most MRSA strains, play an important role in the stability and phenotypic expression of the mecA gene.

The intrinsic resistance of methicillin-resistant (Mc') Staphylococcus aureus (MRSA) depends on a novel penicillin-binding protein (PBP), called PBP 2a or PBP 2', which has reduced affinity for P-lactam antibiotics (4, 9, 12, 13, 28). The structural gene for PBP 2', mecA (26), was recently cloned from a MRSA clinical isolate (15). Introduction of this gene was shown to convert a methicillin-susceptible (Mcs) S. aureus strain to methicillin resistance (26). A genomic DNA clone coding for methicillin resistance was also isolated from a Staphylococcus epidermidis strain (24). This clone was found to be homologous to the mecA gene by restriction map analysis and by hybridization (24). Thus, evidence is accumulating that the mecA gene is the causative genetic element that may explain most of the methicillin resistance of Staphylococcus species. Understanding of the way this gene is regulated and how it is maintained is of crucial importance in view of the still obscure aspects of the expression of methicillin resistance. For instance, the intriguing phenomenon of heterogeneity of resistance expressed by some MRSA strains (2, 8, 19, 23) should be reexamined at the molecular level by using the mecA gene probe now available to us. In this study, using the cloned PBP 2' gene probe, we examined the unstable nature of methicillin resistance. It has long been known that methicillin resistance becomes unstable during long-term culture in drug-free medium (1, 11). To understand the molecular basis of this instability, we investigated the genotype-phenotype correlation of methicillin resistance using three MRSA clones and their methicillinsusceptible subrlones. In the course of this study, we found that stability of resistance was closely associated with the presence of penicillinase (PCase) plasmids, which are harbored by most MRSA clinical isolates (8, 10, 14).

homogeneous in their resistance patterns. The culture of MR6 was composed of subpopulations with various degrees of resistance; that is, it was heterogeneous in its pattern of resistance. The phage types were as follows: MR108 and MR61, nontypable; MR6, type III. The following nine subclones cured of PCase plasmids were used: MR108-1, MR108-2, MR108-3, and MR108-4 were derived from MR108; MR6-1, MR6-2, and MR6-3 were derived from MR6; and MR61-1 and MR61-6 were derived from MR61. The procedure to eliminate PCase plasmids has been described previously (28). Seven Mcs subclones were obtained from three PCase plasmid-negative MRSA clones after 5 days of successive culture in drug-free medium (see below), as follows: MS108-1-1, MS108-1-5, and MS108-1-15 were derived from MR108-1; MS6-2-1 and MS6-2-2 were derived from MR6-2; and MS61-1-1 and MS61-1-3 were derived from MR61-1. Stability test of methicillin resistance. Frozen stocks of MRSA clones and their PCase plasmid-negative subclones were streaked onto heart infusion (HI) agar plates containing 3.13 ,ug of methicillin. A single colony was picked and inoculated into 10 ml of drug-free L broth, followed by overnight culture at 37°C with shaking. The successive subcultures were carried out in 10-ml portions with an inoculum size of 107 CFU at 37°C with shaking. After various periods (in days) of subculture, 0.5 x 102 to 2 x 102 CFU was plated onto HI agar plates. On the next day, colonies were replicated on HI agar plates with or without 3.13 ,ug of methicillin per ml. After incubation overnight at 37°C, colonies growing on drug-free plates but not on methicillin-containing plates were evaluated as Mcs subclones. Determination of MICs of antibiotics. The MIC patterns for MRSA parent strains and their subclones were tested by the plate dilution method with Mueller-Hinton agar at 37°C with an inoculum size of 5 x 10' CFU of bacteria. Growth of the cells was evaluated after incubation for 24 h at 37°C. Population analysis. Frozen stocks of three MRSA isolates and their PCase plasmid-negative subclones were streaked onto HI agar plates containing 5 ,ug of methicillin per ml. A single colony was inoculated into 10 ml of L broth containing 5 jig of methicillin per ml and grown overnight with shaking

MATERIALS AND METHODS

Bacterial strains. PCase-producing MRSA strains MR108, MR6, and MR61 have been described previously (28). Cultures of MR108 and MR61 were composed of subpopulations with the same level of methicillin resistance; they were *

Corresponding author. 600

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at 37°C. About 103 CFU of the overnight culture was spread onto HI agar plates containing various concentrations of methicillin. Colonies were enumerated after incubation for 24 h at 370C. Analysis of PBPs. PBPs were analyzed by the method of Spratt (22), with some modifications (28). A total of 30 ,ul of membrane suspension extracted from about 1010 cells was reacted for 10 min at 30°C with 3 pl of ['4C]benzylpenicillin, the specific activity of which was 50 ,uCi/mmol per ml. The exposure time for fluorography was 14 days at -700C. Cloning of the mecA gene. The genomic DNA fragment containing the mecA gene was cloned into the pUC18 cloning vector (29). The genomic library was constructed by cutting cellular DNA of MR108 with the HindlIl restriction enzyme, followed by ligation into the HindlIl site of pUC18. The library was screened by colony hybridization by using a synthetic oligonucleotide probe, 5'-ATGAAAAAGATA AAAATTGTTCCACTTATT-3', which was synthesized according to sequence data from the initial 30 bases of the mecA structural gene (20). Clone pMR111, which was obtained in this way, had an insert of 4 kilobases (kb) which contained the mecA structural gene, which was proved by comparing the restriction map and the partial sequence with those of the clone reported by Song et al. (20). Southern analysis of the mecA gene. Cellular DNAs of MRSA strains and their subclones were extracted by the method of Dyer and landolo (7). Cellular DNA (1 to 3 ,ug) was digested with EcoRI or HindIII, followed by electrophoresis in a 0.8% agarose gel. DNA was transferred to the nitrocellulose membrane and hybridized with the radiolabeled 4-kb insert of pMR111 as a mecA gene probe (21). The specific activity of the probe was 109 cpm/,ug of DNA. Transduction. The PCase plasmid of MR6 was transduced into MR6-2 by the procedure described by Cohen and Sweeney (5). MR6(29), a subclone of MR6 lysogenized with phage 29 (an international typing phage), was used as the donor of transduction. Recipient MR6-2 cells (1010 CFU) were infected with 109 PFU of transducing phage [induced from MR6(29) by adding mitomycin C] in 1 ml of medium. Transductants were selected by spreading cells onto an HI agar plate containing 12.5 jig of kanamycin per ml. Kanamycin transductants resistance was located on the PCase plasmid of MR6 (data not shown). All experiments included controls for sterility of phage and for kanamycin-resistant progeny of MR6-2 in the absence of phage. None were found in repeated experiments. The presence of the PCase plasmid in the transductants was confirmed by the nitrocefin disk test (17) and gel electrophoresis of plasmid DNA (data not shown). Five independent transductants were obtained; these were designated Ti, T2, T3, T4, and T5. The efficiency of transduction for the PCase plasmid was 1 x 10-9 to 4 x 10-9 in repeated experiments. RESULTS

MICs and patterns of methicillin-resistant subpopulation before and after elimination of PCase plasmid. The possible influence of PCase plasmid elimination on the MIC of methicillin was tested for each clone. The MICs of methicillin for each parent clone were as follows: MR108, 400 ,ug/ml; MR61, 400 ,ug/ml; MR6, 100 jig/ml. The MICs for their PCase plasmid-negative subclones were as follows: MR1081, 400 pug/ml; MR108-2 and MR108-3, 800 ,ug/ml; MR108-4, 200 jig/ml; MR61-1, 400 ,ug/ml; MR61-6, 200 ,ig/ml; MR6-2, 200 ,ug/ml; MR6-1, 100 ,ug/ml; MR6-3, 50 jig/ml. Thus, elimination of the plasmid did not cause an impressive

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alteration of the MIC. However, the distribution of the resistant subpopulations of MR108 and MR61 were influenced more (Fig. 1). In the case of MR108 (Fig. 1A) and MR61 (Fig. 1B), which had homogeneous patterns of resistance, highly resistant subpopulations decreased after elimination of the plasmid. On the other hand, the effect of elimination of the PCase plasmid was variable in the case of MR6, which had a heterogeneous pattern of resistance (Fig. 1C). Two subclones (MR6-1 and MR6-2) showed an increased level of resistance compared with that of MR6, whereas another subclone (MR6-3) had an unchanged or slightly decreased resistance. Preferential appearance of Mcs subclones from cultures of MRSA strains after elimination of the PCase plasmid. Stability of resistance was tested by successive subculturing of MRSA clones and their PCase-free subclones in L broth without methicillin. After 5 days of subculturing, cells were spread onto HI agar plates, and methicillin susceptibility was tested by replica plating. No Mcs subclone was obtained from parental MRSA strains MR108, MR6, or MR61 (8,479, 1,370, and 845 colonies of each strain were tested, respectively). However, Mcs subclones were obtained from subclones MR108-1, MR6-2, and MR61-1, which lacked the PCase plasmids, with frequencies of 0.99% (36 of 3,644), 0.50% (4 of 802), and 0.88% (26 of 2,968), respectively. Time course of appearance of Mcs subclones. Clones MR108 and MR108-1 were subcultured in drug-free L broth; and the frequency of appearance of Mcs subpopulations was calculated on days 4, 7, 15, and 17. The Mcs subpopulation of MR108-1 (free of the PCase plasmid) increased with time: 0.6, 11.3, 96.7, and 98.6% on days 4, 7, 15, and 17, respectively. On the other hand, MR108 (which retained the PCase plasmid) produced no Mcs subclones. Similar time courses were observed with MR61-1 and MR6-2 (data not

shown). Stability of methicillin susceptibility of Mcs subclones. Seven subclones (MS108-1-1, MS108-1-5, MS108-1-15, MS61-1-1, MS61-1-3, MS6-2-1, and MS6-2-2) were chosen from the Mcs subclones obtained in the experiment described above, and the stabilities of their Mcs phenotypes were tested. In the case of MS6-2-1 and MS6-2-2, 12 independent subclones of each were isolated and individually tested for growth on HI agar plates containing 5 p.g of

methicillin per ml. More than half of the 12 subclones grew plate, showing a high tendency of MS6-2-1 and MS6-2-2 to revert to the Mcr phenotype (Table 1). Despite their high tendency of reversion, some subclones remained stably Mcs in repeated reversion tests. Table 1 shows results for two such subclones, MS6-2-1-7 and MS6-2-2-5. With other Mcs subclones derived from MR108 and MR61, no reversion was observed by testing even 1.5 x 108 or 1.4 x 109 CFU of clone progenies. Expression of PBP 2' in correlation with methicillin resistance phenotype. The PBP patterns of MRSA parent clones and their subclones were examined by using labeled penicillin G as an indicator. Mcs subclones MS108-1-1, MS108-1-5, MS108-1-15, MS61-1-1, and MS61-1-3 failed to express PBP 2', whereas their Mcr parents MR108-1, MR61-1, MR108, and MR61 did express it (Fig. 2). As expected from results of the reversion test described above, the unstable clones MS6-2-1 and MS6-2-2, which showed the Mcr phenotype at the time of the study, expressed PBP 2' (Fig. 2, lanes 9 and 10). On the other hand, stable Mcs subclones MS6-2-1-7 and MS6-2-2-6, which were recloned from MS6-2-1 and MS6-2-2, respectively, were found to have lost expression of PBP 2' (Fig. 2, lanes 12 and 13). Therefore, the methicillin resison the

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ANTIMICROB. AGENTS CHEMOTHER.

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0 3.13625125 25 50 100 (ug/ml) Concentration of Methicillin FIG. 1. Effect of elimination of PCase plasmids on methicillin-resistant subpopulations of MRSA strains MR108 (A), MR61 (B), and MR6 0 3.1362512.5 25

50 100

0

25

50 100

(C). About 103 CFU of an overnight culture of each MRSA strain and subclones cured of its PCase plasmid were spread onto HI agar plates with various concentration of methicillin, foliowed by incubation at 37°C. After 24 h, grown colonies were enumerated and plotted. Symbols in panel A: @, MR108; 0, MR108-1; A, MR108-2; A, MR108-3; U, MR108-4. Symbols in panel B: 0, MR61; 0, MR61-1; A, MR61-6. Symbols in panel C: 0, MR6; 0, MR6-1; A, MR6-2; A, MR6-3.

tance phenotype and production of PBP 2' correlated completely. Loss of the mecA gene in Mc' subelones. DNA was extracted from MRSA clones and their subclones as described above, digested with either EcoRI (Fig. 3A) or HindIII (Fig. 3B), and probed with the nick-translated 4-kb insert of pMR111. Four of five Mcs subclones (MS108-1-1, MS1081-5, MS108-1-15, and MS61-1-3) lacked the 4-kb HindIII fragment (Fig. 3A and B, lanes 3 to 5 and 13). As the 4-kb probe contained about 2 kb of flanking genomic DNA in addition to the mecA gene (21), we concluded that the mecA gene plus flanking DNA larger than 2 kb was deleted from the genomes of these subclones. Again, clones MS6-2-1 and

MS6-2-2, which were found to have reverted to the Mcr phenotype, possessed the mecA gene (Fig. 3A and B, lanes 8 and 9), whereas the stable Mc' subclones (MS6-2-1-7 and MS6-2-2-5) of these clones lost the mecA gene (Fig. 3B, lanes 15 and 16). Therefore, among stable Mcs subclones, only MS61-1-1 had the 4-kb DNA fragment that was hybridizable with the mecA gene probe (Fig. 3A and B, lanes 12). That is, six of seven stable Mc' subclones were found to have lost a genomic DNA fragment containing the entire mecA gene. Restoration of resistance stability by transduction of the

TABLE 1. Phenotypic stability of McS subclones derived from three MRSA strains Clones

mecA gene'

No. of colonies tested

No. of Mcr revertants

Frequency (%o)

MS6-2-1 MS&2-2 MS61-1-1 MS61-1-3 MS108-1-1 MS108-1-5 MS6-2-17b MS6-2-2-5C MS61-1-1

+ + +

12 12

6 10 0 0 0 0 0 0 0

50 80 0 0 0 0 0 0 0

-

1.5 x 108 1.5 x 108 1.5 x 108 1.5 x 108

-

1.4

-

1.4

+

1.4

x 109 x 10 x 109

aThe presence (+) and absence (-) of the mecA gene were determined by Southem hybridization of cellular DNA that was extracted from each clone by using pMR111 as a mecA gene probe (see Fig. 3 and the text for details). b

c

A subclone of MR6-2-1. A subclone of MR6-2-2.

FIG. 2. PBP patterns of three MRSA clones and their subclones analyzed by fluorography. The membrane fraction of each clone was incubated with 14C-labeled penicillin G and was subjected to polyacrylamide gel electrophoresis. The exposure time of the fluorograph was 14 days at -70°C. Lanes: 1, MR61-1; 2, MS61-1-1; 3, MS61-1-3; 4, MR108-1; 5, MS108-1-1; 6, MS108-1-5; 7, MS108-1-15; 8, MR6-2; 9, MS6-2-1; 10, MS6-2-2; 11, MR6-2; 12, MS6-2-1-7; 13, MS6-2-2-5. Arrows indicate bands with the characteristic electrophoretic mobility of PBP 2'.

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23.1

0-

2

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FIG. 3. Southern hybridization of cellular DNA extracted from three MRSA clones and their subclones. Cellular DNAs extracted from each clone were digested with restriction enzymes and run in a 0.8% agarose gel electrophoresis. DNA was then transferred to a nitrocellulose membrane sheet and hybridized with the 32P-labeled mecA gene probe. The washed membrane was exposed for 10 h at with an intensifying screen. Cellular DNA was digested with EcoRI (A) or HindIII (B). Lanes 1 through 13, cellular DNA of MR108, MR108-1, MS108-1-1, MS108-1-5, MS108-1-15, MR6, MR62, MS6-2-1, MS6-2-2, MR61, MR61-1, MS61-1-1, and MS61-1-3, respectively; lanes 14 through 16, probe DNA (the insert of pMR111), cellular DNA of MS6-2-1-7, and cellular DNA of MS62-2-5, respectively. Molecular size markers (in kilobases; digest of lambda DNA) are shown.

-70°C

HindIIl

PCase plasmid. To exclude the possible contribution of some genetic elements other than PCase plasmids to the instability of the mecA gene, we reintroduced PCase plasmids into the PCase plasmid-negative subclone and tested the ability of the PCase plasmid to stabilize the resistance phenotype. We could not use MRSA clones MR108 or MR61 or their subclones because they were nontypable strains and were resistant to all typing phages. Since MR6 and their subclones were susceptible to type III phages, we used MR6 as a donor and MR6-2 as a recipient for transduction. We obtained five independent transductants, Ti through T5. These five clones were subcultured in drug-free L broth for 13 days and tested for the appearance of methicillin-susceptible subclones by replica plating. Progenies of these transductants remained uniformly methicillin resistant; there were no Mcs progeny when 87, 67, 64, 74, and 71 colonies of clones Ti, T2, T3, T4, and T5, respectively, were tested. On the other hand, all the progeny of MR6-2 became susceptible; 170 of 170 colonies tested were Mcs. As restoration of methicillin resistance stability was achieved in five independent transductants of PCase plasmids, we concluded that the PCase plasmid itself is responsible for stabilization of methicillin resistance. DISCUSSION

The frequent spontaneous loss of methicillin resistance of MRSA strains during passage in drug-free culture medium

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was first reported by Al Salihy and James (1). They showed that a PCase-producing MRSA strain became susceptible to methicillin when it was subcultured 20 times in drug-free medium at 43°C. This observation coincides well with our results, because the temperature of 43°C used by them was high enough to eliminate PCase plasmids from some cells during culture. Thus, Mcs subclones could have been derived from those cells that lost their plasmids. The longer culture periods than ours that were required for them to detect Mcs subclones might reflect this two-step mechanism of loss of resistance. In fact, Al Salihy and James (1) found that their methicillin-susceptible subclones lost their ability to produce PCase (1). The unstable nature of a PCase nonproducer MRSA strain, L9, was described by Grubb and Annear (11). They observed the appearance of methicillinsusceptible subclones after several weeks of culture at room temperature. This also seems to support the importance of the PCase plasmid in the stability of methicillin resistance. Loss of methicillin resistance in the majority of our clones was accompanied by deletion of the mecA gene. Although there was an exceptional clone, MS61-1-1, which retained the mecA gene, six of seven Mcs subclones analyzed were deleted for the gene. As PBP 2' was not expressed in MS61-1-1, it seems likely that it acquired point mutations or small deletions in the coding region, regulator region, or both of the mecA gene. The pattern of deletion of the mecA gene was characteristic, in that the whole gene was deleted with its flanking DNA in all cases. This reminds us of the nature of the mecA gene, which is that of a mobile genetic element. The presence of insertion sequences in the vicinity of the cloned mec gene has been reported recently (3, 16). Evidence of translocation of the mec gene onto a PCase plasmid from its original chromosomal location has also been reported (25). Thus, loss of the entire mecA gene in our study might be an eduction (cutting out) of a mec transposon. A final conclusion, however, awaits the identification and sequence study of the entire DNA fragments which were deleted from our subclones. This study is now under way. The effect of elimination of PCase plasmids from MRSA strains on their methicillin resistance phenotypes seems to be dependent on their patterns of resistance. Strains with the homogeneous type of resistance were influenced more than those with the heterogeneous type were. Moreover, Mcs subclones derived from these two types of strains also had different stabilities. It is possible that the regulatory role of PCase plasmids differs in these two types of strains. It is interesting, in this context, that the EcoRI-cutting patterns of DNA flanking the mecA gene were different between MRSA clones of the two resistance types (Fig. 3A). In contrast to an evident alteration in the resistant subpopulation profile of clones MR108 and MR61, MICs were not much influenced by the elimination of PCase plasmids. The population analysis seems much more sensitive than the MIC determination to establish the regulatory effect of the PCase plasmid on methicillin resistance. This could be explained by the difference in CFU used in the two experiments; 1 x 103 CFU was used for population analysis and 5 x 104 CFU was used for the MIC determination. It has been known that the PCase plasmid of MRSA strains plays various roles in methicillin resistance. It has been reported that the plasmid confers-the inducibility of PBP 2' (18, 27) and is required for recipient competence for successful transduction of the mec gene (6). The plasmid has also been reported to harbor the second integration site of the mec transposon Tn4291 (25). In addition to these roles, we have shown a new role for the PCase plasmid, that is,

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stable maintenance of the mecA gene. At this stage, we do not know whether these roles of the PCase plasmid are exerted separately. It is possible that the mecA gene-stabilizing function is somehow related to the other functions of the PCase plasmid. More detailed studies on the molecular events which follow elimination of the plasmid are in progress to understand the mechanism of mecA gene stabilization by the PCase plasmid. ACKNOWLEDGMENT We thank S. Yoshikawa, Tokyo University Institute for Medical Science, for valuable discussions and suggestions. 1. 2. 3.

4. 5. 6.

7. 8.

9.

10.

11. 12. 13.

LITERATURE CITED Al Salihy, S. M., and A. M. James. 1972. Loss of methicillinresistance from resistant strains of Staphylococcus aureus. Lancet ii:331-332. Annear, D. I. 1968. The effect of temperature on resistance of Staphylococcus aureus to methicillin and some antibiotics. Med. J. Aust. 1:444 446. Barberis-Maino, L., B. Berger-Bachi, H. Weber, W. D. Beck, and F. H. Kayser. 1987. IS431, a staphylococcal insertion sequence-like element related to IS26 from Proteus vulgaris. Gene 59:107-113. Brown, F. J., and P. E. Reynolds. 1980. Intrinsic resistance to P-lactam antibiotics in Staphylococcus aureus. FEBS Lett. 122:275-278. Cohen, S., and H. M. Sweeney. 1970. Transduction of methicillin resistance in Staphylococcus aureus dependent on an unusual specificity of the recipient strain. J. Bacteriol. 104:1158-1167. Cohen, S., and H. M. Sweeney. 1973. Effect of the prophage and penicillinase plasmid of the recipient strain upon the transduction and the stability of methicillin resistance in Staphylococcus aureus. J. Bacteriol. 116:803-811. Dyer, D. W., and J. J. Iandolo. 1983. Rapid isolation of DNA from Staphylococcus aureus. Appl. Environ. Microbiol. 46: 283-285. Dyke, K. G. H. 1969. Penicillinase production and intrinsic resistance to penicillins in methicillin-resistant cultures of Staphylococcus aureus. J. Med. Microbiol. 2:261-278. Georgopapadakou, N. H., S. A. Smith, and D. P. Bonner. 1982. Penicillin-binding proteins in a Staphylococcus aureus strain resistant to specific P-lactam antibiotics. Antimicrob. Agents Chemother. 22:172-175. Gillespie, M. T., J. W. May, and R. A. Skurray. 1984. Antibiotic susceptibilities and plasmid profiles of nosocomial methicillinresistant Staphylococcus aureus: a retrospective study. J. Med. Microbiol. 17:295-310. Grubb, W. B., and D. I. Annear. 1972. Spontaneous loss of methicillin resistance in Staphylococcus aureus at room temperature. Lancet ii:1257. Hartman, B. J., and A. Tomasz. 1984. Low-affinity penicillinbinding protein associated with f-lactam resistance in Staphylococcus aureus. J. Bacteriol. 158:513-516. Hayes, M. V., N. A. C. Curtis, A. W. Wyke, and J. B. Ward.

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Role of penicillinase plasmids in the stability of the mecA gene in methicillin-resistant Staphylococcus aureus.

The stability of methicillin resistance (Mcr) in three independent clinical isolates, MR108, MR6, and MR61, of methicillin-resistant Staphylococcus au...
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