ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1990, p. 1777-1779

Vol. 34, No. 9

0066-4804/90/091777-03$02.00/0 Copyright © 1990, American Society for Microbiology

NOTES

Insertional Inactivation of the mec Gene in a Transposon Mutant of a Methicillin-Resistant Clinical Isolate of Staphylococcus aureus PETER MATTHEWS AND ALEXANDER TOMASZ*

The Rockefeller University, 1230 York Avenue, New York, New York 10021 Received 12 February 1990/Accepted 29 May 1990

All clinical strains of methiciflin-resistant Staphylococcus aureus (MRSA) examined so far contain the mec and its product, the penicillin-binding protein (PBP) 2A. Yet the same strains show tremendous variation in the phenotypic expression of antibiotic resistance (MIC), which is under the control of a set of additional, auxiliary genes. Thus, the quantitative contribution of the mec gene to the resistance phenotype of MRSA is not known, and no mutants with the lesion located within the mec gene have been described. We subjected a highly resistant MRSA strain to transposon mutagenesis with the erythromycin resistance transposon TnS51, and a mutant expressing greatly decreased methicillin resistance (RUSA4) was selected to characterize the transposon insertion site. The results indicate that the Tn551 insertion site in mutant RUSA4 is between base pairs 1000 and 1400 of the sequence encoding PBP 2A. Thus, the uniform and >200-fold drop in the methicillin MIC (4 tig/ml) for this mutant relative to that for the parent strain (MIC 800 ,Ig/ml) must be related to the inactivation of the PBP 2A gene. The results provide the first unequivocal evidence for the importance of PBP 2A as a quantitative contributor to the MIC for MRSA. gene

overwhelming majority of the cells is close to that for a susceptible bacterium. Thus, while the presence of the mec gene appears to be the sine qua non of methicillin resistance, no precise data concerning either the biochemical or the physiological role of mec or PBP 2A are available. In order to obtain information on this point, we attempted to isolate transposon mutants of a highly and homogeneously resistant S. aureus strain in which the insertion site was within the mec gene. The isolation of such a mutant has already been reported (11), but the characterization was incomplete. Genetic crosses and biochemical analyses have suggested that in mutant RUSA4 (11), Tn551 may be located close to or within the mec gene. Specifically, it was shown that mutant RUSA4 did not produce PBP 2A and did not contain an intact mec marker detectable by genetic transformation. When DNA of RUSA4 was crossed into a novobiocin-resistant and methicillin-susceptible recipient, the transposon marker showed about 40 to 50% linkage to novobiocin resistance. When mutant RUSA4 was used as a recipient for an intact mec gene, all the methicillin-resistant transformants became susceptible to erythromycin, indicating the loss of the transposon due to homologous recombination (11). While strongly suggestive, none of these findings proves unequivocally that the transposon was within the mec gene in RUSA4, since the possibility of polarity effects or complex transposon-induced rearrangements on the chromosome could not be excluded. In addition, transposon mutants with an intact mec gene but greatly decreased methicillin resistance and inhibited PBP 2A production have also been described (2, 7). For these reasons, it was important to perform a detailed molecular characterization of the insertion site in mutant RUSA4. Chromosomal DNA prepared from mutant RUSA4 was subjected to Southern analysis. Agarose electrophoresis

There is general agreement concerning the central impormec gene (a 2.4-kilobase piece of chromosomally located DNA) in the mechanism of intrinsic betalactam resistance of Staphylococcus aureus. The mec gene codes for a'78-kilodalton membrane protein which is capable of binding radioactive penicillin with a very low affinity, the penicillin-binding protein 2A or 2' (PBP 2A). The mec gene must have been imported by staphylococci from an as yet unidentified source, since appropriate DNA probes prepared from mec DNA showed that all strains of methicillin-resistant S. aureus (MRSA) carried this gene, while susceptible isolates did not react with the probe at all (G. L. Archer and E. A. Pennell, Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 673, 1989). Nevertheless, the contribution of mec and its gene product to the resistance phenotype is not clear. When the'cloned mec gene was introduced on a plasmid vector into susceptible strains of staphylococci, the recipients acquired an increased degree of resistance to methicillin (5, 9, 10, 13), but in each case the expression of resistance was heterogeneous, i.e., the cultures were composed of multiple subpopulations of cells for which the antibiotic MICs were vastly different. Similar findings were obtained when the mec marker was transferred to susceptible bacteria by genetic transformation or transduction (11). Furthermore, there was no correlation between the cellular amounts of PBP 2A and the MIC (3, 4, 7), and all the methicillin-susceptible transposon mutants of MRSA that have been described to date have mutations in auxiliary genes that are located at a distance from the mec marker on the staphylococcal chromosome (1, 3, 7). All these mutants carry the intact mec gene, and most, but not all, also contain normal amounts of PBP 2A, in spite of the fact that the methicillin MIC for an

tance of the

*

Corresponding author. 1777

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ANTIMICROB. AGENTS CHEMOTHER.

NOTES

Probe: mec PstI x: z

Probe: Th5S1

Probe: mec

r;:n Ew m

Xba

Xbal x: :9. tl.MWs (k \

S

tXe

44 23, 2

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FIG. 1. Southern analysis of the Tn5SJ insertion site in RUSA4. Total DNA from COL and transposon mutant RUSA4, digested with Pstl (a) or XbaI (b and c), was electrophoresed through 1% agarose, blotted to nitrocellulose, and hybridized with 12P-labeled pMF13, a plasmid containing a Pstl-Xbal fragment of the mec gene (a and b), or pRT1, a plasmid containing an Xbal-Hpal fragment of Tn5SJ (c). The rightmost lane in each autoradiograph contains molecular weight markers (lambda DNA digested with Hindlll). kb, Kilobases.

gels were blotted to nitrocellulose filters and hybridized to 32P-labeled probes by standard procedures (8). S. aureus chromosomal DNA for normal Southern analysis was prepared by the method of Matsuhashi et al. (9). Radiolabeled probes were prepared by nick translation using [32P]dATP, and hybridization was detected by autoradiography. We probed PstI and XbaI digests of whole-cell DNA from RUSA4 and from its parent strain (COL) with 32P-labeled mec DNA, a PstI-XbaI fragment (MF13) from the mec gene of the Australian type strain ANS46 (10), cloned in pTZ19. This fragment is totally internal to the PBP 2A open reading frame, running from nucleotide positions 733 to 1929 in the mec gene sequence described by Song et al. (13). Results of this Southern analysis are shown in Fig. 1. Insertion of TnSSl into COL caused the mec-hybridizing PstI fragment in RUSA4 to increase in size from approximately 15 to 20 kilobases, an amount corresponding to the size of the transposon (5.6 kilobases) (Fig. la). Figure lb shows the XbaI fragments of COL and RUSA4 hybridizing to the probe; insertion of TnSSJ (which has an XbaI site close to one end [3]) evidently introduced an XbaI site into the mec-hybridizing COL XbaI fragment, causing the probe to hybridize with two XbaI fragments of approximately 3.9 and 4.9 kilobases in RUSA4. The results of subsequent reprobing of the RUSA4 XbaI digest with an internal (XbaIHpaI) fragment of TnS5S are shown in Fig. lc. Only one of the mec-hybridizing fragments hybridized to this probe, enabling the orientation and approximate location of the TnSSJ insert within the mec gene to be determined (Fig. 2). From the estimated lengths of the mec-hybridizing fragments and the position of the TnSSJ XbaI site in the highly similar Tn917 sequence (12), the point of insertion of TnSSI within mec can be placed between nucleotides 1000 and 1300 of the PBP 2A gene (mec) sequence described by Song et al. (13). The distinctive features of the phenotype of mutant RUSA4 were the lack of detectable PBP 2A (11) and a uniform drop in the methicillin MIC from that for the parent strain (800 ,ug/ml) to about 4 ,uLg/ml. There was no evidence for heterogeneity, i.e., for a residual subpopulation of cells

in cultures of RUSA4 for which the MICs were higher (11)

(Fig. 3).

It is interesting that the MIC for RUSA4 (4 ,ug/ml) is significantly higher than that for truly methicillin-susceptible staphylococci (0.5 to 1 ,ug/ml) and that it is the same as that for a recently described clinical isolate (CDC 6 [14]) which does not carry the mec gene or P-lactamase but which nevertheless has a uniform intermediate resistance to methicillin. This strain appears to contain modified (low-affinity) forms of PBPs 1 and 2 (14), and it has been suggested that such modified-PBP strains contain a resistance mechanism similar to that obtained by stepwise antibiotic pressure in the laboratory (15). It is conceivable that such modified-PBP strains served as the recipients of the mec gene in the evolution of some strains of MRSA. Some tentative evidence compatible with this suggestion has recently been described (6). When RUSA4 was used as a recipient in genetic transformations of the mec marker and the DNA donors were either heterogeneous or homogeneous strains of -2

-1

'

0 kb

'

1

2

'

3

'

4

14 4,,

'

15

mec probe XbaI

PstI

Pstl

_I XbaI

Psti

TnSSJ probe FIG. 2. Map of the mec region of RUSA4, showing approximate position of the TnS5S insertion, selected endonuclease sites, and probes used for Southern analysis. kb, Kilobases; Chr., chromosomal DNA.

NOTES

VOL. 34, 1990

1779

We thank Barbara Inglis for kind provision of cloned MF13.

UL

0

105 _ RN2677

hetero-1

104 10o3

10~2

0.5 1.0

5

Methicillin

10

50

100

500

(gg/mi)

FIG. 3. Population analysis profile of transposon mutant RUSA4. Analysis of bacterial cultures for the distribution of methicillin MICs was performed by agar dilution, as previously described (4). RN2677 is a methicillin-susceptible laboratory strain, and COL is a highly and homogeneously resistant clinical isolate which was the parent of the Tn551 mutant RUSA4. Also shown is the population analysis profile of a heteroresistant strain (hetero-1). COL and hetero-1 contained normal amounts of PBP 2A; no PBP 2A was detectable in RN2677 and RUSA4.

MRSA, the methicillin-resistant transformants always showed loss of the TnS5S marker (Em) and high and homogeneous levels of methicillin resistance, similar to that seen in the original parent of RUSA4 (11). These data indicate that the dramatic drop in the level of antibiotic resistance of RUSA4 must be attributed to the inactivation of the mec gene and the lack of production of PBP 2A. One may predict, therefore, that agents (e.g., some new beta-lactam compounds to be synthesized in the future) targeted to inactivate PBP 2A should be effective antibiotics against methicillin-resistant staphylococci. This investigation was supported by Public Health Service grant Al 16794 from the National Institutes of Health, a Merck Research Fellowship, and a Fulbright Fellowship (to P.M.).

LITERATURE CITED 1. Berger-Bfichi, B. 1983. Insertional inactivation of staphylococcal methicillin resistance by TnS51. J. Bacteriol. 154:479-487. 2. Berger-Bachi, B., L. Barberis-Maino, A. Strassle, and F. H. Kayser. 1989. FemA, a host-mediated factor essential for methicillin resistance in Staphylococcus aureus: molecular cloning and characterization. Mol. Gen. Genet. 219:263-269. 3. Berger-Bachi, B., A. Strassle, and F. H. Kayser. 1986. Characterization of an isogenic set of methicillin-resistant and susceptible mutants of Staphylococcus aureus. Eur. J. Clin. Microbiol. 5:697-701. 4. Hartman, B. J., and A. Tomasz. 1986. Expression of methicillin resistance in heterogeneous strains of Staphylococcus aureus.

Antimicrob. Agents Chemother. 29:85-92. 5. Inglis, B., P. R. Matthews, and P. R. Stewart. 1988. The expression in Staphylococcus aureus of cloned DNA encoding methicillin resistance. J. Gen. Microbiol. 134:1465-1469. 6. Jordens, J. Z., and L. M. C. Hail. 1988. Characterization of methicillin-resistant Staphylococcus aureus isolates by restriction endonuclease digestion of chromosomal DNA. J. Med. Microbiol. 27:117-123. 7. Kornblum, J., B. J. Hartman, R. P. Novick, and A. Tomasz. 1986. Conversion of a homogeneously methicillin-resistant strain of Staphylococcus aureus to heterogeneous resistance by Tn551-mediated insertional inactivation. Eur. J. Clin. Microbiol. 5:714-718. 8. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 9. Matsuhashi, M., M. D. Song, F. Ishino, M. Wachi, M. Doi, M. Inoue, K. Ubukata, N. Yamashita, and M. Konno. 1986. Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to 3-lactam antibiotics in Staphylococcus aureus. J. Bacteriol. 167:975-980. 10. Matthews, P. R., K. C. Reed, and P. R. Stewart. 1987. The cloning of chromosomal DNA associated with methicillin and other resistances in Staphylococcus aureus. J. Gen. Microbiol. 133:1919-1929. 11. Murakami, K., and A. Tomasz. 1989. Involvement of multiple genetic determinants in high-level methicillin resistance in Staphylococcus aureus. J. Bacteriol. 171:874-879. 12. Perkins, J. B., and P. J. Youngman. 1984. A physical and functional analysis of Tn917, a streptococcus transposon in the TN3 family that functions in Bacillus. Plasmid 12:119-138. 13. Song, M. D., M. Wachi, M. Doi, F. Ishino, and M. Matsuhashi. 1987. Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion. FEBS Lett. 221:167-171. 14. Tomasz, A., H. B. Drugeon, H. M. de Lencastre, D. Jabes, L. McDougall, and J. Bille. 1989. New mechanism for methicillin resistance in Staphylococcus aureus: clinical isolates that lack the PBP 2a gene and contain normal penicillin-binding proteins with modified penicillin-binding capacity. Antimicrob. Agents Chemother. 33:1869-1874. 15. Tonin, E., and A. Tomasz. 1986. ,3-Lactam-specific resistant mutants of Staphylococcus aureus. Antimicrob. Agents Chemother. 30:577-583.

Insertional inactivation of the mec gene in a transposon mutant of a methicillin-resistant clinical isolate of Staphylococcus aureus.

All clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) examined so far contain the mec gene and its product, the penicillin-bindin...
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