Vol.

176,

No.

May

15,

1991

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

SOUTHERN THE

mecA

HYBRIDIZATION

DELETION

FROM

ANALYSIS

Wadat92,

OF

aureus

Yuko Katayamal, Keiichi Takeshi Yokota*

Hiramatsul*

and

1 Department

of Microbiology, 2-l-l Hongo,

Faculty of Medicine, Juntendo Bunkyo-ku, Tokyo, Japan

University,

2 Department

of Bacteriology, 7-3-l Hongo,

Faculty of Medicine, Bunkyo-ku, Tokyo,

of Tokyo,

Received

March

22,

325

METHICILLIN-RESISTANT

Staphylococcus Akihito

1319-l

University Japan

1991

Genomic organization of methicillin-resistant Staphylococcus aureus strains and their methicillin-susceptible subclones were analyzed by pulsed-field gel electrophoresis and Southern hybridization with DNA fragments of methicillin-resistance gene mecA and an insertion element IS431 as probes. The entire mecA gene was deleted in all the seven methicillin-susceptible subclones studied, and the size of the deletion varied from 20 to 100 kilobases depending on each subclone. In six of the seven subclones, however, the downstream deletion end points were confined within a 2.0 kilobase HindIII-Hind111 fragment containing a part of IS431 which was located 2.6 kilobase downstream of mecA gene. The results indicated that the intramolecular transposition of IS43 1 is responsible for the mecA deletion in methicillin-resistant Staphylococcus B 1991Academic FTes*, 1°C. aureus. SUMMARY:

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causative agents of nosocomial infection throughout the world (I). The resistance to methicillin is caused by a novel penicillin-binding protein (PBP), PBP 2’ (or PBP 2a), which has a low binding affinity for (3lactam antibiotics and is specifically expressed in MRSA strains (2-6). The

*To

whom

correspondence

ABBREVIATIONS:

PBP, penicillin-binding electrophoresis.

should

be addressed.

MRSA, methicillin-resistant kb, kilobase( protein;

Staphylococcus aureus; PFGE, pulsed-field gel 0006-291X/91

1319

$1.50

Copyright 0 1991 b? Academic Press. Inc. All rights nj‘ reprodrrctim it? urry j?rtn reserved.

Vol.

176,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNKATIONS

coding gene for PBP 2’, mecA, has been cloned (7) and is shown to be widely distributed in MRSA as well as methicillin-resistant coagulasenegative staphylococci but not in the methicillin-susceptible counterparts of these bacteria (8). However, its origin and transferability between different staphyloccocal species remain unknown. Recently, an insertionlike element IS431, or IS257 (9-l 1) which has almost the same nucleotide sequence as IS43 1, has been found closely associated with mecA gene on the chromosome of MRSA. This element has also been shown associated with other resistance determinants, such as those to both on the chromosome and mercury, tetracycline and trimethoprim plasmids of MRSA (10, 12-14). On the other hand, it has long been known that the resistance phenotype of MRSA strains is unstable when they are passaged in methicillin-free medium (15, 16). We have demonstrated that the loss of the resistance is mostly caused by spontaneous deletion of the entire mecA gene (17). In this paper, we analyzed the deletion in using pulsed-field each methicillin-susceptible subclone gel electrophoresis (PFGE) and Southern hybridization analysis. Using IS43 1 DNA as a probe, the role of the IS element in the mecA deletion was evaluated.

MATERIALS

AND METHODS

Bacterial strains

Penicillinase-producing MRSA strains MR 108 and MR6, and their subclones cured of penicillinase plasmids MR108-1, MR108-3, MR108-4 and MR6-2 have been described previously (6,17). Methicillin-susceptible subclones were obtained from the penicillinase plasmid-negative MRSA clones after 5 days of successive culture in antibiotic-free medium, as follows: MS108-l-l, MS108-1-5 and MS1081-15 were derived from MR108-1; MS108-3-1 from MR108-3; MS108-41 from MRlOS-4; MS6-2-1-7 and MS6-2-2-5 from MR6-2 (17). Southern hybridization analysis and PFGE The DNA extraction from MRSA strains and their subclones, and Southern hybridization analysis were performed as described previously (18). To prepare samples for PFGE, 5 X107 cells were embedded in 40~1 of 0.5% low temperature melting agarose (SeaPlaque, FMC). The samples were incubated with 500mM EDTA, 1% lauroylsarcosine and lOOug/ml lysostaphin (SIGMA) at 37°C for 24 h, and further incubated with 500mM EDTA, 1% lauroylsarcosine and lmg/ml proteinaseK (SIGMA) at 50°C for 48 h. After rinsed twice with TE (10mM Tris-HCl pH8.0, 1mM EDTA) with 1mM Phenylmethylsulfonyl Fluoride (SIGMA), the samples were reacted with 50 units of SmaI (Toyobo) at 25°C for 12 h. They were further treated with lOOpg/ml proteinaseK in TE at 50°C for 1 h, rinsed twice with TE, and then subjected to PFGE with 1% agarose gel (SeaKem HGT, FMC). The PFGE was performed at 8°C for 20 h in 0.5 X TBE running buffer, using CHEF DRII (Bio-Rad) with the electric field strength of 6 1320

Vol.

176,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

V/cm and switching time of 20 sec. Probes used for Southern hybridization, Mc4.3 for mecA gene and 1~0.8 for IS431, are shown in Fig. 1.

RESULTS AND DISCUSSION Restriction enzyme map of the genomic region containing mecA gene and IS431. A restriction map of the genomic region around mecA gene and IS432 of two MRSA strains, MR108 and MR6, is presented in Fig. 1. The map was constructed based on the Southern hybridization data, in which the chromosomal DNAs of MRlO8 and MR6 were digested with various combinations of restriction enzymes and were hybridized with Mc4.3 and 1~0.8 probes (data not shown). It was shown that mecA gene was located 2.6 kb upstream to IS431 in both strains. The restriction map was identical with those of the previous reports (1315). However, the number of IS43 I on the chromosome was one in MRlO8 and two in MR6 (vi& infra) in contrast to four in the previous report (12-14). Genomic rearrangements in methicillin-susceptible subclones. Hind111 digested total DNAs of the MRSA strains and their subclones were probed with Mc4.3 and 1~0.8 (Fig. 2). Mc4.3 hybridized to 4.3 kb fragments of methicillin-resistant clones (MR108, MR 108- 1, MRlOS-3, MR108-4, MR6 and MR6-2) but not to methicillin-susceptible subclones (MS108-l-l, MSlOS-1-5, MS108-1-15, MS108-3-1, MS108-41, MS6-2-1-7 and MS6-2-2-5) (Fig. 2a and Fig. 2~). When the same DNAs were probed with 1~0.8, all the mecA-positive, methicillin-resistant clones showed two bands of 2.7 kb and 2.0 kb in size which corresponded to the HindIII-Hind111 fragments containing the right and left part of IS431, respectively (Fig. 2b and Fig. 2d; also see Fig, 1). On the other

mecA

IS43

1

5’

3’ H I

B I

I

I

H

HHB

H

I

I

I lSO.8

Mc4.3

N

2 kb

c

A restriction map of the genomic region around mecA gene and Fig. 1. IS431 showing mecA gene is located 2.6 kb upstream to IS431 on the same

strand of DNA. Probes for Southern hybidization analysis are shown under the map: Mc4.3, a cloned HindIII-Hind111 fragment containing mecA gene; 1~0.8, the entire sequence of IS431 which was amplified by polymerase chain reaction using synthetic oligonucleotide primer corresponding to 16base inverted repeat of IS431 (9). Open box shows the open reading frame of mecA gene and IS431. Abbreviation for restriction enzymes: B, BgIII; H, HindIII. 1321

Vol.

176,

No.

3, 1991

a

BIOCHEMICAL

AND

b

BIOPHYSKAL

RESEARCH

C

COMMUNICATIONS

d

Fig. 2. Southern hybridization analysis of Hind111 digested DNAs of two MRSA strains and their subclones. The same DNAs were probed with Mc4.3 (a,c) and IsO.8 (b,d). The mecA gene deletion was associated with rearrangements of the genomic fragments containing a part of IS431 in six of seven methicillin-susceptible subclones (arrowheads in b and d). In MR6 and its subclones, one of the two IS431 copies was not involved with the mecA deletion (arrows in d). IsO.8 also hybridized to penicillinase plasmids of MR108 and MR6 (lane 1 in b and lane 1 in d, respectively). a,b) Lanes: 1, MR108; 2, MR108-1; 3, MS108-l-l; 4, MS108-1-5; 5, MS108-l-15; 6, MR108-3; 7, MS108-3-1; 8, MR108-4; 9, MS108-4-1. c,d) Lanes: 1, MR6; 2, MR6-2; 3, MS6-2-1-7; 4, MS6-2-2-5.

hand, the 2.0 kb bands in the methicillin-susceptible subclones (with an exception of MSlOS-l-l) were replaced by new bands with various migration rates (arrowheads in Fig. 2b and Fig. 2d), whereas all other end bands remained intact. These data indicated that the downstream point of the mecA deletion in each subclone is localized within the 2.0 kb HindIII-Hind111 fragments containing the left part of IS43I. In the case of MSlOS-l-1, however, the mecA gene deletion was accompanied by the deletion of IS431 from the chromosome, which was shown by disappearance of both the 2.7 and 2.0 kb bands in Fig. 2b. MR6 and its subclones carried the other copy of IS431 on the chromosome (the bands of 0.6 kb and 0.4 kb indicated by arrows in Fig. 2d). However, this copy of IS43 1 because there was no did not seem to be involved with the mecA deletion change in the migration pattern of the corresponding bands before and after the mecA deletion (Fig. 2d). PFGE analysis of the size of the deletion . MR108 and its subclones were analyzed by PFGE (Fig. 3a and Fig. 3b). Ethidium bromide staining of the gel showed that the 180 kb fragments which are present in MR108, MR108-1, MR108-3 and MR108-4 were replaced by novel fragments with faster migration rates in MS108-l-l, MS108-1-5, MS108l-15, MS108-3-1 and MS108-4-1 (arrowheads), while no detectable change was observed in other fragments. Southern transfer and hybidization experiments revealed that the 180 kb fragments were hybridized with both Mc4.3 and 1~0.8 probes, and that those fragments with faster migration rates of the methicillin-susceptible subclones were 1322

Vol.

176,

No.

3, 1991

BIOCHEMICAL

a

AND

BIOPHYSICAL

RESEARCH

b 1234567

COMMUNICATIONS

C 1234

1234

orlgin

340291-

291 243 194 146 97 48.5

2431941469748.5(W

(W

Fip. 3. PFGE analysis of MRSA strains and their subclones. The fragments with reduced sizes were observed in the methicilIin-susceptible subclones (arrowheads). The sizes of lambda concatemers are indicated at the left side of photographs. a) Lanes: 1, MR108; 2 MR108-1; 3, MS108-l1; 4, MR108-3; 5, MS108-3-1; 6, MR108-4; 7 MS108-4-1. b) Lanes: 1, MR108; 2, MR108-1; 3, MSlOS-I-5; 4, MS108-l-15. c) Lanes: 1 MR6; 2, MR6-2; 3, MS6-2-1-7; 4, MS6-2-2-5.

hybridized with 1~0.8 but not with Mc4.3, except for that of MS108-1-l which hybridized with none of the probes (data not shown). From these data, the size of the deleted fragments were estimated to be 20 kb in MSlOZI-l-1 and MS108-4-1, and 80 kb in MS108-1-5, MS108-l-15 and MSlOS-3-1. In the case of the subclones of MR6, much more reduction in the size of genomic fragment was accompanied by the mecA deletion (Fig. 3~). The size of the deleted fragments were estimated to be 90 kb in MS6-2-1-7 and 100 kb in MS6-2-2-5. Based on these results, we drew a deletion map of the methicillinsusceptible subclones (Fig. 4). In six of seven subclones, the downstream ends of the deleted fragments were confined within the 2.0 kb HindIIIHind111 fragment containing a left part of IS431, while the upstream ends varied from subclone to subclone. The mapping of the deletion of MSlO8-l-1 to the right of MS108-4-1 was based on the following obser-

5’

M

20 kb

mecA

w

IS431

3’

I. t

-

...

------.*MSlOB-l-l MSlOS-1-5 ;g;;;.;.;" MS,0*-4-, MS6-2.,.7 MS6-Z-2-5

........ .................. . -. ......... ...... ........... ......... -. .............................................................................................. ........ . e.. .............. . ......... ........... ......... ..... ) ......... .... ..... ...... --

A deletion map of the methicillin-susceptible Fig. 4. Dotted lines indicate the DNA fragments which were deleted. indicate the end points of the deletion. 1323

subclones. Arrowheads

Vol.

176,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

vations. Firstly, the deletion size of 20 kb was common between the two subclones. Secondly, the cloned 1.4 kb rearranged fragment of MR108-41 containing the left deletion end point of MS108-4-1 (lane 9 in Fig. 2b) was found to hybridize with MS108-l-l (data not shown). Previously, we showed that the loss of methicillin-resistance from MRSA is due to the deletion of mecA gene during passage in antibioticfree medium (17). In this study, we determined the size of the deleted fragments and mapped them on the chromosome. The observation that the deletion occurred at the vicinity of IS431 suggests a possibility that a critical role is played by the IS element in the mecA deletion. Homologous recombination between juxtaposed IS elements does not seemed to be a plausible explanation for the deletion, because there was only one copy of IS431 on the chromosome of MR108, and only one of the two copies of IS431 was involved in the deletion in the case of MR6. Instead, an intramolecular transposition of IS431 was considered as a more likely interpretation of the mecA deletion. Deletion formation caused by the intramolecular transposition of an for some IS IS element of Escherichia coli has been well documented elements such as ISI and IS903 (19, 20). The reported case of deletion caused by ISI in E. coli is quite similar to that observed with IS431 in that the resultant deletions are highly various in size while one end of the deletion is confined in the vicinity of the IS element (19). The deletion mediated by ISZ has been shown to be temperature dependent. The rate of mecA deletion, however, does not seem to be influenced by temperature, because there was no difference in the frequency of appearance of methicillin-susceptible mutants in cultures of MR108-1 which were incubated at 33, 37 and 42’C (unpublished observation). Deletions caused by IS903 have been shown to occur as a result of i.e. the deleted segment of DNA forms a circular replicative transposition, DNA carrying one copy of IS903 on it, while the other copy remaining at the original position (20). If the same function as that of IS903 was effected by IS431 in the mecA deletion, it might be possible that the resultant circular DNA carrying a copy of IS431 serves as a vehicle for intermolecular mecA gene transposition. So far, we have only observed loss of mecA gene from the chromosome of MRSA, and have not detected any evidence in support of mecA gene transposition either to another site of the chromosome or to the resident plasmids. However, as all clinically isolated MRSA strains so far analyzed carry IS432 closely linked to rnecA gene (unpublished observation), the view that the me CA gene is transmitted as a result of IS431 -mediated transposition seems to require a further consideration. This view is now under experimental inquiries.

1324

Vol.

176,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ACKNOWLEDGMENTS We thank supported

Miss E. Suzuki for technical assistance. This work by the grant from Japanese Ministry of Education.

was partially

REFERENCES 1.

2. 3. 4. 5. 6.

Thornsberry, C. (1988) J. Antimicrob. Chemother. 21 C, 9-17. Brown, D. F. J., and Reynolds, P. E. (1980) FEBS Lett. 122, 275-278. Hayes, M. V., Curtis, N. A. C., Wyke, A. W., and Ward, J. B. (1981) FEMS Microbial. Lett. 10, 119-l 22. Georgopapadakou, N. H., Smith, S. A., and Bonner, D. P. (1982) Antimicrob. Agents Chemother. 22, 172- 175. Hartman, B. J., and Tomasz, A. (1984) J. Bacterial. 158, 5 13 -5 16. Utsui, Y., and Yokota, T. (1985) Antimicrob. Agents Chemother.

28, 397-403. 7.

Matsuhashi, M., Song, M. D., Ishino, F., Wachi, M., Doi, M., Inoue, M., Ubukata, K., Yamashita, N., and Konno, M. (1986) J. Bacterial.

167, 975-980. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20.

Tesch, W., Strissle, A., Bager-Bfchi, B., O’Hara, D., Reynolds, P., and Kayser, F. H. (1988) Antimicrob. Agents Chemother. 32, 1494- 1499. Barberis-Maino, L., Berger-Bachi, B., Weber, H., Beck, W. D., and Kayser, F. H. (1987) Gene 59, 107-113. Rouch, D. A., Messerotti, L. J., Loo, L. S. L., Jackson, C. A., and Skurray, R. A. (1989) Mol. Microbial. 3, 161-175. Rouch, D. A., and Skurray, R. A. (1989) Gene 76, 195-205. Matthews, P. R., Reed, K. C., and Stewart, P. R. (1987) J. Gen. Microbial. 133, 19 19- 1929. Gillespie, M.T., Lyon, B. R., Loo, L. S. L., Matthews, P. R., Stewart, P. R., and Skurray, R. A. (1987) FEMS Microbial. Lett. 43, 165-171. Skinner, S., Inglis, B., Matthews, P. R., and Stewart, P. R. (1988) Mol. Microbial. 2, 289-297. Al Salihy, S. M., and James, A. M. (1972) Lancet ii, 331-332. Grubb, W. B., and Annear, D. I. (1972) Lancet ii, 1257. Hiramatsu, K., Suzuki, E., Takayama, H., Katayama, Y., and Yokota, T. (1990) Antimicrob. Agents Chemother. 34, 600-604. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning 2nd ed. Cold Spring Harbor Laboratory Press. Reif, H. J., and Saedler, H. (1975) Mol. Gen. Genet. 137, 17-28. Weinert, T. A., Schaus, N. A., and Grindley, N. D. F. (1983) Science

222,

755-765.

1325