ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1983, p. 450-452 Vol. 24, No.

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0066-4804/83/090450-03$02.00/0
Copyright 0 1983, American Society for Microbiology

Comparative Analysis of Conjugative Plasmids Mediating

Gentamicin Resistance in Staphylococcus aureus
R. V. GOERING* AND E. A. RUFF
Department of Medical Microbiology, Creighton University School of Medicine, Omaha, Nebraska 68178
Received 27 May 1983/Accepted 9 June 1983

Five gentamicin-resistant clinical isolates of Staphylococcus aureus were found

to contain self-transmissible plassaids of 32 to 37 megadaltons in size. Restriction
endonuclease digests of the plasmids were markedly similar to those of reference
plasmids of unrelated geographical origin, thus suggesting the significant contribu-
tion of common conjugal plasmids to the emergence of gentamicin resistance in S.
aureus populations.

Recent reports have documented the conjugal grown overnight on BHI agarose (0.7%). After
transfer of gentamicin resistance (Gm') plasmids ethanol precipitation, the DNA was resus-
in Staphylococcus aureus (4, 7, 11). The propor- pended in 250 ILI of TE buffer (10 mM Tris [pH
tion of staphylococcal Gm' plasmids which are 7.5], 1 mM EDTA) and treated with a 20 ,ug/ml
self-transmissible (Tra+), however, and the ex- concentration of preboiled RNase 1A (1 mg/ml
tent to which conjugation may account for the in 50 mM sodium acetate, pH 5.0) for 30 min at
emergence of Gm' S. aureus, is as yet unknown. 37°C. A 50-,u volume of 5 M potassium acetate
To determine whether self-transmissible plas- was added, and the mixture was clarified (2) by
mids might be contributing toward Gmr S. aure- centrifugation (15,000 x g) for 2 min. The DNA
us encountered sporadically at a local hospital, was reprecipitated from the supernatent fluid
10 clinical isolates of Gm' S. aureus, obtained with 2 volumes of ethanol, cleaved with EcoRI
over a 6-month period, were screened for the restriction endonuclease as outlined by the sup-
presence of Tra+ Gm' plasmids. The results plier, and analyzed on 0.7% agarose gels (15 by
from five strains (Table 1), representative of the 20 cm) at 2.5 V/cm for 18 h.
entire group, are reported here. Bacterial matings were performed by a modifi-
Isolates were screened for plasmid content by cation of the procedure described by Forbes and
the rapid boiling method of Holmes and Quigley Schaberg (4). Overnight BHI agar cultures were
(5), modified for S. aureus. Cells grown over- adjusted in saline to an optical density of 1.0 at
night on brain heart infusion (BHI) agar were 540 nm. Suspensions (4.5 ml) of donor and
suspended in 10 ml of saline (0.5% NaCl) to an recipient cells (S. aureus 80CRS nov rif t3]) were
optical density of 1.0 at 540 nm. The cells were combined and collected by membrane filtration
harvested by centrifuigation, suspended in 0.4 ml (nitrocellulose; 0.45 im). The filters were imme-
of STET buffer (8.0%O sucrose, 5.0% Triton X- diately placed upright on BHI agar plates and
100, 50 mM EDTA, 50 mM Tris; pH 8.0), and were incubated overnight at 37°C. Each filter
transferred to 1.5-mi Eppendorf tubes. After the was then transferred to 10 ml of BHI broth and
addition of 12 ,ul of lysostaphin (Sigma Chemical vortexed thoroughly, and the cells were recov-
Co.; 10 mg/ml in 0.05 M Tris [pH 7.5}-0.145 M ered by centrifugation. The cell pellet was sus-
NaCl), the tubes were placed in boiling water for pended in 1 ml of BHI broth and plated onto
50 s and immediately centrifuged (15,000 x g) BHI agar containing gentamicin (10 p.g/ml), no-
for 8 tnin, and the supernatent fluid was re- vobiocin (10 ,ag/ml) and, in some cases, rifampin
moved to a fresh Eppendorf tube. The nicleic (200 pg/ml) for overnight incubation and count-
acid was precipitated by the addition of 2 vol- ing.
umes of 95% ethanol at ambient temperature, As shown in Fig. 1, all of the strains examined
followed by centrifugation for 5 min. The precip- carried a large plasmid (32 to 37 megadaltons
itated DNA was suspended in 20 ,ul of TES [Md]) and plasmids of either intermediate (ca. 20
buffer (30 mM Tris [pH 8.0], 5 mM EDTA, 50 Md) or low (ca. 1 to 2 Md) molecular mass. All
mM NaCl) and analyzed on 0.7% agarose mini- strains except CRG700 were found to conjugally
gels (7 by 10 cm) in Tris-borate buffer (8) at 9 transfer Gmr at frequencies ranging from 2.5 x
V/cpm for 1.5 to 2 h. 10-' to 2.9 x 10-6 per recipient cell. Cell super-
For res*tiction endonuclease digestion, DNA natants of donor strains mixed with strain 80CRS
was prepared as described above from cells (nov rif) resulted in no transfer of resistance.
450
VOL. 24, 1983 NOTES 451
TABLE 1. S. aureus clinical isolates examined in
this study
Antibiotic Phage lysis
Strain resistancea pattern
CRG500 Pc Gm NTb
CRG700 Pc Gm 80/52/52A
CRG1600 Pc Gm Tc 80/29/52/52A/81
CRG1800 Pc Gm Em NT
CRG1900 Pc Gm NT
a
Pc, Penicillin; Gm, gentamicin; Tc, tetracycline;
Em, erythromycin.
bNT, Nontypable.

Treatment of cells with DNase I (4) had no

influence on the frequency of Gmr transfer.
Electrophoretic analysis of transconjugants
revealed, in each case, the intact acquisition of
the large plasmid of the donor strain. Subse-
quent analysis of these Tra+ plasmids revealed
that pCRG1600 and pCRG1800 (from strains
CRG1600 and CRG1800, respectively) carried
both Gmr and penicillin resistance (Pc') mark-
ers, whereas pCRG500 and pCRG1900 (from
strains CRG500 and CRG1900, respectively) FIG. 2. Agarose gel electrophoresis of plasmid
carried only Gmr (data not shown). Plasmid DNA digested with EcoRI restriction endonuclease.
(A) Lane 1, pCRG1600; lane 2, pCRG1800; lane 3,
pCRG700 also carried only Gmr (see below). pCRG1900; lane 4, pCRG500; lane 5, pCRG700; lane
The 20-Md plasmids of strains CRG700, 6, pUW3626; lane 7, pAM899-1; lane 8, coliphage A
CRG1600, and CRG1900 (Fig. 1, lanes 1, 3, and DNA. (B) Lane 1, pCRG1600; lane 2, pCRG1601.
5) were shown to specify pcr as determined by
electrophoretic analysis of isolates spontaneous-
ly cured of Pcr or of S. aureus CRG153 (152 [9]
cured of its cadmium resistance plasmid) trans-
duced to Pcr (6, 9, 10).
A comparison of EcoRI digests of the 37-Md
Gmr Pcr plasmids (pCRG1600 and pCRG1800;
Fig. 2A, lanes 1 and 2) revealed restriction
patterns differing only in the EcoF fragments
(4.3 and 4.6 kilobases [kb], respectively). The
32-Md Gmr Pcs plasmids (Fig. 2A, lanes 3
through 5) exhibited identical restriction pat-
terns. This last result was surprising in view of
the fact that plasmids pCRG500 and pCRG1900
were clearly Tra+, whereas conjugal transfer
from strain CRG700 to strain 80CR5 was not
detected despite cell counts indicating fully via-
ble donor and recipient cell populations after the
filter mating procedure. When strain CRG153
was transduced to carry plasmid pCRG700,
however, the plasmid was conjugally transferred
at a frequency of 2.7 x 10-6 per recipient cell.
At present, the nature of this host cell influence
on conjugal transfer is unclear.
For purposes of comparison, geograThically
unrelated Gmr plasmids were included in the
FIG. 1. Agarose gel electrophoresis of intact plas- study. Plasmid pUW3626 (Fig. 2A, lane 6) is a
mid DNA from clinical isolates of S. aureus. Lane 1, Gmr pcr plasmid recently reported by Cohen et
strain CRG1600; lane 2, CRG1800; lane 3, CRG1900; al. (1). Although pUW3626 was not originally
lane 4, CRG500; lane 5, CRG700; lane 6, molecular examined for conjugal transfer, we found it to be
weight standards pII147, pK545, and pC194. Tra+, transferring at a frequency (1.4 x 10-6 per
452 NOTES ANTIMICROB. AGENTS CHEMOTHER.
recipient cell) comparable to that of pCRG1800. LITERATURE CITED
Plasmid pAM899-1 (Fig. 2A, lane 7) is a Tra+ 1. Coben, M. L., E. S. Wong, and S. Falkow. 1982. Common
Gmr Pcs staphylococcal plasknid recently report- R-plasmids in Staphylococcus aureus and Staphylococcus
ed by Forbes and Schaberg (4). Both plasmid epidermidis during a nosocomial Staphylococcus aureus
outbreak. Antimicrob. Agents Chemother. 21:210-215.
restriction patterns were similar to those of the 2. DavIks, R. W., D. Botstein, and J. R. Roth. 1980. A
plasmids isolated here, with the most striking manual for genetic engineering: advanced bacterial genet-
correlation being between pUW3626 and ics, p. 123. Cold Spring Harbor Laboratory, Cold Spring
pCRG1800 (Fig. 2A, lanes 2 and 6.). Harbor, N.Y.
3. Engel, H. W. B., N. Soedlrman, J. A. Rost, W. J. van
A final point of comparson concerns a deriva- Leeuwen, and J. D. A. van Embden. 1980. Transferability
tive of strain CRG1600 which was found to have of macrolide, lincomycin, and streptogramin resistances
spontaneously lost Pcr. Electrophoretic analysis between Group A, B, and D streptococci, Streptococcus
of this isolate (not shown) revealed loss of both pneumoniae, and Staphylococcus aureus. J. Bacteriol.
142:407-413.
the 20-Md pcr plasmid and the pcr determinant 4. Forbes, B. A., and D. R. Schaberg. 1983. Transfer of
associated with plasmid pCRG1600. Restriction resistance plasmids from Staphylococcus epidermidis to
enzyme analysis (Fig. 2B) revealed that this new Staphylococcus aureus: evidence for conjugative ex-
of resistance. J. Bacteriol. 153:627-634.
Gmr Pcs plasmid (designated pCRG1601) result- 5. changeHohnes, D. S., and M. Quigley. 1981. A rapid boiling
ed from a 7.6-kb deletion in the EcoB fragment method for the preparation of bacterial plasmids. Anal.
(12.4 kb) of pCRG1600. Although the cause of Biochem. 114:193-197.
this selective deletion is not understood, a corre- 6. Kloos, W. E., and P. A. Pattee. 1965. Transduction analy-
sis of the histidine region in Staphylococcus aureus. J.
lation between Pc' and the presence of this 12.4- Gen. Microbiol. 39:195-207.
kb EcoRI fragment was invariably found with all 7. McDonnell, R. W., H. M. Sweeney, and S. Coben. 1983.
plasmids examined in this study, including those Conjugal transfer of gentamicin resistance plasmids intra-
from other geographical locations. It is tempting and interspecifically in Staphylococcus aureus and Staph-
to speculate that a 4.8-kb EcoRI fragment ylococcus epidermidis. Antimicrob. Agents Chemother.
23:151-160.
(uniquely present in all the Gmr Pcs plasmids 8. Meyers, J. A., D. Sanchez, L. P. Elwell, and S. Falkow.
examined) may have resulted from a deletion 1976. Simple agarose gel electrophoretic method for the
event similar to that occurring in the formation identification and characterization of plasmid deoxyribo-
acid. J. Bacteriol. 127:1529-1537.
of plasmid pCRG1601. Confirmation of this sug- 9. nucleic
Pattee, P. A., and J. N. Baldwin. 1961. Transduction of
gestion would support the recent speculation of resistance to chlortetracycline and novobiocin in Staphy-
others (1) regarding the possible clonal origin of lococcus aureus. J. Bacteriol. 82:875-881.
Gmr plasmids in S. aureus. Studies are currently 10. Pattee, P. A., and J. Baldwin. 1962. Transduction of
in progress to address these questions. resistance to some macrolide antibiotics in Staphylococ-
cus aureus. J. Bacteriol. 84:1049-1055.
We thank B. Forbes and D. Schaberg, University of Michi- 11. Schaberg, D. R., D. B. Clewedl, and L. Glatzer. 1982.
gan Medical School, for sharing their unpublished results. Conjugative transfer of R-plasmids from Streptococcus
This study was supported in part by a grant-in-aid from the faecalis to Staphylococcus aureus. Antimicrob. Agents
American Heart Association, Nebraska Affiliate. Chemother. 22:204-207.