AGENTS AND p.
0066-4804/81/090359-07$02.00/0
Penicillin
and Tetracycline Resistance Plasmids
in
Staphylococcus
epidermidis
PAT A. TOTTEN,t* LUCY VIDAL, AND JACK N. BALDWIN
Department of Microbiology, University of Georgia, Athens, Georgia30602
Received29August 1980/Accepted17June 1981
The geneticnatureofpenicillin (Pc) and tetracycline (Tc) resistance plasmids
in Staphylococcus epidermidis were studied and compared with those in S. aureus.Of10S.epidermidisstrains transduced forpenicillin resistance,wecould
isolate Pc plasmids from only 3. One of these plasmids also encoded for cadmium resistance and another encoded for resistance to ethidium bromide, traits also associated with S. aureus Pc plasmids. Endonuclease fingerprinting of the Pc
plasmids from the twospecies revealed extensive heterogeneity. Two S. epider-midis strains were also transduced for tetracycline resistance. Both harbored
plasmids indistinguishable from S.aureusTcplasmidsasjudged by endonuclease
fingerprinting. These datasuggestthatgenetic exchange between S. aureusand S.epidermidisoccursin vivo.
Althoughthepenicillin (Pc) and tetracycline (Tc) resistanceplasmids of Staphylococcus
au-reushave been well characterized (8), little is
knownaboutthese resistancedeterminantsinS.
epiderinidis. Only recently has a Pc' plasmid
been identified in S. epidermidis (18). Before this time,the only evidence for S. epidermidis pcr plasmidswascotransduction or loss of
pen-icillinresistancewith other traits(14, 19, 21, 22). The Tcr plasmids of S. epidermiidis have been studied ingreaterdetail andarethesamesizeas
those of S. aureus (12, 20). Recently the Tcr plasmids of S. epidermidis andS. aureus were
compared bydeoxyribonucleic acid (DNA)
ho-mologyandrestrictionenzymeanalysisand
ap-pearedtobesimilar butnotidentical (5). Antibiotic resistances in S. epidermidis are
clinically important fortwo reasons: (i) S. epi-dermidishas beenincreasingly implicatedasan
opportunistic pathogen (9, 10, 25), and the
spread of antibiotic resistance markers within this species could seriously complicate
chemo-therapy;and (ii) if staphylococci exchange plas-mids invivo,antibiotic resistances harboredby
S.epidermidis mayspreadtocoresidentS.
au-reusstrains. Theimportance ofthis interspecific exchange is evident when one considers that more strains of S. epidermidis are antibiotic resistant than are strains of S. aureus isolated
undercomparableconditions (1, 10).
Todetermine whetherS. epidermidis and S.
aureus strainsexchange plasmid DNA invivo, we characterized the Tcr and pcr plasmids of
tPresent address: Department ofMicrobiology and
Im-munology, SC-42, University of Washington, Seattle, WA 98195.
thesetwospecies.Sirnilaritiesofphenotypicand
physicalcharacteristicsweredemonstrated,and thesignificanceof these
findings
wasexamined.MATERIALS AND METHODS
Media. Yeast extract broth (YETS) contained a
0.3% yeastextract (Difco Laboratories)and 3%
Tryp-ticase soybroth (BBLMicrobiology Systems).Allagar mediaused contained 1.5% agar(Difco).
Bacteria and bacteriophages. S. epidermidis
typing phages andpropagating strains ofS.
epider-midis have been described(29) andwerekindly
sup-pliedby J.T. Parisi. Table1lists strains of S.
epider-midisand theirsources.S. aureusstrainsweretaken from the stock culture collection of J. N. Baldwin.
Staphylococciwere identified by the Gram stain
re-action,catalase production, and theabilitytoproduce acid from glucose anaerobically after incubation at
37°Cfor7days (27). Thecoagulasetest wasusedto
identify the species of the isolates.
Spectinomycin-resistant strainswere isolated onYETS plates
con-taining200,ugofspectinomycinperml(Trobicin;The
UpjohnCo.).
Transduction. Transductionwasaccomplished as described by Olson et al. (18), using transducing phages 29, 108, and 113.
Transductants
wereselectedonplates containing0.16,g ofpenicillinG or 10 ,ug of
tetracyclineperml andwereconfirmedby their
spec-tinomycinresistance and cryptic plasmidcontent as
detectedbv theBrij minilysis technique (33). Phenotypic testing.Antibioticsusceptibilitywas
determinedby usingamodification of the standardized discdiffusion test (11). YETS was used in place of Mueller-Hinton agarasthe testmedium. Penicillin-andtetracycline-resistant colonies were scored after
growthfor48honYETSplatescontaining 10,ugof
tetracyclineor0.16 ugofpenicillinperml.Penicilinase
productionwasdetectedby the starch iodineoverlay technique (1).Aerobicproduction of acid from
360 TOTTEN, VIDAL, AND BALDWIN TABLE 1. Characteristics and source of strains of S.
epidermidis used intransduction experiments" Phenotypic response'
Strain - Source
Cd Asa Hg PMA EB Pc Tc Bla
Recipient JB216 R NT R R S SS - UGa JB248 S S S S S S NT - JTP Donor PS29 S S S S R R R + JTP PS108 R S S S S R S + JTP 12228 S S 5 S R R + ATCC JB117 R S R R S R R + UGa JB20 S S R R S R S + UGa JB41 R S R R S R R + UGa JB44 R S R R S R S + UGa JB36 S R S S S R NT + UGa JB49 S R S S S R NT + UGa JB104 _S R S S S R NT + UGa aCd, Cadmium; Asa, arsenate; Hg, mercury; PMA, phen-ylmercuric acetate; EB, ethidium bromide; Pc,penicillin;Tc, tetracycline; Bla, beta-lactamase production; R,resistant;S,
susceptible; +, positive test; -, negative test; UGa, from nasal cultures ofUniversity of Georgia students; JTP, J. T. Parisi; ATCC,American Type Culture Collection.
nitol and ribose was determined by the method of
Schaefler(22). Deoxyribonucleasetestagarand
Chris-tiansonureaagarwereobtained from Difco andwere
used according to the manufacturer's directions. Ethidium bromide (EB) resistance was scored on
YETS platescontaining19jigof EB.
Heavy-metal resistances were screened by thewell diffusion method ofSummers and Jacoby (28) with thefollowing solutions of heavy metals:0.1 MCdC12,
0.1 M HgCl2, 5 ,uM phenylmercury acetate, and a
saturated solution of sodium arsenate. Mercury and cadmium resistanceswerefurther testedbythe pro-cedurespublishedby Weissetal.(31)and Novick and Roth (17). Zones of inhibition were compared with those ofS. aureusstrains8325-4 and8325-4 (p1258),
thesusceptibleand resistantcontrols.
Isolation and analysis ofplasmid DNA. The Brij minilysis procedure (33) was used forscreening
strains forplasmid content. For isolation ofplasmid
DNA, thelysostaphin-cleared lysis procedure (26) fol-lowed by CsCl-EB centrifugation wasused with the
following modifications. The medium used for over-nightgrowth ofpenicillin-resistant strainscontained 5Mugofmethicillinperml and 0.5%glycine. The con-centrations oflysostaphin usedwere 15Mug/mlforS. aureus and 30 jg/ml for S. epidermidis. These
in-creasedconcentrations oflysostaphinandglycine gave
betterlysis of S. epidermidis strains. Plasmid DNA
isolated from CsCl-EBgradientswasexhaustively
di-alyzed against TE buffer [10 mM
tris(hydroxy-methyl)aminomethane-1 mM ethylenediaminetetra-acetic acid, pH 7.5] and was subsequently concen-tratedthreefoldbyevaporation. Forelectron
micros-copy, DNA was further purified by chloroform-iso-amyl alcohol (24:1, vol/vol) extraction followed by
ethanolprecipitationandresuspensioninTEbuffer.
Agarose gel electrophoresis. The procedure of
Wilson and Baldwin(32)wasused.Gel concentrations
varied from 0.7to1.0%,dependingontheexperiment. Electron microscopy. Open circular plasmids
wereobtained by limited digestion with deoxyribonu-clease (30) and were prepared for electronmicroscopy bythe technique ofKleinschmidt (7). Contour lengths of20 to 25molecules of each plasmid were determined along with an internal standard, bacteriophage PM2 (6.4 megadaltons [Mdal] [4]), kindly donated by R. Wilson.
Compatibility. Compatibility of phenotypically different plasmids was determined by the method of Cabello et al. (2). Transductants were selected on mediacontaining19Mgof EB per ml or 60 mMCdC12.
After two serialsingle-colony isolations on the appro-priate selective media, transductants were analyzed for plasmid content by agarose gel electrophoresis. Displacementoftheresident plasmid by the selected transduced plasmid was the behavior expected for the incompatible plasmids.
Restriction enzyme analysis. EcoRI, HaeIII,
HindIII, andHpaIIwereobtained from New England Biolabs (Beverly, Mass.), and plasmids were digested aspublished (32). Standards used to determine molec-ular weights of restriction fragments were lambda and PM2, both cut with HindIII aspublished (32).
RESULTS
Transductionandphenotypic testing. Ta-ble 1 summarizes the phenotypic properties of
S.epidermidisdonorsandrecipientsusedinthe transduction experiments. All donor strains could transduce both recipients. Strain JB248
was susceptible to heavy metals, and
cotrans-duction ofthesetraitswithpenicillinresistance
couldbetestedin thisrecipient.However, JB248 harbored multiple plasmids. Therefore, the re-strictionanalysis and electronmicroscopywere carried out with transductants of the other re-cipient, JB216.
Strainsweretested forcotransduction of
pen-icillin resistance with erythromycin (Em) and
heavy-metalresistancetraits associatedwith S.
aureus pCr plasmids. The four donor strains
resistantto
Hg2+
andphenylmercuryacetatedidnot cotransduce these traits with
penicillin
re-sistance. Ofthe fourCddonorstrains,only one (PS108) cotransduced this trait with penicillin
resistance. Alldonorstrainswereerythromycin
susceptible. Therefore, Emrgenes were not
pres-ent onthesepcrplasmidsaspreviouslyreported for some S. aureus strains (8). Cotransduction of EB with penicillin occurred with PS29, the
only EB-resistantstrain tested. AllPcr
transduc-tantsproduced penicillinase.
Cotransduction of Pcr with other traits
re-portedtobeassociated with S.epidermidispcr
plasmids could not be tested with our strains. Bothrecipient strains wereureaand ribose pos-itive; therefore, cotransduction of these traits with penicillin resistance could not be tested. Becauseallpcrdonors didnotfermentmannitol
orproduce nuclease,thesetraits were not linked withpenicillinresistance inthesestrains.
PLASMIDS IN S. EPIDERMIDIS 361 Tcr transductants were obtained by using
JB216astherecipient and strains JB12228 and PS29 as the donors. No other donor strainswere
tested for transduction oftetracycline resistance. Plasmid content of transductants. When
CsCl-EB-purified plasmidDNAwas
electropho-resed on agarosegels,the pcrtransductants from donor strains PS29, PS108, and 12228 showed additional plasmid bands; the other seven pcr transductants didnot.The results ofone
trans-ductionexperiment (Fig. 1) show thataplasmid (pAJ1003) contained bythe donor, 12228 (slot
4), wasalso harboredbythe transductant(slots
2 and 3). Therecipient, JB216 (slot 1),didnot
containaplasmidof this size. Plasmidswerealso found in pcrtransductants when either JB216or
JB248wasusedastherecipient.The otherseven
strains servedasdonors ofpenicillin resistance,
but no newplasmidsweredetected in the
recip-ients eitherby theBrijminilysis techniqueorby lysostaphinlysis followed by CsCl-EB
centrifu-gation. Analysis of the Tcr transductants
re-vealedplasmids (pAJ1004andpA51005)which
migratedsimilarlytotheS.aureusTcrplasmid
(pCW6).
Electronmicroscopy. Molecular massesof pcr plasmid DNA isolated from the transduc-tantsJB217, JB218,andJB219 and five strains of S. aureus were determined by electron mi-croscopy(Table 2).S.epidermidisTcr plasmids
o*
LF
C
FIG. 1. Agarose gel electrophoresis ofBrij minily-satesobtained from transduction with JB216 as re-cipient and Pcr the marker transduced. (1) JB216
(recipient), (2 and3) JB219(transductant), and (4)
JB12228(donor).P,Pcrplasmid(pAJ1003); LF,
lin-earfragments (chromosome);C, crypticplasmid.
were not sized by electron microscopy, since their migration on agarose gels was similar to that of the2.9-MdalS. aureus Tcr plasmid.
Compatibility
testing. Plasmid pAJ1001 was transduced into strain JB218 (harboring pAJ1002) by selecting for EB resistance. Both plasmids appeared on electrophoresis of the transductant and were therefore compatible. Similarly, plasmid pAJ101wastransduced into strain JB219 (harboring pAJ1003) but, due to the similarsiie, the presence of both plasmids could not be resolvedelectrophoretically. How-ever, HaellI digests of the transductant revealedfragmentsof bothplasmids,indicating that these
plasmidswerecompatible.Compatibilitytesting between plasmids pAJ1002 and pAJ1003 gave uninterpretable results.
Restriction endonuclease digest pat-terns. Selected pcr plasmids of S. aureus after digestion with restriction enzymes were com-pared with digests ofpcr plasmids from S. epi-dermidis transductants JB217, JB218, and JB219. JB216 servedas acontroltoposition the digest products of the cryptic plasmid present in the transductants. Figure 2 shows comparisons of S. aureus and S. epidermidis plasmids di-gested with EcoRI. Slot9contained the cryptic plasmidcontrol.Althoughplasmids of S. aureus
(slots 1 through 5) and S. epidermidis (slots 6 through 8) appearedquiteheterogeneous, a few exceptions were noted. Slots 2 and 5 contained digests which appeared to have three bands in
common. Three other digests (slot 4, 5, and 8) had 1.5-and 0.74-Mdal bands in common. It is also interesting to note that digests of four of the five S. aureus plamids tested (slots 2 through 5) had 1.4-Mdal fragments. This region of the
gelwasmaskedbythe cryptic plasmid in strains ofS.epidermnidis.However, since the molecular size totals of the fragments produced from pAJ1001, pAJ1002, and pAJ1003 were >13.7, 10.0,and 16.8 Mdal,respectively, and the molec-ular sizes calculated by electron microscopy were 15.5, 9.9, and 17.2 Mdal, a 1.4-Mdal band wasprobablynotpresent in digests of the latter
twoplasmids.
HaeIII digestion patternsofS. aureus and S.
epidermidis Pcr plasmids were also compared
(Fig. 3). The cryptic plasmid present in the S.
epidermidisstrainswasnotdigested by HaeIII;
only the covalently closed and open circular formswerepresentongels. The position of these bands relative to linear digest products of the
pcrplasmids could be changed by altering the agarose concentrations (compare Fig. 3A and 3B), allowing us to detect bands previously masked.Althoughtheoveralldigestionpatterns
ofthe pcr plasmidswere quite different, many of theplasmids appearedtohavebands in
TABLE 2. Characteristics ofplasmids used in endonucleasestudiesa Molecular
Plasmid Resistance mass Donor strain Transductant Source
(Mdal) S. epidermridis pAJ1001 PCr, EBr 15.5±0.4 PS29 JB217 pAJ1002 pcr,Cdr 9.9±0.3 PS108 JB218 pAJ1003 Pc' 17.2±0.5 12228 JB219 pAJ1004 Tcr 2.9 PS29 JB227 pAJ1005 Tcr 2.9 12228 JB228
Notdetected pcr 7strains:JB117, JB20,JB41, 7strains JB44, JB36, JB49,JB104
S.aureus
pAJI Pcr, Asar, Hg' 21.2 Wild-type
strain
pAJ2 Pcr,Emr 26.1
pAJ3 Pcr,Cd',Asar,Emr 10.6 Wild-type
strain
pAJ5 Pcr, Cd,Asar,Hg' 19.5
Trans-formant of 8325-4 p1258 pCr,Cd,Asa',Hg, 18.6 Trans-Em' formant of 8325-4 pCW6 Tcr 2.9 Trans-formant of 8325-4
"Molecularmasses weredetermined on transductants of JB216. Heavy-metal resistances were determined ontransductants
ofJB248. Tcplasmidmolecular masses were determined byendonuclease fragment sizes on agarose gels. Em, Erythromycin.
0.74_.,
FIG. 2. EcoRI digests of plasmid DNA from
strainsofS.aureusandS.epidermidis. Digests ofS.
aureus pcrplasmids are in slots 1 through 5: (1) pAJ3, (2)pI258, (3) pAJ1, (4) pAJ2, and(5) pAJ5. Digests ofS.epidermidisPc'plasmidsareinslots 6
through 8: (6)pAJ1001,(7) pAJ1002,and(8) pAJ1003.
Theseplasmidswereisolatedfrom transductants of JB216,and thedigests thereforeinclude the1.6-Mdal
cryptic plasmid (slot 9).Molecularmasses(in mega-daltons) of significant fragmentsareindicatedonthe
left.
mon.Forinstance,a1.9-Mdal bandwaspresent
indigestsofalltheS.
epidermnidis
andS. aureus pcr plasmids tested.Unlike HaeIII digestion,
plasmids
of S.epi-dermidis and S. aureus
digested
with HindIII andelectrophoresedonagarosegelsappearedtocontainveryfewfragmentsincommon(datanot shown).
Tcrplasmids of S.epidermidisand S. aureus
werealsocompared(Fig. 4). Again,JB216 served
toposition thefragments
generated
by
enzymedigestion ofthe
cryptic
plamid.HindIlI (Fig.
4A) and HpaII (Fig. 4B)
digestion
products
ofpCW6 (from S. aureus, slot
3)
andpJB1005
(from S.epidermidis, slot1)werethesamesize.
Because theintactplasmidsmigratedtosimilar positions on agarose
gels
(notshown),
weas-sumed that themolecular
weights
of theseplas-mids wereverysimilar.
Therefore,
afragment
ofthe size that would be masked by the
cryptic
plasmid could not be present in the enzyme
digests.
DISCUSSION
Inthisstudy,Pcr plasmidswerefound in three strains of S.epidermidis. These plasmidswere
identified by transductioninto
penicillin-suscep-tible recipientsandhad molecularsizes of 15.5, 9.9, and 17.2 Mdal. No Pc' plasmids could be
PLASMIDS 363
.~~~~~;*
C(
A
FIG. 3. HaeIIIdigests ofPc'plasmids. (A)Digestswereelectrophoresedon 1%agarosefor11 hat32V.
The molecularmassesofthe control(PM2cutwithHindIII, slot2) arelisted(inmegadaltons)on theleft. PlasmidDNAofS. epidermidis digestedwith HaeIII is in slots 3through6.Thepositionsofthecovalently closed circular(CCC)andopencircular(OC) forms of cryptic plasmid (slot3)areindicatedbyarrows.Slots
containingPc'plasmids in additiontothecrpticplasmid were: (4)pAJ1003, (5)pAJ10(X2, and(6)pAJ1001.
Digests ofthePc'plasmidsofS.aureusarein thefollowingslots: (1and10) pAJ3, (7) pAJ5, (8)pAJ1,and (9)pI258. (B)HaeIIIdigestselectrophoresed under conditions (0.8%agarose,15h,25V) different fromthose
of(A)in ordertoexposelinearfragmentsmaskedbycrypticplasmidDNA.S.aureusPcrplasmiddigestsare:
(1)pI258, (2) pAJ1,and(3) pAJ3. Theposition ofthecrypticplasmid ofS.epidermidiscanbeseenin slot 7 and is indicated. S.epidermidis plasmid digestswere(4) pAJ1001,(5)pAJ1X002,and(6)pAJ1(003.A1.9-Mdal
fragmentwhichwasmaskedinFig.5Acannowbeseenin slots4, 5,and 6.
FIG. 4. S.aureusandS.epidermidis Tc'plasmids digestedwith HindIII (A) andHpaII (B). Slots in
bothsections containdigests of: (1)pAJ1005,the S. epidermidis Tcrplasmid and the resident cryptic
plasmid,(2)thecrypticplasmid oftheS.epidermidis recipient,and(3)pCW6,theS.aureusTcr plasmid.
demonstrated in transductants-by using seven
other donor strains and the technique of lyso-staphin-mediatedBrij lysisfollowedbyagarose gel electrophoresis. This procedure should de-tectplasmidDNA similartotheDNAofthePcr plasmidsofS.aureus orofpcrplasmids
identi-fiedinthe otherstrains of S.epidermidis.
Per-hapsthe Pcrgenesinthese strainsare on
plas-mids whichwere notisolatedbyour technique
because theywerepresent in onlyafew copies percellor wereextremelylabile. It is also
pos-sible thatalarge plasmidinthe donorcouldbe fragmented whentransduced and incorporated
into therecipient chromosome. Although chro-mosomal pcr genes are rare in S. aureus (8),
their presence inthese sevenstrains of S. epi-dermidis would alsoexplain the results.
Donor strains and transductants were also
tested forproperties reportedly associatedwith penicillin resistance in S. epidernidis. These
includeaerobic acidproduction from riboseand
mannitol, ureaseproduction,and nuclease
pro-duction (14, 22). No association between those traits andpenicillinresistancewasfound inour
strains. The S. epiderrnidis pcr plasmids were
alsocomparedbycompatibilitytesting andwere
foundtobe inatleasttwocompatibilitygroups, indicating possibledifferences in replication con-trol (2) ormembrane attachment sites (6) for
thesemolecules.
The pcr plasmids ofS.epidermnidiswere
sim-ilar to those found in S. aureus in several
re-spects.One S. epidermidis plasmidencoded for EB resistance and another encoded for
resist-ancetocadmium,traitsalsoassociated S.aureus
pcr plasmids. Theplasmidsofthesetwospecies 3.S-.-0.27
o.24::
i
t VOL. 20,1981 _12 ,CC364 TOTTEN, VIDAL, AND BALDWIN
were in the same size range (see Table 2).
Al-thoughthesestaphylococcalplasmidsappeared
heterogeneous byendonuclease digestion, they sharedseveralfragments of the samesize after digestion with EcoRI. A 1.9-Mdal fragment gen-erated by HaeIII digestion (see Fig. 3) was shared by all staphylococcal pcrplasmids and could possibly indicate a conserved region in these plasmids. Recently, Novick et al. also re-ported the physical heterogeneity of S. aureus
pcr plasmids (16). These differences could be
partially attributed totransposons and several transpositional "hot spots" identified on these
plasmids (13, 15). Possible conserved regions
such as the 1.9-Mdal HaeIII fragments could representaplasmid core onto whichtransposons
haveinserted. TheSouthern blotting technique (24) couldbeused tofurtherexaminetheareas ofpossible homologyin theplasmidsused in our study.
The two Tcr S. epidermidisplasmids in our
study hadindistinguishable patternsafter
diges-tion with HindIII and HpaII, indicating that these twospeciesmayharbor the sameplasmid. Surprisingly,asjudgedby thesamecriteria,the
Tcr plasmidsof S. aureusaresimilarinseveral
otherstrains tested (23, 32). Thereasonfor the
conservation ofthe sequences of the Tcr plas-mids opposed to the heterogeneity ofthe pcr
plasmids of S. aureusand S. epidermidisis not known.Recently Groves (5) found extensive
ho-mologybutslightlydifferent restrictionenzyme patterns between theTcr plasmids of S. aureus and S. epidermidis. Although these plasmids
mayhave sequencedifferenceswhich would
ex-plain the divergent restriction enzyme
finger-prints, possible restriction-modification differ-encesbetweentheS.aureusandS.epidermidis
hostswould alsoexplaintheseresults.
Lacey (8) haspostulated that S. epidermidis
mayact as areservoirofresistancedeterminants for the more clinically significant S. aureus. In
vitrostudieshaveshownthatstaphylococcican
exchange DNA by transduction or protoplast
fusion (34;M.L. Stahland P. A.Pattee,Abstr. Annu. Meet. Am. Soc. Microbiol. 1980, H32, p. 113). Inthisstudy, we have shown thatplasmid
DNA may be exchanged between these two
staphylococcal speciesinvivo basedonthe sim-ilarity of theirTcrplasmids. However, some S.
epidermidisstrainsalso harbor resistance deter-minants notfrequentlyfound in S. aureus. For instance, 7 ofthe 10 strains of S.epidermidisin ourstudydid nottransduce pcrplasmids
phys-icallysimilar to those found in S. aureus. Also,
Wilsonetal.(33)surveyedtetracycline-resistant
strains of S. epidermidis and found that 2.9-Mdal plasmidswere missingin many of them. Perhaps transfer of resistance determinants in vivoisconfined mainly toplasmid species,and
thereforechromosomal pcr andTcrgenesin S.
epidermidis have not spread to S. aureus
strains. Future studiesin which these two
spe-ciesareisolated from thesamehostor
environ-ment may allow the detection ofmore strains whichhavesimilar resistance determinants.
The data presented here do not refute the
theory that Pcr, Tcr, and other plasmid genes may beexchangedbetweenstaphylococciin vivo viatransduction. Further studies by DNA/DNA hybridizationorheteroduplex analysismay
clar-ify thephysical relationships of
staphylococcal
plasmids and couldbeused tofurther examine
theirepidemiology.
ACKNOWLEDGMENTS
WethankRonWilson and Barbara Brownfortheir tech-nical advice,suggestions, and criticisms.We arealsoindebted
toDanVapnek and Bruce Carlton fortherestriction enzymes
sokindlysupplied.
ThisinvestigationwassupportedinpartbyPublic Health Service grant AI15442from theNationalInstituteofAllergy and Infectious Diseases.
LITERATURE CITED
1. Baldwin, J. N.,R.H.Strickland,and M. F. Cox.1969.
Somepropertiesof thebeta-lactamase genesin Staph-ylococcusepidermidis. Appl.Microbiol.18:628-630. 2. Cabello, F.,K.Timmis,andS.N.Cohen.1976.
Repli-cationcontrolincompositeplasmid constructed byin
vitrolinkage oftwodistinctreplicons. Nature(London) 259:285-290.
3. Causey,S.C.,and L. R. Brown.1978.Transconjugant analysis: limitations on the use of sequence-specific endonucleases forplasmididentification. J. Bacteriol. 135:1070-1079.
4. Franklin,R. M.1974.Structureandsynthesisof
bacte-riophage PM2, withparticular emphasison the viral
lipid bilayer. Curr. Top.Microbiol. Immunol. 68:107-158.
5. Groves, D.J. 1979. Interspecific relationships of anti-bioticresistance in Staphylococcus sp.: isolation and comparison ofplasmids determiningtetracycline resist-anceinS.aureusandS.epidermidis.Can.J.Microbiol. 25:1468-1475.
6. Jacob, F., S. Brenner, and F. Cuzin. 1963. On the
regulation of DNA replication in bacteria. ColdSpring
HarborSymp. Quant. Biol. 28:329-348.
7. Kleinschmidt,A. K.1968.Monolayer techniquesin
elec-tron microscopy of nucleic acid molecules. Methods
Enzymol.12B:361-377.
8. Lacey, R. W. 1975. Antibiotic resistance plasmids of
Staphylococcusaureusand their clinical importance.
Bacteriol.Rev.39:1-32.
9. Males,B.M., W.A.Rogers, andJ. T.Parisi. 1975.
Virulencefactors ofbiotypesofStaphylococcus epider-midisfromclinicalsources.J. Clin.Microbiol.
1:256-261.
10.Marsik, F. J., andJ. T. Parisi. 1973. Significanceof
Staphylococcusepidermidisin theclinicallaboratory.
Appl. Microbiol. 25:11-14.
11.Matsen, J. M., and A. L. Barry. 1974. Susceptibility testing:diffusiontestprocedures, p. 418-427. In E. H.
Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manualofclinicalmicrobiology,2nded.American So-cietyforMicrobiology,Washington,D.C.
12. Minshew,B.H., andE. D.Rosenblum.1973.Plasmid
fortetracyclineresistance inStaphylococcus
epidermi-dis.Antimicrob. AgentsChemother.3:568-574. 13. Murphy, E., andR. P.Novick.1979.Physicalmapping
of Staphylococcus aureuspenicillinaseplasmidpI524: characterization of an invertibleregion. Mol. Gen. Ge-net. 175:19-30.
14. Nazar, K., P. B.Heczko, and G. Pulverer.1977. Trans-duction ofpenicillin resistance together with the ability toferment mannitol and ribose inStaphylococcus epi-dermidis. J. Gen. Microbiol. 99:449-452.
15. Novick, R. P., I.Edelman,M. D.Schwesinger,A. D. Gruss, E. C. Swanson, and P. A. Pattee. 1979. Ge-netic translocation in Staphylococcus aureus. Proc. Natl. Acad.Sci. U.S.A. 76:400-404.
16.Novick, R. P., E. Murphy, T. J.Gryczan, E. Baron, andL. Edelman.1979.Penicillinaseplasmidsof Staph-ylococcusaureus:restriction-deletion maps. Plasmid 2:
109-129.
17. Novick, R.P., and C. Roth. 1968. Plasmid-linked resist-ance toinorganic salts in Staphylococcusaureus. J. Bacteriol. 95:1335-1342.
18.Olson, W.C.,Jr., J. T.Parisi,P.A.Totten,and J. W.
Baldwin. 1979.Transduction ofpenicillinase
produc-tion inStaphylococcusepidermidis and nature of the genetic determinant. Can. J. Microbiol.25:508-511.
19.Posten, S.M., and T. J. Palmer. 1977. Transduction of
penicillinaseproduction and other antibiotic resistance markers in Staphylococcus epidermidis. J. Gen. Micro-biol. 103:235-242.
20. Rosendorf, L. L.,and F. H.Kayer.1974.Transduction andplasnid deoxyribonucleic acidanalysis in a multiply antibiotic-resistant strain ofStaphylococcus epidermi-dis. J.Bacteriol.120:679-686.
21. Schaefler,S. 1971.Staphylococcus epidermidis BV: an-tibiotic resistance patterns, physiological characteris-tics,and bacteriophage susceptibility. Appl. Microbiol. 22:693-699.
22.Schaefler, S. 1972.Polyfunctionalpenicillinase plasmid
inStaphylococcus epidermidis:bacteriophage restric-tionand modification mutants. J. Bacteriol. 112:697-706.
23. Shafferman, A., Z. Shalita, and L. Hertman. 1978. Cleavage maps of a tetracycline plasmid from
Staphy-PLASMIDS IN
lococcus aureus. J. Bacteriol. 134:345-348.
24. Southern, E. M. 1975. Detection of specific sequences among DNAfragments separated by gel electrophore-sis. J.Mol. Biol. 98:503-517.
25. Speller, D.C. E., and R. G. Mitchell. 1973.Coagulase negative staphylococci causing endocarditis after
car-dial surgery. J.Clin. Pathol. 26:517-522.
26. Stuffier,P.W., H. M.Sweeney, and S. Cohen. 1974. Co-transduction ofplasmids mediating resistance to tetracycline and chloramphenicol in Staphylococcus aureus.J. Bacteriol. 120:934-944.
27. Subcommittee on Taxonomy ofStaphylococciand Micrococci. 1965. Recommendations. Int. Bull. Bac-teriol. Nomen. Taxon. 15:109-110.
28. Summers, A. O., and G.A. Jacoby. 1977. Plasmid-determined resistancetotelluriumcompounds.J. Bac-teriol. 129:276-281.
29. Talbot, HI W., Jr., and J. T. Parisi. 1976. Phage typing of Staphylococcus epidermidis.J. Clin. Microbiol. 3: 519-523.
30. Vinograd, J., J. Lebowitz, R.Radloff, R. Watson, and P. Laipis. 1965. The twisted circular form of polyoma viral DNA. Proc. Natl. Acad. Sci. U.S.A. 53:
1104-1111.
31. Weiss,A.A., S. D. Murphy, and S. Silver. 1977. Mer-cury and organomercurial resistances determined by
plasmidsinStaphylococcus aureus. J. Bacteriol. 132: 197-208.
32. Wilson,C. R., and J. N. Baldwin. 1978. Characterization and construction ofmolecular cloning vehicles within Staphylococcus aureus. J. Bacteriol. 136:402-413.
33. Wilson,C.R.,P.A.Totten,and J.N. Baldwin. 1978. Rapid procedure for the detection of plasmids in Staph-ylococcusepidermidis. Appl. Environ. Microbiol. 36: 368-374.
34. Yu, L., and J. N. Baldwin. 1971. Intraspecific
transduc-tion in Staphylococcus epidermidis and interspecific transduction between Staphylococcus aureus and
Staphylococcusepidermidis.Can. J. Microbiol. 17:767-773.
