Vol. 149, No. 3JOURNAL OF BACTERIOLOGY, Mar. 1982, p. 995-10040021-9193/82/030995-10$02.00/0

Heterogeneity of Tetracycline Resistance Determinants inStreptococcus

VICKERS BURDETT,* JULIA INAMINE, AND SHRINIVAS RAJAGOPALAN

Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710

Received 13 August 1981/Accepted 2 November 1981

We found that naturally occurring tetracycline resistance in streptococci isencoded by more than one genetic determinant. Two of these distinct determi-nants were cloned, and the regions that are necessary and sufficient for expressionof tetracycline resistance were defined by deletion analysis. These cloneddeterminants were further characterized by DNA-DNA hybridization experi-ments which also identified a third genetically unrelated tetracycline resistancedeterminant. Some of these genetic differences appear to represent mechanisticdifferences. The tetL determinant was associated with small nonconjugativeplasmids and mediated resistance to tetracycline. The tetM determinant was mostoften "nonplasmid" associated and mediated resistance to minocycline as well astetracycline. The tetN determinant was represented on a large conjugativeplasmid and was genetically distinct from tetL and tetM, although phenotypicallyit resembled tetM.

Resistance to tetracycline occurs at high fre-quency among clinical isolates of both gram-positive and gram-negative bacteria. The mecha-nism and genetics of tetracycline resistance-havenot been studied in streptococci (including S.pneumoniae), although the overwhelming major-ity of clinical isolates are tetracycline resistant.Although tetracycline resistance in gram-nega-tive bacteria is predominantly plasmid mediated,this does not appear to be the case amongclinical isolates of streptococci (2, 19). For ex-ample, in surveys of tetracycline-resistant groupB streptococci and oral streptococci, the major-ity of isolates tested do not contain any detect-able plasmid, although conjugal transfer of tetra-cycline resistance from some of these strains hasbeen demonstrated by several laboratories (20,37). Plasmid-mediated tetracycline resistanceoccurs in these organisms since tetracyclineresistance plasmids have been isolated fromgroup B streptococci (2) as well as from S.faecalis (6, 11, 22, 40).We describe here an initial genetic analysis of

plasmid- and "nonplasmid"-mediated tetracy-cline resistance in streptococci. The term non-plasmid will be used to designate tetracyclineresistance determinants where tetracyclineresistance plasmids have not been demonstrat-ed, although the existence of plasmids cannot beexcluded. To facilitate this analysis, we success-fully cloned one plasmid and one nonplasmidresistance determinant into Escherichia coli.This work demonstrates the presence of threegenetically unrelated tetracycline resistance de-terminants in streptococci, all of which are dis-

tinct from those characterized in other gram-positive and gram-negative organisms. Inaddition to genetic dissimilarities, mechanisticdifferences were identified based on the suscep-tibilities to the lipophilic tetracyclines minocy-cline and chelocardin.

MATERIALS AND METHODS

Bacterial strains. The bacterial strains used, theirrelevant phenotypes, and plasmid contents are shownin Table 1. Transformations into E. coli employedSK1592 as the recipient strain (24). The E. coli cloningvector pVH2124 was a derivative of ColEl-amp (38)which has undergone a spontaneous deletion in themob region (10) of ColEl. The resulting vector was8,640 base pairs (bp) and contained a single EcoRI sitein the colicin gene.

Protease (type VI, pronase P), tetracycline, minocy-cline, and ampicillin were obtained from Sigma Chemi-cal Co.; restriction endonucleases were from BethesdaResearch Laboratories; bacterial alkaline phosphataseand lysozyme were from Worthington DiagnosticsCorp.; agarose (standard low Mr) was from Bio-RadLaboratories; and [a-32P]deoxyribonucleotides werefrom New England Nuclear Corp. Culture media werepurchased from Difco Laboratories. Chelocardin (alsocalled cetocycline or cetotetrine) was a gift fromAbbott Laboratories. T4 DNA ligase, DNA polymer-ase I, and EcoRI endonuclease were generous giftsfrom P. Modrich, and BAL31 exonuclease was a giftfrom R. Burns.

Cell growth. L-broth (26) and M9-Casamino Acidsmedia (34) were employed for routine cultivation of E.coli, whereas brain heart infusion broth (Difco) wasemployed for streptococcal isolates. Pneumococcalstrains were grown in brain heart infusion broth con-taining 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate) (pH 7.4) and 5% heat-inactivat-

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996 BURDETT, INAMINE, AND RAJAGOPALAN

ed horse serum. Resistance levels were determined byusing serial dilutions of antibiotic (42) and cells grownin antibiotic medium 3 (Difco).DNA isolation. Plasmid DNA from E. coli and Strep-

tococcus spp. was isolated as previously described (2,18). Briefly, spheroplasts were lysed by the addition ofsodium dodecyl sulfate (SDS) to 1Cc, followed byprecipitation of bulk cellular DNA by I M NaCI at 0°C.In some cases, DNA was purified by centrifugation inCsCI ethidium bromide density gradients. Alternative-ly, a rapid procedure for isolation of plasmid DNAfrom E. coli (1) was used to detect plasmids fromtransformants. This procedure facilitated restrictionanalysis of deletion mutants.

Cellular DNA from streptococci was isolated from100 ml of overnight cultures grown in brain heartinfusion broth containing 6.5 mM cysteine and 20 mMDL-threonine. Cells were washed in 10 mM Tris-hydrochloride-10 mM EDTA (pH 8.0) and resus-pended in 25 ml of this same buffer. After treatmentwith 50 mg of lysozyme for 3 h at 37°C (1 h for S.fiaecalis), the cells were centrifuged and resuspendedin 8 ml of this buffer. Lysis was brought about by theaddition of 1 ml of 20% SDS, and the lysate wastreated with 6 mg of protease at 37°C for I h. Pneumo-

cocci were lysed with deoxycholate-SDS (35). Alllysates were gently extracted with an equal volume ofchloroform-isoamyl alcohol (24:1), and the DNA wasrecovered from the aqueous layer by ethanol precipita-tion. The extraction and precipitation steps were re-peated several times.Recombinant DNA methods. Restriction endonucle-

ase digestions were performed according to conditionsrecommended by the manufacturer. After cleavage,samples which were to be ligated were heat inactivatedat 68°C for 10 min and slowly cooled to room tempera-ture. T4 DNA ligase reactions contained 66 mM Tris(pH 7.6), 50 mM NaCl, 10 mM MgCl2, 0.1 mM ATP,10 mM dithiothreitol, and 2 U of T4 DNA ligase per ml(43). Incubation was at 12°C for 12 to 18 h (43).Bacterial alkaline phosphatase was used to remove 5'-phosphates from the linearized vehicle to prevent self-ligation and to enhance ligation with the DNA to becloned (16, 44). BAL31 exonuclease (25) was used toorder the Hincdl fragments of pJI2.

Deletions in hybrid plasmids were generated bypartial digestion with Hincll, followed by heat inacti-vation of the endonuclease and ligation with T4 DNAligase. E. coli transformation was performed by themethod of Cohen et al. (5) or Kushner (24). All

TABLE 1. Bacterial strains and relevant properties'

Strain

S. JtuecalisJH2-2JH2-2 (pMV158)JH2-2 (pMV163)JH2-2 (pAMod)JH2-2 (pMV120)BM6201 (pIP614)OGlRF (pAM211)

S. agalactiaeMV762MV107MV158MV159MV160MV206B109

S. pnelumoniaeN77

Cellular genotype

tI/f'rif,is

./i.ti.s

rif fis

riuf's

Plasmid content

pMV158 (TCr)pMV]63 (Tcr)pAMcsI (Tc'-)pMV120 (Tc', trca+)pIP614 (Tcr. tra )pAM211 (pADI::Tn916; Tc'-, tra')

ri/' strTc-Cmr Tc'-Cmr TcrCmr- TcrTc'-Cm' Tc'- Em'

Cm' Tc'

E. coliD15-7C600 (R6W)C600 (pSC101)D7-1C600 (pVH2124)SK1 592SK1592 (pVB AIS)SK1592 (pVB * Bl1)SK1592 (pVB * Cl )SK1592 (pJI2)

RPI (Tc'- Km' Apr)R6 (Tcr- Cmr Kmr)pSCI01 (Tcr)RAl (Tcr-)pVH2124 (cea', Ap-)nonepVH2124:pMVI58 (Tcr- Apr)pVH2124:pMV163 (Tcr Apr)pVH2124:pAMcxl (Tcr Apr)pVH2124: tet B109

Reference

2. 4, 11

615

9

20

31

303030303824This paperThis paperThis paperThis paper

'l rif, fius, and sttr are cellular mutations conferring resistance to rifampin (rif), fusidic acid (fiis), andstreptomycin (str); Cmr, Tc', Em'r, Sm', Kmr, and Apr are naturally occurring resistances to chloramphenicol viachloramphenicol acetyl transferase, tetracycline, erythromycin, streptomycin, kanamycin. and ampicillin,respectively; cea and trar refer to colicin El production (cea') and conjugative properties (tra+).

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STREPTOCOCCAL tet DETERMINANTS 997

recombinant DNA procedures were carried out inaccordance with the National Institutes of HealthGuidelines for Recombinant DNA Research.

Gel electrophoresis. Electrophoresis of DNA restric-tion fragments in agarose gels was carried out in Tris-borate buffer (17) or E buffer (28) at 8 V/cm. The DNAbands were stained with ethidium bromide, visualizedwith UV light, and photographed through a Wrattanno. 23 filter. Phage lambda DNA digested with HindIlIor EcoRI or both provided molecular weight markers(7).

Blot hybridization. DNA fragments were transferredto nitrocellulose filters from agarose gels after electro-phoresis by the protocol of Southern (39) as modifiedby Wahl et al. (41). Nick-translated [32P]DNA (33)(>107 cpm/,Lg) was denatured for 10 min at 100°C andwas placed in a solution of 6x SSC, 1 x Denhardt (8)0.5% SDS, and 50 jig of salmon sperm DNA per ml at65°C. Hybridization was carried out at 65°C for 24 h.The transfers were washed for 1 h at 65°C in 3 x SSC,lx SSC, 0.3x SSC, and then 0.1x SSC each with0.1% SDS. SSC contains 0.15 M NaCl and 0.015 Msodium citrate. For autoradiography, the dried filterwas exposed to Kodak XR-5 film, using a DupontCronex Lightning Plus intensifying screen at -20°C.

RESULTSWe recently described the isolation of several

tetracycline resistance plasmids (pMV120,pMV158, and pMV163) from S. agalactiae (2)which can be introduced into S. sanguis bytransformation. Because of the small sizes(5,200 and 5,400 bp, respectively) and high copynumbers of pMV158 and pMV163 along with aphenotypically selectable marker, they are at-tractive candidates for cloning vectors. Wetherefore initiated experiments to elucidatesome of the functionally important regions ofthese plasmids. In an initial analysis, we com-pared the pattern of DNA fragments generatedby a number of restriction endonucleases andconstructed detailed restriction endonucleasecleavage maps for pMV158 and pMV163. Therestriction maps demonstrated that these plas-mids had very little in common. Only one or twobands of similar size were seen, using severalmulti-hit restriction endonucleases.To test for limited regions of homology, we

analyzed pMV158, pMV163, and pAMcxl, a 6.0-megadalton tetracycline resistance plasmid pre-viously characterized in S. faecalis (4, 11), byusing the DNA transfer method of Southern(39). Plasmid DNAs digested with HincIl werefractionated by agarose gel electrophoresis,transferred to nitrocellulose membrane filtersand hybridized with plasmid [32P]DNA preparedby nick translation. After hybridization andautoradiography, results of the type shown inFig. 1 were obtained. When 32P-labeled pMV158DNA (as a chimera with pVH2124 see below)was hybridized with HinclI-cut plasmid DNAs,it was found that pMV163 fragments B, D, and E

and pAMal fragments A, D, and E share se-quence homology with pMV158, whereas nohomology with a tetracycline-sensitive deletionof pAMal (pAMalAT) was found. Reciprocalhybridizations with pMV163 and pAMoxl DNAsas probes indicated similar regions of homology(Table 2). In heterologous reactions, hybridiza-tion was always localized to the same few bands,as shown in Fig. 1. These data taken togetherwith the mapping data suggested that the regionof homology might be the region responsible fortetracycline resistance.

Deletion mapping. Deletion analysis was car-ried out to test whether the regions of homologyare associated with tetracycline resistance. Thiswas facilitated by constructing hybrids betweenpVH2124. These hybrids replicate and expresstetracycline resistance in E. coli.and express tetracycline resistance in E. coli.

Plasmid DNAs from pMV158, pMV163, andpAMal were treated with EcoRI to generateunit-length linear molecules and ligated withEcoRI-cleaved pVH2124 DNA. Ampicillin-re-sistant transformants of E. coli SK1592 weretested for inability to produce colicin El, resist-ance to tetracycline, and the presence of extra-chromosomal DNA. The majority of the trans-formants which were unable to produce colicinEl were also resistant to tetracycline. The step-tococcal plasmids alone were not able to trans-form E. coli recipient strains.When plasmid DNA isolated from tetracy-

cline-resistant transformants was characterizedby using EcoRI endonuclease, a fragment corre-sponding to the linear vector pVH2124 wasalways obtained. The other EcoRI fragmentsfrom hybrid plasmids corresponded to fragmentsgenerated from the streptococcal plasmid. In thecase of pAMal, tetracycline-resistant recombi-nant plasmids contained DNA corresponding tothe EcoRI fragment B (smaller) or EcoRI frag-ments A plus B. For pMV158, both EcoRIfragments A plus B were cloned, although frag-ment A was sufficient to mediate tetracyclineresistance. Upon analysis of plasmid DNAsfrom a number of transformants from each ex-periment, using different restriction endonucle-ases, it was found that streptococcal tetracyclineresistance was expressed when the plasmid wasinserted in either orientation relative topVH2124. Plasmid DNAs isolated from transfor-mants were able to transform E. coli to tetracy-cline resistance at a high frequency (about 105/jig). Transformants thus obtained were resistantto 12.5 ,ug of tetracycline per ml. The hybridplasmids containing intact pMV158, pMV163,and pAMotl chosen for further study were desig-nated pVBRAl5, pVB B11, and pVB Cll, re-spectively.

Specific deletions of pVB Bll were generated

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998 BURDETT, INAMINE, AND RAJAGOPALAN

FIG. 1. Sequences common to pMV158 and other small resistance plasmids. (I) Agarose gel electrophoresisof plasmid DNA digests as visualized by ethidium bromide stain. (II) The DNA was transferred to nitrocellulosefilters (33) and hybridized to 32P-labeled pVB-A15. pMV158 (A), pMV163 (B), pAMol (C), and pAMolIAT (D)were digested with Hincll; pMV120 (E) and pIP614 (F) were digested with HpaI. Lane G is lambda DNAdigested with EcoRI and HindIll and mixed with EcoRI-cut PVH2124. The exact sizes of the HincII fragments ofpMV158, pMV163, and pAMoal are shown in Table 2.

TABLE 2. Results of Southern hybridizations"

Hincll fragment (kb)

2.851.000.790.51

1.851.000.930.860.78

3.451.161.121.050.910.830.420.41

pVB-A15++ti-

+1+

+- +~

+-

Probe

pVB BI1 pVB-C11

+1+++

++

+ +-+ +-

+-

+±A-A-

++++A-A-

A-+

Plasmid

pMV158

pMV163

pAMoc1

a 32P-labeled probes were hybridized to Hinclh-cut plasmid DNAs transferred to nitrocellulose filters as in Fig.1. Autoradiograms from the homologous reactions always coincided with the fluorescence pattern. Thehybridization to individual bands was assigned values of + +± +, or -, indicating whether an intense band, lessintense band, or no band, respectively, was visible on the autoradiogram.

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STREPTOCOCCAL tet DETERMINANTS 999

with restriction endonuclease HinclI. Since theE. coli vector pVH2124 contains two HincIlsites, one of which is within the ,B-lactamasegene, and since pMV163 has five HincII sites,partial HincIl digests of pVBWB11 were used totransform E. coli. Two classes of transformantswere characterized: those which were ampicillinresistant (retained from pVH2124) but tetracy-cline sensitive (lost from the pMV163 region),and tetracycline-resistant clones which were nolonger ampicillin resistant. Examples of deletionplasmids derived from pVB*B11 are shown inFig. 2.

Plasmids from tetracycline-sensitive transfor-mants had all lost the 860-bp fragment alone orin combination with other HincIl fragments.Tetracycline-resistant (ampicillin-sensitive)transformants all retained the 860- and 1,000-bpfragments which hybridized with the probe butloss of the 3,800-bp fusion fragment (whichincludes approximately 650 bp of the 780-bpfragment that "blots") did not affect expressionof tetracycline resistance (this 780-bp fragmentis split by the insertion of the vector at theEcoRI site of pMV163). Over 40 Tcr Aps cloneswere tested, and in no case was the 1,850-bpfragment lost, suggesting that this fragment maybe important for the expression of tetracyclineresistance and that any region of homology wastoo small to be seen under the hybridizationconditions used. The cellular phenotype associ-ated with a particular deletion plasmid was al-ways confirmed by retransformation.

Deletions of pVBA15 and pVB-C11 were alsogenerated by using HinclI and PstI. As with

HR H H(A) bI

3200 2800

(B) TETRACYCLINE SENSITIVEI

31

I

pVB-B11, removal of any of the restriction frag-ments corresponding to the region of homologyresulted in loss of tetracycline resistance. Re-striction maps of pMV158, pMV163, andpAMotl (Fig. 3) include the tetracycline resist-ance region, as defined by the hybridization,deletion, and cloning data. For example, thesmall EcoRI fragment of pAMox is sufficient tomediate tetracycline resistance.

Hybridization with other Tcr DNAs. A numberof other streptococcal plasmids, transposons,and conjugative elements have been describedwhich mediate resistance to tetracycline (2, 6,11, 15, 19, 20, 22, 37, 40). Using the clonedpMV158 plasmid (pVB-A15), the 32P-labeledprobe was prepared by nick translation andhybridized with a number of other plasmid andcellular DNAs derived from other tetracycline-resistant organisms (Table 3). The results ofthese DNA-DNA hybridization tests were uni-formly negative. Controls demonstrated that<0.5 copies per chromosomal equivalent of ho-mologous plasmid was readily detectable. Thisanalysis also indicated that the E. coli vector(pVH2124) shares no homology with any of thestreptococcal DNAs tested. The streptococcalprobe DNAs also failed to hybridize with any ofthe four tetracycline resistance determinantsrepresented by pSC101, RP1, RA1, and R6 fromE. coli (30), pT181 from S. aureus (21), orpJP3106 from B. sphaericus (J. Polak, submittedfor publication). Thus, the small nonconjugativeplasmids (pMV158, pMV163, and pAMotl) ap-parently share a novel tetracycline resistancedeterminant.

R H H H H HI777 f"777"l pVB. B11

g00 930 1850 860 1000 (Base Pairs)

I I pVB.B11.21

I I

I I

I

pVB. B11.3

pVB. B11.33

IL I pVB.B11.30

pVB. B11.45

TETRACYCLINE RESISTANT

l

l II I

I

l I I I

I IIW

FIG. 2. Deletion analysis of pVB-B11. (A) Map of pVB-B11 showing relevant restriction endonucleasecleavage sites: R (EcoRI) and H (HincIl). 173, The region containing the fragments which hybridize withheterologous probes: () pVH2124 vector; ( ) pMV163. (B) Tetracycline-resistant and tetracycline-sensitive deletions were analyzed as described in the text. The open regions represent the fragments deleted;vertical bars represent remaining HincII sites.

pVB. Bll. 7

pVB. B 11.8

pVB. Bll. 15

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1000 BURDETT, INAMINE, AND RAJAGOPALAN

though conjugal transfer of drug resistance de-terminants (including Tc, Cm, and Em) has beendemonstrated, no plasmids have been observedin this strain (20). Total cellular DNA from B109

Xho I was cleaved with EcoRI and ligated to plasmidXPh I pVH2124 linearized with EcoRI. Tetracycline-

resistant transformants of E. coli SK1592 werefound to have, as unselected markers, ampicillin

-Hha I resistance and colicin El immunity, with lack ofcolicin El production characteristic of insertionsSau96 into the EcoRI site of the pVH2124 vectorplasmid. Plasmid DNA isolated from one such

6 transformant contained a 20-kilobase (kb) EcoRIfragment in addition to a fragment correspond-ing to pVH2124. A restriction map of this plas-mid, designated pJI2, has been constructed (Fig.4A).By employing the same strategy described

above, we were able to define the region of pJI2that was necessary and sufficient for tetracyclineresistance. Partial HincII digests of pJI2 wereused to transform E. coli. Tetracycline-resistant-PSt I transformants always retained the 5.0-kb frag-ment (Fig. 4B). One plasmid, pJI2.7, is a doubledeletion having lost the HinclI fragments adja-

-Sal I cent to the 5.0-kb fragment on both sides. Con-versely, tetracycline-sensitive derivatives ofpJI2 each have lost the 5.0-kb fragment often inconjunction with other fragments.The 5.0-kb fragment of plasmid pJI2 was used

Eco RI

Htinc II Hl0Hinc 1

ftlnd /SI

Hl'ndIII I a

Pst I / HIndmSa! I

FIG. 3. Maps of pMV158, pMV163, and pAMNalshowing cleavage sites for the restriction endonucle-ases studied. The filled portion on the inner circleindicates the tet region, and the inner circle is subdi-vided by lines representing the Hincli sites.

Cloning of tetracycline resistance determinantfrom B109. As an initial approach to analysis ofnonplasmid-mediated tetracycline resistance,we cloned a tetracycline resistance determinantfrom S. agalactiae B109 (20) into E. coli. Al-

TABLE 3. Summary of Southern hybridization data

Radioactive probeSource of DNA electrophoresed DNAon gel

pVB-A15 pJI2

Tcr plasmidspMV158, pMV163, pAMal +pMV120 - -pAM211, pIP614 - +

Tcr S. agalactiae'B109, MV107, MV158, - +MV159, MV160, MV206

Tcr S. faecalisbDS16p° - +

Tcr S. pneumoniae'N77 - +

Tcs Streptococcus spp. dJH2-2D, MV762B -

a S. agalactiae B109 (France [20]), MV107 (Canada[9]), MV158, MV159, MV160 (Los Angeles [2]), andMV206 (North Carolina [2]).

b S. faecalis DS16p° (Michigan). This strain wascharacterized in some detail by Francke and Clewell(15) and contains Tn9I6.

S. pneumoniae N77 (Japan [31]).d No homology was seen to the resistance region.

Hha I.

Hl'ncIIBstE II-

Hpa II

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STREPTOCOCCAL tet DETERMINANTS 1001

(A)

(B) TETRACYCLINE RESISTANT

I

CI

l tl ~ l ll30 2000u 3230

I I I 1I3900 2000 940' 3230

.

41-

l

zi~

5000

c

5600

ppJI2.1

pJI2.2

pJI2.9

pJI2 .56

pJI2. 7

TETRACYCLINE SENSITIVE

II pJI2.13

I pJI214

pJI2 24

pJI2.36

FIG. 4. Analysis of pJI2. (A) Map of pJI2 showing restriction endonuclease cleavage sites. (in) pVH2124vector region; ( ) inserted DNA. (B) Tetracycline-sensitive and tetracycline-resistant deletions of pJI2 were

analyzed as described in the text. The open regions represent the fragments deleted; the vertical bars representremaining HincIl sites.

as a probe in Southern hybridization experi-ments to test for homology with cellular andplasmid DNAs. Hybridization with HinclI-cleaved cellular DNAs from tetracycline-resis-tant strains was observed with all of the tetracy-cline-resistant strains tested (Fig. 5). Despitesome heterogeneity in the fragment sizes ob-served, all of the tetracycline-resistant strainscontained fragments sharing extensive sequencehomology with the 5.0-kb fragment. None of thetetracycline-resistant strains tested containedany detectable plasmid DNA (2, 19, 20) by anumber of criteria. In the absence of the exactlocalization of the tetracycline resistance gene(s)within the 5.0-kb fragment, we cannot excludethe possibility that the homologies being ob-served are because of homologous sequencessurrounding dissimilar tetracycline resistancegenes. This seems unlikely since the plasmidspAD211 containing the conjugative transposonTn916 and pIP614 both contain sequences incommon with this determinant. There was nohomology with pMV158, pMV163, or pMV120from S. agalactiae, pT181 form S. aureus,pJP3106 from B. sphaericus, or any of the fourplasmid-borne tetracycline resistance determi-nants from E. coli.

Since there is no currently accepted nomen-clature describing determinants in this system,we designated the streptococcal tetracycline

resistance determinants described in this workas tetL, tetM, and tetN. This nomenclature willhopefully avoid confusion of the streptococcaltetracycline determinants with the analogousloci of Bacillus (45) and E. coli (30), which, in atleast several cases, are genetically distinct fromthose described here.A summary of all of the hybridization data is

presented in Table 3. The two probes represent-ed by pMV158 and pJI2 specify two geneticallydistinct tetracycline resistance determinants andhave been designated tetL and tetM. The tetra-cycline resistance determinant of pMV120shares no homology with either tetL or tetM andmight be designated tetN. Colony hybridizationhas shown that pJH1 (22) shares homology withtetL and not tetM, although this large conjuga-tive plasmid mediates tetracycline resistance.The tetL determinant has been found to beassociated with several small nonconjugativeplasmids, whereas tetM is most often found inresistant strains where plasmids have not beendemonstrated.

Levels of resistance to tetracycline and analogs.Since a number of genetically nonhomologousresistance determinants exist in streptococci, wetested differences in the level of resistance totetracycline, minocycline, and chelocardin, us-ing liquid cultures with different antibiotic con-centrations. The resistance level mediated by

0-4-p J I 2(base pairs)

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1002 BURDETT, INAMINE, AND RAJAGOPALAN

plasmids was determined in strains containingno other resistance determinant. Two patternsof resistance were detected. The small noncon-jugative plasmids pMV158, pMV163, andpAM(x1 (tetL) showed resistance to 12.5 p.g oftetracycline per ml but wild-type sensitivity tominocycline (<0.1 pLg/ml) and chelocardin (3.1jLg/ml). The nonplasmid-associated tetracyclineresistance determinants tetM and pMV120(tetN) mediated resistance to 25 to 50 jLg oftetracycline per ml, as well as to 3.1 to 6.2 jig ofminocycline per ml, but were unable to provideany resistance to chelocardin. No differences inresistance levels for any of these drugs wereobserved between tetM- and tetN-containingstrains.The sensitivity of Streptococ cus clinical iso-

lates to minocycline (14) and chelocardin (32)has been reported, and the overall sensitivity ofour strains was comparable to those previouslyreported. The differences observed in suscepti-bility to minocycline among tetracycline-resis-tant strains of streptococci had not been report-ed previously and are reminiscent of thedifferences in resistance to analogs observedamong tetracycline-resistant isolates of E. coli(3, 30).

DISCUSSIONWe defined three naturally occurring tetracy-

cline resistance determinants (tetL, tetM, and

FIG. 5. Sequences common to the tetracyclineresistance regions of pJI2 and cellular DNAs. DNAwas extracted from a number of tetracycline-resistantand tetracycline-sensitive strains of streptococci. TheDNAs were cut with HincII, and the cleavage prod-ucts were separated on 1% agarose gels in E buffer(23). The DNA was transferred to nitrocellulose filters(33) and hybridized to the 5.0-kb fragment of pJI2,which was labeled with 32p. (A) Plasmid pJI2: (B andC) S. pneumoniae N77 and BM6001; (D, E, L, and N)S. faecalis JH6, DS16p°, pIP614, and JH2-2; (F to Kand M) S. agalactiae MV107, MV158, MV159,MV160, MV206, B109, and MV762. The approximatesizes of the fragments which show homology in N77,MV107, and pIP614 are as indicated.

tetN) in Streptococcus spp. by DNA-DNA hy-bridization techniques. Further, we have shownthat some of these genetic differences reflectmechanistic differences at the level of resistanceto tetracycline and minocycline.The tetracycline resistance determinants have

been defined by both hybridization and deletionanalysis. The tetL determinants associated withsmall nonconjugative plasmids (pMV158,pMV163, and pAM(a1) were cloned into E. coliplasmids, where they were found to expresstetracycline resistance. When these clones wereused as probes in Southern hybridization experi-ments, the three streptococcal plasmids sharedonly a limited region of homology. This regionwas shown to be involved in tetracycline resist-ance by deletion analysis of the hybrids. Theregion of homology in each case was found to beboth necessary and sufficient for tetracyclineresistance. We previously observed that S. san-guis Challis is transformed by pMV158 andpMV163 but not pAMcx1 (2). Since these plas-mids all carry tetL, the failure of pAMal totransform S. saunguiis is probably not due to afailure of pAMocl to express its resistance totetracycline but probably because it is unable tobecome established in this host.A tetM determinant was cloned from total

cellular DNA of S. agaluictiae B109. This strainis capable of transfer of its resistance markers enbloc in the absence of plasmids by a DNase-resistant process (20). Analysis of the recombi-nant plasmid pJI2 by deletion analysis hasshown that the tetM determinant itself resideswithin a 5.0-kb region of the hybrid. Further,when the 5.0-kb fragment of pJI2 was utilized asa probe in Southern hybridization experiments,it was found that this DNA region was commonto a number of tetracycline-resistant strains in-cluding S. pnelunoniae N77, S. fuaecalis(DS16p°, pAM211::Tn9l6, pIP614), and S. aga-lactiae B109, MV107, MV158, MV159, MV160,and MV206. Tetracycline-sensitive streptococci(JH2-2D or MV762B) contained no sequenceshomologous with the tetracycline resistance re-gion. DNA from tetracycline-resistant transcon-jugants in JH2-2D exhibit an identical pattern ofhybridization as several of the naturally occur-ring isolates tested. This strongly suggests thatthey all share a common resistance determinant,although strong homology with only outside(non-tet) sequences cannot yet be rigorouslyexcluded.

Neither of the probes which were constructedcontained sequences homologous to the conju-gative tetracycline resistance plasmid pMV120isolated from S. agalactiae. Analysis of thisdeterminant (tetN) is currently under way. Wehave also shown that tetL and tetM of strepto-cocci do not hybridize with tetracycline resist-

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STREPTOCOCCAL tet DETERMINANTS 1003

ance determinants from any other organism test-ed to date. The ability to identify these geneticdeterminants will allow epidemiological studieson the spread of tetracycline resistance determi-nants in different populations.

In the nonstreptococcal systems which havebeen tested, the expression of tetracyclineresistance is inducible. The basic mechanism ofresistance in all these systems appears to bealtered tetracycline transport (29), although oth-er reports differ as to whether this can accountfor the degree of resistance observed (27, 36). InStreptococcus spp., resistance is expressed con-stitutively in strains carrying tetL, M, or N alone(2, 3; V. Burdett, unpublished data). This, to-gether with the genetic dissimilarity of thesedeterminants, may indicate underlying mecha-nistic differences in tetracycline resistance instreptococci. We are currently testing this possi-bility.The expression of heterologous tetracycline

resistance determinants has been demonstratedby several laboratories (12, 13, 23). Ehrlich (13)was able to stably introduce staphylococcalresistance plasmids into Bacillus subtilus. Ec-cles et al. (12) and Kreft et al. (23) clonedtetracycline resistance plasmids from B. subtilusinto E. coli, where the hybrids conferred less ofan increase in resistance than the same hybrid inthe natural host (12).The experiments reported here demonstrate

that two genetically distinct tetracycline resist-ance determinants (tetL and tetM) from Strepto-coccus spp. can confer tetracycline resistanceon E. coli. The introduction of tetL into Strepto-coccus spp. or E. coli increases the resistance ofeither host by 50- to 60-fold. On the other hand,a single tetM determinant increases the resist-ance level of streptococci by 250- to 500-fold,whereas in E. coli, resistance increases by only10-fold. The reasons for these differences arenot clear.

ACKNOWLEDGMENTSThis work was supported by Public Health Service grant

A115619 from the National Institutes of Health. JI. wassupported by a Graduate Fellowship from the National Sci-ence Foundation.We thank Paul Modrich and Dale Blazey among others for

useful discussions.

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