Plasmids mediating multiple drug resistance in group B streptococcus: Transferability and molecular properties

PLASMID

2, 137- 149 (1979)

Plasmids Mediating Streptococcus:

Multiple Drug Resistance in Group Transferability and Molecular Properties

B

VICKERS HERSHFIELD Department

of Microbiology, Duke University Medical Durham, North Carolina 27710

Center,

Accepted September 22, 1978 Group B P-hemolytic streptococci isolated from a number of geographic locations were investigated with regard to the genetic basis of their drug-resistance determinants. Strains have been identified carrying plasmid-mediated resistance to erythromycin, lincomycin, and streptogramin B (MLS resistance). These MLS-resistance plasmids all have molecular weights of approximately 17 x lo6 and are capable of transfer during mixed incubation by a mechanism that appears similar to conjugation. These plasmids were transferable to group B, D, F, and H recipients. Transfer could also be shown from groups F and H to group D recipients. Plasmids DNA isolated from recipient strains following transfer was identical with that of the donor. The similarity of restriction fingerprints of MLS-resistance plasmids from group A, B, and D isolates obtained suggest that they constitute a family of related plasmids.

Certain characteristics of plasmids found are shared by in the Enterobacteriaciae streptococcal plasmids. But, despite a considerable amount of information on drugresistance factors in enterobacteria and interactions of these plasmids, the literature on drug-resistance plasmids in streptococci is still scarce. It is known that both multiple drug resistance (Jacob and Hobbs, 1974; Marder and Kayser, 1977; VanEmbden et al., 1977)and bacteriocin production (Dunny and Clewell, 1975; Jacob et al., 1975) can be mediated by conjugative plasmids in Streptococcusfaecalis. These plasmids are able to mediate their own transfer and, in certain cases, to mobilize nonconjugative R-plasmids present in the same cell (Dunny and Clewell, 1975)as well as chromosomal markers (Franke et al., 1978). Further, transfer of plasmids from S. faecalis to other streptococcal species has been demonstrated in vitro (VanEmbden et al., 1977); the finding in certain cases that certain plasmids from different species are of similar size, encode the same antibiotic resistances, and are es-

sentially homologous with each other (El Sohl et al., 1978; Yagi et al., 1975) strongly suggests that interspecific transfer also occurs in vivo. Evidence for drug resistance in group B streptococcus (S. agalactiae) has been reported (Dixon and Lipinski, 1974; Horodniceanu et al., 1976), and the incidence of appearance of strains resistant to erythromycin, lincomycin, and tetracycline has increased in the last few years (Dixon and Lipinski, 1974; Eickhoff et al., 1%4). Rplasmids mediating erythromycin and lincomycin resistance have been isolated (Horodniceanu et al., 1976; this work). These plasmids are in the same size range (MW, 1718 x 106) as erythromycin-resistance plasmids from S. faecalis (Clewell et al., 1974; Courvalin et al., 1974) and S. pyogenes (Malke et al., 1976). The similarities among these plasmids suggested the possibility of genetic exchange among these strains. This laboratory is interested in the ecology and dissemination of resistance genes among /3-hemolytic streptococci. As a preliminary

137

0147-619X/79/010137-13$02.00/0 Copyright All rights

8 1979 by Academic Press, Inc. of reproduction in any form reserved.

138

VICKERS

HERSHFIELD

TABLE

1

BACTERIAL STRAINS Species (or serogroup)

Relevant resistance characteristics”

D25303

Group

B

MLS,

Tc

pMV103

(MLS)

MV141

Group

B

MLS,

Tc

pMVl41

(MLS)

MV154

Group

B

MLS,

Cm, Tc

pIP501 (MLS,

DS5

S. faeculir

(D)

Ben, MLS,

JH201

S.frrecalis

(D)

JHZ-2

S. fhecalis

(D)

MV250

s. songu,s var Challis (H)

Strain

Tc

Plasmids (resistances)

Source other designatmns

Cm)

M. Dixon,

Canada

C. Baker,

USA

D. Bouanchaud (Horodniceanu el al., 1976) France BM610I

pAMor1, (Tc); pAMpI, (ML%; pAMy (Ben)

D. CIewell

gent

“One

This work, gent” mutant of JH2 (Jacob and Hobbs, 1974)

Rif, Fus

none

A. Jacob (Jacob and Hobbs, 1974)

none

none

D. LeBlanc,

NCTC

7868

MV251

Group

F

“OW

“O”=Z

D. LeBlanc

MV481

Group

B

Rif

none

This work.

spontaneous

JH201

Group

D

MLS,

This work.

by mating

BH6201 PIP613

Group

D

MLS

pIP613 (MLS)

P. Courvalin (Courvalin 1974) France

D10535

Group

A

MLS

~10535 (MLSI

M. Dixon

gent

pAM@

(MLS)

ATCC

12393 RifR with

DS5

PAMPI

’ MLS resistance Cm, chloramphenicol:

includes Em (erythromycin-macrotides), Lm (lincomycin-lincosamides) Rif, rifampicin; Fus, fusidic acid; gent, gentamycin: Ben, bacteriocin.

step, we have developed a plasmid transfer system in group B streptococcus as a means for further study of plasmids in streptococci. Evidence is presented that R-plasmids (with molecular weights of 17-20 x 106) mediating erythromycin resistance in group B and D streptococci are capable of mediating their own transfer. Further, all erythromycin resistance plasmids from streptococci so far examined appear to constitute a family of DNA molecules with similar patterns of cleavage by restriction endonucleases.

and streptogramin

B antibiotics.

er ol.,

Tc. tetracycline:

(MV481, MV250Rif, and MV251Rif) were obtained by several cycles of selection on Brain Heart Infusion (BHI) agar (Difco) plates containing increasing concentrations of Rif. Minimum inhibitory concentrations (MICs) were determined on Todd-Hewitt (Difco) agar plates containing doubling concentrations of antibiotic, using a Steers multiple inoculator (Steers et al., 1959). Strain JH201 pAM/31 was obtained from a cross (using the filter-mating procedure outlined below) between DS5 (donor) and JH201 (recipient). MLS-resistant (to erythromycin, MATERIALS AND METHODS lincomycin, and streptogramin B) transconStrains. The bacterial strains used are jugants were rare (2 x 10e7)but derivitives listed in Table 1. Group B streptococci are could be isolated which contained the originally from clinical specimens and the pAMP1 plasmid alone. Media. Strains were grown in BHl broth resistant strains originated in different countries. Strain MV154 is included as a (Difco) except where stated. Solid medium was made by adding 1.3% agar to broth. partially characterized plasmid-containing strain. Rifampicin (Rif)-resistant mutants For isotopic labeling modified M9 medium

MULTIPLE

DRUG RESISTANCE

(Jacob et al., 1975) was supplemented with 0.5% casamino acids, 0.3% yeast extract, 20 pg/ml deoxyadenosine, 0.1% cysteine, and 20mM DL-threonine to facilite spheroplast formation. This medium is referred to as M9-YE-CYS. Curing procedures. Curing of plasmids was accomplished at elevated temperature as follows: An 18-hbroth culture was diluted, usually l:lO, into fresh broth and incubation continued for 18 h at either 37 or 42°C before plating onto antibiotic-free media. Acridine orange was also tried as a curing agent over a range of 0.5 to 10 &ml. Logphase cultures were diluted 1:10 into acridine orange broth. After overnight incubation in the dark, cultures grown in the highest concentration not inhibitory to growth were plated and tested. Control cultures grown in BHl broth with no acridine orange were also plated. Other chemical agents known to be mutagenic were avoided. Individual colonies resulting from these treatments were tested for antibiotic susceptibility by replicate plating onto plates containing 10 pg/ml erythromycin (Em) or chloramphenical (Cm). Determination of type of resistance. The method employed for determining whether resistance to erythromycin was inducible or constitutive is a modification of the quantitative growth assay of Hyder and Streitfeld (1973). These assays were carried out in BHl broth. Drug-induced cultures were prepared by mixing 0.2 ml of overnight culture with 5.8 ml of broth containing 0.07 pg/ml of erythromycin. An uninduced culture was prepared in an identical manner without antibiotics. When the cultures reached early log phase (2-3.5 h), 0.6-ml volumes of each culture were added to assay tubes containing 5.3 ml of broth. The incubation was continued for 1 h and then 120 pg of antibiotic was added to tubes to be challenged and broth added to the controls. Growth of all cultures was followed at 30-min intervals on a Klett.-Summerson calorimeter at 540 nm. Mating procedure. Fresh overnight cultures (5 ml) in BHl broth were diluted 1: 10

IN GROUP B STREPTOCOCCUS

139

and mixed in a ratio of approximately one donor (0.05 ml) to 10 recipients (0.5 ml). The cells were collected on a Millipore filter (HAWP, 0.45 pm) which were then placed on BHl agar plates and incubated at 37°C for up to 24 h. The organisms on the filters were then resuspended in 5 ml BHl broth and the mixture was plated onto BHl plates selective for transconjugants. The mating mixture was also plated on appropriate medium to determine the total number of donors and recipients present. Controls consisting of donors and recipients alone were treated in the same way. Frequency is expressed as the number of transconjugants per donor colony-forming unit. Colonies were counted after 48 h. Transformations. The transformable Challis strain of S. sanguis and the transformable group F streptococcus were used as recipient strains for plasmid transformation. Overnight cultures grown in Todd-Hewitt broth, 0.15% bovine serum albumin, plus 0.3% glucose, were maintained in log phase by 6 serial 1:lO dilutions over a period of several days. After a final dilution of 1:5 followed by 1 h additional growth, O.l-ml aliquots were mixed with DNA samples (-0.2 pg/ml final concentration) in a total volume of 0.2 ml. Incubation was continued at 37°C for 15 min. Transformation reactions were stopped by the addition of 200 kg of pancreatic deoxyribonuclease (DNase) in 20mM MgSO,. After DNase treatment for 15 min the cultures were incubated for 2 h before plating on BHI agar supplemented with 10 pg/ml Em or Cm and on antibiotic free medium. Labeling, lysis, isolation of plasmid DNA.

Cells were grown as follows: A l-ml 18 h culture in M9-YE-CYS was diluted 1:10 into fresh broth and grown for 6-8 h at 37°C. This broth culture was used to inoculate 100 ml of M9-YE-CYS containing 0.2 ml of [methyl-3HJthymidine (1 mCi/ml; 55 Ci/mmol). Group B streptococcus cultures were grown in the presence of 5% CO,. Lysis and dye-buoyant density centrifugation were carried out essentially as described

140

VICKERS

HERSHFIELD

by LeBlanc et al. (1976) except in the following respects. After washing, cells were heat shocked at 65°C for 20 min prior to lysozyme addition. DNA was precipitated with 0.54-vol of isopropanol after the phenol and the chloroform-isoamyl alcohol extraction steps, DNA was collected by low-speed centrifugation and resuspended in TES (50 mM Tris-HCl, 5mM EDTA, 50 mM NaCl, pH 8.0). CCC plasmid DNA was separated from contaminating chromosomal DNA by centrifugation in a CsCl density gradient containing 400 @g/ml of ethidium bromide (EB) (Radloff et al., 1967) for 60 h at 33000 rpm in a Ti50 rotor at 18°C in a Beckman L2-50 ultracentrifuge. Gradients were fractionated from the bottom by puncturing with a 20-gauge needle. Twenty-microliter samples of each fraction were spotted onto Whatman No. 3 paper, dried, and counted for radioactivity. The fractions containing supercoiled plasmid DNA were pooled, the EB extracted with CsCl-saturated isopropanol, and the samples dialyzed against TES buffer. Agarose gel electrophoresis. Plasmid molecular weights were estimated by electrophoresis in agarose gels in Tris-borate buffer (Greene et al., 1974) at 100 V for 3 h. Gels were stained in 0.5 pg/ml ethidium bromide for 30 min and DNA bands were visualized with a short-wave ultraviolet lamp (Ultra-Violet Products, Inc., San Gabriel, Calif.). The gels were photographed using Polaroid type 665 film through Wrattan No. 23 plus uv filters. Chemicals. CsCl was a product of Kawecki-Berylco Company (Revere, Pa.). Other chemicals were obtained from Mallinkrodt Chemical Corporation (St. Louis, MO.). Lysozyme, Pronase, and deoxyribonuclease were purchased from Sigma Chemical Company (St. Louis, MO.). Antibiotics were also obtained from Sigma. Lincomycin was a gift from the Upjohn Company (Kalamazoo, Mich.) and streptogramin B was a gift from B. Weisblum. Restriction endonucleases were purchased from New England Biolab, (Beverly, Mass.). EcoRl was a gift from

Paul Modrich. Agarose was ME grade from SeaKem (Rockland, Mass.). RESULTS

Genetic Stability of Drug-Resistance Traits in Group B Streptococci The stability of the antibiotic-resistance traits of strains D25303, MV 154, and MV 141 was tested by screening for the appearance of antibiotic-sensitive segregant clones by replica-plating techniques. Several culture conditions and chemical agents known to promote plasmid curing in other systems were tested (see Methods). No loss of resistance was observed after growth in broth at 37°C or followed growth in the presence of acridine orange. However, incubation of cultures at 42°C resulted in loss of resistance to all MLS antibiotics tested (Em, Lm, Sg). Although the frequency of curing by the elevated temperature procedure varied with the strain and from experiment to experiment, this procedure was always effective. In tests of approximately 1000 colonies, the frequency of isolation of erythromycin-sensitive colonies ranged from 1 (MV141) to 15% (D25303). Cured derivitives had minimum inhibitory concentrations for both erythromycin and lincomycin comparable with sensitive wild-type strains (0.05 pg/ml) and colony morphology and /3-hemolysis on blood agar were not effected. Tetracycline resistance was never lost. In the case of MV154, chloramphenicol (Cm) resistance was always lost when erythromycin resistance was lost. Linkage of CmR with MLSR has been reported for MV154 (Horodniceanu et al., 1976). One sensitive segregant of each strain was tested for reversion to high-level resistance. No reversion to Em (10 &ml), Lm (10 E.L~/ ml) or Cm (15 pg/ml) resistance was observed when drug-containing plates were seeded with lo9 CFU. Isolation of Plasmid-Specific DNA from 025303, MVl41, and MV154 The spontaneous loss of resistance to MLS antibiotics led to the suggestion that re-

MULTIPLE

DRUG RESISTANCE IN GROUP B STREPTOCOCCUS

sistances to these drugs may be mediated by plasmids. To test this possibility, DNA was extracted from the parent strains and one of the cured derivitives and analyzed by dye-buoyant density centrifugation (Radloff et al., 1967). All MLSR parental strains contained a satellite DNA banding at a density heavier than chromosomal DNA. Illustrated in Fig. 1 is the banding profile of DNA isolated from D25303 and D25303.1 (a cured derivative). The absence of any satellite DNA from the cured derivative suggests that this component in the parental strain is related to the MLS-resistance character and that tetracycline resistance, which could not be cured, is most likely to be chromosomal. Similar CsCl-ethidium bromide banding profiles were obtained for MV154 and MV141 and their cured derivatives (data not shown). Satellite DNA fractions obtained from D25303, MV141, and MV154 were subjected to restriction endonuclease cleavage by HpaI and agarosegel electrophoresis (Fig. 2). Included in the gel was HpaI digest of PAM/~ 1 plasmid DNA as an internal marker of known molecular weight (17.0 x 106),and A Hind111 digest as molecular weight markers. Thus, the molecular weight of each plasmid was obtained by summing the fragments. We estimate the size of the plasmids from group B streptococcus to be 17.7, 16.7, and 19.8 x lo6 for satellite DNA isolated from strains D25303, MV141, and MV154, respectivey. The plasmids in these strains have been designated, in order, pMV103, pMV141, and pIP501 (Horodiceanu et al., 1976). pIPSO has been reported to have a molecular weight of 20.0 x IO6 and it carries Cm as well as MLS markers thus allowing the possibility that pIPSO carries a 3 x lo6 Cm gene in addition to the 17 x IO6 dalton MLS plasmid. The two major types of erythromycin resistance (inducible and constitutive) long recognized in staphylococcus have also been reported in strains of group A streptococcus (Hyder and Streitfeld, 1973).These types of erythromycin resistance were also

FRACTION

NO.

FIG. 1. Dye-buoyant density gradient of DNA extracted from strain D25303(A) and the cured derivitive D25303.1 (B) Cells from 100 ml of culture grown in [3H]thymidine were harvested, lysed, and the DNA centrifuged to equilibrium in a C&I-EB density gradient. Fractions were collected and 20 ~1 of each were spotted and counted as described.

found among the strains of group B streptococcus studied here. Strains MV154 and MV141 are representative of the inducible type of resistance. Their growth in broth was temporarily inhibited by 20 pg/ml of erythromycin unless they had been preincubated with an inducing, subinhibitory concentration of drug (0.05 &ml). In the uninduced culture of MV154, growth was interrupted by the erythromycin challenge for 3.5 h after which growth resumed at the original rate (Fig. 3). MV141 exhibited a much longer lag before growth resumed at the previous exponential rate. Heterogeneity within the cultures could not accuont for this kind of response since the efficiency of plating on agar containing 20 pg/ml of erythromycin was the same as that seen on antibiotic-free medium. Presumably these streptococci are induced in situ by high antibiotic concentrations and therefore their inducible nature

142

VICKERS HERSHFIELD

FIG. 2. Agarose gel electrophoresis of plasmid DNA isolated from JH201 pAM/31, D25303, MV141, and MV154, cleaved by HpaI. Plasmid DNAs isolated in dye-buoyant density gradients and cleaved by HpaI were run for 3.0 h in a 0.7% at 100 V. Tracks 1, 2, 3, and 4 are uncleaved plasmids pAMp1, pMV103, pMV141, and pIPSOl, respectively; tracks 6, 7, 8, and 9 are those same plasmids cleaved by HpaI. Tracks 5 and 10 are A Hind111 fragments: 17.6 x lo6 (left end plus right end associated), 14.96 x 106, 6.07 x 106, 4.1 x 106, 2.71 x 106, 1.35 x 106, and 1.14 x 106.(The 0.34 X IO6 fragment cannot be seen on this gel.) The reader should note that fragment b (track 6) and fragments c and d (track 8) are doublets.

does not become apparent by oridinary plating procedures. In contrast, strain D25303 showed no interruption of growth after the addition of 20 ,ug of erythromycin/ml regardless of whether they had been previously grown in broth containing a subinhibitory concentration (Fig. 4). This response is representative of a constitutive type of resistance. Another approach was taken to determine whether the extrachromosomal DNA of D25303 and MV154 is related to resistance. S. sang& var Challis and group F strep-

tococcus are known to be suitable recipient strains for transformation by plasmid pAMP1 DNA from S. fuecalis (LeBlanc and Hassel, 1976; LeBlanc, et al., 1978). Plasmid DNA obtained from dye-buoyant density gradients was added to competent cultures of either recipient strain and transformants were selected on plates containing 10 pg/ ml erythromycin. Erythromycinresistant transformants were obtained; the frequencies of transformation with pMV103 and pIPSO DNA were in the range of lOA7per CFU, somewhat lower than might have been

MULTIPLE

DRUG RESISTANCE IN GROUP B STREPTOCOCCUS

expected. The transformed strains were similar to the plasmid donor strains in exhibiting high levels of resistance to the unselected plasmid markers lincomycin (pMV 103, pIPSOl, and PAM@) and chloramphenicol (pIPSOl). Transformants using pIP501 DNA could also be selected using 10 pg Cm/ml. Examination of DNA extracted from several of these transformants in dye-CsCl gradients revealed that all the transformants had acquired a satellite DNA band at a density identical to that of the original parental strains. In agreement with previous published results (LeBlanc and Hassel, 1976; LeBlanc et al., 1978), no satellite DNA was observed in the recipient Challis or group F strains (data not shown). The satellite DNA behaved on agarose gels in a manner identical to that of the parental plasmid DNAs from D25303 and MV154 both in size and cleavage patterns obtained with the Hind111 restriction endonuclease (Fig. 5). These results confirm the plasmid linkage of MLS resistance and suggest that the plasmids from group B streptococcus can be stably maintained by S. sanguis and group F streptococci. I

IOL

I

A

I

1

I2345670

TIME

1

’

1

11

(hours)

FIG. 3. Growth rates of induced and uninduced cultures of strain MV154 as affected by the addition (arrow) of a high concentration of erythromycin. Closed symbols represent the growth of control cultures receiving no challenge of erythromycin.

TIME

143

(hours)

FIG. 4. Growth rates of induced and uninduced cultures of strain D25303 as affected by the addition (arrow) of a high concentration of erythromycin. Closed symbols represent the growth of control cultures receiving no challenge of erythromycin.

Transfer of Multiple Antibiotic Resistance to Other Streptococci It has been shown that some large antibiotic-resistance plasmids (MW > 38 x 106d) from group D streptococci are self-transmissible by conjugation to other group D streptococci as well as to group A and group B recipients (VanEmbden et al., 1977). All three of our resistant group B streptococci were able to donate Em resistance conjugally to MV481, a wild-type group B isolate (Table 2). MV481 is an antibiotic-sensitive clinical isolate made resistant to rifampicin (Rif) which is free of plasmid DNA asjudged by dye-CsCl density gradient analysis (data not shown). Further, as shown in Table 2, the Em-resistant group B strains were also able to transfer the Em resistance to a group D recipient (JH2-2). Although the transfer frequencies of these filter matings was always in the range of 10-l to lop4 per donor, the frequency of transfer in liquid medium from any of the group B donors was below

144

VICKERS HERSHFIELD

1

2

3

4

5

6

FIG. 5. Hind111 fingerprints ofplasmid DNA isolated from S.fueculis pAMPI, MV103, MVI54, and S. sanguis var Challis transformants. Plasmid DNAs were isolated from dye-buoyant density gradients. The cleavage patterns of plasmids from the parent strains (pAMp1, pMV103, and pIPSOl) (tracks 1, 3, and 5, respectively) are presented next to plasmids isolated from the corresponding transformant (tracks 2,4, and 6). Electrophoresis was for 3 h in a 1% agarose gel at 100 V.

the level of detection. All of the group B Mechanism of Transfer strains studied transferred all of the other Transfer of plasmids from group B resistances as unselected markers that have streptococcus probably occurs by some sort been shown to be plasmid mediated. Tetraof “conjugative” mechanism. Cell-free filcycline resistance was never transferred. trates from all donor strains did not transfer The S. sanguis (group H) strains transformed by plasmids pMV103 and pIPSO were able drug resistance to recipient strains in either to transfer their resistances at high frequency broth or filter matings. The cell-free filtrates to JH2-2 on filters (Table 2). The trans- were prepared by passing an overnight broth formable strains (made Rif resistant) were culture through a membrane filter (0.45 pm; also used as recipients in mating experi- Millipore Corp.). These filtrates were also ments. In these matings, transconjugants tested for the presence of bacteriophage by were obtained at low frequencies (IO-’ per spotting onto plates overlaid with a numdonor) when D25303 was used as the donor. ber of presumptive indicator strains. The

MULTIPLE

DRUG

RESISTANCE

strains that acted as conjugal recipients were tested as indicators and no plaques were ever obtained. The presence of 50 &ml of deoxyribonuclease I and 5 mM MgS04 during the mating procedure and in the selective plates had no significant effect on the number of transconjugants obtained. Pretreatment of donors (group B) with 0.25% sodium hypochlorite (Chlorox) prevented transfer of plasmids pMV103, pIPSOl, and pMV141. Donor strains were treated by adding 0.1 ml of Chlorox (5.25% sodium hypochlorite) to 2 ml of culture and incubating for 30 min at 37°C. The Chlorox was removed by washing the cells twice with 2 vol of medium. No transfer of Em resistance was detected after incubation of treated donors with the JH2-2 recipient (<3 X log). No viable donor cells remained after this treatment although cell morphology remained unchanged as judged by Gram stain. Similar treatment of group B donors with chloroform, toluene, 0.2% KCN, or 0.2% sodium azide had no effect on viability or the ability to act as donors. We have examined the kinetics of transfer from a group B donor into JH2-2. Aliquots were filtered and the filters were incubated on nonselective plates for intervals up to 20 h before resuspending and plating the bacteria on agar supplemented with apTABLE

2

FREQUENCYOF TRANSFEROF RESISTANCE DETERMINANTSTO JH2-2 (GROUPD) AND MV481 (GROUPB) RECIPIENTS Recipient

Donor

strain

DZS303 (pMV103) ML’154 (pIPSOl) MV141 (pMV141) S. senguis (pMV103) s. sanguis (pIPsol) S. sanguis (pAMpI) S. foecalis (PAM@) BH6201 (pIP613)

Group B B B H H H D D

0.6-l 6.7 5 x 6 x 5 x 2 x 1 x 1 x

JHZ-2

MV481

CD)

(W

x lo-* x 1O-3 10-s 1O-3 10-p lo+ 10-t 10”

5 x lo0.8 x 10-Z 1.3 x IO“ 7 x 10-S -

’ Donors and recipients were incubated together on filters at a ratio of I donor to every 10 recipients for 18 h. Frequencies are expressed as the number of resistant colonies observed per number of donor colony-forming units present at the end of mating.

IN GROUP B STREPTOCOCCUS

01

I2

1

’ 34 TIME

(

’ 5

145

1 6

.I 20

(hours)

FIG. 6. Kinetics of acquisition of erythromycin resistance of strain D25303 by JH2-2. Donor and recipients were mixed at time zero in a 1 to 10 ratio as described under Materials and Methods. The matings were performed on filters at 3PC. Filters were withdrawn at various times and suitable dilutions were plated on agar to select for donors (O), recipients (A), and recipients having acquired antibiotic resistance (W); (+) below the level of detection. Colonies were counted after 24 h and transconjugant colonies were purified and tested for the presence of unselected plasmid markers. Transconjugant colonies were also tested for the ability to grow on Enterococcosel agar (BBL). S. faecalis (JH2-2) will grow on this differential medium while group B streptococci are inhibited.

propriate antibiotics. The results of these platings were used to estimate donor and recipient viable counts and the number of recipients having received the Em-resistance characteristic. Figure 6 shows that the majority of the transfer takes place during the first 90 min. It can also be noted that during this time there is little increase in the number of potential donors in the mating mixture relative to the increase in transconjugants. Plasmid DNA identical in size to that in the donor could be isolated from all JH2-2 transconjugants tesed. Further, these plasmids had restriction endonuclease fingerprints identical with donor DNAs. Figure 7 shows Hind111(A) and HincII (B) fingerprints of the following plasmids obtained from

2

3

4

5

6

123456

B

FIG. 7. Hind111(A) andHincl1 (B)fingerprints ofsix MLS-resistance plasmids. The fingerprintsofthreegroup B plasmids pMV103, pMV141, and plP501, (tracks 3.4, and 5) arecompared withplasmids oftwogroup D(pIP613, track6andpAMP1, track2)andonegroup A strain(p10535, track 1). Electrophoresis in a 1% agarose gel was carried out for 3 h at 100 V. The Hind111 and HincII fragments range in size from 4 x 106d. to 0.15 x 106d,

A

MULTIPLE

DRUG RESISTANCE IN GROUP B STREPTOCOCCUS

transconjugants: PAM@, pMV103, pMV141, pIPSOl, and pIP613 (Hind111 digests of the parent plasmids pAMP1, pMV103, pIPSO are shown in Fig. 5). Plasmid ~10535 from S. pyogenes (group A) is included for comparison as this plasmid hybridizes with pAMP1 (Yagi et al., 1974). Consistent with the DNA hybridization results pAMP1 and ~10535 have a large number of digest bands in common, as do pIP613 and pIPSO which are homologous. The three plasmids from group B (pMV103, pMV141, and pIPSOl) also share bands with each other as well as with the plasmids from group D and group A. These fingerprints indicate that the 17 x lo6 MW MLS-resistance plasmids found in a number of natural isolates of streptococcus are highly conserved. DISCUSSION

The major conclusion we draw from our data is that genes mediating resistance to MLS antibiotics in the strains of group B streptococcus studied are borne on selftransmissible plasmids of similar molecular weights. The three plasmids characterized (pMV103, pMV141, and pIPSOl) all have molecular weights around 17 x 106.The criteria used to assign these resistances to a plasmid are several. Resistance to all MLS antibiotics (erythromycin, lincomycin, and streptogramin B) was lost simultaneously during incubation of a culture at elevated temperature. The loss of resistance from these strains coincided with the loss of all plasmid DNA that could be isolated as a satellite in CsCl-EB gradients. Further, these plasmid DNAs could be introduced into S. sanguis var. Challis by transformation; the resistances were acquired simultaneously no matter which drug was used for selection of transformants. The plasmids characterized in these studies of group B streptococci are similar in size to erythromycin-resistance (MLS) plasmids isolated from other species of streptococcus. In S. faecalis (group D), the plasmids PAM/~ 1 and pIP6 13have been reported

147

to have MW of 17.0 x lo6 and 17.6 X 106, respectively. Two strains of S. pyogenes (group A) have also been reported to have plasmids of -17 megadaltons. And in at least one instance, a group D (PAM/? 1) and a group A (~10535) MLS plasmid have been shown to share extensive nucleotide sequence homology (Yagi et al., 1975). More recently, several group B and group D plasmids have been shown to be similar (ElSohl et al., 1978) by hybridization. These data taken together suggest that these MLS plasmids have a single ancestral origin and have become disseminated among different groups of streptococcus. Since the MLS plasmids all seem to have similar molecular weights and several plasmids in different groups appear to be related, the question arose as to how these plasmids might be transmitted to different host backgrounds in nature. We have shown that MLS-resistance plasmids from group B and group D are transferable to a wide range of streptococcal hosts by a mechanism resembling conjugation among the enterobacteria. This transfer mechanism is not dependent on the host, since group B, D, H, and F strains can serve as donors of resistance to the same range of recipients. Plasmids pMV103, pMV141, and pIPSO all transfer at frequencies greater than 10e4 and their transfer is not dependent on the original host strain (Table 2). The mechanism of transfer is inferred to be similar to conjugation. There was no transfer from killed donor cells. Transfer was insensitive to treatment with high levels of DNase I and no bacteriophage could be detected capable of growth on a number of recipient test strains. These results are strongly against transduction and transformation as means of plasmid transfer. Previous studies of conjugation among streptococci have dealt only with plasmids isolated from group D strains. Our results extend the knowledge of naturally occurring transmissible plasmids to group B streptococcus. The transmissible plasmids reported to date in group D are all at least

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VICKERS HERSHFIELD

38 x lo6 daltons in size (Dunny and Clewell, 1975; Jacob and Hobbs, 1974; Jacob et al., 1975;Marder and Kayser, 1977;VanEmbden et al., 1977)and many determine bacteriocin production (Dunny and Clewell, 1975;Jacob et al., 1973; VanEmbden et al., 1977). The class of 17-megadalton MLS-resistance plasmids described here probably represent another class of sex factors analogous to an incompatability group Escherichia coli (Datta, 1975). Further evidence that the MLS plasmids form a cohesive, yet evolving family of plasmids can be derived from the Hind111 and HincII cleavage patterns (Fig. 7). The similarities of the patterns generated by these enzymes suggest that all of these MLS-resistance plasmids are related. pMV103, pMV141, and pIPSO appear to share, at least, a core of related sequencies since they have about half their fragments in common. They also have fragments in common with MLS plasmids from S. faecafis (pIP613, pAMP1) and S. pyogenes (~10535). pAMPI and ~10535, known to be homologous by hybridization (Yagi et al., 1975), have the same pattern of enzyme fragments. pIPSO and pIP613 are also indistinguishable by these two criteria. The transmissible nature of the group D and group B plasmids, and the similarity of these plasmids with ~10535 from group A suggest strongly that ~10535 is also a self-transmissible plasmid. This possibility is under investigation in our laboratory. The fact that a wide variety of plasmids are now known to mediate their own transfer will enable us to study the host range of these plasmids and their molecular evolution. These properties also allow their introduction into a uniform host background such that isogenic strains can then be used in the study of plasmid-plasmid interactions and the nature of plasmids in streptococcus. ACKNOWLEDGMENTS This research was supported by Grant CA 14236 from the National Cancer Institute, GM 12551 from National Institute for General Medical Sciences, and by

the Walker P. Inman Fund. The excellent technical assistance of Jennifer A. Teunissen is gratefully acknowledged. We thank R. 0. Bums and P. Modrich for their interest and support in this work.

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