Zbl. Bakt. 273, 369-377 (1990) © Gustav Fischer Verlag, StuttgartlNew York

Common Antibiotic Resistance Plasmids in Staphylococcus aureus and Staphylococcus epidermidis from Human and Canine Infections ST. SCHWARZ, M. CARDOSO, S. GROLZ-KRUG, and H. BLOBEL Institut fiir Bakteriologie und Immunologie der Justus-Liebig-Universitat Giessen, 0-6300 Giessen

With 5 Figures· Received December 14, 1989 . Accepted in revised form March 1, 1990

Summary The plasmids of a multiresistant "canine" Staphylococcus epidermidis-culture were investigated. Two small plasmids, the 4.55 kB chloramphenicol resistance (CmR-) plasmid pSC 4 and the 4.45 kB tetracycline resistance (TetR-) plasmid pST 3 could be isolated. Detailed restriction maps of pSC 4 and pST 3 were constructed by double restriction endonuclease digests. The restriction maps revealed extensive structural homologies between pSC 4 from "canine" S. epidermidis and the CmR-plasmid pC 221 from "human" S. aureus as well as between pST 3 from "canine" S. epidermidis and the TerR-plasmid pT 181 from "human" S. aureus. These data suggested that an exchange of small plasmids between S. epidermidis and S. aureus might be possible. Zusammenfassung Aus einer multiresistenten Staphylococcus epidermidis-Kultur vom Hund konnten 2 kleine Plasmide isoliert werden: das 4.55 kB Chloramphenicolresistenz-Plasmid pSC 4 und das 4.45 kB Tetrazyklinresistenz-Plasmid pST 3. Detaillierte Restriktionskarten dieser beiden Resistenzplasmide wurden durch Doppelverdauung mit Restriktionsendonukleasen erstellt. Diese Restriktionskarten zeigten ausgedehnte strukturelle Homologien zwischen den Chloramphenicolresistenz-Plasmiden pSC 4 aus S. epidermidis vom Hund und pC 221 aus S. aureus vom Mensch sowie zwischen den Tetrazyklinresistenz-Plasmiden pST 3 aus S. epidermidis vom Hund und pT 181 aus S. aureus vom Mensch. Diese Ergebnisse lassen einen Austausch von kleinen Resistenzplasmiden zwischen S. epidermidis und S. aureus als moglich erscheinen. Introduction

Staphylococcus epidermidis is considered to be an opportunistic pathogen for humans and animals (30, 33). In humans S. epidermidis is frequently associated with

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St. Schwarz, M. Cardoso, S. Grolz-Krug, and H. Blobel

nosocomial infections (24, 30); in animals mainly with mastitis, cystitis and wound infections (25, 31). S. epidermidis has a high antimicrobial resistance which complicates its effective chemotherapeutic control (30, 32). So far, the genetic basis of antibiotic resistance in S. epidermidis has not been studied extensively, in contrast to that of S. aureus. However, plasmids could be identified in some S. epidermidis-isolates from "human" infections as carriers of antibiotic resistance determinants (4, 5, 9, 15, 39,40). This presentation describes the isolation of a chloramphenicol resistance (CmR-) and a tetracycline resistance (TetR-) plasmid from a multiresistent S. epidermidis-culture of "canine" origin. Both plasmids could be characterized by restriction endonuclease digestion and compared with Cm R- and TetR-plasmids of other staphylococcal species from infections of humans and animals.

Fig. 1. Agarose gel electrophorogram of the CmR-plasmid pSC 4 from "canine" S. epider-

midis.

Lane M: marker DNA; sizes of the visible fragments from the top: 5664, 4133, 3146, 2528, 1713, 1310, 890 basepairs (bp), lane 1: Cia I-linearized pSC 4, lane 2: Hpa II-digested pSC 4, lane 3: uncleaved pSC 4 in its supercoiled (sc) and open circular (oc) form. Fig. 2. Agarose gel electrophorogram of the TetR-plasmid pST 3 from "canine" S. epider-

midis.

Lane M: marker DNA; sizes of the visible fragments, from the top: 5664, 4133,3146, 2528, 1713 bp, lane 1: uncleaved pST 3 in its supercoiled (sc) and open circular (oc) form, lane 2: Cia I-linearized pST 3.

Common Antibiotic Resistance Plasmids in Staphylococcus

371

Material and Methods

Bacterial cultures. The culture of S. epidermidis used was isolated from an urinary tract infection of a dog. It was identified biochemically with the API-STAPH system (bio Merieux, La Balme Les Grottes, France) (1). Growth conditions of the bacteria were as previously described (37). Plasmid isolation and gel electrophoresis. For plasmid peparation, a single colony of the "canine" S. epidermidis-culture was inoculated into 2 ml brain heart infusion broth (BHI, Gibco, Paisley, Scotland) and grown for 18 h at 37°C on a rotary shaker (75 rpm). Plasmids were isolated from the staphylococci by a modification of the alkaline lysis procedure (34). This modification consisted in the removal of the staphylococcal cell walls denaturation by treatment with 40 !-tg/mllysostaphin (Sigma, Deisenhofen, FRG) prior to the alkaline denturation. The plasmids were subjected to electrophoresis for 2 hat 10 V' cm- 1 in 1% (wtlvol) agarose gels (37). After staining with 10 !-tg/ml ethidium bromide (Boehringer, Mannheim, FRG), plasmid DNA was visualized in the gels by ultraviolett light illumination and photographed (film 667, Polaroid, St. Albert, UK). Antibiograms. The S. epidermidis-culture was examined for its antimicrobial resistance patterns by the agar diffusion method (2) using discs, containing 25 !-tg ampicillin, 30 !-tg chloramphenicol, 10 !-tg clindamycin, 30 !-tg doxycycline, 15 !-tg erythromycin, 10 !-tg gentamicin, 30 !-tg kanamycin, 30 !-tg neomycin, 10 !-tg penicillin G, 10 !-tg streptomycin, 23.75 !-tg sulfamethoxazole or 30 !-tg tetracycline. After incubation for 18-24h at 37°C the antibiograms were evaluated (2). Plasmid transformation. Protoplast transformation experiments were performed as previously described (37). S. aureus RN 4220, a derivative of S. aureus RN 8325-4 (21), served as recipient, since it was susceptible to all tested antimicrobial agents and did not carry any plasmid. Of this recipient strain, protoplasts were produced by treatment with 40 !-tg/ml lysostaphin for 2 h at 3 rc. These protoplasts were transformed with the entire plasmid content of the "canine" S. epidermidis-culture in the presence of polyethylene glycol (40% wt/vol, Mr 6000, Sigma, Deisenhofen, FRG) in an osmotically stable medium (3). Subsequently, the transformed protoplasts were selected on DM 3- regeneration plates (3) supplemented with 15 !-tg/ml chloramphenicol or 15 !-tg/ml tetracycline respectively. Clones which appeared on these selective media were screened for plasmid DNA. Restriction endonuclease digests. Respectively 5 units of the restriction endonucleases Ace I, Bam H I, Bgl II, Bst E II, Cia I, Eco R I, Hae III, Hpa II, Hind III, Kpn I, Mbo I, Sac I, Taq I and Xba I (Boehringer, Mannheim, FRG) were used (37). The plasmid fragments were analysed in 0.7-2.0% (wt/vol) agarose gels (37) or 7.5% (wt/vol) polyacrylamide gels (18), according to their sizes. The sizes of the linearized plasmids and the respective plasmid fragments could be estimated on the basis of logarithmic plots against marker DNA, which consisted of Hae III-digested Adv 1 DNA (12) and pBl DNA cleaved separately with Pst I (5664 bp), Hind III (4133, 845,686 bp) and Pst I/Bam HI (3136,2528 bp).

Results The S. epidermidis-culture fermentated glucose, fructose, mannose, maltose, lactose and sucrose. It produced alkaline phosphatase and urease, reduced nitrate and produced acetyl-methyl-carbinol from sodium pyruvate. Thus, the identity of the isolated staphylococcal culture as S. epidermidis could be established with a certainty of 99.9% (1). The S. epidermidis-culture proved to be resistant to ampicillin, chloramphenicol, clindamycin, doxycycline, erythromycin, gentamycin, kanamycin, neomycin, penicillin G, streptomycin, sulfamethoxazole and tetracycline. The culture contained 6 plasmids. Of these 2 small plasmids could be identified in interspecies protoplast transformation

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St. Schwarz, M. Cardoso, S. Grolz-Krug, and H. Blobel

experiments. One encoded resistance to chloramphenicol, the other to tetracycline. The chloramphenicol resistance (CmR)-plasmid, designated as pSC 4 and the tetracycline resistance (TetR)-plasmid, designated as pST 3 were subjected to restriction endonuclease analyses. Both plasmids could be linearized with the enzyme CIa I (Fig. 1, 2), allowing estimations of their size: 4.55 kB for pSC 4 and 4.45 kB for pST 3. The 2 plasmids differed in their restriction endonuclease digestion patterns with Ace I, Bst E II, Eco R I, Hind III, Hpa II, Kpn I, Mbo I, Sac I, Xba I and Taq I (Table 1). Neither pSC 4 nor pST 3 had cleavage sites for the enzymes Bam H I, Bgi II and Hae III. Detailed restriction maps could be constructed for pSC 4 (Fig. 3) and pST 3 (Fig. 4) on

pC 221

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MOBS.

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Fig. 3. Comparative analysis of the restriction maps of the CmR-plasmid pC 221 from "human" S. aureus (6), pSC 4 from "canine" S. epidermidis, pSC 1 from "canine" S. intermedius (27) and pSC 2 from "porcine" S. hyicus (26). Restriction endonuclease cleavage sites are: Ac (Ace I), Bs (Bst ElI), C (CIa I), E (Eco R I), H (Hind III), Hp (Hpa II), M (Mbo I), Sc (Sac I), T (Taq I), X (Xba I). Below each map there is a distance scale in kB; in the map of pC 221 the regions encoding replication functions (REP), chloramphenicol acetyltransferase (CAT), and mobilization function (MOB A and MOB B) are indicated. The position of the Taq I-cleavage sites marked with tor t are not determinable exactly, but the only 2 possible positions are indicated alternatively.

Common Antibiotic Resistance Plasmids in Staphylococcus

I( I,

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Fig. 4. Comparative analysis of the restriction maps of the TetR-plasmids pT 181 from "human" S. aureus (8), pST 3 from "canine" S. epidermidis and pST 1 from "porcine" S. hyicus (24). The restriction endonuclease cleavage sites are: C (Cia I), H (Hind III), Hp (Hpa II), K (Kpn I), M (Mbo I), T (Taq I), X (Xba I). Below each map there is a distance scale in kB and in the map of pT 181 the regions encoding replication functions (REP) and tetracycline resistance (TetR) are indicated.

Table 1. Numbers and sizes of the plasmid fragments, generated by restriction endonuclease digestion of the CmR-plasmid pSC4 and the TerR-plasmid pST3, both from "canine" S. epi-

dermidis

Restriction endonucleases

Acc I Bst E II Cia I Eco R I Hind III Hpa II Kpn I Mbo I Sacl Xba I Taq I

pSC4 No. of fragments 2 1 1 1 1 2 3 1 2 11

Sizes of fragments (in kB) 2.53,2.02 4.55 4.55 4.55 4.55 2.70, 1.85 2.00, 1.35, 1.20 4.55 2.45,2.10 1.20,0.75,0.65,0.48 0.46, 0.30, 0.20, 0.17 0.15,0.10,0.05

pST3 No. of fragments

Sizes of fragments (in kB)

1

4.45

3 1 1 4

2.38, 152, Q.56 4.46 4.46 2.20, 1.34, 0.54, 0.38

1 6

4.46 1.55, 1.52, 0.65, 0.44 0.22,0.08

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St. Schwarz, M. Cardoso, S. Gr6Iz-Krug, and H. Blobel

the basis of double digests. These restriction maps proved to be suitable for comparative analyses of pSC 4 and pST 3 with other staphylococcal plasmids also encoding Cm R or TetR. pSC 4 from "canine" S. epidermidis and pC 221 from "human" S.aureus were nearly indistinguishable by their sizes and their restriction maps (Fig. 3). These homologies included the regions encoding replication proteins, mobilization functions and chloramphenicol acetyltransferase. The S. epidermidis plasmid pST 3 showed extensive structural conformity with the prototype TetR-plasmid pT 181 from "human" S. aureus, also including the regions encoding replication functions and tetracycline resistance (Fig. 4).

Discussion

Staphylococcus epidermidis occurs frequently on the skin of humans (11, 13) and only occasionally on the skin of animals that live in close contact with humans (33). S. epidermidis, formerly considered to be a non pathogenic inhabitant of the skin, is now recognized as an opportunistic pathogen for both humans and animals (23, 30). In humans, it can colonize prosthetic heart valves (7, 26), orthopedic prostheses and intravascular catheters (33). It could also cause wound or urinary tract infections (19, 28), endophthalmitis (41) and otitis media (6). In animals, S. epidermidis was found to be associated with mastitis and wound infections (25, 31). Little information has been available on the antimicrobial resistance patterns and the plasmid profiles of these staphylococci from infections of animals, contrarily to the wealth of informations on S. epidermidis from "human" infections (4, 5, 24, 39, 40) which generally indicated a high antimicrobial resistance of S. epidermidis (24, 27, 29, 32). "Human" S. epidermidis was found to carry a variety of plasmids (16, 24), many of them considered to be cryptic, since they could not be associated with any phenotypic characteristics. Other plasmids encoded resistance to p-lactam antibiotics, chloramphenicol, gentamicin tetracycline and macrolide-lincosamide-streptogramin-antibiotics (5, 9, 15, 39). It appeared, that the antibiotic resistance plasmids of S. epidermidis might spread to other coresident staphylococcal species (20, 40). Thus, S. epidermidis could possibly serve as a reservoir of resistance determinants for other staphylococci (13). Evidence for an exchange of resistance plasmids between S. epidermidis and S. aureus from human infections has already been derived from the isolation of common PenR/Gm R(40), Cm R-(39) TetR-plasmids (9,40). In our investigations a multiresistant "canine" S. epidermidis-culture was examined for antibiotic resistance flasmids. This led to the isolation of 2 small antibiotic resistance plasmids: the Cm -plasmid pSC 4 and the TetR-plasmid pST 3. The pSC 4plasmid had a size of approximately 4.55 kB and carried only the determinant for resistance to Cm. The restriction map of pSC 4 revealed a high degree of homology to that of the CmR-~lasmid pC 221 from "human" S. aureus (8). However, structural homoglies to Cm -plasmids of other staphylococcal species from animal infections, such as pSC 1 from "canine" S. intermedius (36, 38) and pSC 2 from "porcine" S. hyicus (37) were limited to the REP/CAT-region. This could also be confirmed by restriction endonuclease fingerprinting of pSC 4 and pSC 1 using the restriction enzyme Taq I (Fig. 5). The common plasmid fragments were located exclusively in the REP-region. Because of their common REP/CAT-region pSC 1, pSC 2 and pSC 4 were considered to belong to the pC 221-family of CmR-plasmids.

Common Antibiotic Resistance Plasmids in Staphylococcus

375

Fig. 5. Restriction endonuclease fingerprinting of the CmR-plasmids pSC 4 from "canine" S. epidermidis (lane 1) and pSC 1 from "canine" S. intermedius (lane 3) using the enzyme Taq I. Lane M contains the marker DNA (10). The sizes of the marker DNA fragments, beginning from the top, are: 1713, 1319, 845,535,460, 360/352,272,223,213/212,178,142,131,83,40 bp. The 40 bpfragment of the marker DNA and the 50 bp fragment of pSC 4 are not visible. Common Taq I-fragments located in the REP-region are marked with arrows. The TetR-plasmid pST 3 with a size of 4.45 kB exhibited a distribution of restriction endonuclease cleavage-sites, similar to that of the TetR-plasmid pT 181 from "human" S. aureus (10). It was also structurally related to the TetR-plasmid pST 1 from S. hyicus (35). These findings corresponded closely to previous analyses of TetR-plasmids from S. aureus and S. epidermidis, both isolated from infections of humans (9). These TetRplasmids corresponded in size and differed only slightly in their restriction endonuclease cleavage patterns. Our results suggested the possibility of interspecies plasmid exchanges between different staphylococcal species from human and animal hosts (14). However, in which direction this transfer could occur remains to be answered. In addition to protoplast transformations or protoplast fusions, transducing phages were found to be involved in the spreading of small antibiotic resistance plasmids, such as the S. aureus TetR-plasmid pT 181 (17). Furthermore, mobilization of small plasmids by large conjugative plasmids presented another possible way of transmission. Conjugative plasmids had been demonstrated in S. aureus (17) as well as in S. epidermidis (40) and regions encoding mobilization functions (mob A, mob B) could be located in the restriction map of the CmR-plasmid pC 221 from "human" S. aureus (22).

Acknowledgements. The authors like to thank Mrs. Ute Neuschulz for her excellent

assistance.

References

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