Veterinary Microbiology, 24 (1990) 113-122 Elsevier Science Publishers B.V., Amsterdam

113

Isolation and restriction endonuclease analysis of a tetracycline resistance plasmid from

Staphylococcus hyicus St. Schwarz and H. Blobel lnstitut J~r Bakteriologie und Immunologie der Justus-Liebig- Universiti~t Giefien, Frankfurterstr. 107, 6300 Gieflen (F.R. GO (Accepted 20 December 1989)

ABSTRACT Schwarz, St. and Blobel, H., 1990. Isolation and restriction endonuclease analysis of a tetracycline resistance plasmid from Staphylococcus hyicus. Vet. Microbiol., 24:113-122.

A plasmid of 4.550 kb, conferring resistance to tetracycline, was demonstrated in Staphylococcus hyicus cultures from piglets with exudative epidermitis. The plasmid-encoded properties were determined both by curing and interspecific protoplast transformation experiments. The tetracycline resistance (TET ®) plasmid, designated pST 1, was characterized by restriction endonuclease analysis and a preliminary restriction map was constructed. The pST1 plasmid was demonstrated in 19 (57.6%) of 33 S. hyicus cultures by Southern blot hybridization. It was also detectable by electron microscopy.

INTRODUCTION

Staphylococcus hyicus causes exudative epidermitis in pigs (Rolle and Mayr, 1984; Eich, 1985; Gyles and Thoen, 1986 ). Antibiotics, especially penicillins, tetracyclines and sulfonamides, have been used extensively for control of this infection (Eich, 1985 ). This might have led to the high antimicrobial resistance of S. hyicus (Schwarz and Blobel, 1989), In other pathogenic staphylococcal species, antimicrobial resistance is often associated with extrachromosomal nucleic acids (Lacey, 1975; Groves, 1979; Lyon and Skurray, 1987 ). In this connection the antimicrobial resistance patterns and plasmid content of 33 S. hyicus cultures from piglets with exudative epidermitis were studied, with particular reference to their tetracycline resistance. 0378-1135/90/$03.50

© 1990 - - Elsevier Science Publishers B.V.

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MATERIALS AND METHODS

Bacterial cultures Of the 33 S. hyicus cultures used in this study 32 were isolated from piglets with exudative epidermitis from different farms in several geographical areas of Germany between 1983 and 1986; the remaining culture was the S. hyicus reference strain NCTC 10350. All cultures were identified as S. hyicus following the recommendations of Schleifer ( 1986 ).

Plasmid isolation and gel electrophoresis For plasmid preparation single colonies of the 33 S. hyicus cultures were inoculated into 2 ml brain-heart infusion broth (BHI, Gibco, Paisley, Great Britain) containing 30/~g/ml tetracycline. The staphylococci were grown for 18 h at 37 ° C on a rotary shaker ( 175 rpm). Plasmids were isolated by a modification of the alkaline lysis procedure (Schwarz and Blobel, 1989). This modification consisted in the removal of the staphylococcal cell walls by treatment with 40 #g/ml lysostaphin (Sigma, Deisenhofen, F.R.G. ) prior to the alkaline denaturation. The isolated plasmids were subjected to electrophoresis in 0.7-1.5% (w/v) agarose gels (Schwarz et al., 1989a). After staining with 10/zg/ml ethidium bromide (Boehringer, Mannheim, F.R.G. ), plasmid DNA was visualized in the gels by exposure to ultraviolet light, and photographed (film 667, Polaroid, St. Albans, Great Britain).

Antibiograms All 33 S. hyicus cultures were examined for their antimicrobial resistance patterns by the agar diffusion method (Barry and Thornsberry, 1985 ), using discs, containing 25/tg ampicillin, 30/~g chloramphenicol, 10/tg clindamycin, 30/zg doxycycline, 15/tg erythromycin, 30/zg kanamycin, 10/tg streptomycin, 23.75/zg sulfamethoxazole or 30/~g tetracycline. The antibiograms were evaluated after incubation for 18-24 h at 37 ° C (Barry and Thornsberry, 1985 ).

Curing experiments A tetracycline-resistant S. hyicus culture containing the plasmid in question was incubated for 48-72 h at an elevated temperature of 42 °C in the presence of 10/~g/ml ethidium bromide. The staphylococci were then plated on nonselective trypticase-soy-agar plates (TSA, Oxoid, Basingstoke, Great Britain) and incubated for 18 h at 37 °C. The resulting clones were then replicaplated on selective TSA plates containing 15/~g/ml tetracycline. Apparently cured clones were screened for plasmid DNA.

Plasmid transformation Protoplast transformation experiments were performed according to the method of Chang and Cohen (1979). S. aureus RN 4220, a derivative of S.

ISOLATION AND ANALYSIS OF A TETRACYCLINE RESISTANCE PLASMID FROM S. HYICUS

1 15

aureus RN 8325-4 (Novick, 1967), was chosen as the recipient since it was susceptible to all tested antimicrobial agents and did not carry any plasmid. The transformed protoplasts were selected on DM 3 regeneration plates (Chang and Cohen, 1979 ) supplemented with 15/tg/ml tetracycline and also screened for plasmid DNA.

Restriction endonuclease digests Five units of the restriction endonucleases AccI, BamHI, BglII, BstEII, ClaI, EcoRI, HaeIII, HpaII, HindIII, KpnI, MboI, PvuII and TaqI (Boehringer, Mannheim, F.R.G.) were used (Schwarz et al., 1989a). The plasmid fragments were analysed in 0.7-2.0% (w/v) agarose gels (Schwarz et al., 1982) or 7.5% (w/v) polyacrylamide gels (Maniatis et al., 1982 ), according to their size. The sizes of the linearized plasmid and the respective plasmid fragments were estimated on the basis of logarithmic plots against marker DNA. The marker DNA consisted of HaeIII-digested 2dv 1 DNA (Kr~Sger et al., 1984) and pBl DNA cleaved separately with PstI (5664 bp), HindIII (4133, 845, 686 bp) andPstI/BamHI (3136, 2528 bp).

Southern blot hybridization This was performed according to the capillary blot procedure using a Gene Screen Plus hybridization transfer membrane (Du Pont, Boston, U.S.A. ). The large HindlII fragment of the TET ® plasmid from S. hyicus was used as probe. Radioactive labelling, prehybridization and hybridization were performed as previously described (Schwarz et al., 1989c).

Electron microscopy The method of Kleinschmidt (1968 ) was used to demonstrate the TET ® plasmid of S. hyicus electron microscopically. The plasmid DNA was shadowed with platinum-iridium on copper grids. Photographs were taken using a Zeiss EM 10 electron microscope (Zeiss, Oberkochen, F.R.G. ). RESULTS

The identity of the 33 S. hyicus cultures was confirmed morphologically and biochemically. Plasmids were detected in 22 (66.7%) of these cultures, varying in numbers from 1 to 4 per culture (Schwarz and Blobel, 1989). The 22 plasmid-positive S. hyicus cultures were much more resistant to antimicrobial agents than the 11 plasmid-negative S. hyicus cultures (Table 1 ). Our interest concentrated on the tetracycline resistance in 19 of the 22 plasmid-positive S. hyicus cultures. Curing experiments and interspecific protoplast transformation were used in order to determine whether tetracycline resistance was plasmid-borne. For curing experiments a S. hyicus culture was used which contained only the putative TET ® plasmid. After 3 days at 42°C in the presence of the nucleic acid-binding compound ethidium bromide, ap-

ST. SCHWARZAND H. BLOBEL

l 16

TABLE 1 Antimicrobial resistance patterns of 22 plasmid-positive and 11 plasmid-negative S. hyicus cultures Numbers of resistant S. hyicus cultures Plasmid-positive Ampicillin Chloramphenicol Clindamycin Doxycycline Erythromycin Kanamycin Streptomycin Sulfamethoxazole Tetracycline

5 (22.7%) 3 (13.6%) 1 (4.5%) 3 (13.6%) 1 (4.5%) 1 (4.5%) 12 (54.5%) 22 (100.0%) 19 (86.4%)

Plasmid-negative 3 (27.3%) 3 (27.3%) 111 (100.0%) -

qncluding S. hyicus reference strain NCTC 10350.

1

2

3

4

chr

Fig. 1. Uncleaved plasmid content in crude lysates of a tetracycline-resistant 3". hyicus culture and its respective cured and transformed clones. Lanes 1 and 3, plasmid content of the original TET-resistant S. hyicus culture. Lane 2, plasmid content of the same TET-sensitive culture after curing. Lane 4, plasmid content of the TET-resistant transformed S. aureus clone, chr: chromosomal DNA. proximately 65% of the resulting clones appeared to be cured. They lost their plasmid (Fig. 1, lane 2) and became tetracycline-sensitive exhibiting a large zone of growth inhibition in the respective antibiograms. For the interspecific protoplast transformation the putative TET ® plasmid from the same S. hyicus culture was taken. The resulting transformants became resistant to tetracycline and carried a small plasmid, as did the original plasmid-donor S. hyi-

ISOLATIONAND ANALYSISOF A TETRACYCLINERESISTANCEPLASM1DFROMS. HYICUS

1 17

cus culture (Fig. 1, lane 4). This TET ® plasmid was designated pST1 and further characterized by restriction endonuclease analysis, using the enzymes

AccI, BamHI, BglII, BstEII, ClaI, EcoRI, HaeIII, HpaII, HindIII, KpnI, MboI, PvuII and TaqI. Digests with ClaI, HpaII and KpnI linearized this plasmid with 4550 bp (Fig. 2). Approximate sizes of the pST1 fragments obtained after digestion with HindIII, MboI and TaqI are listed in Table 2. The restriction enzymes AccI, BamHI, BgIII, BstEII, EcoRI, HaeIII and PvuII had no cleavage sites in the TET ® plasmid from S. hyicus. A preliminary restriction map of pST 1 was constructed by double restriction endonuclease digests (Fig. 3 ). Comparison of pST1 from S. hyicus with the S. aureus TET ® plasmid pT 181 on the basis of restriction enzyme cleavage sites revealed great similarities between pST1 and pT181 (Table 2). The large HindIII fragment of pST 1, containing the tetracycline resistance gene in pT 18 l, was therefore radioactively labelled and used as probe for Southern blot hybridization (Fig. 4). The probe recognized its complementary sequences in all 19 tetracyclineresistant S. hyicus cultures, whereas the supercoiled form and at least 1 of the open circular forms of the TET ® plasmid were marked (Fig. 4b). The additional hybridization signals in Fig. 4b represented further open circular forms of the TET ® plasmid. However, because of their small quantities, they were not visible in all lanes of the less sensitive ethidium bromide-stained agarose gels. A previously described chloramphenicol resistance plasmid (Schwarz et al., 1989b ) occurring in 2 of these 19 S. hyicus cultures, as well as some small 1

M

2

OC

4. 550 kb SC

Fig. 2. Agarose gel electrophoresis of uncleaved and restriction endonuclease-digested pST1. Lane M, marker DNA (sizes of the visible fragments beginning from the top: 5664, 4133, 3136, 2528, 1713 and 1310 bp. Lane l, uncleaved pST 1 exhibiting supercoiled (sc) and open circular (oc) forms. Lane 2, Clal-digested, linearized pST1.

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ST. SCHWARZAND H. BLOBEL

TABLE 2 Comparative analysis of the plasmid fragments of pSTI and pTl 81 obtained after single restriction endonuclease digests pSTI from S. hyicus

pT181 from S. aureus

No. of fragments

Sizes of fragments (bp)

No. of fragments

Sizes of fragments (bp)

KnpI HpaII

1 1 1

4550 4550 4550

I 1 2

HindIII

3

3

Mbo!

4

2430 1575 540 2250 1440 540 310

lbqI

6

1650 1515 650 450 210 75

7

4437 4437 4421 16 2353 1525 560 2116 1422 529 346 24 1569 1566 654 384 200 5l 23

Restriction endonuclease

Clal

5

Mbol~ Hlpall~Taql Hindlll~

Taql --'~

/~Hind III Mbol

Fig. 3. Preliminary circular restriction m a p of the T E T ® plasmid pST 1 from S. hyicus.

cryptic plasmids, served as controls, to ensure the specificity of the hybridization. None of these plasmids was recognized by the D N A probe. Furthermore, pST 1 could be demonstrated by electron microscopy (Fig. 5 ).

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ISOLATION AND ANALYSIS OF A TETRACYCLINE RESISTANCE PLASM1D FROM S. HYICUS

a M 1 2 3 4 5 6 7i8

M 9 R 1011 12131A 14 C 15 16 17 18 19

.;f,... • .+,,... lln

b M 1 2 3 4 5 6 718 M 9 R

~ N

....

g

1011 1213

A 14C1516171819

m

| Fig. 4. (a) Agarose gels. (b) Southern blot hybridization. Southern blot hybridization of all plasmids from the 19 tetracycline-resistant S. hyicus cultures, the TET-cured S. hyicus clone (R) and 2 S. hyicus cultures, which were ampicillin (A)- or chloramphenicol-resistant (C). Lane M, marker DNA, generated by partial EcoRI digest of 2dv 21/8 DNA. The visible fragments from the bottom are 1310, 1713, 3153, 6306 and 9459 bp. Lanes 1-19, plasmid content of the TET®-S. hyicus cultures 1-19. Lane R, plasmid content of the TET-cured S. hyicus clone. Lane A, plasmid content of a S. hyicus culture exhibiting only ampicillin resistance. Lane C, plasmid content of a S. hyicus culture exhibiting only chloramphenol resistance.

Fig. 5. Electron microscopial demonstration ofpST1 in its open circular form ( ×435 000).

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DISCUSSION

Tetracycline resistance in staphylococci is commonly mediated by small plasmids ranging in size from 4.0 to 4.5 kb (Lyon and Skurray, 1987 ). These plasmids are structurally closely related (Lyon and Skurray, 1987). A high degree of similarity between the TET ® plasmids from different staphylococcal species of human origin has been demonstrated on the basis of restriction endonuclease digests (Cooksey and Baldwin, 1985; Groves, 1979 ). The TET ® plasmid pT 181 first isolated by Iordanescu ( 1976 ) from human S. aureus has been studied very intensively. Its complete nucleotide sequence was published by Khan and Novick (1983). However, little is known about TET* plasmids from staphylococci isolated from animals (Kloos et al., 1981 ). Attempts to determine plasmid-mediated tetracycline resistance in S. hyicus have already been made by Noble et al. (1988). They defined the plasmid-encoded properties solely by curing experiments and indicated a molecular weight of 2.7 MDa for a plasmid supposed to carry a determinant for resistance against tetracycline. However, curing experiments alone may result in misleading information, particularly if the "cured" derivative has lost 2 or more plasmids. This would prevent a correct correlation between the phenotypical marker and the respective plasmid. Therefore, in addition to curing experiments, we conducted the more specific protoplast transformations. This led to clones which carried only the putative TET ® plasmid and became resistant to tetracycline. Thus, the extrachromosomal nature of tetracycline resistance in S. hyicus was demonstrated by two different experimental methods. The fact that tetracycline resistance in different staphylococcal species was mediated mainly by closely related plasmids raises the questions of the transmission and origin of these plasmids. Experiments attempting in vitro transfers of resistance genes between S. aureus cultures of human and animal origin showed that such transfer occurred in either direction at a low frequency, and only within a narrow range of recipients (Lacey, 1980). Moreover, the TET ® plasmids were too small to encode their own conjugative transfer apparatus (Lyon and Skurray, 1987). On the basis of these findings a common reservoir of resistance determinants for staphylococci from both humans and animals appeared to be unlikely (Lacey, 1980). On the other hand, restriction endonuclease analysis of the TET ® plasmid pST 1 from porcine S. hyicus cultures revealed a high degree of conformity with the TET ® plasmids from human S. aureus, especially with pT 181. The origin of these TET* plasmids and their evolutionary movement within the genus Staphylococcus remain to be clarified. It seems improbable that such closely related TET ® plasmids developed independently from one another in different staphylococcal species. More likely is the assumption of a common ancestor for staphylococcal TET ® plasmids, (Chopra and Howe, 1978), possibly spreading by transduction with only slight alterations. Transfer by transduc-

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121

ing phages has been demonstrated for the S. a u r e u s TET ® plasmid pT 181 (Lyon and Skurray, 1987). Finally, the pervasiveness of TET ® plasmids in different staphylococcal species from human and animal sources may be associated with frequent therapeutic administration of tetracyclines. This would demand a more judicious administration of antibiotics for more effective control of staphylococcal infections. ACKNOWLEDGEMENTS

The authors would like to thank Mrs. S. Gr61z-Krug and Mrs. U. Neuschulz for their excellent technical assistance, and Prof. Dr. G. Hobom for providing marker DNA. REFERENCES Barry, A. and Thornsberry, C., 1985. Susceptibility tests: diffusion test procedures. In: E.H. Lenette, A. Balows, W.H. Hausler, Jr. and H.J. Shadomy (Editors), Manual of Clinical Microbiology, 4th edn. Am. Soc. Microbiol., Washington DC, pp. 978-987. Chang, S. and Cohen, S.N., 1979. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Molec. Gen. Genet., 168:111-115. Chopra, I. and Howe, T.G.B., 1978. Bacterial resistance to the tetracyclines. Microbiol. Rev., 42: 707-724. Cooksey, R.C. and Baldwin, J.N., 1985. Relatedness of tetracycline resistance plasmids among species of coagulase-negative staphylococci. Antimicrob. Agents Chemother., 27: 234-238. Du Pont Company, 1985. Catalog N. NEF 976. Protocols for electrophoretic and capillary transfer of DNA and RNA, DNA and RNA hybridization and DNA and RNA rehybridization. Du Pont Company, Boston, U.S.A. Eich, K.O., 1985. Handbuch Schweinekrankheiten. Verlagsunion Agrar, 2. Auflage. MfinsterHiltrup, pp. 172-175. Groves, D.J., 1979. Interspecific relationships of antibiotic resistance in Staphylococcus sp.: isolation and comparison of plasmids determining tetracycline resistance in S. aureus and S. epidermidis. Can. J. Microbiol., 25: 1468-1475. Gyles, C.L. and Thoen, C.O., 1986. Pathogenesis of Bacterial infections in Animals. 1st edn., Iowa State University Press, Iowa, pp. 14-20. Iordanescu, S., 1976. Three distinct plasmids originating in the same Staphylococcus aureus strain. Arch. Roum. Pathol. Exp. Microbiol., 35:111-118. Khan, S.A. and Novick, R.P., 1983. Complete nucleotide sequence of pTl81, a tetracycline resistance plasmid from Staphylococcus aureus. Plasmid, 10:251-259. Kleinschmidt, A.K., 1968. Monolayer techniques in electron microscopy of nucleic acids. In: L. Grossman and K. Moldave (Editors), Methods in Enzymology, Vol. 12. Academic Press, New York, pp. 361-376. Kloos, W.E., Orban, B.S. and Walker, D.D., 1981. Plasmid composition of Staphylococcus species. Can. J. Microbiol., 27: 271-278. Kr6ger, M., Hobom, G., SchiJtte, H. and Mayer, H., 1984. Eight new restriction endonucleases from Herpetosiphon giganteus - divergent evolution in a family of enzymes. Nucleic Acids Res., 12: 3127-3141. Lacey, R.W., 1975. Antibiotic resistance plasmids of Staphylococcus aureus and their clinical importance. Bacteriol. Rev., 39: 1-32.

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Lacey, R.W., 1980. Rarity ofgene transfer between animal and human isolates of Staphylococcus aureus in vitro. J. Gen. Microbiol., 119: 437-442. Lyon, B.R. and Skurray, R., 1987. Antimicrobial resistance of Staphylococcus aureus. Genetic basis. Microbiol. Rev., 51: 88-134. Maniatis, T., Fritsch, E.F. and Sambrook, J., 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 150-178. Noble, W.C., Rahmann, M.J. and Lloyd, D.H., 1988. Plasmids in Staphylococcus hyicus. J. Appl. Bact., 64: 145-149. Novick, R., 1967. Properties of cryptic high frequency transducing phage in Staphylococcus aureus. Virology, 33:155-166. Rolle, M. and Mayr, A., 1984. Medizinische Mikrobiologie, Infektions- und Seuchenlehre. 5. Aufl. Enke-Verlag, Stuttgart, pp. 693-701. Schleifer, K.H., 1986. Gram-positive cocci. In: P.H.A. Sneath, N.S. Mair, M.E. Sharpe and J.G. Holt (Editors), Bergey's Manual of Systematic Bacteriology, 2. Williams and Wilkins, Baltimore, pp. 1013-1035. Schwarz, St. and Blobel, H., 1989. Plasmids and resistance to antimicrobial agents and heavy metals in Staphylococcus hyicus from pigs and cattle. Zbl. Vet. Med., B., 36: 669-673. Schwarz, St., Cardoso, M. and Blobel, H., 1989a. Plasmid-encoded resistance to chloramphenicol in canine Staphylococcus intermedius isolates. Med. Sci. Res., 17:451-453. Schwarz, St., Cardoso, M. and Blobel, H., 1989b. Plasmid-mediated chloramphenol resistance in Staphylococcus hyicus. J. Gen. Microbiol, 135: 3329-3336. Schwarz, St., Spies, U., Reitz, B., Seyfert, H.M., L~immler, Ch. and Blobel, H., 1989c. Detection and interspecies transformation of a fl-lactamase-encoding plasmid from Pasteurella haernolytica. Zbl. Bakt. Hyg., A, 270: 462-469.

Isolation and restriction endonuclease analysis of a tetracycline resistance plasmid from Staphylococcus hyicus.

A plasmid of 4.550 kb, conferring resistance to tetracycline, was demonstrated in Staphylococcus hyicus cultures from piglets with exudative epidermid...
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