Acquisition of Chloramphenicol Resistance by the Linearization and ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2000, p. 393–395 0066-4804/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 44, No. 2

NOTES Acquisition of Chloramphenicol Resistance by the Linearization and Integration of the Entire Staphylococcal Plasmid pC194 into the Chromosome of Streptococcus pneumoniae CAROL A. WIDDOWSON, PETER V. ADRIAN,

AND

KEITH P. KLUGMAN*

Medical Research Council, South African Institute for Medical Research, WITS Pneumococcal Diseases Research Unit, Johannesburg, South Africa Received 17 May 1999/Returned for modification 22 July 1999/Accepted 1 November 1999

Chloramphenicol resistance in Streptococcus pneumoniae was associated with cat, which has 100% identity with catpC194 from Staphylococcus aureus. Inverse PCR with primers specific for pC194 confirmed that in some isolates the entire staphylococcal plasmid was present in the S. pneumoniae chromosome, with linearization having occurred between catpC194 and the origin of replication. are shown in Table 1. To determine whether catpC194 was located within the chromosomes of these six strains, a Southern blot of a pulsed-field gel of SmaI restricted and unrestricted DNA was probed with a whole-gene probe for catpC194 (random primed DNA labelling kit; Boehringer Mannheim, Mannheim, Germany). The largest SmaI restriction fragment (⬎300 kb) from each of the six chloramphenicol-resistant strains hybridized to the catpC194 probe. In the unrestricted samples, only the chromosomal DNA hybridized to the catpC194 probe. Extrachromosomal DNA in the form of small cryptic plasmids was detected in two chloramphenicol-resistant strains and did not hybridize to the catpC194 probe. Sequence analysis. Double-stranded PCR products were sequenced in both directions with the fmol cycle sequencing kit (Promega, Madison, Wis.) according to the manufacturer’s recommendations. The nucleotide sequence of cat from strain 1 was determined with primers F2 and R3 (Fig. 1). In addition, internal cat primers were used for sequencing. The nucleotide sequence of cat from this strain shared 100% identity with catpC194. Further PCR analysis showed that pC194 was present in this strain (data not shown) and that the plasmid sequence was linearized between catpC194 and reppC194. To determine the position of linearization and the flanking sequence of pC194, inverse PCR was performed with primers R2 and F4 (Fig. 1) on EcoRI-restricted and -ligated fragments. A 3.8-kb amplicon which could be restricted with EcoRI into fragments of 1.6 and 2.2 kb was obtained (data not shown). Nucleotide sequencing of the inverse PCR product with primers R2 and F4 showed that pC194 was linearized at a position 1088 nucleotides from the published MboI site (8) (accession no. J01754) (Fig. 1). The nucleotide sequence flanking pC194 (346 bp upstream and 265 bp downstream; Fig. 1) did not show any significant homology with other sequences in the EMBL and GenBank databases. PCR amplification of these flanking sequences with primer pairs F1-R1 and F5-R5 showed that these flanking sequences were not present in the sensitive controls and suggests that they are not wild-type pneumococcal chromosomal DNA, as reported previously (18). The 5⬘ sequence flanking pC194 was detected in all six chloramphenicol-resistant strains and appears to be in the identical position with respect to catpC194, as determined with strain 1 (Table 1). The flanking sequence 3⬘ of

Resistance to chloramphenicol in Streptococcus pneumoniae is due to the acetylation of the antibiotic by the production of a chloramphenicol acetyltransferase (CAT) (4, 11). In S. pneumoniae, cat is carried on the conjugate transposon Tn5253 (18), a composite transposon consisting of the tetracycline resistance transposon Tn5251 and Tn5252, a 47-kb transposon carrying the chloramphenicol resistance determinant (1). Tn5252 is capable of conjugal transfer between pneumococci and Streptococcus agalactiae, Streptococcus gordonii, and Enterococcus faecalis (1, 17), most likely through site-specific recombination (17). Chloramphenicol-resistant S. pneumoniae has been shown to contain sequences homologous to catpC194 and other flanking sequences from the Staphylococcus aureus plasmid pC194 (5, 14). S. pneumoniae does not naturally have plasmids that confer antibiotic resistance. pC194 has been introduced into S. pneumoniae in the laboratory by transformation, but the plasmid is lost at a rate of 2% per generation when it is grown in the absence of chloramphenicol (2). The aim of the study described here was to determine the nature of and putative origins of cat in S. pneumoniae. Bacterial isolates. S. pneumoniae R6 and ATCC 49619 were used as chloramphenicol-sensitive controls. Twenty-five chloramphenicol-resistant (MICs, 8 to 16 ␮g/ml) clinical isolates of S. pneumoniae were obtained between 1990 and 1995 from the South African Institute for Medical Research, Johannesburg, South Africa. MICs were determined on Mueller-Hinton agar supplemented with 5% horse blood by an agar dilution method as described by the National Committee for Clinical Laboratory Standards (12). Detection of the cat gene. PCR amplification with primers F3 and R3 (Fig. 1) was used to detect catpC194 in all 25 clinical S. pneumoniae isolates. Pulsed-field gel electrophoresis profiles (9) showed that more than half of the isolates (including strain 1) were closely related to the Spain23F-1 clone. Six genotypically unrelated strains were selected for further study (data not shown). The resistance profiles and serotypes of these strains * Corresponding author. Mailing address: Pneumococcal Diseases Research Unit, SAIMR, P.O. Box 1038, Johannesburg 2000, South Africa. Phone: 27 11 489 9010. Fax: 27 11 489 9012. E-mail: keithk @mail.saimr.wits.ac.za. 393

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ANTIMICROB. AGENTS CHEMOTHER.

FIG. 1. Nucleotide sequences of the 5⬘ and 3⬘ ends of pC194 (capital letters) (8) and the flanking DNA (lowercase letters). The sequences of the primers used for PCR and sequencing reactions are shown in boldface type, and the 5⬘ to 3⬘ direction is indicated (3). Regions of secondary structure (inverted repeats 34) at the 5⬘ and 3⬘ ends of pC194 are indicated. The nicked site within the origin of replication (underlined) is indicated (10). Insertions into the pC194 sequence described previously (8) are shaded, and the T-to-C substitution in the origin of replication is indicated.

linearized pC194 was not detected by PCR in the other five chloramphenicol-resistant strains, of which two strains appeared to have deletions from pC194 at the 3⬘ end (Table 1). pC194 in eight of eight Spain23F-1-related clones had 5⬘ and 3⬘ flanking sequences identical to those in strain 1 (data not shown). The 3⬘ variants may be the result of subsequent deletion or recombination of the element harboring catpC194 in the pneumococcus, possibly in association with the pC194 origin of replication which has been identified as a hot spot for deletion formation (10). There are a number of possible mechanisms by which pC194 has integrated into a putative conjugable element. The 5⬘ and 3⬘ ends of linearized pC194 in strain 1 are characterized by a number of inverted repeats at both ends (Fig. 1). It is possible that these structures may be involved in some type of recom-

bination, especially since these features have previously been identified to play roles in recombination between plasmids and the chromosome (13). Another possible mechanism of integration is via some form of illegitimate recombination as a result of defective replication of the plasmid. pC194 replicates via a rolling-circle replication mechanism during which large amounts of single-stranded plasmid are produced (15, 16). Such a mechanism has been shown to greatly enhance illegitimate recombination by such plasmids (7); however, the site of integration in strain 1 was 100 bp from the recognized nick site of pC194 (10). The integration site of pC194 is also different from previously identified site-specific recombination sites RSA and RSB described in staphylococcal plasmids (6). The presence of the entire plasmid, including a functional origin of replication and RepH, suggests that it may be possible

TABLE 1. Genetypically unrelated chloramphenicol-sensitive and -resistant strains of S. pneumoniae, the serotype, resistance profile, and the presence of regions of pC194 and the flanking structures determined by PCR with the primers described in Fig. 1 Strain

Serotype

R6 ATCC 49619 1 4 6 37 65 70

1 23F 19A 11 14 19A 19A

a

MIC (␮g/ml)a

Amplicon production with the following primer pairs:

Pen

Chl

Tet

Erm

Cln

F2-R3

F2-R4

F1-R1

F1-R2

F4-R5

F5-R5

F4-R2

0.03 0.25 1 0.5 0.13 0.5 1 1

1 1 8 16 16 8 16 8

1 1 32 1 32 64 32 32

0.25 0.25 0.25 0.25 0.25 ⬎16 ⬎16 16

0.25 0.25 0.25 0.25 0.25 32 ⬎32 ⬎32

⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹

⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

Pen, Penicillin; Chl, chloramphenicol; Tet, tetracycline; Erm, erythromycin; Cln, clindamycin.

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NOTES

for the plasmid to replicate independently from the chromosome, allowing the production of increased levels of CAT. RepH is able to identify a different origin of replication and as a result ligates the strand prematurely, resulting in deletion plasmids (3). Such deletion plasmids resulting from replication of the linear pC194 may be produced and could enhance the abilities of such strains to survive in the presence of chloramphenicol. These plasmids would most likely not be able to replicate in S. pneumoniae since the origin of replication would be disrupted. The integration of pC194 into a conjugative element represents a means by which chloramphenicol resistance and potentially other plasmid-mediated resistance genes can be introduced, maintained, and disseminated in S. pneumoniae. REFERENCES 1. Ayoubi, P., A. O. Kilic¸, and M. N. Vijayakumar. 1991. Tn5253, the pneumococcal ⍀(cat tet) BM6001 element, is a composite structure of two conjugative transposons, Tn5251 and Tn5252. J. Bacteriol. 173:1617–1622. 2. Ballester, S., P. Lopez, J. C. Alonso, M. Espinosa, and S. A. Lacks. 1986. Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids. Gene 41:153– 163. 3. Ballester, S., P. Lopez, M. Espinosa, J. C. Alonso, and S. A. Lacks. 1989. Plasmid structural instability associated with pC194 replication functions. J. Bacteriol. 171:2271–2277. 4. Dang-Van, A., G. Tiraby, J. F. Acar, W. V. Shaw, and D. H. Bonanchaud. 1978. Chloramphenicol resistance in Streptococcus pneumoniae: enzymatic acetylation and possible plasmid linkage. Antimicrob. Agents Chemother. 13:557–583. 5. David, F., G. de Ce´spe`des, F. Delbos, and T. Horaud. 1993. Diversity of chromosomal genetic elements and gene identification in antibiotic-resistant strains of Streptococcus pneumoniae and Streptococcus bovis. Plasmid 29:147– 153.

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6. Gennaro, M. L., J. Kornblum, and R. P. Novick. 1987. A site-specific recombination function in Staphylococcus aureus plasmids. 196:2601–2610. 7. Gruss, A., and S. D. Ehrlich. 1989. The family of highly interrelated singlestranded deoxyribonucleic acid plasmids. Microbiol. Rev. 53:231–241. 8. Horinouchi, S., and B. Weisblum. 1982. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bacteriol. 150:815–825. 9. Lefevre, J. C., G. Faucon, A. M. Sicard, and A. M. Gasc. 1993. DNA fingerprinting of Streptococcus pneumoniae by pulsed-field gel electrophoresis. J. Clin. Microbiol. 31:2724–2728. 10. Michel, B., and S. D. Ehrlich. 1986. Illegitimate recombination occurs between the replication origin of plasmid pC194 and a progressing replication fork. EMBO J. 5:3691–3696. 11. Miyamura, S., H. Ochiai, Y. Nitahara, Y. Nakagawa, and M. Tereo. 1977. Resistance mechanism of chloramphenicol in Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis. Microbiol. Immunol. 21:69–76. 12. National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved standard M7-A4. National Committee for Clinical Laboratory Standards, Wayne, Pa. 13. Peeters, B. P., J. H. de Boer, S. Bron, and G. Venema. 1988. Structural plasmid instability in Bacillus subtilis: effect of direct and inverted repeats. Mol. Gen. Genet. 212:450–458. 14. Pepper, K., G. de Ce´spe`des, and T. Horaud. 1988. Heterogeneity of chromosomal genes encoding chloramphenicol resistance in streptococci. Plasmid 19:71–74. 15. te Riele, H., B. Michel, and S. D. Ehrlich. 1986a. Are single-stranded circles intermediates in plasmid DNA replication? EMBO J. 5:631–637. 16. te Riele, H., B. Michel, and S. D. Ehrlich. 1986b. Single-stranded plasmid DNA in Bacillus subtilis and Staphylococcus aureus. Proc. Natl. Acad. Sci. USA 83:2541–2545. 17. Vijayakumar, M. N., and S. Ayalew. 1993. Nucleotide sequence analysis of the termini and chromosomal locus involved in site-specific integration of the streptococcal conjugative transposon Tn5252. J. Bacteriol. 175:2713–2719. 18. Vijayakumar, M. N., S. D. Priebe, and W. R. Guild. 1986. Structure of a conjugative element in Streptococcus pneumoniae. J. Bacteriol. 166:978–984.