Staphylococcal tetracycline–MLSB resistance plasmid pSTE2 is the product of an RSA-mediated in vivo recombination

Tomasz Hauschild1,{dagger}, Petra Lüthje2,{dagger} and Stefan Schwarz2,*

1 Department of Microbiology, Institute of Biology, University of Bialystok, 15-950 Bialystok, Swierkowa 20 B, Poland; 2 Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany


* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; E-mail: stefan.schwarz{at}fal.de

Received 2 March 2005; returned 19 April 2005; revised 29 April 2005; accepted 26 May 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: The complete nucleotide sequence of the 6913 bp plasmid pSTE2 from Staphylococcus lentus, which mediates inducible resistance to tetracyclines, macrolides and lincosamides, was determined. The plasmid was analysed for potential reading frames and structural features to gain insight into its development from potential ancestor plasmids.

Methods: Plasmid pSTE2 was transformed into Staphylococcus aureus RN4220. Suitable restriction fragments were cloned into E. coli plasmid vectors and sequenced. In vitro susceptibility testing was performed to confirm the resistance phenotype mediated by this plasmid.

Results: Plasmid pSTE2 consisted of two parts, each of which corresponded closely to previously identified staphylococcal plasmids. The initial 4439 bp represented a pT181-analogous tet(K)-carrying tetracycline resistance plasmid, whereas the remaining 2474 bp represented a pPV141-related erm(C)-carrying macrolide-lincosamide-streptogramin B resistance plasmid. Both putative parental plasmids harboured the staphylococcal recombination site A (RSA) and the pT181-like plasmid also carried the recombinase gene pre whose product acts at RSA. Analysis of the junctions of the pT181-like and the pPV141-like homologous parts in pSTE2 suggested that plasmid pSTE2 developed from pT181- and pPV141-like ancestor plasmids by cointegrate formation at RSA.

Conclusion: Plasmid pSTE2 is the first completely sequenced plasmid from S. lentus and represents the product of an in vivo derived RSA-mediated recombination between two compatible plasmids.

Keywords: site-specific recombination , cointegrate formation , Staphylococcus , recombination sites


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Staphylococcus lentus is a common inhabitant of the physiological skin flora of animals including various mammals, e.g. ruminants, carnivores and rodents, but also birds. Members of this staphylococcal species are considered as rarely pathogenic. Previous studies have identified a number of antimicrobial resistance genes in S. lentus isolates most of which are located on plasmids. Small plasmids carrying either the tetracycline resistance genes tet(K) or tet(L),1 or the macrolide-lincosamide-streptogramin B (MLSB) resistance genes erm(B)2 or erm(C)3 have been identified occasionally in S. lentus isolates. During a recent survey of transferable antimicrobial resistance among staphylococci from rodents and insectivores, we identified a S. lentus isolate (No. 32) from a common shrew (Sorex araneus). This isolate carried a small plasmid, designated pSTE2, that mediated resistance to tetracyclines, macrolides and lincosamides.

The aim of this study was to analyse the structure and organization of plasmid pSTE2 to gain insight into the processes that led to its formation.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The S. lentus isolate was identified to species level using the ID32 Staph system (bioMérieux, Nürtingen, Germany). Plasmid profiling and electrotransformation into the recipient strain Staphylococcus aureus RN42204 followed previously described protocols. MIC determination by broth microdilution was performed and evaluated according to the document M31-A2 of the Clinical and Laboratory Standards Institute (CLSI; formerly known as NCCLS).5 The resistance genes present on plasmid pSTE2 were identified by PCR.3,4 Plasmid pSTE2 was mapped with restriction endonucleases and suitable ClaI and HindIII fragments were cloned into pBluescript II SK+ (Stratagene, Amsterdam, The Netherlands). Sequence analysis of these fragments was conducted on both strands by primer walking, starting with the M13 reverse and forward primers and completed with primers derived from sequences obtained with these standard primers (MWG, Ebersberg, Germany). Sequence comparisons were performed with the BLAST programs blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST/) and with the ORF finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html; both last accessed 2 March 2005). The nucleotide sequence of plasmid pSTE2 has been deposited in the EMBL database under accession no. AJ888003.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Plasmid pSTE2 from S. lentus was found to be 6913 bp in size. PCR assays showed that this plasmid carried a tet(K) gene for tetracycline resistance and an erm(C) gene for resistance to MLSB antibiotics. Resistance to tetracyclines, macrolides and lincosamides was confirmed by in vitro susceptibility testing (Table 1). It is assumed that this plasmid also mediates resistance to streptogramin B antibiotics, however experimental proof was not possible due to the lack of commercially available streptogramin B antibiotics. Comparison of the pSTE2 sequence revealed two parts of this plasmid which showed extended similarities to other plasmid sequences deposited in the databases (Figure 1).


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Table 1. MIC values of various antimicrobial agents for the original S. lentus strain No. 32, the recipient strain S. aureus RN4220 and S. aureus RN4220::pSTE2 transformants, either non-induced or induced by growth in the presence of 1 mg/L tetracycline (TET) or 1 mg/L erythromycin (ERY)

 


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Figure 1. Structural comparison of plasmid pSTE2 from S. lentus (accession no. AJ888003) with the plasmids pT181 from S. epidermidis (accession no. AE015930) and pPV141 from S. chromogenes (accession no. U82607). Restriction endonucleases are abbreviated as follows: B (BclI), C (ClaI), H (HindIII), Hp (HpaII), K (KpnI) and X (XbaI). A distance scale in kb is presented below each map. The genes tet(K), repC, pre, erm(C) and repL are presented as arrows with the arrowhead indicating their direction of transcription. The black box symbolizes the recombination site RSA the 24 bp core sequence of which is displayed in capital letters above or below the corresponding maps. The first eight bases of the adjacent sequences are shown in lower case letters. The grey shaded regions indicate ≥98% nucleotide sequence identity between pSTE2 and either pT181 or pPV141.

 
The first part (bases 1–4439) closely resembled a number of small staphylococcal tet(K)-carrying plasmids. The best match was the 4439 bp pT181-like tet(K)-carrying plasmid found in Staphylococcus epidermidis strain ATCC 12228 (database accession no. AE015930) from which the initial 4439 bp of plasmid pSTE2 differed by only 1 bp. This part of pSTE2 comprised the tet(K) gene (positions 1439–114) which codes for a 459-amino-acid membrane-associated efflux protein consisting of 14 transmembrane domains. It was preceded by a small reading frame for a 16-amino-acid peptide (positions 1587–1537) which together with two inverted repeated sequences represents the translational attenuator responsible for the tetracycline-inducible expression of tet(K). Growth in the presence of 1 mg/L tetracycline resulted in a moderate 2-fold increase in the MIC of tetracycline for the pSTE2 transformant (Table 1). The repC gene (positions 2599–1655) codes for a 314 amino acid plasmid replication protein and the pre gene (positions 4367–3126) codes for a recombinase protein of 413 amino acids which acts at the staphylococcal recombination site RSA. The RSA site was located at positions 4416–4439 in pSTE2. In addition to RSA, pT181-like tet(K)-carrying plasmids usually also harbour a second recombination site, RSB. The RSB site in pSTE2 was detected at positions 3096–3128 and includes the translational termination codon of the pre gene.

The second part of pSTE2 (positions 4440–6913) exhibited a high degree of similarity to small erm(C)-carrying MLSB resistance plasmids from staphylococci and bacilli with a best match of 98% identity to the 2410 bp plasmid pPV141 from Staphylococcus chromogenes (database accession no. U82607). This segment of pSTE2 contained the erm(C) gene (positions 4781–5515) which codes for a ribosomal RNA methylase of 244 amino acids. The erm(C) gene was also preceded by a complete translational attenuator which is required for the inducible expression of erm(C).3 In the non-induced state, the original S. lentus strain as well as the S. aureus RN4220::pSTE2 transformant exhibited resistance to the 14-membered macrolide erythromycin, but susceptibility to the non-inducers, e.g. 16-membered macrolides spiramycin and tilmicosin as well as to the lincosamide clindamycin (Table 1). The high MIC (≥128 mg/L) of tulathromycin—which is a 1:9 mixture of 13- and 15-membered azalides—indicated that tulathromycin is also an efficient inducer of erm(C) gene expression. When grown in the presence of 1 mg/L erythromycin, the transformant also showed high-level resistance to the 16-membered macrolides and clindamycin (Table 1). This change in the resistance phenotype is indicative of inducible expression of the erm(C) gene.6 An increase in the MIC of erythromycin after induction with erythromycin was not detectable due to the range of concentrations tested. Within the erm(C) regulatory region, a small open reading frame for a 19 amino acid peptide (positions 4662–4721) and two pairs of inverted repeated sequences were detected. The difference in size between the 2474 bp segment of pSTE2 and the 2410 bp plasmid pPV141 was mainly due to a 58 bp deletion seen in the translational attenuator of the constitutively expressed erm(C) gene of pPV141.3 The repL gene which codes for a 158-amino-acid plasmid replication protein was seen at positions 5963–6439 and a second RSA site was detected at positions 6890–6913 in the pSTE2 sequence.

The recombination site RSB is present on many small staphylococcal plasmids.7 RSB-mediated cointegrate formations were seen only after co-transduction of these plasmids and a phage-determined recombination system is believed to play a role in these processes.8 In contrast, RSA is found only rarely on small staphylococcal plasmids and cointegrates were obtained under laboratory conditions with strains carrying different RSA-containing plasmids in the absence of phages.7,9 It has been shown that the plasmid recombination protein Pre mediates a site-specific recombination which involves RSA, but not RSB.9 Heterologous cointegrates between the 4.4 kb tetracycline resistance plasmid pT181 and the 3.7 kb erm(C)-carrying plasmid pE194, both of which carry RSA sites and pre genes, have already been derived in vitro.8,9

In the case of plasmid pSTE2, both partner plasmids had an RSA site, but only the pT181-like partner harboured a pre gene. It is likely that Pre from one such plasmid can mediate cointegrate formation between different co-resident plasmids provided that they have RSA sites. It has been suggested that such heterologous cointegrates might play a role as intermediates in the evolution of larger plasmids carrying multiple antibiotic resistance genes.9 One such model for an RSA-based cointegrate formation between an aadD-encoding kanamycin-neomycin-bleomycin pUB110-like resistance plasmid and a pNS1981-like tet(L)-encoding tetracycline resistance plasmid has been proposed although the intermediate form was not detected.10 Analysis of the sequences flanking the RSA sites in pSTE2 compared with the corresponding sequences in pT181 and pPV141 confirmed that recombination at the RSA sites has led to the cointegrate formation displayed in Figure 1. Moreover, two intact plasmid replication genes, repC and repL, were detected in pSTE2 and their interaction in terms of a coordinated replication and stable maintenance of pSTE2—as seen in the original S. lentus as well as in the S. aureus laboratory host—remains to be clarified.

The presence of genes coding for resistance to the two most frequently used classes of antimicrobials in veterinary medicine, tetracyclines and macrolides, on the same plasmid is of relevance with regard to the spread of resistance. Since this plasmid carries two different replication genes, it might be able to replicate in a broader range of bacterial hosts than its parental plasmids. Moreover, a plasmid such as pSTE2 might be able to incorporate other resistance plasmids or parts of them via its RSA and/or RSB sites. Although cointegrates of pT181-like plasmids into larger plasmids via the insertion sequence IS257 have been described,11 this is to the best of our knowledge the first report of a naturally occurring RSA-cointegrate involving tet(K)- and erm(C)-carrying parental plasmids.


    Footnotes
 
{dagger} These authors contributed equally to this study. Back


    Acknowledgements
 
This work is part of project 158 of the German–Polish Cooperation in Agricultural Research. P. L. is supported by a Georg-Christoph-Lichtenberg-scholarship of the county Lower Saxony.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1. Schwarz S, Roberts MC, Werckenthin C et al. Tetracycline resistance in Staphylococcus spp. from domestic animals. Vet Microbiol 1998; 63: 217–27.[CrossRef][ISI][Medline]

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3. Werckenthin C, Schwarz S, Westh H. Structural alterations in the translational attenuator of constitutively expressed ermC genes. Antimicrob Agents Chemother 1999; 43: 1681–5.[Abstract/Free Full Text]

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5. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals—Second Edition: Approved Standard M31-A2. NCCLS, Wayne, PA, USA, 2002.

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8. Novick RP, Projan SJ, Rosenblum W et al. Staphylococcal plasmid cointegrates are formed by host- and phage-mediated rec systems that act on short regions of homology. Mol Gen Genet 1984; 195: 374–7.[CrossRef][ISI][Medline]

9. Gennaro ML, Kornblum J, Novick RP. A site-specific recombination function in Staphylococcus aureus plasmids. J Bacteriol 1987; 169: 2601–10.[ISI][Medline]

10. Schwarz S, Gregory PD, Werckenthin C et al. A novel plasmid from Staphylococcus epidermidis specifying resistance to kanamycin, neomycin and tetracycline. J Med Microbiol 1996; 45: 57–63.[Abstract]

11. Werckenthin C, Schwarz S, Roberts MC. Integration of pT181-like tetracycline resistance plasmids into large staphylococcal plasmids involves IS257. Antimicrob Agents Chemother 1996; 40: 2542–4.[Abstract]