School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK
Keywords: ABC transporters , macrolide resistance , ketolide resistance , staphylococci
Sir,
The antibiotic resistance determinants Msr(A), MsrC and Msr(D) are all members of a group of incomplete or Class 2 ABC transporters, characterized by the presence of two fused nucleotide-binding domains but lacking any identifiable transmembrane domains, and are typically involved in cellular processes other than transport.1
The msr(A) gene is carried on large staphylococcal plasmids, and has been shown to confer high-level inducible resistance to 14- and 15-membered ring macrolides and type B streptogramins (MSB antibiotics) in Staphylococcus aureus in the absence of any additional plasmid-encoded determinants.2 A single copy of msrC is found on the chromosome of Enterococcus faecium. Insertional inactivation of msrC in this species resulted in increased susceptibility to MSB antibiotics.3 We recently reported the cloning of msrC into S. aureus, where it was found to confer a greatly enhanced level of protection against MSB antibiotics compared with that in E. faecium.4 Both determinants confer high-level MSB-resistance in S. aureus, whether cloned on a high copy number vector or integrated in single copy into the chromosome.4,5 The homologous msr(D) determinant, found on large chromosomal genetic elements in streptococci, was recently shown to confer low-level resistance to 14-membered ring macrolides and ketolides in Streptococcus pneumoniae.6 Msr(D) shares a high level of amino acid sequence similarity with Msr(A) and MsrC (75% and 78%, respectively).
We report here the cloning of msr(D) into S. aureus RN4220 on a multicopy shuttle vector. The msr(D) gene and its promoter were amplified from Streptococcus pyogenes ATCC® BAA-946,7 cloned into the PCR cloning vector pDrive (Qiagen Ltd) and subcloned into the KpnI site of the staphylococcal vector pSK265.8 The cloning vectors pDrive and pSK265 carry ampicillin- and chloramphenicol-resistance determinants, respectively. All molecular cloning was carried out in Escherichia coli JM109. Transformants carrying this shuttle vector construct, designated pMsrD, were selected on LB agar containing 50 mg/L ampicillin and 5 mg/L chloramphenicol. S. aureus RN4220 was transformed with pMsrD by electroporation, with selection of transformants on agar containing 5 mg/L chloramphenicol.8
S. aureus RN4220 transformed with pMsrD was found to have enhanced resistance to erythromycin, a 14-membered ring macrolide (MIC 48 mg/L), and telithromycin, a ketolide antibiotic (MIC 0.1250.25 mg/L), representing 16-fold and 4-fold increases in MICs, respectively (Table 1). The MIC of telithromycin was increased further to 1 mg/L upon induction with 1 mg/L erythromycin (Table 1). However, pMsrD transformants remained susceptible to 16-membered ring macrolides, lincosamides and to streptogramins A and B, even following induction with erythromycin. Daly et al.6 recently reported that msr(D) similarly confers resistance to macrolides and inducible resistance to ketolides in S. pneumoniae.
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The msr(A) gene is preceded by a control region containing a number of inverted repeat sequences and an open reading frame encoding a short leader peptide (LP).2 Deletion of this control region resulted in constitutive resistance to MSB antibiotics in S. aureus.5 We found that MICs of telithromycin for S. aureus RN4220 carrying msr(A) which was deleted for its control region (2 mg/L) were not affected by the addition of erythromycin, indicating that the msr(A) control region may also be required for inducible resistance to telithromycin. A similar control region has been identified upstream of the msrC gene in E. faecium,3 and is likely to be responsible for the inducible nature of resistance conferred by this determinant. We located an open reading frame preceded by a ShineDalgarno sequence 110 nt upstream of the msr(D) coding sequence that could potentially encode a short LP (Met-Tyr-Leu-Ile-Phe-Met). A series of inverted repeat sequences were identified in this putative control region, but none of these was found to encompass the ribosome binding site preceding msr(D). Moreover, consensus 10 and 35 promoter sequences were identified within the open reading frame of the proposed LP. No promoter sequences were found upstream of the LP. The nucleotide sequence upstream of msr(D) did not closely resemble the control regions upstream of msr(A) and msrC, which are thought to be involved in the regulation of gene expression by translational attenuation.2,3 It is therefore unlikely that msr(D) expression is regulated by the same mechanism. Although msr(D) was expressed from its own promoter in S. aureus in this study, it may be noted that other researchers have demonstrated the co-transcription of mef(A) and msr(D).9 In this case, the LP sequence upstream of msr(D) would also be co-transcribed, and its expression could potentially regulate translation of the msr(D) transcript.
It remains unclear how such incomplete ABC transporters provide the bacterial cell with protection against antibiotics. It was originally proposed that active efflux of the drugs could be orchestrated by hijacking and altering the specificity of the transmembrane domains of a chromosomally-encoded ABC transporter.5 This would require the recognition and transport of the structurally distinct MSB antibiotics by a pre-existing membrane component of an ABC transporter system.
An alternative mechanism could involve an ATP-dependent conformational change in the ribosomal binding sites of MSB antibiotics, causing dissociation of bound drugs and allowing their diffusion out of the bacterial cell. Macrolides, their ketolide derivatives and type B streptogramins are all known to share overlapping binding sites on the bacterial ribosome.10 The fact that msr(D) and mef(A) are co-transcribed9 does not automatically signify an efflux mechanism for Msr(D): it is not uncommon to find concomitant carriage of determinants that confer resistance to the same antibiotics by different mechanisms (e.g. erm and mef genes in streptococci11).
A recent report described the efflux of telithromycin from S. pyogenes strains carrying the mef(A) determinant.11 Cloning of mef(A) and msr(D) on separate plasmids in S. pneumoniae showed that resistance to telithromycin was linked to carriage of msr(D), rather than to the mef(A) gene itself.6 We can speculate that the telithromycin efflux phenotype would also be observed if the drug was removed from its binding site on the ribosome, allowing it to diffuse passively out of the cell. This efflux phenotype was found to be disrupted by the addition of sodium arsenate,11 an ATP inhibitor. This indicates that Msr(D), an ABC transporter, acts in an energy-dependent manner, either to pump antibiotics from the cell or to displace them from their binding sites. In the absence of any additional plasmid-encoded determinants, msr(A), msrC and msr(D) all confer resistance to antibiotics that share overlapping binding sites on the ribosome, suggesting that the latter mechanism is more probable.
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None to declare.
Acknowledgements
This work was supported by a University of Leeds Research Scholarship to E. D. R. Telithromycin was kind provided by Leonard Katz, Kosan Biosciences.
References
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Cantón R, Mazzariol A, Morosini MI et al. Telithromycin activity is reduced by efflux in Streptococcus pyogenes. J Antimicrob Chemother 2005; 55: 48995.
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