Enhanced resistance to erythromycin is conferred by the enterococcal msrC determinant in Staphylococcus aureus

Elinor Reynolds and Jonathan H. Cove*

School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK


* Corresponding author. Tel: +44-113-343-5630; Fax: +44-113-343-5638; Email: j.h.cove{at}leeds.ac.uk

Received 26 July 2004; returned 28 October 2004; revised 18 November 2004; accepted 19 November 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives:

The msrC gene, found on the chromosome of Enterococcus faecium, shares a high degree of similarity with the staphylococcal erythromycin resistance determinant msr(A). The enterococcal determinant was cloned into Staphylococcus aureus to determine whether msrC could confer antibiotic resistance in staphylococci.

Methods:

A shuttle vector comprising pBluescript and pSK265 was used to introduce multiple copies of msrC into S. aureus RN4220. The integration vector pCL84 was employed to insert a single copy of msrC into the S. aureus chromosome. MICs were determined by the broth microdilution method.

Results:

Expression of msrC from both chromosomal and plasmid loci in erythromycin-susceptible S. aureus RN4220 (MIC 0.25 mg/L) gave rise to enhanced protection against erythromycin, with an MIC of 32–64 mg/L for S. aureus RN4220 containing msrC in multiple copies and an MIC of 16–64 mg/L with msrC inserted as a single copy in the S. aureus chromosome.

Conclusions:

MsrC mediates high-level resistance to erythromycin in S. aureus.

Keywords: macrolide resistance , ABC transporter , molecular cloning , staphylococci , enterococci


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The species-specific msrC gene of Enterococcus faecium is thought to confer low-level protection against 14-membered-ring macrolides and type B streptogramins (MSB).1 Insertional inactivation of msrC, which is located on the chromosome of E. faecium, was shown to result in increased susceptibility to MSB antibiotics in this species.1 MsrC shares significant sequence identity with Msr(A), a plasmid-encoded ATP-binding cassette (ABC) transporter that confers MSB resistance in staphylococci.13 Both Msr(A) and MsrC are Class 2 ABC transporters, a group lacking any identifiable transmembrane domains, which are generally involved in cellular processes other than transport. A number of similar ‘incomplete’ ABC systems involved in antibiotic resistance have been identified, both in Gram-positive pathogens and antibiotic-producing organisms.4 Msr(A) was initially thought to confer erythromycin resistance by active efflux of the drugs,3 but alternative mechanisms of action, such as energy-dependent displacement of the antibiotic from the ribosome, have been proposed.4

The high level of sequence similarity and common antibiotic substrate specificities of Msr(A) and MsrC gave rise to the suggestion that the two may have evolved from a single determinant.1 There is, however, a substantial difference in the level of resistance to antibiotics conferred by the two determinants: the MIC of erythromycin for E. faecium is <1 mg/L,1 a sharp contrast to the level of erythromycin resistance (MICs of up to 128 mg/L) found in Staphylococcus aureus strains expressing msr(A).2

In order to determine whether the presence of msrC causes reduced susceptibility to MSB antibiotics in S. aureus, we have cloned the msrC determinant together with its promoter and control region from E. faecium TX2465 into S. aureus RN4220. We report here the finding that the level of MSB resistance is greatly enhanced by expression of msrC in S. aureus, and that this phenomenon appears to be independent of gene copy number.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bioinformatics tools

Sequence alignments were carried out using the T-COFFEE multiple sequence alignment tool.5 Codon usage was calculated using the Graphical Codon Usage Analyser tool on http://www.gcua.de and codon usage database http://www.kazusa.or.jp/codon/

Bacterial strains and cloning vectors

The msrC gene was cloned from E. faecium TX24651 into S. aureus RN42206 via a high copy-number shuttle vector, designated pMsrC, constructed from the Escherichia coli vector pBluescript SK+ (Stratagene Cloning Systems, La Jolla, CA, USA) and the staphylococcal vector pSK265, which encodes the cat chloramphenicol resistance gene.7

The shuttle vector pCL848 was used for insertion of a single copy of msrC into the chromosome of S. aureus CYL316,8 a derivative of RN4220. This vector is able to replicate in E. coli but not S. aureus, and utilizes the site-specific recombination system from the staphylococcal phage L54a to integrate into the attB site of the lipase structural gene (geh) on the staphylococcal host chromosome.8 The tetracycline resistance marker on pCL84 was used for selection of staphylococcal transformants carrying the integrated vector. The resulting strain containing a single chromosomal copy of msrC was designated CYL316(csMsrC). All genetic manipulations involving shuttle vectors were carried out in E. coli JM109 (Promega, Southampton, UK).

PCR amplification, cloning and sequencing of the msrC gene

The msrC gene including the upstream control region (GenBank ID AY004350) was amplified by PCR using forward (5'-AAG CGG ATC CAG TTG CCA GAG G-3') and reverse (5'-ATT ACT TTG AAT TCC TTT TAG CAC AC-3') primers, engineered to contain BamHI and EcoRI restriction sites, respectively (in italics), to allow cloning into pBluescript and pCL84 at these sites. The msrC:pBluescript construct was cloned into the EcoRI and KpnI sites of pSK265 to generate the shuttle vector pMsrC. Restriction endonucleases (Invitrogen, Paisley, UK) and DNA ligase (Promega) were used in accordance with the manufacturers' instructions.

Nucleotide sequence determination of the cloned msrC gene was carried out using the DYEnamic ET Terminator Cycle Sequencing kit (Amersham Biosciences, UK) at the Automated DNA Sequencing Facility at the Centre for Biomolecular Sciences, School of Biochemistry and Microbiology, Leeds, UK.

Transformation of E. coli and S. aureus with plasmid DNA

E. coli JM109 competent cells were purchased from Promega and transformed following the supplier's instructions. S. aureus strains were transformed by electroporation.9 S. aureus cells transformed with the shuttle vectors pMsrC and pCL84:MsrC were selected on 6 mg/L chloramphenicol and 3 mg/L tetracycline, respectively.

Determination of susceptibility to antibiotics

S. aureus strains carrying msrC were not exposed to MSB antibiotics before antibiotic susceptibility testing. MICs were determined by broth microdilution according to NCCLS guidelines.10 Erythromycin was incorporated into MIC culture media at 5 mg/L for induction of resistance to type B streptogramins and at 0.1 mg/L for all other antibiotics, as described previously for strains carrying msr(A).2 The same inducing concentrations were found to be optimal for msrC-mediated resistance in S. aureus.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Sequence analysis

Alignments of the amino acid sequences of MsrC and Msr(A) showed 42% sequence identity and >70% similarity overall. The similarity between the two sequences is not confined to the nucleotide binding domains, but extends along the entire length of the sequence, including the Q-linker region which tethers the two ATP-binding domains together.

An alignment of the upstream control regions of msrC and msr(A) is shown in Figure 1. Both sequences contain similar consensus promoter sequences, Shine–Dalgarno sequences, a putative short peptide, and a number of inverted repeat sequences.1,3 Deletion of the entire control region of msr(A) was found previously to result in constitutive expression of MSB resistance,11 indicating that the inducible phenotype is dependent on the presence of the control region. Upstream of both msr(A) and msrC are sequences that could encode short polypeptides with almost identical sequences over the first eight amino acids (Figure 1). The staphylococcal leader peptide has been shown not to be essential for MSB resistance mediated by msr(A), but it has been proposed that it may be involved in the regulation of gene expression by a process of translational attenuation.11 The high degree of similarity between the control regions of msrC and msr(A) (Figure 1) suggests a similar regulatory mechanism in E. faecium.



View larger version (35K):
[in this window]
[in a new window]
 
Figure 1. Nucleotide sequence alignment of the upstream control regions of msrC and msr(A). Matching nucleotides are indicated by asterisks (*). The –10 and –35 promoter sequences and inverted repeat (IR) sequences are underlined. Shine–Dalgarno (SD) sequences are indicated by bold type. Amino acid sequences of the leader peptides and N-terminal sequences of MsrC and Msr(A) are given in single-letter code outside corresponding nucleotide sequences.

 
The GC ratios of msrC (37.9%) and msr(A) (30.4%) are close to the overall values for E. faecium and Staphylococcus species (approximately 39% and 33%, respectively). Codon usage in Msr(A) and MsrC was not found to differ significantly from overall usage in Staphylococcus species or E. faecium. An annotated draft genome sequence for E. faecium (GenBank ID AAAK00000000) was analysed for the presence of any mobile genetic elements near msrC that may have been involved in horizontal transfer of the gene, but no such sequences could be identified in the regions 20 kb upstream or downstream of msrC. There is currently no information available regarding the chromosomal location of msrC in other strains of E. faecium.

Cloning of msrC and nucleotide sequence determination

The msrC determinant of E. faecium TX2465 was successfully cloned into S. aureus. The shuttle vector pMsrC, made up of pBluescript, pSK265 and msrC, was used to introduce multiple copies of msrC into S. aureus RN4220. The integration vector pCL848 was employed for insertion of a single copy of msrC into the staphylococcal chromosome.

Sequencing through the entire cloned msrC gene revealed a number of base changes from the published sequence for msrC.1 Six of these nucleotide substitutions would result in changes at the amino acid level. Sequencing through the msrC gene that had been amplified directly from E. faecium TX2465 revealed the same set of base substitutions. Furthermore, the nucleotide sequence of the msrC gene in the draft genome sequence for E. faecium (GenBank ID AAAK00000000) was found to be identical to the sequence obtained in this study. This confirmed that the cloned msrC gene was identical to that found in the parent strain TX2465, and that any observed differences from the previously published sequence were not a result of expression in S. aureus, nor were they PCR artefacts.

Activity of MsrC in S. aureus

The enterococcal determinant msrC was found to confer high-level resistance to erythromycin in S. aureus, whether expressed from a high copy-number plasmid, or in single copy from a chromosomal locus (Table 1). The MIC of erythromycin for S. aureus RN4220 was increased from 0.25 to 16–64 mg/L (≥64-fold increase) by carriage of the msrC gene. In contrast, the MIC of erythromycin for E. faecium TX2465 was reduced from 0.5–0.75 mg/L to 0.06–0.09 mg/L (eight-fold decrease) by inactivation of msrC.1 The determinant was additionally found to confer inducible resistance to other 14- and 15-membered-ring macrolides and type B streptogramins in S. aureus (Table 1), consistent with the finding that MICs of these antibiotics were decreased following inactivation of msrC in E. faecium.1 Interestingly, disruption of msrC in E. faecium was reported to cause a small decrease in resistance (16–8 mg/L) to tylosin, a 16-membered-ring macrolide;1 however, msrC did not confer any reduction in susceptibility to tylosin in S. aureus (Table 1). Resistance to type A streptogramins or lincosamides was not found to be enhanced by carriage of msrC (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Antibiotic resistance spectra for msrC and msr(A) in S. aureus

 
Transfer of pMsrC to susceptible RN4220 was found to occur stably and at high frequency, with erythromycin MICs for transformants remaining identical to those for the original clone.

In conclusion, the species-specific msrC gene of E. faecium displays a high level of sequence similarity to msr(A), an acquired MSB resistance determinant of staphylococci. The chromosomally-located msrC gene was shown previously to confer a small reduction in susceptibility to MSB antibiotics in E. faecium.1 In this study, we have shown that expression of msrC resulted in high-level protection against erythromycin and other MSB antibiotics in S. aureus. This intriguing discovery that msrC confers high-level resistance in S. aureus, but not in its enterococcal host species, could shed new light on its mechanism of action and allow identification of additional factors required for resistance.


    Acknowledgements
 
We thank Dr Barbara Murray and Professor Tim Foster for providing E. faecium TX2465 and the pCL84 integration system, respectively. This work was supported by a University of Leeds Research Scholarship to E.R.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Singh, K. V., Malathum, K. & Murray, B. E. (2001). Disruption of an Enterococcus faecium species-specific gene, a homologue of acquired macrolide resistance genes of staphylococci, is associated with an increase in macrolide susceptibility. Antimicrobial Agents and Chemotherapy 45, 263–6.[Abstract/Free Full Text]

2 . Ross, J. I., Farrell, A. M., Eady, E. A. et al. (1989). Characterisation and molecular cloning of the novel macrolide-streptogramin B resistance determinant from Staphylococcus epidermidis. Journal of Antimicrobial Chemotherapy 24, 851–62.[Abstract]

3 . Ross, J. I., Eady, E. A., Cove, J. H. et al. (1990). Inducible erythromycin resistance in staphylococci is encoded by a member of the ATP-binding transport super-gene family. Molecular Microbiology 4, 1207–14.[ISI][Medline]

4 . Reynolds, E., Ross, J. I. & Cove, J. H. (2003). Msr(A) and related macrolide/streptogramin resistance determinants: incomplete transporters? International Journal of Antimicrobial Agents 22, 228–36.[CrossRef][ISI][Medline]

5 . Notredame, C., Higgins, D. G. & Heringa, J. (2000). T-Coffee: a novel method for fast and accurate multiple sequence alignment. Journal of Molecular Biology 302, 205–17.[CrossRef][ISI][Medline]

6 . Fairweather, N., Kennedy, S., Foster, T. J. et al. (1983). Expression of a cloned Staphylococcus aureus {alpha}-hemolysin determinant in Bacillus subtilis and Staphylococcus aureus. Infection and Immunity 41, 1112–7.[ISI][Medline]

7 . Jones, C. L. & Khan, S. A. (1986). Nucleotide sequence of the enterotoxin B gene from Staphylococcus aureus. Journal of Bacteriology 166, 29–33.[ISI][Medline]

8 . Lee, C. Y., Buranen, S. L. & Ye, Z.-H. (1991). Construction of single-copy integration vectors for Staphylococcus aureus. Gene 103, 101–5.[CrossRef][ISI][Medline]

9 . Schenk, S. & Laddaga, R. A. (1992). Improved method for electroporation of Staphylococcus aureus. FEMS Microbiology Letters 94, 133–8.[CrossRef][ISI]

10 . National Committee for Clinical Laboratory Standards. (2003). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Sixth Edition: Approved Standard M7-A6. NCCLS, Wayne, PA, USA.

11 . Ross, J. I., Eady, E. A., Cove, J. H. et al. (1996). Minimal functional system required for expression of erythromycin resistance by msrA in Staphylococcus aureus RN4220. Gene 183, 143–8.[CrossRef][ISI][Medline]