Insertional inactivation of mutS in Staphylococcus aureus reveals potential for elevated mutation frequencies, although the prevalence of mutators in clinical isolates is low

Alexander J. O’Neill and Ian Chopra*

Antimicrobial Research Centre and Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

Received 13 December 2001; returned 8 March 2002; revised 3 May 2002; accepted 17 May 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Populations defective in mismatch repair that exhibit elevated mutation frequencies to antibiotic resistance have been reported amongst pathogenic Gram-negative bacteria. Whether such mutators occur widely in clinical isolates of Gram-positive species, and in important pathogens such as Staphylococcus aureus, is unknown. Insertional inactivation of the mutS gene of S. aureus RN4220 by targeted plasmid integration produced a strain with mutation frequencies for antibiotic resistance up to 78-fold greater than those exhibited by RN4220, thereby providing proof of the concept that staphylococcal mutators could arise. Subsequently, 493 clinical S. aureus isolates were examined for the presence of mutators. However, no strain exhibited a >=10-fold increase in mutation frequency compared with laboratory strain 8325-4. Detailed phenotypic and genotypic analysis of vancomycin-intermediate S. aureus strain Mu50 was performed, since the published genome sequence of this organism suggests that mutS is inactive as a result of a frameshift. However, elevated mutation frequencies were not observed in Mu50, and re-sequencing of a portion of mutS from this strain indicated that this gene was intact. Transient increases in mutation frequency during the stationary phase of growth occur in other bacteria, although no such increases were observed in S. aureus. We conclude that neither permanent increases in the basal mutation frequency, nor transient increases in mutation frequency under starvation, are likely to play a significant role in the development of antibiotic resistance in S. aureus.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutation plays a central role in the evolution of bacterial resistance to antibiotics by refining existing resistance determinants, or by giving rise to variant drug targets with reduced affinity for antibiotics.1 Current concepts of bacterial adaptation and survival in the presence of antibiotics are primarily based on the assumption that resistance arises in cells possessing normal mutation frequencies.2 However, strains exhibiting elevated mutation frequencies have recently been reported amongst populations of pathogenic Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Helicobacter pylori.35 In some cases mutation frequencies to antibiotic resistance are 1000-fold higher than normal strains and such hypermutators can occur with frequencies of up to 20% of clinical isolates.5 The majority of hypermutator populations identified in these clinical isolates harbour defects in the methyl-directed mismatch repair (MMR) system,4,5 a post-replicative DNA repair pathway that functions to identify and correct mismatched DNA duplexes. For reasons that are not clear, mutS is the most frequent mutator allele in natural coliform and pseudomonad populations,4,5 with large DNA deletions often responsible for deficiency of this allele.4

In addition to a significantly elevated frequency of mutation, MMR-deficient strains also present reduced barriers to interspecies gene exchange.4 Thus, the potential importance of mutator strains in the development and spread of antibiotic resistance within bacterial populations is evident.

Staphylococcus aureus is a versatile pathogen in which multiple resistance to antibiotics has emerged, often as a consequence of mutation in drug targets.6 It has been suggested that mutators also exist in naturally occurring populations of S. aureus.7,8 However, because of the small population group, the true clinical prevalence of such strains is unknown. Furthermore, whether staphylococcal mutators (like their counterparts in E. coli, S. enterica and P. aeruginosa) also contain MMR defects is unknown. In this paper we report the results of several experiments designed to assess the importance of hypermutation in S. aureus for the evolution of antibiotic resistance in this organism.

The presence in S. aureus of the typical Gram-positive mutSL operon analogous to that found in other Gram-positive organisms911 suggests an active MMR repair system in staphylococci. Since disruption of mutS is an important mechanism for achieving mutator status,4,5,9 we inactivated the mutS gene in S. aureus to establish whether this confers a mutator phenotype. We also surveyed a large collection of clinical isolates for the presence of mutator phenotypes. This collection included 49 S. aureus strains recovered from the lungs of patients with cystic fibrosis (CF), a niche that strongly predisposes P. aeruginosa to hypermutability.5 Furthermore, in response to DNA sequence data which suggest that vancomycin-intermediate S. aureus strain Mu50 may be hypermutable,11,12 we have re-analysed this strain both genotypically and phenotypically to determine whether it is indeed hypermutable. Finally, we have determined whether mutation frequencies are enhanced in the stationary phase of growth in S. aureus, as reported for Mycobacterium smegmatis.13


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotics

Rifampicin, fusidic acid and norfloxacin were from Sigma-Aldrich (Poole, UK), Monotrim (trimethoprim) from Solvay Healthcare (Southampton, UK) and lithium mupirocin was a gift from Glaxo SmithKline (Harlow, UK).

Bacterial strains, plasmids and media

The clinical S. aureus isolates (n = 493) employed in this study comprised: (i) community-derived isolates from patients attending a general practitioner in Morley, West Yorkshire, UK (n = 82); (ii) a collection of methicillin-sensitive S. aureus (MSSA) isolated in 1997 by the Routine Diagnostic Laboratory (RDL) of the Leeds General Infirmary (LGI), Leeds, UK (n = 45); (iii) mixed MSSA and methicillin-resistant S. aureus (MRSA) isolated during the period 1998–1999 from both hospital and community sources by the RDL and LGI (n = 293); (iv) a collection of MRSA from around the world,14 which included the original Jevons strain15 and prevalent UK epidemic MRSA types 15 and 16 (n = 17); (v) hetero- and homogeneous vancomycin-intermediate resistant S. aureus (VISA) isolates (n = 7) comprising strains Mu3, Mu5016 (provided by Dr D. Livermore, PHLS, Colindale, UK), 9/642(2), 9/642(26),17 99.3759V,18 992NJ and 963sm;19 and (vi) sputum isolates from CF patients (n = 49, of which 32 were obtained from The Royal Brompton Hospital, London and 17 were from the LGI, Leeds).

S. aureus 8325-420 was used as a non-clinical control strain for mutation frequency determinations and as a source of staphylococcal chromosomal DNA. E. coli JM109 (Promega, Southampton, UK) and S. aureus RN422021 were used as hosts for cloning. Plasmid pCR-Blunt (Invitrogen, Paisley, UK) and pGEM-T Easy (Promega) were used for introducing DNA into E. coli, and the broad host-range replicon, pJIM2246,22 was used for introducing DNA into S. aureus. Thermosensitive vector pG+host923 was used for directed plasmid integration in S. aureus.

For routine culture, strains were grown in Luria–Bertani (LB) broth (Fisher, Loughborough, UK) or on LB agar (Fisher) at 37°C.

Antibiotic susceptibility testing

MICs were determined using an agar dilution method24 in Iso-Sensitest agar (Oxoid, Basingstoke, UK), using inocula in Iso-Sensitest broth (Oxoid) of 106 cfu/spot. Vancomycin MICs were determined by agar dilution in brain–heart infusion agar (Oxoid). After 24 h aerobic incubation at 37°C, the MIC was defined as the lowest concentration of antibiotic that prevented visible growth of bacteria.

Standard procedure for determination of mutation frequencies

Bacteria were grown in Iso-Sensitest broth until they reached late logarithmic phase (OD600 c. 1). Undiluted aliquots were then plated on to antibiotic-containing (4 x MIC) (selective) Iso-Sensitest agar. Aliquots of diluted cultures were spread on antibiotic-free (non-selective) Iso-Sensitest agar to give a total viable count. Three independent cultures were sampled in triplicate to minimize error caused by inter- and intra-sample variation. Mutation frequency was calculated as the number of resistant mutants recovered on the selective plates as a proportion of total viable cells after 24 h incubation.25

Screening of mutation frequencies in clinical isolates

An initial screen was performed to evaluate the mutation frequency of all 493 strains, which involved plating single cultures on to agar containing 4 x MIC of rifampicin. Strains exhibiting an apparent two-fold or greater elevation in mutation frequency in this screen relative to 8325-4 were re-evaluated for their mutation frequencies to at least two other drugs (usually fusidic acid and norfloxacin or trimethoprim) at 4 x MIC using the standard procedure described above. This was necessary to distinguish strains that exhibit a true elevated frequency of mutation from those that harbour a pre-existing subpopulation resistant to only one of the drugs.

Mutation frequencies in stationary phase cultures

Strains were grown in three 60 mL batch cultures at 37°C with shaking until culture turbidity (OD600) reached a plateau (stationary phase). The beginning of stationary phase was designated time zero, and samples were processed as described above under standard procedure for determination of mutation frequencies. Further samples were processed at 24 h intervals until viable cell numbers dropped to the point at which resistant mutants could no longer be recovered.

PCR and DNA sequencing

Oligonucleotide primers were designed using Oligo 5.0 (MBI, Cascade, USA) and synthesized by MWG Biotech (Milton Keynes, UK). Primers were based either on raw sequence data from the S. aureus 8325 sequencing project (http://www.genome.ou.edu/staph.html) or on sequence data from the Mu50 genome.11

Standard PCR amplifications utilized PCR Master Mix (ABgene, Epsom, UK) and crude cell lysates. Long and accurate (LA)-PCR was performed using either PfuTurbo (Stratagene, Amsterdam, The Netherlands) or Extensor Hi-fidelity PCR Master Mix (ABgene) on high-purity genomic DNA templates prepared using the QIAamp kit (Qiagen, Crawley, UK) in accordance with the manufacturer’s instructions. Successful PCR amplification was confirmed by agarose gel electrophoresis.26 DNA sequencing was carried out by Lark Technologies Incorporated (Saffron Walden, Essex, UK) on an Applied Biosystems 377 DNA sequencer.

DNA manipulation

Recombinant DNA procedures were performed as described previously.26 Restriction endonucleases, calf intestinal alkaline phosphatase and the LigaFast Rapid DNA Ligation System were from Promega. Purification of DNA following PCR or restriction was achieved using the QIAquick PCR Purification Kit or the Qiagen MinElute Kit (both from Qiagen). Plasmid DNA was purified from E. coli using the Plasmid Mini Kit (Qiagen) in accordance with the manufacturer’s instructions. The same kit was used to purify plasmid DNA from S. aureus, although lysostaphin (100 mg/L) (Sigma-Aldrich) was added to the resuspension buffer (P1) to facilitate cell lysis.

Transformation

Chemically competent E. coli JM109 cells were transformed by heat-shock in accordance with manufacturer’s instructions. Electro-competent S. aureus cells were prepared and transformed as described previously.27

Directed plasmid integration

The strategy for gene disruption by targeted plasmid integration was based on procedures described previously.28,29 An 820 bp internal gene fragment of mutS was PCR amplified from 8325-4 using primers MutS1 (5'-CGCATATCGAGGATGTTGTTCAATA) and MutS2 (5'-GCTTTGTCTTCCGCACCTAAAAT), ligated to pGEM-T Easy and transformed into E. coli. Once the identity of the insert had been confirmed by sequencing, it was excised on an EcoRI restriction fragment and ligated to EcoRI-linearized pG+host9. Following ethanol precipitation, this construct was electroporated into RN4220, and transformants selected at 27°C on NYE agar27 containing 10 mg/L erythromycin.

Transformants were screened for the presence of plasmid containing the mutS gene fragment by sizing inserts from EcoRI-digested plasmid mini-preps on agarose. DNA inserts of the correct size were eluted from the gel and subjected to PCR analysis to confirm their identity. The broth culture of one successful transformant containing pG+host9::{Delta}mutS was serially diluted to 10–5 and spread on to a series of LB agar plates each containing 10 mg/L erythromycin. Plates were incubated at 42°C to force integration of the thermosensitive replicon into the chromosome at the mutS locus. In parallel, RN4220 transformed with pG+host9 containing no insert was subjected to identical platings. This demonstrated that non-specific integration of the plasmid into the S. aureus chromosome did not occur.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sequencing of the mutSL operon of 8325-4

To verify that the control strain for mutation frequency determinations contained an intact mutSL operon, and to provide a source of mutSL for further experiments, a 5072 bp region spanning this operon was long-PCR amplified from 8325-4 using primers MutS/L I (5'-TTTTATTAGGTTTGAGGCGTTTTC) and MutS/L II (5'-CCTTTTGGCTTGTATTGCTGAAT). This amplicon was ligated into pCR-Blunt, propagated in E. coli and subjected to DNA sequencing.

The complete DNA sequence of this operon (GenBank accession no. AF378369) is identical to that of the 8325 genome sequencing project and comprises intact open reading frames for mutS and mutL. The predicted S. aureus MutS protein exhibits 53%, 46% and 38% identity to MutS from Bacillus subtilis, HexA from Streptococcus pneumoniae and MutS from E. coli, respectively, whereas the staphylococcal MutL is 50%, 41% and 27% identical to its counterpart in these species, respectively.

Identification and characterization of a mutS insertion mutant in S. aureus RN4220

Putative mutS strains of S. aureus RN4220 were generated by the directed plasmid integration procedure outlined in Materials and methods. The absence of non-integrated (temperature-insensitive) plasmid in the putative integrants was verified by plasmid purification procedures. Integrants were subjected to preliminary mutation frequency analysis, resulting in the identification of several integrants possessing at least 50-fold increases in mutation frequency for rifampicin resistance compared with RN4220.

One integrant with an increased mutation frequency was chosen at random for more detailed evaluation. An LA-PCR-based strategy was employed to confirm that the site of plasmid integration was within mutS. High-purity DNA templates from the integrant, and RN4220 as control, were subjected to long PCR with primers intI (5'-ATTACGACGATAAACCCTAACGAAGTTGTT) and intII (5'-CAAATCATCTGCCGCACCTATTCTA), which amplify a 1492 bp fragment of mutS spanning the 820 bp locus employed for targeted integration. A c. 1.5 kb product was obtained when using RN4220 template DNA, as expected, but a c. 6.1 kb product (1.5 kb of native target + 4.6 kb of integrated plasmid) was amplified from the integrant (Figure 1). This confirms that the site of plasmid integration is mutS. However, the PCR also generated a small quantity of a c. 1.5 kb product from the integrant (Figure 1), probably indicating that low-level spontaneous excision of the integrated plasmid occurs in the absence of selection pressure.



View larger version (111K):
[in this window]
[in a new window]
 
Figure 1. Gel electrophoresis of LA-PCR on RN4220 and RN4220 mutS using primers intI and intII.

 
Phenotypic examination of the integrant confirmed that the mutS gene had been successfully disrupted. In addition to an elevated mutation frequency for rifampicin (78-fold increase) this strain also displayed enhanced mutation frequencies for resistance to fusidic acid (15-fold increase), norfloxacin (42-fold increase) and mupirocin (44-fold increase) (Table 1), and was designated RN4220 mutS.


View this table:
[in this window]
[in a new window]
 
Table 1.  Antimicrobial susceptibilities and mutation frequencies of S. aureus strains
 
Complementation analysis

A plasmid construct for complementing deficiency of the mutSL operon was generated to verify that disruption at this locus was responsible for the observed increase in mutation frequency. The mutSL operon (including the promoter region) was excised from pCR-Blunt::mutSL by restriction with SacI/NotI and ligated to pJIM2246 digested similarly. This construct was initially transformed into JM109, before recovery and introduction into RN4220 mutS. Supplying mutSL in trans on pJIM2246 complemented disruption of MMR in RN4220 mutS, returning the mutation frequency for rifampicin resistance to 1.47 ± 0.66 x 10–7. This is identical to that originally observed for RN4220 (Table 1).

Mutation frequencies in clinical isolates

Determination of mutation frequency to rifampicin resistance is a sensitive method for examining bacterial mutation frequencies.4 Therefore, MIC determinations were performed on all 493 S. aureus isolates (Figure 2) to determine whether rifampicin could be used to quantify mutation frequencies in these clinical strains.



View larger version (15K):
[in this window]
[in a new window]
 
Figure 2. Activity of rifampicin against 493 clinical isolates of S. aureus. Shading indicates level of resistance according to the breakpoints proposed by Aubry-Damon et al.30 White bars, susceptible (MIC <= 0.5 mg/L); grey bars, intermediate resistant (MIC 1–4 mg/L); black bars, resistant (MIC >= 8 mg/L).

 
Rifampicin retained activity against the majority of clinical isolates. On the basis of the low (<=0.5 mg/L) and high (>=8 mg/L) rifampicin resistance breakpoints proposed by Aubry-Damon et al.,30 452 (91.7%) of the isolates in the collection retained susceptibility to rifampicin, 14 (2.8%) exhibited intermediate resistance and only 27 (5.5%) (MIC >= 8 mg/L) were resistant (Figure 2).

Resistant strains exhibiting MICs <= 64 mg/L remained sufficiently susceptible to rifampicin to perform mutant selections at 4 x MIC. When high-level resistance prevented the use of rifampicin for determining mutation frequencies, selections were performed using alternative drugs (fusidic acid, norfloxacin or trimethoprim) at 4 x MIC.

Strains apparently exhibiting elevated mutation frequencies (>=2-fold) relative to 8325-4 were identified in the initial screen, although subsequent accurate mutation frequency determinations with rifampicin and the other antibiotics failed to detect any true hypermutable phenotypes (data not shown). Only one clinical strain (G21) exhibited a small, but consistent, elevation in mutation frequency to more than one drug, displaying reproducible three- to four-fold increases in mutation frequency for resistance to rifampicin, fusidic acid and norfloxacin compared with the laboratory strain 8325-4 (Table 1); however, the mutation frequencies exhibited by strain G21 were substantially lower than those observed for RN4220 mutS (Table 1).

Subsequent attempts to characterize the genetic basis of elevated mutation frequencies in G21 proved unsuccessful since the mild mutator phenotype initially detected in this strain was lost on long-term cryogenic storage (data not shown).

Lack of a mutator phenotype in VISA strain Mu50

Genome sequence data originally generated from VISA strain Mu50 and the vancomycin-sensitive progenitor (N315) by Kuroda et al.,11 and recently analysed in a pair-wise fashion by Avison et al.,12 indicate that the mutS gene of Mu50 may be inactive. An apparent frameshift towards the N-terminus of the gene truncates the predicted product encoded by this locus from the 872-amino-acid MutS protein to a 100-amino-acid polypeptide. Given the likely multi-locus nature of vancomycin resistance in S. aureus,12 an elevated mutation frequency in Mu50 could provide a basis for understanding the development of vancomycin resistance in staphylococci.

However, Mu50 was not identified as hypermutable in our screen. To confirm this finding, Mu50 was subjected to accurate mutation frequency determinations for resistance to fusidic acid and mupirocin. These drugs were employed as they are amongst the very few agents to which Mu50 remains susceptible, and for which mutants are generated at readily quantifiable frequencies. The mutation frequencies for Mu50 were not significantly different from those obtained with control strains 8325-4 and RN4220, but were significantly lower than the frequencies observed for the mutS-disrupted derivative of RN4220 (Table 1).

The lack of a hypermutable phenotype in Mu50 suggested that the strain we examined might differ from that previously sequenced, or that a sequencing error occurred during the original analysis of the mutS gene. Although a non-hypermutable phenotype for Mu50 has been independently verified (M. Avison, personal communication), phenotypic and genotypic tests were performed to ensure that the strain we analysed was indeed the Mu50 previously sequenced. The genotypic tests involved PCR amplification and sequencing of a portion of rpoB (primers F3 and F430), uhpT [pri- mers SAV0222F (5'-CGAAACGTGGCCGATACTTA) and SAV0222R (5'-ATCGTATTAACTGCATCGCCTTTAC)] and the entire bleO gene [primers SAV0034F (5'-AAGGATCCAAAGGATTAATTATGAGC) and SAV0034R (5'-ATGGATCCTCGGTTTTCTAGTGTAAC)]. In all instances, the data obtained for the Mu50 we examined were identical to those expected of the sequenced strain (Table 2).


View this table:
[in this window]
[in a new window]
 
Table 2.  Comparison of phenotypic and genotypic properties of the sequenced Mu50 strain and that used in the analysis reported here
 
PCR amplification [using primers MutS F (5'-TTGAGCTCGGTTTGAGGCGTTTTCT) and MutS R (5'-GTGAGCTCATTTTCCCCATTTTGCAACAC)] and DNA sequencing [using primers MutS F and MutS S (5'-TTTGTTTATCATCTAC)] of part of the mutS gene from Mu50 were therefore carried out to determine whether or not the published sequence of this gene was in error. Although redundancy of sequencing reads usually renders mistakes in completed genomes rare, a number of errors have already been reported in the Mu50 genome.12 Sequencing from primer MutS S revealed no frameshift in the mutS gene. However, sequencing from MutS F suggested a frameshift, with three adenines (nucleotide positions 272–274 in SAV1296) in place of two (positions 272/273), exactly as found in the published sequence of mutS from Mu50 (Figure 3). Closer examination of the chromatogram from this sequencing read suggested that this apparent frameshift was an error. Thus, the adenine peak comprises only two, not three, peaks (Figure 3), corroborating the sequence determination from the reverse primer.



View larger version (20K):
[in this window]
[in a new window]
 
Figure 3. Chromatogram from the DNA sequence determination of mutS. Sequences in Mu50 are shown from forward (lower trace) and reverse (upper trace) primers at the locus where the apparent frameshift occurs. The lower rows of letters in each case are the automatic base calls, and the artefactual adenine leading to the apparent frameshift is circled.

 
These results indicate that mutS in Mu50 is intact, and that this strain is not hypermutable. In addition, they provide a rationale for the misread included in the Mu50 genome sequence; the region of mutS carrying the apparent frameshift appears liable to sequencing error.

Determination of mutation frequency in stationary phase

Mutation frequencies for resistance to fusidic acid, rifampicin and norfloxacin were evaluated for strains G21 and 8325-4 at several time points during stationary phase growth. No significant increases in mutation frequency to any of the drugs were observed in either strain as they progressed into stationary phase (Figure 4).



View larger version (17K):
[in this window]
[in a new window]
 
Figure 4. Changes in mutation frequency that accompany progression into stationary phase of (a) G21 and (b) 8325-4. Frequencies of spontaneous mutation to fusidic acid, rifampicin and norfloxacin resistance were determined as described in Materials and methods. Time 0 indicates the end of exponential growth. Frequencies of mutation have been converted to logarithms (base 10) to allow accurate calculation of t-test-based confidence limits. Samples were taken at exactly 24 h intervals, although individual values for different antibiotics have been artificially staggered to enable error bars to be visualized. Narrow lines, norfloxacin; bold lines, fusidic acid; dashed lines, rifampicin.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutator phenotypes have been described in a wide variety of prokaryotic and eukaryotic cells. The existence of hypermutable populations in diverse bacterial genera and especially in bacteria recovered from stressful environments3,5 strongly suggests that hypermutability represents a fundamental bacterial strategy to accelerate evolution and enhance adaptivity. Since S. aureus is an important pathogen exhibiting a remarkable degree of adaptivity, particularly in respect of antibiotic resistance, we have carried out several experiments on this species to assess the potential importance of mutability in shaping the adaptive process.

Examination of 493 clinical strains of S. aureus utilizing a sensitive mutation frequency screen failed to detect any strong mutators. Assuming such phenotypes do exist in natural S. aureus populations, the data reported here argue for an extremely low clinical prevalence (<0.25%). The likely rare occurrence of hypermutators in S. aureus contrasts with a higher prevalence of hypermutable strains in other genera.4,5 This implies that hypermutability is not a major factor in the evolution of staphylococcal drug resistance.

Significantly, no mutators were identified amongst S. aureus strains isolated from CF patients. The stressful environment of the CF lung is thought to provide a niche where hypermutability offers colonists an adaptive advantage, a finding borne out by the high prevalence (c. 20%) of hypermutable P. aeruginosa isolates recovered from this site.5 S. aureus is an extremely common colonizer of the CF lung, particularly in younger CF patients, and is likely to be subject to many of the stresses also encountered by P. aeruginosa. However, failure to identify hypermutable S. aureus isolates from this niche suggests that hypermutability might represent a specific pseudomonal response to the CF lung, and not one that occurs in S. aureus.

The genome sequence of VISA strain Mu50 indicates that this organism appears to be deficient in MutS.11,12 However, mutation frequency determinations and re-sequencing of a portion of the mutS gene have revealed that Mu50 is not hypermutable and in fact contains an intact mutS gene. Since we have also been unable to detect hypermutability in several other VISA isolates analysed here, it appears that permanent changes in basal mutability have not contributed to the development of vancomycin resistance in these strains, or in Mu50.

Despite our inability to detect a natural strong mutator strain of S. aureus, this study demonstrates that the potential for a mutator phenotype nevertheless exists in this organism. Accordingly, we have shown that the products of the staphylococcal mutSL operon are active and serve to reduce the accumulation of mutations in S. aureus. We have also demonstrated that this repair pathway is not essential for staphylococcal viability. Therefore, it is feasible that clinical S. aureus strains may occur that possess defects in DNA repair and, consequently, elevated mutation frequencies.

The strength of the mutator phenotype associated with a disrupted MMR pathway in S. aureus appears similar to that reported for the closely related Gram-positive organism B. subtilis. Thus, a 78-fold increase in mutation frequency to rifampicin resistance was observed in RN4220 mutS, an increase similar to the 60-fold increase reported for a mutSL mutant of B. subtilis.9 However, these increases in mutation frequency are low compared with those observed in Gram-negative organisms lacking MMR.4,5 It therefore seems that Gram-positive organisms may be less reliant on the MMR system to ensure genome fidelity.

Aside from permanent genetic changes that lead to a mutator phenotype, transient mutator phenotypes may also arise in bacteria, particularly during periods of environmental stress.1 This strategy enables an organism to profit temporarily from the adaptive benefits of an elevated mutation rate without incurring continuous accumulation of deleterious mutations. Mutation frequencies for antibiotic resistance in M. smegmatis increase up to 104-fold during the stationary phase.13 This increase may result from a deficit of MMR components, as expression of both MutS and MutH is heavily down-regulated during the stationary phase in E. coli.31 However, neither S. aureus 8325-4 nor G21 demonstrated an increase in mutation frequency on progression into stationary phase.

Apart from providing fundamental information on the MMR system in S. aureus, the mutS mutant described here may be a useful laboratory tool for evaluation of new antimicrobial agents. The elevated mutation frequency of this strain may help to assess the potential for emergence of bacterial resistance to novel antibiotics in development.2,32


    Acknowledgements
 
E. coli VE6838, the source of pG+host9, was provided by Dr E. Maguin (INRA, France), vector pJIM2246 by Dr P. Renault (INRA) and strain 99.3759V by Dr D. Morrison (Glasgow Royal Infirmary, Glasgow, UK). We thank Dr F. M’Zali (Leeds University) for providing a large number of clinical S. aureus strains and Dr M. H. Wilcox (LGI, Leeds) and M. Chadwick (The Royal Brompton Hospital, London) for providing isolates from CF patients. We thank Artur Morais Moita and Julian Hurdle for technical assistance, and Dr M. H. Wilcox for helpful discussions.


    Footnotes
 
* Corresponding author. Tel: +44-113-233-5604; Fax: +44-113-233-5638; E-mail: i.chopra{at}leeds.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Martinez, J. L. & Baquero, F. (2000). Mutation frequencies and antibiotic resistance. Antimicrobial Agents and Chemotherapy 44, 1771–7.[Free Full Text]

2 . Miller, K., O’Neill, A. J. & Chopra, I. (2002). Response of Escherichia coli hypermutators to selection pressure with antimicrobial agents from different classes. Journal of Antimicrobial Chemotherapy 49, 925–34.[Abstract/Free Full Text]

3 . Bjorkholm, B., Sjolund, M., Falk, P. G., Berg, O. G., Engstrand, L. & Andersson, D. I. (2001). Mutation frequency and biological cost of antibiotic resistance in Helicobacter pylori. Proceedings of the National Academy of Sciences, USA 98, 14607–12.[Abstract/Free Full Text]

4 . LeClerc, J. E., Li, B. G., Payne, W. L. & Cebula, T. A. (1996). High mutation frequencies among Escherichia coli and Salmonella pathogens. Science 274, 1208–11.[Abstract/Free Full Text]

5 . Oliver, A., Canton, R., Campo, P., Baquero, F. & Blazquez, J. (2000). High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 288, 1251–4.[Abstract/Free Full Text]

6 . Maranan, M. C., Moreira, B., BoyleVavra, S. & Daum, R. S. (1997). Antimicrobial resistance in staphylococci—epidemiology, molecular mechanisms, and clinical relevance. Infectious Disease Clinics of North America 11, 813–49.[ISI][Medline]

7 . Schmitz, F. J., Fluit, A. C., Hafner, D., Beeck, A., Perdikouli, M., Boos, M. et al. (2000). Development of resistance to ciprofloxacin, rifampin, and mupirocin in methicillin-susceptible and -resistant Staphylococcus aureus isolates. Antimicrobial Agents and Chemotherapy 44, 3229–31.[Abstract/Free Full Text]

8 . Cuny, C. & Witte, W. (2000). In vitro activity of linezolid against staphylococci. Clinical Microbiology and Infection 6, 331–3.[ISI][Medline]

9 . Ginetti, F., Perego, M., Albertini, A. M. & Galizzi, A. (1996). Bacillus subtilis mutS mutL operon: identification, nucleotide sequence and mutagenesis. Microbiology 142, 2021–9.[Abstract]

10 . Prudhomme, M., Mejean, V., Martin, B. & Claverys, J. P. (1991). Mismatch repair genes of Streptococcus pneumoniae: HexA confers a mutator phenotype in Escherichia coli by negative complementation. Journal of Bacteriology 173, 7196–203.[ISI][Medline]

11 . Kuroda, M., Ohta, T., Uchiyama, I., Baba, T., Yuzawa, H., Kobayashi, I. et al. (2001). Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357, 1225–40.[ISI][Medline]

12 . Avison, M. B., Bennett, P. M., Howe, R. A. & Walsh, T. R. (2002). Preliminary analysis of the genetic basis for vancomycin resistance in Staphylococcus aureus strain Mu50. Journal of Antimicrobial Chemotherapy 49, 255–60.[Abstract/Free Full Text]

13 . Karunakaran, P. & Davies, J. (2000). Genetic antagonism and hypermutability in Mycobacterium smegmatis. Journal of Bacteriology 182, 3331–5.[Abstract/Free Full Text]

14 . Kumari, D. N. P., Keer, V., Hawkey, P. M., Parnell, P., Joseph, N., Richardson, J. F. et al. (1997). Comparison and application of ribosome spacer DNA amplicon polymorphisms and pulsed-field gel electrophoresis for differentiation of methicillin-resistant Staphylococcus aureus strains. Journal of Clinical Microbiology 35, 881–5.[Abstract]

15 . Jevons, M. P. (1961). ‘Celbenin’-resistant staphylococci. British Medical Journal I, 124–5.

16 . Hiramatsu, K. (1998). Vancomycin resistance in staphylococci. Drug Resistance Updates 1, 135–50.[ISI]

17 . Howe, R. A., Bowker, K. E., Walsh, T. R., Feest, T. G. & MacGowan, A. P. (1998). Vancomycin-resistant Staphylococcus aureus. Lancet 351, 602.[ISI][Medline]

18 . Hood, J., Edwards, G. F. S., Curran, E., Thakker, B., Lockhart, M., Morrison, D. et al. (2000). Vancomycin-intermediate resistant Staphylococcus aureus in Scotland. Fourth Decennial International Conference on Nosocomial Infections and Healthcare, Atlanta, GA, USA.

19 . Anonymous. (1997). Update: Staphylococcus aureus with reduced susceptibility to vancomycin—United States, 1997. Morbidity and Mortality Weekly Report 46, 813–5.[Medline]

20 . Novick, R. (1967). Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology 33, 155–66.[ISI][Medline]

21 . Fairweather, N., Kennedy, S., Foster, T. J., Kehoe, M. & Dougan, G. (1983). Expression of a cloned Staphylococcus aureus alpha-hemolysin determinant in Bacillus subtilis and Staphylococcus aureus. Infection and Immunity 41, 1112–7.[ISI][Medline]

22 . Renault, P., Corthier, G., Goupil, N., Delorme, C. & Ehrlich, S. D. (1996). Plasmid vectors for Gram-positive bacteria switching from high to low copy number. Gene 183, 175–82.[ISI][Medline]

23 . Maguin, E., Prevost, H., Ehrlich, S. D. & Gruss, A. (1996). Efficient insertional mutagenesis in lactococci and other Gram-positive bacteria. Journal of Bacteriology 178, 931–5.[Abstract]

24 . British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing; report of the working party on antibiotic sensitivity testing of the British Society for Antimicrobial Chemotherapy. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1–50.[ISI][Medline]

25 . O’Neill, A. J., Cove, J. H. & Chopra, I. (2001). Mutation frequencies for resistance to fusidic acid and rifampicin in Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 47, 647–50.[Abstract/Free Full Text]

26 . Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

27 . Schenk, S. & Laddaga, R. A. (1992). Improved method for electroporation of Staphylococcus aureus. FEMS Microbiology Letters 73, 133–8.[Medline]

28 . Kaatz, G. W., Seo, S. M. & Foster, T. J. (1999). Introduction of a norA promoter region mutation into the chromosome of a fluoroquinolone-susceptible strain of Staphylococcus aureus using plasmid integration. Antimicrobial Agents and Chemotherapy 43, 2222–4.[Abstract/Free Full Text]

29 . Foster, T. J. (1998). Molecular genetic analysis of staphylococcal virulence. Methods in Microbiology 27, 433–54.[ISI]

30 . Aubry-Damon, H., Soussy, C. J. & Courvalin, P. (1998). Characterization of mutations in the rpoB gene that confer rifampin resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 42, 2590–4.[Abstract/Free Full Text]

31 . Feng, G., Tsui, H. C. & Winkler, M. E. (1996). Depletion of the cellular amounts of the MutS and MutH methyl-directed mismatch repair proteins in stationary-phase Escherichia coli K-12 cells. Journal of Bacteriology 178, 2388–96.[Abstract]

32 . O’Neill, A. J., Chopra, I., Martínez, J. L. & Baquero, F. (2001). Use of mutator strains for characterization of novel antimicrobial agents. Antimicrobial Agents and Chemotherapy 45, 1599–600.[Free Full Text]