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
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Abstract |
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Introduction |
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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
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Materials and methods |
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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 19981999 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 LuriaBertani (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 brainheart 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 manufacturers 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 manufacturers 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 manufacturers 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::mutS was serially diluted to 105 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.
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Results |
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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.
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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 107. 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.
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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).
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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).
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Discussion |
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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
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Acknowledgements |
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Footnotes |
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References |
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