Intact mutS in Laboratory-Derived and Clinical Glycopeptide-Intermediate Staphylococcus aureus Strains

The mutS gene of the methyl-directed mismatch repair systemwas sequenced in 10 parent and glycopeptide-intermediate Staphylococcusaureus strains. The mutS gene was intact in all strains studied.Hence, mutations in this gene had played no role in the developmentof vancomycin resistance in these strains.

INTRODUCTION

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Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) strains arecommon causes of hospital-acquired infections, and recentlytheir prominence as causes of community-onset infections hasincreased (2, 5). For many years, the glycopeptide antibioticvancomycin was the agent to which all MRSA strains were uniformlysusceptible. However, since 1997, clinical isolates with reducedsusceptibility to glycopeptide antibiotics (glycopeptide-intermediateS. aureus [GISA]) have arisen in various countries around theworld (11). Treatment failure has been a common result of infectionswith such strains. A typical vancomycin MIC for GISA strainsis 8 µg ml^-1. Several laboratories have reported on laboratorypassage-selected GISA strains for which, for the most part,the MICs are similar (7, 20, 22). Recently, the first fullyvancomycin-resistant S. aureus strain (for which the MIC is>128 µg ml^-1) was reported. Full resistance was dueto the acquisition of van genes from an enterococcal source(4).

In contrast to van-mediated resistance, the mechanism of vancomycinresistance in GISA strains is not fully understood. A varietyof alterations in peptidoglycan structure compared to that ofsusceptible S. aureus have been reported for GISA strains (3,9, 23). The role of a thickened cell wall in vancomycin resistancein GISA strains has been emphasized (6). Vancomycin resistanceis believed to evolve in multiple mutational steps (10, 20,24). Recently Avison et al. (1) reported on an in silico investigationcomparing the genomes of clinical vancomycin-intermediate strainMu50 with those of several unrelated vancomycin-susceptiblestrains. Several loss-of-function mutations affecting cell wallbiosynthesis and intermediary metabolism genes were identifiedin Mu50.

Antibiotic resistance can arise by mutations in different chromosomalloci, and this process is affected by the mutation rate (14).Mutator strains that show significantly increased mutation ratesare present in many bacterial populations (15). Such strainscan develop a variability of alleles that may allow them tosurvive better in the host environment (13) or in the presenceof antibiotics (18). The methyl-directed mismatch repair systemsof mutator strains are typically defective (13, 18). Methyl-directedmismatch repair is dependent on the MutS, MutL, and MutH proteinsand corrects mismatched base pairs arising during replication(16). O'Neill and Chopra (19) insertionally inactivated themutS gene of S. aureus strain RN4220 and observed increasedmutation frequencies with rifampin, fusidic acid, norfloxacin,and mupirocin. Schaaff et al. (21) have recently reported onthe development of vancomycin resistance in a mutator derivativeof strain RN4220, created through a knockout of mutS. Vancomycinresistance evolved more rapidly and to a higher level in themutS knockout strain than in the parent with an intact mutSgene.

Pfeltz et al. (20) subjected 12 S. aureus strains of variousgenetic backgrounds and methicillin resistance levels to selectionfor vancomycin resistance. Six strains acquired vancomycin resistancerapidly, four strains acquired it slowly, and two strains didnot acquire it at all. MRSA strains tended to acquire the vancomycinresistance phenotype more rapidly and to a higher level thanrelated susceptible strains. Schaaff et al. (21) suggested thatmutator clones might be present in MRSA populations at a highfrequency and that they may play a role in the ease of acquisitionof vancomycin resistance.

We decided to test this hypothesis by determining the statusof mutS in strains of S. aureus that gave rise to vancomycin-resistantstrains rapidly, slowly, and not at all. The mutS gene sequencewas determined by sequencing PCR products of the mutS gene.Second, we report on the status of mutS in GISA strains derivedfrom these parent strains. In addition, we report the sequencesof the mutS genes in two clinical GISA strains. The mutS genewas intact in all strains sequenced, and hence our results didnot support a major role for mutS mutations in the developmentof GISA strains.

Strains and growth conditions.

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Strains and Growth conditions.

The strains used in the study and their relevant characteristicsare given in Table 1 (20). The strains were stored at -80°Cin 30% (vol/vol) glycerol and periodically streaked onto trypticsoy agar (Difco Laboratories, Detroit, Mich.) to provide workingplates that were stored at 4°C. Cultures on tryptic soyagar were grown at 37°C. Tryptic soy broth (Difco Laboratories)was used for liquid cultures, which were grown at 37°C withshaking at 250 rpm.

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TABLE 1. Strains used

DNA isolation.

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Dna isolation.

Overnight-grown 5-ml cultures were used to isolate genomic DNAwith a Wizard genomic DNA isolation kit (Promega Corporation,Madison, Wis.). GISA strains which are less susceptible to lysostaphin(20) were lysed by increasing the concentration of lysostaphinfrom 100 to 500 µg ml^-1 in 0.05 M Tris-HCl (pH 7.5) containing145 mM NaCl.

Amplification and sequencing of mutS in S. aureus.

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Amplification and Sequencing of...

Primers MutS-1 (5'-TTCAAGACTTCTTCATTAAACCGTTAATG-3') and MutS-9(5'-GAGTTCTTTAATTTTCCCCATTTTGC-3') (21) were constructed (Sigma-Genosys,TheWoodlands, Tex.) to amplify the complete mutS gene as wellas its putative promoter and ribosomal binding site. PCR amplificationwas carried out with the GeneAmp amplification system (AppliedBiosystems, Foster City, Calif.) by using Taq DNA polymerase(Promega Corporation). The amplified product was purified witha QIAquick PCR purification kit (QIAGEN Inc, Valencia, Calif.)for direct PCR product sequencing. The purified PCR product( $~$ 3.2 kb) was completely sequenced by using a primer walkingstrategy. The terminator cycle sequencing method with ABI PrismBigDye v3.0 chemistry was used to sequence the mutS PCR productin an ABI Prism310 genetic analyzer (Applied Biosystems).

Sequence analysis.

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Sequence analysis.

Multiple alignment of the translated mutS gene sequences ofthe strains studied, including strains Mu50 (a well-studiedclinical GISA strain), N315 (a hospital MRSA strain) (12), andMW2 (a community MRSA strain) (2), was carried out by usingthe CLUSTAL W 1.8 multiple-sequence alignment tool. The sequencesof mutS genes from Mu50, N315, and MW2 were obtained from theNational Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov).

Only minor changes were observed in the nucleotide sequencesof the mutS genes from the various strains (Table 2). At positions1424 and 2423, the clinical GISA strains had G nucleotides,compared to T and A nucleotides, respectively, in the otherstrains. At position 2642, the clinical GISA strains had a Cnucleotide instead of the T nucleotide found in the other strainsstudied. At position 2418, strain BB399V₁₂ had a C nucleotideinstead of the A nucleotide in the other strains. At position2820, an A was substituted for G in strains RN450 and BB270V₁₅.This substitution was responsible for the substitution of lysine(K) for glutamic acid (E) in these two strains.

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TABLE 2. Nucleotide sequence changes in mutS and its putative promoter

The only difference in nucleotide sequence observed in the putativepromoter sequence was a G nucleotide at position 235 in strainsMu50, N315, HIP5836, and HIP5827 instead of the A nucleotidein the other strains.

The translated mutS sequences were highly conserved. Only twoamino acid changes were detected. First, a proline (P) residuewas substituted for a threonine (T) residue at position 706in strain HIP5836. This change results in what is consideredto be a weaker conservation group (STPA) according to the GonnetPam250 matrix. Second, strains RN450 and BB270V₁₅ had a K residueinstead of an E residue at position 840. This change also resultsin a weak conservation group (NDEQHK). The same amino acid substitutionin strains RN450 and BB270V₁₅ is consistent with the fact thatboth strains are of the 8325 lineage. However, being of thislineage appears to play no role in the acquisition of vancomycinresistance, because strain RN450 failed to develop vancomycinresistance.

In none of the translated mutS sequences was a mutation thatwould lead to a nonfunctional MutS protein detected. It is possiblethat mutations in the mutL and mutH genes may be present inour strains, although in naturally occurring enteric bacterialmutators (13) and Pseudomonas aeruginosa (17), the preponderanceof mutations is in the mutS gene. It was originally believedthat mutS in GISA strain Mu50 was inactive, as the originalpublished genome sequence of this organism indicated that aframeshift mutation was present (12). However, the resequencingof a portion of mutS in Mu50 indicated that the gene was intact(19). It is possible that there may be differences in levelsof mut gene expression between different strains. Downregulationof MutS and, to a lesser extent, MutH is known to occur in stationary-phaseand starved bacteria (8). However, O'Neill and Chopra (19) foundno increase in mutation frequency in stationary-phase S. aureus.Thus, we have been unable to support the hypothesis that mutatorclones are present in MRSA isolates at a high frequency andthat they are responsible for the greater ease with which GISAstrains arise from such parent strains (20, 21).

A further implication of our results is that mutators do notplay a major role in the development of antibiotic resistancein S. aureus, which is in accord with the findings of O'Neilland Chopra (19). Those authors have speculated that gram-positivebacteria in general are less reliant on the methyl-directedmismatch repair system to ensure genetic fidelity. Recently,Willems et al. (25) found amino acid substitutions in MutSLof oxazolidinone-resistant or -susceptible strains of the gram-positivebacterium Enterococcus faecium but no evidence of hypermutablephenotypes.

Nucleotide sequence accession numbers.

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Nucleotide sequence accession...

The mutS sequences from the strains used in this study havebeen deposited in the GenBank database under accession numbersAY324086 (RN450), AY324087 (COL), AY324088 (COLV₅), AY324089(COLV₁₀), AY324090 (13136p^-m⁺), AY324091 (13136p^-m⁺V₅), AY324092(13136p^-m⁺V₂₀), AY324093 (BB270V₁₅), AY324094 (BB568V₁₅), AY324095(BB399V₁₂), AY324096 (HIP5836), and AY324097 (HIP5827).

ACKNOWLEDGMENTS

This work was supported by grants AI049964 and AI43970 fromthe National Institutes of Health.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120. Phone: (309) 438-7244. Fax: (309) 438-3722. E-mail: bjwilkin{at}ilstu.edu.

REFERENCES

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