The spread of multidrug-resistant Staphylococcus aureus (MRSA) strains in the clinical environment has begun to pose serious limits to treatment options. Yet virtually nothing is known about how resistance traits are acquired in vivo. Here, we apply the power of whole-genome sequencing to identify steps in the evolution of multidrug resistance in isogenic S. aureus isolates recovered periodically from the bloodstream of a patient undergoing chemotherapy with vancomycin and other antibiotics. After extensive therapy, the bacterium developed resistance, and treatment failed. Sequencing the first vancomycin susceptible isolate and the last vancomycin nonsusceptible isolate identified genome wide only 35 point mutations in 31 loci. These mutations appeared in a sequential order in isolates that were recovered at intermittent times during chemotherapy in parallel with increasing levels of resistance. The vancomycin nonsusceptible isolates also showed a 100-fold decrease in susceptibility to daptomycin, although this antibiotic was not used in the therapy. One of the mutated loci associated with decreasing vancomycin susceptibility (the vraR operon) was found to also carry mutations in six additional vancomycin nonsusceptible S. aureus isolates belonging to different genetic backgrounds and recovered from different geographic sites. As costs drop, whole-genome sequencing will become a useful tool in elucidating complex pathways of in vivo evolution in bacterial pathogens.

No abstract

A highly vancomycin-resistant mutant (MIC ‫؍‬ 100 g/ml) of Staphylococcus aureus, mutant VM, which was isolated in the laboratory by a step-pressure procedure, continued to grow and synthesize peptidoglycan in the presence of vancomycin (50 g/ml) in the medium, but the antibiotic completely inhibited cell wall turnover and autolysis, resulting in the accumulation of cell wall material at the cell surface and inhibition of daughter cell separation. Cultures of mutant VM removed vancomycin from the growth medium through binding the antibiotic to the cell walls, from which the antibiotic could be quantitatively recovered in biologically active form. Vancomycin blocked the in vitro hydrolysis of cell walls by autolytic enzyme extracts, lysostaphin and mutanolysin. Analysis of UDP-linked peptidoglycan precursors showed no evidence for the presence of Dlactate-terminating muropeptides. While there was no significant difference in the composition of muropeptide units of mutant and parental cell walls, the peptidoglycan of VM had a significantly lower degree of crosslinkage. These observations and the results of vancomycin-binding studies suggest alterations in the structural organization of the mutant cell walls such that access of the vancomycin molecules to the sites of wall biosynthesis is blocked.Multidrug-resistant strains of methicillin-resistant Staphylococcus aureus (MRSA) in which the therapeutic choice is often reduced to a small number of antibiotics, primarily vancomycin, have spread worldwide during the late 1980s and mid1990s. The appearance of vancomycin resistance among clinical isolates of enterococci has raised concern about transfer of the resistance genes to highly virulent strains of MRSA with obvious dire implications for chemotherapy. While current concern is directed primarily to interspecific transfer of enterococcal resistance genes, attention has also been paid to other, alternative vancomycin resistance mechanisms that may emerge among S. aureus and coagulase-negative strains of nosocomial staphylococci as a consequence of the extensive use of vancomycin in the hospital environment worldwide (for a review, see reference 42). Moderately increased vancomycin (and teicoplanin) MICs have indeed been noted among some clinical isolates of coagulase-negative staphylococci (3,6,20,31,32,40), and glycopeptide-resistant variants or mutants of staphylococci have also been isolated in several laboratories by the usual step-pressure procedures (5,7,8,16,18,40).While studying the effect of cell wall synthesis inhibitors on the expression of methicillin resistance in S. aureus, we observed rare staphylococcal cells that were able to form colonies on agar containing 6 and even 12 g of vancomycin per ml. Serial passages of such colonies in vancomycin-containing media (step-pressure procedure) resulted in the emergence of stable variants (mutants) with even higher levels of vancomycin resistance. One mutant for which the vancomycin MIC was 100 g/ml had the unique capacity to quantitatively remove vancomycin f...

Custom-designed gene chips (Affymetrix) were used to determine genetic relatedness and gene expression profiles in Staphylococcus aureus isolates with increasing MICs of vancomycin that were recovered over a period of several weeks from the blood and heart valve of a patient undergoing extensive vancomycin therapy. The isolates were found to be isogenic as determined by the GeneChip based genotyping approach and thus represented a unique opportunity to study changes in gene expression that may contribute to the vancomycin resistance phenotype. No differences in gene expression were detected between the parent strain, JH1, and JH15, isolated from the nares of a patient contact. Few expression changes were observed between blood and heart valve isolates with identical vancomycin MICs. A large number of genes had altered expression in the late stage JH9 isolate (MIC ‫؍‬ 8 g/ml) compared to JH1 (MIC ‫؍‬ 1 g/ml). Most genes with altered expression were involved in housekeeping functions or cell wall biosynthesis and regulation. The sortase-encoding genes, srtA and srtB, as well as several surface protein-encoding genes were downregulated in JH9. Two hypothetical protein-encoding genes, SAS016 and SA2343, were dramatically overexpressed in JH9. Interestingly, 27 of the genes with altered expression in JH9 grown in drug-free medium were found to be also overexpressed when the parental strain JH1 was briefly exposed to inhibitory concentrations of vancomycin, and more than half (17 of 27) of the genes with altered expression belonged to determinants that were proposed to form part of a general cell wall stress stimulon (S. Utaida et al., Microbiology 149:2719-2732, 2003). Staphylococcus aureus strains with decreased susceptibility to vancomycin (so-called vancomycin-intermediate S. aureus[VISA] strains) have been identified in clinical specimens in several countries during the past decade (10,11,25). Although several abnormal physiological properties have been described in VISA isolates, the mechanisms of resistance and whether or not it involves acquisition of genes has remained unclear. A major problem in mechanistic studies has been the lack of availability of isogenic vancomycin-susceptible strains that could be considered the parental strains of the VISA isolates. Even when a VISA isolate shared a common multilocus sequence type with a fully sequenced vancomycin-susceptible strain, the clinical origins and times of isolation were far apart, excluding the possibility of attributing genetic and phenotypic differences to the antibiotic susceptibility phenotype.A recently described series of methicillin-resistant S. aureus (MRSA) isolates (JH1 through JH15) were recovered from a single patient during extensive chemotherapy with vancomycin (24). Bacterial isolates recovered at various times during therapy showed increasing vancomycin MICs which in isolates JH9 and JH14 eventually reached 8 g/ml, a value typical of many clinical VISA isolates. The first isolate chronologically, JH1, and all of the subsequent isolates sha...

A series of isogenic methicillin-resistant Staphylococcus aureus isolates recovered from a bacteremic patient were shown to acquire gradually increasing levels of resistance to vancomycin during chemotherapy with the drug (K. Sieradzki, T. Leski, L. Borio, J. Dick, and A. Tomasz, J. Clin. Microbiol. 41:1687-1693, 2003). We compared properties of the earliest (parental) vancomycin-susceptible isolate, JH1 (MIC, 1 g/ml), to two late (progeny) isolates, JH9 and JH14 (vancomycin MIC, 8 g/ml). The resistant isolates produced abnormally thick cell walls and poorly separated cells when grown in antibiotic-free medium. Chemical analysis of the resistant isolates showed decreased cross-linkage of the peptidoglycan and drastically reduced levels of PBP4 as determined by the fluorographic assay. Resistant isolates showed reduced rates of cell wall turnover and autolysis. In vitro hydrolysis of resistant cell walls by autolytic extracts prepared from either susceptible or resistant strains was also slow, and this abnormality could be traced to a quantitative (or qualitative) change in the wall teichoic acid component of resistant isolates. Some change in the structure and/or metabolism of teichoic acids appears to be an important component of the mechanism of decreased susceptibility to vancomycin in S. aureus.Methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin (so called VISA strains) have been detected among clinical isolates in several countries (5, 13, 15, 22; M. C. Ploy, C. Grelaud, C. Martin, L. de Lumley, and F. Denis, Letter, Lancet 351:1212, 1998), raising serious concern about the impact of such a resistance mechanism on the chemotherapy of multidrug-resistant staphylococci. A number of studies have described properties of various VISA isolates (4,7,9,18,22). However, attempts to identify the mechanism of resistance in these VISA strains remain problematic because of conflicting observations concerning the properties of various VISA isolates and also because isogenic vancomycin-susceptible (parental) strains were not available for comparison. This problem is bypassed by the recent identification of a series of isogenic MRSA isolates with gradually increasing vancomycin MICs (23). The isolates, named JH1 through JH15, were recovered in consecutive samples from a single bacteremic patient who underwent extensive chemotherapy with vancomycin during a 2-month period. The JH isolates shared an identical pulsed-field gel electrophoretic pattern, spaA type, and multilocus sequence type and carried the same staphylococcal cassette chromosome mec type III (23). The MIC of vancomycin for the first isolate, JH1, was 1 g/ml, which increased to 8 g/ml for the final isolates, JH9 and JH14.In this communication, we compare the resistant VISA isolates JH9 and JH14 to the parental strain, JH1, in order to identify physiological and biochemical properties that are associated with the mechanism of reduced susceptibility to vancomycin. Examination of these strains should provide unique...

Microb. Drug Resist. 4, 159 -168). We now report that the distorted peptidoglycan composition is related to defects in penicillin-binding protein 4 (PBP4); no PBP4 was detectable by the fluorographic assay in membrane preparations from the mutants, and comparison of the sequence of pbp4 amplified from the mutants indicated disruption of the gene by two types of abnormalities, a 17-amino acid long duplication starting at position 305 of the pbp4 gene was detected in the vancomycin-resistant mutant, and a stop codon was found to be introduced into the pbp4 KTG motif at position 261 in the mutant selected for teicoplanin resistance. Additional common patterns of disturbances in the peptidoglycan metabolism of the mutants are indicated by the increased sensitivity of mutant cell walls to the M1 muramidase and decreased sensitivity to lysostaphin, which is a reversal of the susceptibility pattern of the parental cell walls. Furthermore, the results of high performance liquid chromatography analysis of lysostaphin digests of peptidoglycan suggest an increase in the average chain length of the glycan strands in the peptidoglycan of the glycopeptide-resistant mutants. The increased molar proportion of muropeptide monomers in the cell wall of the glycopeptide-resistant mutants should provide binding sites for the "capture" of vancomycin and teicoplanin molecules, which may be part of the mechanism of glycopeptide resistance in S. aureus.

A number of methicillin-resistant Staphylococcus aureus (MRSA) isolates were recovered over a period of several weeks from blood samples and from the heart valve of a patient who underwent extensive vancomycin chemotherapy for persistent S. aureus bacteremia. Consecutive isolates showed gradually decreasing growth rates during in vitro cultivation and increasing vancomycin MICs, from an MIC of 1 g/ml for the initial isolate to an MIC of 8 g/ml for the final MRSA isolates, which also became tolerant to vancomycin. Major changes were observed in the oxacillin resistance phenotype of several of the isolates-apparently related to in vivo exposure to imipenem, which was also used during a period of chemotherapy. Both the gradually increasing vancomycin MICs and the changes in oxacillin resistance could be reproduced by appropriate exposure of the initial MRSA isolate to antibiotics in vitro. All isolates had the same pulsed-field gel electrophoresis pattern, spaA type, and multilocus sequence type (MLST), which was identified as a single-locus variant of ST5, the MLST characteristic of previously characterized MRSA isolates with reduced susceptibility to vancomycin in the United States and Japan.

We tested the effect of a number of mechanistically distinct antibacterial agents on the expression of methicillin resistance in a highly and homogeneously resistant strain of methicillin-resistant Staphylococcus aureus. The antibiotics, used at 0.25 x MIC, included inhibitors of early steps in peptidoglycan synthesis (fosfomycin, beta-chloro-D-alanine, D-cycloserine); bacitracin; teicoplanin and vancomycin; beta-lactam inhibitors chosen on the basis of their relatively selective affinities for penicillin-binding proteins 1, 2, 3 and 4 of S. aureus (imipenem, cefotaxime, cephradine and cefoxitin); compounds that inhibit various steps in protein synthesis (tetracycline, chloramphenicol, gentamicin, erythromycin and quinupristin/dalfopristin) and an inhibitor of DNA gyrase (temafloxacin). All inhibitors of early cell wall synthesis caused reduction of methicillin resistance and change from the homogeneous to the heterogeneous methicillin-resistant phenotype. Similar effects were obtained with only cephradine out of the four beta-lactams tested, and with erythromycin and quinupristin/dalfopristin as well. The other inhibitors of protein synthesis and DNA gyrase had no effect.