Received 2 October 2002/Returned for modification 29 November 2002/Accepted 7 January 2003

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2003, p Vol. 41, No /03/$ DOI: /JCM Copyright 2003, American Society for Microbiology. All Rights Reserved. Evolution of a Vancomycin-Intermediate Staphylococcus aureus Strain In Vivo: Multiple Changes in the Antibiotic Resistance Phenotypes of a Single Lineage of Methicillin-Resistant S. aureus under the Impact of Antibiotics Administered for Chemotherapy K. Sieradzki, 1 T. Leski, 1 J. Dick, 2 L. Borio, 2 and A. Tomasz 1 * The Rockefeller University, New York, New York 10021, 1 and John Hopkins Medical Institutions, Baltimore, Maryland Received 2 October 2002/Returned for modification 29 November 2002/Accepted 7 January 2003 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. Clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to vancomycin socalled VISA isolates have been identified in several countries, but identification of the mechanism of resistance has been made difficult by conflicting observations concerning the properties of individual VISA isolates (3, 9, 11, 15) and by the lack of isogenic isolates with different levels of vancomycin susceptibility. Here we describe the physiological properties of consecutive MRSA isolates recovered from a single patient during extensive vancomycin therapy (D. Flayhart, A. Hanlon, T. Wakefield, T. Ross, L. Borio, and J. Dick, Abstr. 101st Gen. Meet. Am. Soc. Microbiol., abstr. A-39, 2001), which was accompanied by gradually increasing vancomycin MICs for the isolates. Our observations strongly suggest that these VISA isolates represent the progeny of a single MRSA strain that emerged in vivo under the selective pressure of vancomycin therapy. The purpose of the studies described here was to compare the properties of the VISA isolates as they emerged during various stages of the patient s chemotherapy in order to better understand the mechanism of evolution of drug resistance in vivo. MATERIALS AND METHODS Strains and growth conditions. The S. aureus isolates recovered from clinical material in Baltimore, Md., are listed in Table 1. Bacteria were grown in tryptic soy broth (TSB) (Difco, Detroit, Mich.) at 37 C with aeration. Growth was monitored by measuring the optical density at 620 nm with an LKB spectrophotometer (Pharmacia LKB Biotechnology, Inc., Uppsala, Sweden). Viable titers * Corresponding author. Mailing address: The Rockefeller University, 1230 York Ave., New York, NY Phone: (212) Fax: (212) tomasz@mail.rockefeller.edu. were also determined routinely at sampling times by plating diluted cultures on tryptic soy agar (TSA) (Difco). Antibiotic susceptibility. Susceptibility testing was performed by the agar dilution method according to NCCLS recommendations (10). Oxacillin and vancomycin susceptibility profiles of bacterial cultures were also tested by population analysis (18). Selection of highly resistant subpopulations was achieved by picking single colonies from the population analysis plates containing elevated concentrations of antibiotics and using these as inocula for overnight cultures grown in antibiotic-free TSB. Time-kill curves. Kill rates were measured for exponentially growing cultures that at zero time (corresponding to an optical density at 620 nm of 0.1, or about 10 7 CFU per ml) received concentrations of vancomycin ranging from 2 to 100 times the corresponding MIC. Control cultures received no antibiotic. Portions of the cultures (200 l) were removed at various intervals, serially diluted, and plated on TSA. Colonies were counted after 48 h of incubation at 37 C. DNA manipulations. Chromosomal DNA for pulsed-field gel electrophoresis (PFGE) was prepared as described before (4). Chromosomal DNA for PCR was prepared as described before (2), except that the proteinase K digestion step was preceded by treatment of the bacterial cells with lysostaphin. MLST and spaa typing. Multilocus sequence typing (MLST) based on the sequences of seven housekeeping genes (6, 8) and typing based on the polymorphic region of protein A (spaa typing) (6, 13) were performed by published procedures. Detection of meca. The presence of meca was determined by PCR with 20 pmol of primers PF-MECP (5 -GATTGGGATCATAGCGTCATT-3 ) and PR- MECP (5 -GTTTTTCGAGTCCCTTTTTACC-3 ), which were used to amplify a 481-bp internal fragment of the gene. PCR was carried out for 30 cycles of 30 s at 95 C, 52 C for 30 s, and 72 C for 1 min and one final extension step at 72 C for 4 min. RESULTS AND DISCUSSION Characterization of JH VISA isolates. All JH isolates were resistant to erythromycin, clindamycin, levofloxacin, and rifampin and susceptible to tetracycline, linezolid, and quinupristin-dalfopristin, and all isolates carried the meca gene 1687

2 1688 SIERADZKI ET AL. J. CLIN. MICROBIOL. Isolate Date obtained (mo/day) Source TABLE 1. Genetic profiles of analyzed MRSA isolates Oxacillin (hetero class a ) MIC, g/ml, of: Vancomycin b MLST spaa type PFGE pattern JH1 7/20 Blood 0.75 (1) TJMBMDMGMK A0 JH2 9/20 Blood 25 (3) TJMBMDMGMK A0 JH3 9/24 Blood 100 (3) TJMBMDMGMK A0 JH5 10/01 Blood 0.75 (1) TJMBMDMGMK A0 JH6 10/06 Blood 1.5 (1) TJMBMDMGMK A0 JH9 10/13 Blood 0.75 (1) TJMBMDMGMK A0 JH14 10/17 Heart valve 1.5 (1) TJMBMDMGMK A0 JH15 10/23 Nares of contact 0.75 (1) TJMBMDMGMK A0 PA TJMBMDMGMK A1 PC TJMBMDMGMK A1 Mu TJMBMDMGMK A2 M TJMGMK A1 NJ TMBMDMGMK A3 a As defined in reference 18. b As determined by the E-Test. (data not shown). The dates of recovery of the MRSA isolates and antibiotic treatments are shown schematically in Fig. 1. All JH strains had an indistinguishable PFGE pattern which was similar but not identical to the PFGE patterns of several previously described VISA isolates from the United States and Japan (Fig. 2). Table 1 summarizes the date and clinical source of isolation, oxacillin and vancomycin MICs, and the results of three additional molecular typing techniques used for the characterization of the bacteria. Also included for comparison are the relevant properties of several other VISA isolates from the United States and strain Mu50 from Japan. All JH isolates including the earliest isolate, JH1, subsequent isolates JH2 through JH14, and isolate JH15 (recovered from the nares of a family contact) shared a common multilocus sequence type and a common, invariant spaa type. The MLST of the JH isolates appears to be a hitherto-undescribed single-locus variant of ST5, which is the characteristic MLST of the New York clone of MRSA (6) and of several previously described MRSA isolates from the United States and Japan including VISA isolates PC3, MI, NJ (1, 12, 15), and Mu50 (9). Changes in oxacillin and vancomycin susceptibilities as a function of the chronological date of isolation of JH strains. Cultures of the earliest isolate, JH1, and surveillance culture JH15 showed heterogeneous oxacillin resistance phenotypes when tested for population analysis profiles (PAPs). For the FIG. 1. Schematic representation of antibiotic exposure and recovery of MRSA isolates as a function of calendar dates (month/day/year). Antibiotic concentrations are given in micrograms per milliliter.

3 VOL. 41, 2003 EVOLUTION OF A VISA STRAIN IN VIVO 1689 FIG. 2. PFGE patterns of JH isolates recovered at different times during the patient s clinical history. Chromosomal DNA was prepared, SmaI restricted, and separated by PFGE. For identification of the isolates, see Table 1. Downloaded from great majority of bacteria ( 99.9%), oxacillin MICs were 0.75 g/ml. The cultures also contained subpopulations of highly resistant cells for which oxacillin MICs were 400 g/ml, but these subpopulations were present at a low (10 6 ) frequency in the cultures. A major increase in the oxacillin MICs for the majority of cells and a less heterogeneous PAP were detected in cultures of the isolates obtained next chronologically, JH2 and JH3; these isolates were recovered from the patient after a therapeutic regimen of imipenem treatment for noscomial pneumonia. Cultures of isolates recovered after this time (JH6 through JH14) showed heterogeneous oxacillin phenotypes; the MICs for the majority of cells were reduced to 1 to 3 g/ml (Fig. 3). Similar but much less extensive changes were also apparent with respect to the vancomycin susceptibility of the JH isolates. While the vancomycin MICs for the early isolates (JH1, JH2, and JH3) were within the range of susceptibility breakpoints, those for the late isolates (JH6, JH9, and JH14), recovered from the patient after an extended course of vancomycin therapy, gradually increased from 1 g/ml to 4, 6, and eventually 8 g/ml for isolates JH9 and JH14 (Fig. 3). Moreover, all of these isolates were capable, at low frequencies, of forming colonies on agar containing 8.0 or sometimes even 16 g of vancomycin/ml. If the changing oxacillin and vancomycin phenotypes were the products of selective antibiotic pressure in vivo, then it might be possible to reconstruct these phenotypic changes by applying similar antibiotic selection to the earliest isolate, JH1, in vitro. Figures 4 and 5 demonstrated this to be the case. A culture of strain JH1 was plated on agar containing increasing concentrations of imipenem (the -lactam antibiotic used in the chemotherapy of the patient), and a rare colony capable of growing on plates containing 50 g of imipenem/ml was used as an inoculum for an overnight TSB culture (strain JH1IMP50 in Fig. 4). Plating of the overnight culture on agar containing either imipenem or oxacillin at increasing concentrations demonstrated the selection of a more highly and homogeneously -lactam-resistant culture from strain JH1 that mimicked the properties of strains JH2 and JH3 (Fig. 4). With a similar procedure, it was also possible to generate from cultures of strain JH1 plated on agar containing increasing concentrations of vancomycin bacterial cultures that showed increased resistance to vancomycin, as indicated by a shift in the shape of the PAPs toward higher vancomycin MICs (see the PAPs of JH1 and JH1P4 in Fig. 5). Subculturing of such bacteria in liquid medium containing one-half the MIC of vancomycin allowed the selection of bacteria (JH1P4T4 in Fig. 5) for which vancomycin MICs were even higher, approaching those of in vivo-selected strains JH9 and JH14. A careful comparison of the shapes of the PAP curves illustrating the oxacillin and vancomycin susceptibility profiles for the JH strains (Fig. 3) suggests that increasing vancomycin MICs were accompanied by a reverse change in the -lactam antibiotic susceptibility profiles. For instance, for strains JH2 on April 2, 2019 by guest

4 1690 SIERADZKI ET AL. J. CLIN. MICROBIOL. Downloaded from FIG. 3. Oxacillin (top panel) and vancomycin (bottom panel) susceptibility profiles for JH isolates. Bacterial cultures were grown and plated on agar containing various concentrations of antibiotics for population analysis. and JH3, for which oxacillin MICs were relatively high, vancomycin MICs were lower; in contrast, for strains JH9 and JH14, vancomycin MICs were the highest but oxacillin MICs were relatively low for the majority of the bacteria in cultures. This inverse relationship between -lactam and vancomycin susceptibility profiles could be reproduced in vitro. A colony of strain JH3 capable of growing on agar containing 8 g of vancomycin/ml was used as an inoculum to select a homogeneous culture of these bacteria by overnight growth in TSB. In such cultures (JH3V8), the vancomycin MIC for the majority of the bacteria increased from 2 to 16 g/ml. The phenotype of FIG. 4. Enrichment of the JH1 culture for a subpopulation of bacteria with elevated oxacillin resistance. Isolate JH1 was plated for population analysis on agar containing increasing concentrations of imipenem. Bacterial cells capable of forming colonies on agar containing 50 g of imipenen/ml (arrow) were picked, diluted in drug-free TSB, grown overnight, and plated for population analysis on both imipenem- and oxacillin-containing plates. JH3V8 cultures was stable during growth in nonselective medium. Plating of cultures of JH3 and JH3V8 on oxacillincontaining agar plates produced PAPs with phenotypes that were the inverse of the vancomycin susceptibility profiles of these strains: the oxacillin MIC decreased from about 100 g/ml for the majority of cells of JH3 to as low as 0.75 g/ml for cells of JH3V8 (Fig. 6). Changes in growth rate and susceptibility to the bactericidal effect of vancomycin as a function of the chronological dates of on April 2, 2019 by guest

5 VOL. 41, 2003 EVOLUTION OF A VISA STRAIN IN VIVO 1691 FIG. 5. Enrichment of the JH1 culture for subpopulations of bacteria for which vancomycin MICs were elevated. Isolate JH1 was plated for population analysis on agar containing increasing concentrations of vancomycin. Bacterial cells capable of forming colonies on agar containing 4.0 g of vancomycin/ml (arrow) were picked, diluted in drug-free TSB, grown overnight, and plated for population analysis (mutant JH1P4). Extended exposure of mutant JH1P4 to gradually increasing concentrations of vancomycin in growth medium eventually allowed for the selection of mutant JH1P4T4, for which the vancomycin MIC was 8.0 g/ml. isolation of JH strains. The growth rates of the JH strains were determined by monitoring the rate of increase in the optical density during aerobic incubation at 37 o C for TSB cultures in vitro. The doubling times of the cultures increased from 30 min for JH1 to 45 min for JH2 and JH3 and approximately 60 min for JH6, JH9, and JH14. Cultures of JH1 and JH14 were compared for their susceptibility to the bactericidal effect of vancomycin during exposure of TSB cultures to various concentrations of the antibiotic administered at different MIC equivalents. Figure 7 shows that cultures of JH1 exposed to vancomycin at concentrations corresponding to 2, 5, and 10 times the MIC (2, 5, and 10 g of vancomycin/ml, respectively) underwent an extensive loss of the viable titer (from about 10 8 CFU per ml to CFU per ml) during a 4-h incubation period. There was no detectable decrease in the viable titer of cultures of JH14 during exposure to a similar range of vancomycin MIC equivalents (corresponding to 16, 40, and 80 g of vancomycin/ml) (Fig. 7). After exposure of the cultures to 10 times the respective vancomycin MICs for 24 h, the viable titers of JH1 and JH14 were reduced to and CFU per ml, respectively. Vancomycin susceptibility of MRSA strain PA237. Interestingly, the JH strains share a common and hitherto-undescribed MLST, , which is a single-locus variant of ST5, the genetic background of previously characterized VISA isolates from the United States and Japan (6). The JH series is closely related but not identical to the previously characterized FIG. 6. Suppression of phenotypic expression of resistance to oxacillin by selection of vancomycin resistance in isolate JH3. Bacterial cultures were grown in TSB to stationary phase and plated at several dilutions on TSA containing various concentrations of vancomycin. Single colonies that could grow at concentrations of vancomycin above the MICs for the majority of the cells were picked and suspended in growth medium supplemented with the same drug concentrations. After several cycles of selection, a mutant (JH3V8) for which the vancomycin MIC was 16 g/ml was obtained. VISA isolates. The close genetic relatedness of the JH series to other vancomycin-resistant strains (MI, NJ, PC3, and Mu50) and, in turn, of those strains to an MRSA clone that is widespread in hospitals in the New York metropolitan area is of obvious concern. Sieradzki et al. previously showed that several isolates belonging to this MRSA clone, although still susceptible to vancomycin, contained subpopulations of bacteria

6 1692 SIERADZKI ET AL. J. CLIN. MICROBIOL. FIG. 7. Changes in susceptibility to the bactericidal effect of vancomycin. The viability of isolates JH 1 and JH14 was determined during exposure to vancomycin at concentrations corresponding to 1, 2, 5, and 10 times the MIC. Exponentially growing cultures were exposed to the antibiotic (arrow), and aliquots were removed at various times to determine the viable titer of bacteria as described in Materials and Methods. for which vancomycin MICs approached and even surpassed those for the majority of cells of various VISA isolates (15). Strain PA237 was such an isolate recovered from a hospitalized patient in Pennsylvania. This strain belonged to the New York MRSA clone (ST5) and was analyzed for vancomycin susceptibility by population analysis (Fig. 8). Strain PA237 showed a heterogeneous vancomycin phenotype, since it contained subpopulations (at a frequency of approximately 10 4 ) FIG. 8. Vancomycin susceptibility of strain PA237. The strain was grown overnight in TSB and then plated for population analysis on TSA plates containing various concentrations of vancomycin (E). A colony of strain PA237 growing on an agar plate containing 4 g of vancomycin/ml was picked (arrow) and used as an inoculum for drugfree TSB to generate a new culture (PA237P4), the PAP for which is also shown (F). of bacteria which could grow on agar containing 4.0 g of vancomycin/ml. A colony picked from an agar plate containing 4.0 g of vancomycin/ml (Fig. 8) and used as an inoculum for drug-free TSB was grown overnight and replated for population analysis. Now the vancomycin MIC for the majority of the bacteria was 4.0 g/ml, and the population profile was similar to that of other VISA isolates. The similar genetic background of all VISA isolates described so far suggests that some determinant carried in the New York MRSA clone may predispose the bacteria to the development of vancomycin resistance. All of the JH isolates characterized in this study were recovered from a single patient at various times after the onset of a prolonged course of chemotherapy with vancomycin (for the MRSA infection) and a shorter course of treatment with imipenem (for noscomial pneumonia) (Flayhart et al., Abstr. 101st Gen. Meet. Am. Soc. Microbiol.). All of the JH isolates had identical PFGE patterns as well as identical MLST and spaa types, suggesting that the JH isolates represented the progeny of the earliest MRSA isolate, JH1, and that the changing phenotypes of the subsequent isolates mirrored the selective pressure of chemotherapeutic agents molding the antibiotic susceptibility profiles of the bacteria. The extremely low and heterogeneous oxacillin-resistant phenotype of JH1 was shown to change to a more homogeneous and more highly resistant phenotype in isolates JH2 and JH3 apparently as a consequence of the brief imipenem therapy given to the patient. Subsequently, in later isolates, such as JH6, JH9, and JH14 (isolated after the therapy with imipenem had been discontinued), the oxacillin PAP again changed to low and heterogeneous resistance, similar to the phenotype of

7 VOL. 41, 2003 EVOLUTION OF A VISA STRAIN IN VIVO 1693 the original isolate, JH1. This change might have been the result of continued vancomycin chemotherapy selecting for gradually increasing vancomycin MICs which in turn might have brought about the decline in oxacillin resistance. Such a seesaw-like effect between vancomycin resistance and -lactam resistance was originally observed for in vitro-selected vancomycin-resistant S. aureus mutants (14) and was subsequently also noted for some clinical VISA isolates (15). The experiments illustrated in Fig. 6 actually reconstructed this phenomenon for isolate JH3 and its more highly vancomycin-resistant subpopulation. It was also possible to reconstruct the increased and more homogeneous oxacillin-resistant phenotype in vitro by applying selective imipenem pressure to isolate JH1 in the laboratory (Fig. 5). An interesting property of the VISA isolates was the gradually decreasing growth rate of the bacteria, which seemed to parallel the increase in the vancomycin MIC. A similar phenomenon was noted for vancomycin-resistant mutants isolated in the laboratory (14). The slow growth rate for JH14 may be one of the factors responsible for the vancomycin tolerance of this isolate, as demonstrated in Fig. 8. The dependence of the bactericidal activity of antimicrobial agents on the rate of growth of target bacteria has been demonstrated repeatedly in the past (5, 7, 19). ACKNOWLEDGMENTS Partial support for this work was provided by a grant from the National Institutes of Health (RO1-AI45738). The help of Marilyn Chung with multiplex PCR of the staphylococcal chromosomal cassette mec (SCCmec) is gratefully acknowledged. ADDENDUM Since the submission of the manuscript, we also determined the structural type of SCCmec carried by isolates JH1, JH4, JH9, and JH14 by using a multiplex PCR method (10a). All isolates contained the same type II SCCmec, further confirming that these bacteria are close relatives of the New York- Japan pandemic MRSA clone (10b). REFERENCES 1. Aires-de-Sousa, M., H. de Lencastre, I. S. Sanches, K. Kikuchi, K. Totsuka, and A. Tomasz Similarity of antibiotic resistance patterns and molecular typing properties of methicillin-resistant Staphylococcus aureus isolates widely spread in hospitals in New York city and in a hospital in Tokyo, Japan. Microb. Drug Resist. 6: Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.) Current protocols in molecular biology, p John Wiley & Sons, Inc., New York, N.Y. 3. 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