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. Tomasz1*
The Rockefeller University, New York, New York 10021,1 John Hopkins Medical Institutions, Baltimore, Maryland 212872
*Corresponding author. Mailing address: The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: (212) 327-8277. Fax: (212) 327-8688.
Received October 2, 2002; Revised November 29, 2002; Accepted January 7, 2003.Abstract
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 μg/ml for the initial isolate to an MIC of 8 μg/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—so-called 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.
Material 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 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.
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 107 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 μl) were removed at various intervals, serially diluted, and plated on TSA. Colonies were counted after 48 h of incubation at 37°C.
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 (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 1-4-1-4-12-1-28 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 great majority of bacteria (>99.9%), oxacillin MICs were 0.75 μg/ml. The cultures also contained subpopulations of highly resistant cells for which oxacillin MICs were >400 μg/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 μg/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 μg/ml to 4, 6, and eventually 8 μg/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 μg 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 β-lactam antibiotic used in the chemotherapy of the patient), and a rare colony capable of growing on plates containing 50 μg 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 β-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 β-lactam antibiotic susceptibility profiles. For instance, for strains JH2 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 β-lactam and vancomycin susceptibility profiles could be reproduced in vitro. A colony of strain JH3 capable of growing on agar containing 8 μg 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 μg/ml. The phenotype of JH3V8 cultures was stable during growth in nonselective medium. Plating of cultures of JH3 and JH3V8 on oxacillin-containing 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 μg/ml for the majority of cells of JH3 to as low as 0.75 μg/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 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 37oC 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 μg of vancomycin/ml, respectively) underwent an extensive loss of the viable titer (from about 108 CFU per ml to 8 × 106 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 μg 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 6 × 103 and 5 × 104 CFU per ml, respectively.
Vancomycin susceptibility of MRSA strain PA237
Interestingly, the JH strains share a common and hitherto-undescribed MLST, 1-4-1-4-12-1-28, 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 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 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) of bacteria which could grow on agar containing 4.0 μg of vancomycin/ml. A colony picked from an agar plate containing 4.0 μg 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 μg/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 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 β-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).
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).
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.
Aires-de-Sousa, M., H. de Lencastre, I. S. Sanches, K. Kikuchi, K. Totsuka, and A. Tomasz. 2000. 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. 6:253-258.
Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1995. Current protocols in molecular biology, p. 241-242. John Wiley & Sons, Inc., New York, N.Y.
Centers for Disease Control and Prevention. 1997. Update: Staphylococcus aureus with reduced susceptibility to vancomycin—United States, 1998. Morb. Mortal. Wkly. Rep. 46:813-815.
Chung, M., H. de Lencastre, P. Matthews, A. Tomasz, I. Adamsson, M. Aries de Sousa, T. Camou, C. Cocuzza, A. Corso, I. Couto, A. Dominguez, M. Gniadkowski, R. Goering, A. Gomes, K. Kikuchi, A. Marchese, R. Mato, O. Melter, D. Oliveira, R. Palacio, R. Sa-Leao, I. Santos Sanches, J. H. Song, P. T. Tassios, and P. Villari. 2000. Molecular typing of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis: comparison of results obtained in a multilaboratory effort using identical protocols and MRSA strains. Microb. Drug Resist. 6:189-198. [PubMed].
Cozens, R. M., E. Tuomanen, W. Tosch, O. Zak, J. Suter, and A. Tomasz. 1986. Evaluation of the bactericidal activity of beta-lactam antibiotics on slowly growing bacteria cultured in the chemostat. Antimicrob. Agents Chemother. 29:797-802. [Free Full text in PMC].
Crisostomo, M. I., H. Westh, A. Tomasz, M. Chung, D. C. Oliveira, and H. de Lencastre. 2001. The evolution of methicillin resistance in Staphylococcus aureus: similarity of genetic backgrounds in historically early methicillin susceptible and resistant isolates and contemporary epidemic clones. Proc. Natl. Acad. Sci. USA 98:9865-9870. [Free Full text in PMC].
Eng, R. H., F. T. Padberg, S. M. Smith, E. N. Tan, and C. E. Cherubin. 1991. Bactericidal effects of antibiotics on slowly growing and nongrowing bacteria. Antimicrob. Agents Chemother. 35:1824-1828. [Free Full text in PMC].
Enright, M. C., N. P. Day, C. E. Davies, S. J. Peacock, and B. G. Spratt. 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38:1008-1015. [Free Full text in PMC].
Hiramatsu, K., H. Hanaki, T. Ino, K. Yabuta, T. Oguri, and F. C. Tenover. 1997. Methicillin resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J. Antimicrob. Chemother. 40:135-136. [PubMed] [Free Full Text].
NCCLS. 2000. Performance standards for antimicrobial disk susceptibility test. NCCLS, Wayne, Pa.
Oliveira, D. C., and H. de Lencastre. 2002. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 46:2155-2161. [Free Full text in PMC].
Oliveira, D. C., A. Tomasz, and H. de Lencastre. 2002. The secrets of success of a human pathogen: molecular evolution of pandemic clones of methicillin-resistant Staphylococcus aureus. Lancet Infect. Dis. 2:180-189. [PubMed].
Ploy, M. C., C. Grelaud, C. Martin, L. de Lumley, and F. Denis. 1998. First clinical isolate of vancomycin-intermediate Staphylococcus aureus in a French hospital. Lancet 351:1212.
Roberts, R. B., M. Chung, H. de Lencastre, J. Hargrave, A. Tomasz, and the Tri-State MRSA Collaborative Study Group. 2000. Distribution of methicillin-resistant Staphylococcus aureus clones among health care facilities in Connecticut, New Jersey, and Pennsylvania. Microb. Drug Resist. 6:245-251. [PubMed].
Shopsin, B., M. Gomez, S. O. Montgomery, D. H. Smith, M. Waddington, D. E. Dodge, D. A. Bost, M. Riehman, S. Naidich, and B. N. Kreiswirth. 1999. Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. J. Clin. Microbiol. 37:3556-3563. [Free Full text in PMC].
Sieradzki, K., and A. Tomasz. 1999. Gradual alterations in cell wall structure and metabolism in vancomycin-resistant mutants of Staphylococcus aureus. J. Bacteriol. 181:7566-7570. [Free Full text in PMC].
Sieradzki, K., R. B. Roberts, S. W. Haber, and A. Tomasz. 1999. The development of vancomycin resistance in a patient with methicillin-resistant Staphylococcus aureus. N. Engl. J. Med. 340:517-523. [PubMed] [Full Text].
Sieradzki, K., and A. Tomasz. 1997. Inhibition of cell wall turnover and autolysis by vancomycin in a highly vancomycin-resistant mutant of Staphylococcus aureus. J. Bacteriol. 179:2557-2566. [Free Full text in PMC].
Sieradzki, K., M. G. Pinho, and A. Tomasz. 1999. Inactivated pbp4 in highly glycopeptide-resistant laboratory mutants of Staphylococcus aureus. J. Biol. Chem. 274:18942-18946. [PubMed] [Free Full Text].
Tomasz, A., S. Nachman, and H. Leaf. 1991. Stable classes of phenotypic expression in methicillin resistant clinical isolates of staphylococci. Antimicrob. Agents Chemother. 35:124-129. [Free Full text in PMC].
Tuomanen, E., R. Cozens, W. Tosch, O. Zak, and A. Tomasz. 1986. The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth. J. Gen. Microbiol. 132:1297-1304. [PubMed].