Stable Classes of Phenotypic Expression in Methicillin-Resistant
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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1991, p /91/ $02.00/0 Copyright 1991, American Society for Microbiology Vol. 35, No. 1 Stable Classes of Phenotypic Expression in Methicillin-Resistant Clinical Isolates of Staphylococci ALEXANDER TOMASZ,* SHARON NACHMAN,t AND HOWARD LEAFt The Rockefeller University, 1230 York Avenue, New York, New York Received 5 July 1990/Accepted 10 October 1990 A collection of coagulase-positive and -negative clinical strains of staphylococci, all of which gave a positive reaction with a mec-specific DNA probe, was analyzed for the mode of phenotypic expression of methicillin resistance by using population analysis on agar plates containing different concentrations of the antibiotic. Strains could be divided into four arbitrary expression classes. Cultures of class 4 strains were composed of uniformly and highly resistant bacteria (MIC pg/ml). In contrast, cultures of strains belonging to classes 1, 2, and 3 were heterogeneous: they were composed of two or more subpopulations of cells that differed from one another in MICs and frequencies. In cultures of strains belonging to expression class 1, most of the cells had methicillin MICs of 1.5 to 3,ug/ml, i.e., only two to three times higher than those for truly susceptible strains. In cultures of strains belonging to expression classes 2 and 3, the methicillin MICs for the majority of bacteria ranged from 6 to 12 and up to 50 to 200,Lg/ml, respectively. While the definition of the expression classes was arbitrary, the modes of phenotypic expression were specific and reproducible: randomly picked colonies of a given strain produced identical population profiles. The strain-specific mode of expression was also retained after numerous single-colony picks and sequential passages in antibiotic-free medium. We suggest that these classes represent stages in an evolutionary sequence leading to progressively improved phenotypic expression of methicillin resistance in staphylococci. The phenotypic expression of methicillin resistance in staphylococci presents several intriguing features. Methicillin-resistant strains appear to be uniform in that they all contain the unique, foreign-born mec gene and its product, the 78-kDa penicillin-binding protein 2A or 2' (for a recent review, see reference 3). On the other hand, the degree of antibiotic resistance (i.e., MIC, as determined by the conventional techniques of susceptibility testing) varies tremendously from one strain to another, spanning the range of a few micrograms per milliliter (values not far above the MIC for susceptible Staphylococcus aureus) to several milligrams per milliliter. By using a technique of higher resolution (population analysis), one can also demonstrate an even more peculiar feature of these bacteria: broth cultures of most methicillin-resistant S. aureus (MRSA) strains are not composed of cells ofuniform MICs, but rather they are made up of several subpopulations of bacteria that differ widely in their degrees of antibiotic resistance (4). Thus, there exists a surprising degree of nonuniformity in the phenotypic expression of antibiotic resistance, both from one strain to another and also within the progeny of a single MRSA isolate. While both genetic and environmental factors are known to influence the phenotypic expression of methicillin resistance, the mechanistic basis of this diversity is not understood. To see if some pattern in this complexity can be recognized, we undertook a careful analysis of the phenotypic expression of antibiotic resistance in a number of methicillin-resistant staphylococcal strains (both coagulase-positive and -negative isolates). We found that the frequency and resistance level of progeny cells in cultures of MRSA isolates were * Corresponding author. t Present address: Department of Pediatrics, Children's Medical Center at Stony Brook, State University of New York, Stony Brook, NY t Present address: Division of Infectious Diseases, New York Veterans Administration Medical Center, New York, NY highly reproducible and characteristic of the particular strain, implying a genetic control in the population structure of these bacteria. MATERIALS AND METHODS Strains. Coagulase-positive and -negative staphylococcal isolates, classified as methicillin resistant by criteria used in clinical microbiology laboratories, were kindly supplied by colleagues in the United States and Portugal. The sources included Edward Buttone (Mt. Sinai Hospital, New York, N.Y.), Paul Ellner (Columbia University-Presbyterian Hospital Medical Center, New York, N.Y.), Barry Hartman (The New York Hospital-Cornell Medical Center, New York), Ken Van Horn and Gary Wormser (Westchester County Medical Center, Valhalla, N.Y.), Henry Isenberg (Long Island Jewish Medical Center, New Hyde Park, N.Y.), Patrick Cassidy (Merck Research Laboratories, Rahway, N.J.), Odette Ferreira (Faculdade de Farmacia, Lisbon, Portugal), Seixas Antao (Sao Jose Hospital, Lisbon, Portugal), Torres-Pereira (Instituto Camara Pestana, Lisbon, Portugal), and M. Vitoria Vaz Pato (Instituto Ricardo Jorge, Lisbon, Portugal). Strains received were grown in tryptic soy broth (TSB; Difco, Detroit, Mich.), and stocks were prepared from stationary-phase cultures which were frozen after the addition of sterile glycerol (final concentration, 10%6) and stored at -70 C. For the susceptibility testing and other physiological experiments, 100-,ul portions of stock cultures (about 106 to 107 viable cells) were inoculated into 2 or 10 ml of TSB and incubated at 37 C with or without aeration for 16 to 18 h to provide turbid cultures which were then used directly for population analysis (4). Serial dilutions of such cultures were plated on tryptic soy agar (TSA; Difco) containing a range of concentrations (usually from 0.5 to 800,ug/ml) of methicillin (Sigma Chemical Co., St. Louis, Mo.) with or without a constant concentration (4,ug/ml) of clavulanic acid 124
2 VOL. 35, 1991 (Beecham Co., Bristol, Tenn.). Plates were incubated at 37 C for 48 h before the colonies were counted. Omission of clavulanic acid did not alter the shape of the population analysis curves. DNA probe. The mec-specific DNA probe was obtained from John Kornblum; it consists of a 1.3-kb Pstl fragment cloned into Escherichia coli vector Bluescript and contains 201 bp of the penicillin-binding protein 2A open reading frame (2). A DNA preparation of this plasmid was obtained by use of established techniques (7). A radiolabeled probe was prepared by using a random priming kit and [32P]dATP (Amersham Corp., Arlington Heights, Ill.) as described before (12). Dot blot hybridization. Whole-cell lysates were prepared by using 0.5-ml stationary-phase cultures. Cells were centrifuged and suspended in 1 ml of TE, and cells walls were digested by the addition of 10,u of lysostaphin (Sigma; 10 mg/ml) and 5 RI of muramidase (Sigma; 10 mg/ml). The spheroplasts were then lysed with 10,ul of sodium dodecyl sulfate (10%) and 2 RI of proteinase K (10 mg/ml). DNA was denatured at alkaline ph by the addition of 10 pi of 3 M NaOH and incubation at room temperature for 10 min. The samples were then chilled at 0 C, and the ph value was neutralized by the addition of 110 pi of 2 M ammonium acetate. Lysates were kept on ice, and serial dilutions were made in TE at 0 C. Samples (50 pi) were then loaded into dot blot wells. A Bio-Rad dot blot apparatus and Gene Screen membranes were used. Radiolabeled probe was added to Gene Screen membranes after prehybridization and hybridization at 60 C by standard methods (7). RESULTS Testing of clinical isolates with a mec-specific DNA probe. Stocks of clinical isolates were grown in liquid culture and tested for the presence of the mec gene by using the DNA probe and culture lysates containing about 4 x 10' cell equivalents of DNA. Not all strains classified as methicillin resistant by the clinical laboratory criteria gave a positive signal with the DNA probe. Of the isolates that gave a positive signal, 11 strains of S. aureus and 5 coagulasenegative strains were selected for detailed study. Modes of phenotypic expression of methicillin resistance. For a preliminary screening of the expression mode, stationary-phase cultures (109 to 1010 CFU/ml) aerobically grown overnight (16 h) were plated at two dilutions, 10' and 1i-, on a series of agar plates containing serial (twofold) dilutions of methicillin at concentrations ranging from 0 and 0.5 to 200,ug/ml. All plates also contained clavulanic acid (4,ug/ml) to eliminate the possible contribution of penicillinase to the MIC. Plates were incubated at 37 C for 48 h before the colonies were counted. The appearance of the plates in such a preliminary test is illustrated in Fig. 1. To obtain more accurate and reproducible results, it was important to plate bacteria at several concentrations, including samples of undiluted stationary-phase cultures (up to 109 CFU per plate). The number of bacteria capable of forming colonies was plotted against the concentration of methicillin for each strain, and these curves (population analysis profiles [PAPs]) were then compared. Both coagulase-positive and -negative strains could be subdivided into four arbitrary expression classes on the basis of their PAPs (Fig. 2A and B). The great majority of cells (99.9 or 99.99%) in cultures of class 1 strains had MICs (1.5 to 3 jig/ml) not much greater than those for susceptible staphylococci (0.75,ug/ml), but each culture also contained a EXPRESSION OF METHICILLIN RESISTANCE 125 FIG. 1. Analysis of the expression of methicillin resistance by plating MRSA strains on antibiotic-containing agar plates. S. aureus SA 1 (class 1), SA 7 (class 2), SA 43 (class 3), and SA 10 (class 4) were grown into stationary-phase cultures and plated on methicillincontaining agar, each at two cell concentrations: 108 CFU (on the left half of each plate) and approximately 103 CFU (on the right half of each plate). The numbers along the top of the photograph indicate the concentration of methicillin in micrograms per milliliter. The numbers along the left side refer to the expression classes. S, Susceptible strain. varying very low number (10-7 to 10-8) of bacteria that could form colonies even in the presence of methicillin at up to 25 pug/ml or more. In cultures of class 2 strains, the majority of cells (.99.9%) had more substantial MICs (6 to 12 pug/ml), and in these cultures the frequency of highly resistant cells (capable of growing in the presence of methicillin at 25,ug/ml or more) was also higher (10-6 to 10-4) than in the class 1 strains. The PAPs of most class 2 strains also showed the presence of several subpopulations with MICs intermediate between those for the cell majority and those for the most highly resistant bacteria. In class 3 strains, cultures were composed of bacteria 99 to 99.9% of which had high resistance levels (MIC, 50 to 200,ug/ml) plus usually a single subpopulation of very highly resistant cells (10-3 to 10-2) capable of forming colonies even at 300 to 400 pug of methicillin per ml. Class 4 cultures were composed of cells of uniform and very high methicillin resistance (MIC, 400 to 1,000,ug/ml) (Table 1). Strain specificity and stability of PAPs. Two or more strains were selected from each expression class to test the stability of phenotypic expression of methicillin resistance. Representative strains were plated on nonselective agar (TSA), and three or more single colonies of each strain were picked at random, grown in liquid cultures (TSB), and tested. Such single-colony subcultures produced PAPs that were surprisingly similar to that of the original strain (Fig. 2A and B). In the case of the class 1 strain shown in Fig. 2, single colonies were picked sequentially for at least 15 times over a period of a half year. Again, subcultures gave PAPs virtually indistinguishable from that of the original strain. Antibiotic resistance levels in cultures composed of multiple subpopulations. PAPs of cultures of class 2 strains often suggested the presence of several "waves," i.e., three or four, minority subpopulations of bacteria with gradually increasing apparent MICs. To test the uniqueness of resistance levels in these cells, individual colonies were picked from agar plates containing antibiotic at concentrations of 8, 120, and 500 pug of methicillin per ml. Colonies were dispersed in saline, serially diluted, and replated immediately for a PAP. As expected, all cells could grow at the concentration of methicillin that was present in the TSA plate from which the particular colony was picked. Additional cells
3 126 TOMASZ ET AL. M 5 UL. 1x10 Ex105 strains Methicillin (jg/ml) 0 Class 4 1x104 1x103 I di Mathicilin (jgml) FIG. 2. Population analysis of MRSA strains. Stationary-phase cultures of class 1, 2, 3, and 4 strains and a susceptible strain were plated at several dilutions on a series of agar plates containing increasing concentrations of methicillin. The strains were the same as those described in the legend to Fig. 1. The symbols indicate the results obtained from three randomly picked colonies of each strain. with higher MICs were also present with frequencies predictable from the PAP of the original strain (Fig. 3). Selection of the most highly resistant subpopulations in the presence of antibiotic. Several heterogeneous isolates (Table 1, expression classes 1 to 3) were grown in TSB, and 107 ANTIMICROB. AGENTS CHEMOTHER. CFU was inoculated in 10 ml of TSB containing methicillin (100,ug/ml) and clavulanic acid (4,ug/ml) and incubated at 37 C. Each strain grew to turbid culture within 48 h. Such cultures were composed of bacteria that showed high-level and homogeneous resistance to methicillin. These bacteria represent the progeny of the highly resistant minority population present in the original heterogeneous strain selected by antibiotic pressure. In a previous publication, such cultures were referred to as HOM* cultures in reference to their instability frequently observed in the absence of drug selection (4). Stability of the highly resistant subpopulation (HOM*) in the absence of drug pressure. The stability of HOM* cultures was tested by passage in antibiotic-free medium in the following manner. A 100-,lI volume (containing approximately 107 CFU) of highly turbid (stationary-phase) cultures was inoculated into 10 ml of drug-free TSB and incubated at 37 C until turbidity had again reached the maximum, at which point the culture was back diluted again and passage in drug-free medium was repeated for approximately 50 to 100 cell doublings. At that point, various dilutions of the cultures were plated on TSA and TSA-containing methicillin (100,ug/ml) plus clavulanic acid (4,ug/ml). The HOM* derivatives of individual strains differed from one another: some retained their homogeneity upon passage in drug-free medium, the rest reverted to heterogeneity (Table 1). One unstable HOM* strain was analyzed in more detail for the rate of reversion from the HOM* to the heterogeneous state. After each 10 generations of growth in drug-free medium, the culture was tested for its expression of resistance by population analysis. Figure 4 shows that, in the case of this particular strain, reversion from the uniform (homogeneous) to a heterogeneous (class 2) mode of expression occurred in a stepwise fashion: after the first 30 to 40 cell divisions in the absence of antibiotic, it was the most highly resistant group of bacteria that dropped in frequency, giving rise to a transient PAP that resembled the shape of a PAP of a class 3 strain. After further passages in drug-free medium, the frequency of the most highly resistant bacteria further declined as did the frequency of cells with an intermediate (MIC, 25 to 50,ug/ml) resistance level, until finally the PAP of the culture reached a shape reminiscent of the PAP of the original heterogeneous resistant strain. Stability of the expression mode in class 4 (homogeneous) strains. Since the strains used in these studies originated in a clinical specimen, the possibility had to be considered that at least some of the homogeneously resistant strains obtained were actually unstable HOM* cultures selected during antibiotic therapy in the hospital setting. Strains classified as class 4 were put through the stability test (serial passage in antibiotic-free medium for 40 to 50 generations). While most strains retained homogeneity, one (Table 1, strain SA 10) reverted to a heterogeneous (class 2) phenotype after 50 serial subcultures in drug-free medium. DISCUSSION Although the beta-lactam MICs for MRSA strains may reach extremely high levels (e.g., several milligrams of methicillin per milliliter), it is only in relatively rare, socalled homogeneous isolates that every cell in a culture of an MRSA strain shares such high levels of resistance. Instead, previous studies (4, 9) indicate that most clinical isolates of MRSA show the so-called heterogeneous form of resistance. Cultures of such heterogeneous strains have sometimes been described as being composed of two bacterial populations:
4 VOL. 35, 1991 EXPRESSION OF METHICILLIN RESISTANCE 127 TABLE 1. Expression classes of methicillin-resistant staphylococci amic (jg/ml) for Frequency of cells Frequency of Stability of the most Expression Straina StranaOrigin Origin' MICe cell (maj majorityc fority' not majority inhibited MICd by highly tant cellse resis- resistant tion aftersubpopula- selection" class SA 1 CDC 3 1o-4 1o-7_1o-8 Stable 1 SA 80 N.Y. Hospital 3 1o _10-8 Unstable SE 81 N.Y. Hospital _10-8 ND9 SE 83 N.Y. Hospital _10-8 ND SA 4 WCMC Unstable 2 SA 7 WCMC Unstable SA 17 WCMC Unstable SE 32 L.I.J ND SA 43 Mt. Sinai (400)h Stable 3 SA 89 ICP (400) ND SA 103 FF 50 lo-3 1o-3 (400) ND SE 115 SJ (400) ND SA 10 WCMC 400 Unstable 4 SA 19 LIJ 400 Stable SA 111 SJ 800 Stable SE 133 SJ 800 Stable a SA, Staphylococcus aureus; SE, coagulase-negative staphylococci. b Abbreviations: CDC, Centers for Disease Control, Atlanta, Ga.; N.Y. Hospital, New York Hospital-Cornell University Medical Center, New York; WCMC, Westchester County Medical Center, Valhalla, N.Y.; LIJ, Long Island Jewish Hospital New Hyde Park, N.Y.; Mt. Sinai, Mt. Sinai Hospital, New York; ICP, Instituto Camara Pestana, Lisbon; FF, Faculdade de Farmacia, Lisbon; SJ, San Jose Hospital, Lisbon. C MIC (of methicillin) that inhibits the growth of 99 to 99.9% of cells. d Frequency of cells capable of growing at the MIC for the majority of cells. Frequency of cells capable of growing in the presence of 100 jlg of methicillin per milliliter. f Stability of the HOM* population after 100 generations of growth in the absence of antibiotics (see text). g ND, Not done. h Frequency of cells capable of growing in the presence of 400 jig of methicillin per milliliter. one with a low and one with an extremely high level of resistance (11). However, a detailed population analysis of at least some isolates (e.g., Fig. 3) suggests the presence of multiple subpopulations with a virtually continuous spectrum of intermediate MICs between the MIC for the majority of the cells and the MIC for the most highly resistant bacteria. Often, the majority of cells (i.e., >90% CFU) have a relatively low MIC, only two to five times that of a typical susceptible strain, and cells with extremely high resistance levels (MIC > 100,ug/ml) are rare (10-3 to 10-7). A quantitative and pictorial expression of this peculiar phenotypic expression of a bacterial property is provided by population analysis curves in which the frequencies of bacteria with various MICs are plotted against the concentration of antibiotic. The most surprising and novel finding described in this paper is the apparent reproducibility of such PAPs for a given strain from one cultivation to another. While MRSA strains have frequently been examined by population analysis in the past (5, 6, 10), the strain specificity and stability of these PAPs seemingly were not recognized until now. The critical evidence described in this paper supporting the stability of PAPs was the experiment in which strains belonging to expression class 1, 2, or 3 were plated on nonselective agar, three or more colonies were picked from each at random, and these colonies were then grown in liquid cultures and analyzed by population analysis. Each colony reproduced a PAP characteristic of the original strain. Our definitions used in the classification are arbitrary. Nevertheless, classes 1 through 4 together form a progression of gradually improved phenotypic expression of antibiotic resistance which can include both coagulase-positive and -negative staphylococci. At one end of this spectrum are what we propose to call class 1 heterogeneous strains in UA. C) 1xE0 Methicillin (jg/mi) FIG. 3. Antibiotic susceptibility of subpopulations with intermediate resistance levels in a culture of a class 2 MRSA strain. Population analysis of the class 2 strain (original strain) is shown (0). A single colony was picked from the agar plates containing 8 (0), 120 (U), and 500 (*),ug of methicillin per ml; the cells were dispersed in water and immediately replated for population analysis ---). The positions of the intermediate resistance colonies picked from the original population analysis curve are shown ( ).
5 128 TOMASZ ET AL. ANTIMICROB. AGENTS CHEMOTHER. E0 cycle Origina Cycle A MthIcIlln (Wgml) FIG. 4. Stepwise loss of the homogeneous expression of resistance during the cultivation of a HOM* culture in antibiotic-free medium. The HOM* population of an originally heterogeneous MRSA was passaged in antibiotic-free medium as described in the text. One cycle of passage involved approximately 8 to 10 cell divisions. The population analysis curves of the original strain (L), of the HOM* derivative (x), and of the culture after 5 (0), 7 (A), and 10 (0) cycles of passage in antibiotic-free medium are shown. which over 99.99% of the cells in a culture have MICs not far above the MIC for a typical contemporary methicillinsusceptible strain. The frequency of highly resistant cells in class 1 cultures can be extremely low (10-v to 10-8). In spite of their extremely low frequency, the most highly resistant subpopulations of MRSA can overgrow a culture under conditions of antibiotic pressure. With appropriately sized inocula, every one of the clinical isolates, including class 1 isolates, was able to give rise to a culture of highly resistant bacteria (HOM*) in medium containing high concentrations of methicillin (25 or 100 jxg/ml) and clavulanic acid (4,ug/ml) (Table 1). The practical implication of this finding is that every MRSA, irrespective of its type of expression of resistance, could potentially cause treatment failure in vivo. However, individual heterogeneous isolates differed in the stability of the highly resistant (HOM*) subpopulation after the release of antibiotic pressure. The majority of HOM* cultures examined here'reverted to a heterogeneous expression of resistance, while some strains retained uniform and high MICs after 50 to 100 generations of growth in nonselective medium. At the other extreme of progression of phenotypic expression classes' are the class 4 (homogeneous) strains, in which all cells in a culture share uniform and high MICs. Class 2 and class 3 heterogeneous strains exhibit PAPs that are intermediate between those of class 1 and class 4 isolates. The collection examined in this article had no epidemiological bearing, and therefore the relative frequencies of strains with class 1, 2, or 3 phenotypes cannot be evaluated from the data described here. The stability of the PAP patterns was well established for the strains shown in Fig. 2. Similar reproducibility of population structures has also been observed in transposon mutants and their appropriate genetic transformants (unpublished findings). To what extent such stability holds true for all MRSA isolates is not known. Clearly, instability of phenotypic expression can exist in many of the HOM* cultures left to grow without antibiotic selection. Interestingly, this was also true for at least one of the strains that initially (upon receipt from the clinical laboratory) showed a class 4 (homogeneous) resistance but subsequently reverted to a heterogeneous expression. In this case, selection of HOM* may have occurred in the hospital environment. Inquiry at the clinical source confirmed our suspicion that the patients were receiving antibiotic therapy (a cephalosporin) at the time of culture. The process of reversion may occur occasionally in a stepwise manner, generating transient (unstable) class 3 or class 2 expression modes (Fig. 4) before some more stable form of phenotype is established. Thus, it is important to purify clinical isolates by streaking and single-colony isolation before undertaking more detailed physiological studies. The reproducibility of the complex strain-specific patterns of the phenotypic expression of antibiotic resistance in progeny cultures generated from randomly picked colonies of MRSA is a surprising and remarkable phenomenon. The major conclusion of this finding is that the mode of phenotypic expression of methicillin resistance is staphylococci is genetically controlled at the population level as well. Since all strains carried the mec gene (and also, presumably, its gene product, the penicillin-binding protein 2A) irrespective of their expression class, the genetic determinants controlling the mode of expression must be the auxiliary genes that have already been identified through the analysis of the phenotypes of transposon mutants (1, 4, 8). ACKNOWLEDGMENTS These investigations were supported in part by U. S. Public Health Service grant R01 Al from the National Institutes of Health. S.N. was supported by a fellowship from the Association of Pediatric Department Chairmen, Pediatric Scientist Training Program, which is funded by the National Institute of Child Health and Human Development. We acknowledge the excellent technical assistance of Marilyn Chung and numerous helpful discussions with H. de Lencastre. REFERENCES 1. Berger-Bachi, B., L. Barberis-Maino, A. Strassle, and F. H. Kayser. FemA, a host-mediated factor essential for methicillin resistance in Staphylococcus aureus: molecular cloning and characterization. Mol. Gen. Genet. 219: Galetto, D., J. Froggatt, J. Kornblum, and G. Archer Abstr. Annu. Meet. Am. Soc. Microbiol. 1988, A59, p Hackbarth, C. J., and H. F. Chambers Methicillinresistant staphylococci: detection methods and treatment of infections. Antimicrob. Agents Chemother. 33: Hartman, B. J., and A. Tomasz Expression of methicillin resistance in heterogeneous strains of Staphylococcus aureus. Antimicrob. Agents Chemother. 29: Heneine, N., and P. 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6 VOL. 35, 1991 EXPRESSION OF METHICILLIN RESISTANCE Maniatis, T., E. F. Fritsch, and J. Sambrook Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 8. Murakami, K., and A. Tomasz Involvement of multiple genetic determinants in high-level methicillin resistance in Staphylococcus aureus. J. Bacteriol. 171: Sabath, L. D Chemical and physical factors influencing methicillin resistance of Staphylococcus aureus and Staphylococcus epidermidis. J. Antimicrob. Chemother. 3(Suppl. C): Sabath, L. D., and S. J. Wallace Factors influencing methicillin resistance in staphylococci. Ann. N.Y. Acad. Sci. 182: Thornsberry, C., and L. K. McDougal Successful use of broth microdilution in susceptibility tests for methicillin-resistant (heteroresistant) staphylococci. J. Clin. Microbiol. 18: Tomasz, A., H. B. Drugeon, H. M. de Lencastre, D. Jabes, L. McDougal, and J. Bille New mechanism for methicillin resistance in Staphylococcus aureus: clinical isolates that lack the PBP 2a gene and contain normal penicillin-binding proteins with modified penicillin-binding capacity. Antimicrob. Agents Chemother. 33:
Krzysztof Sieradzki and Alexander Tomasz* The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
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