Multiple Mechanisms of Methicillin Resistance and Improved Methods for Detection in Clinical Isolates of Staphylococcus aureus

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1991, p. 632-639 0066-4804/91/040632-08$02.00/0 Copyright D 1991, American Society for Microbiology Vol. 35, No. 4 Multiple Mechanisms of Methicillin Resistance and Improved Methods for Detection in Clinical Isolates of Staphylococcus aureus HERMINIA DE LENCASTRE,1t AGNES M. SA FIGUEIREDO,1 CARL URBAN,2'3 JAMES RAHAL,2'4 AND ALEXANDER TOMASZ1* The Rockefeller University, New York, New York 10021-63991; Booth Memorial Medical Center, Flushing, New York 113552; New York University School of Medicine, New York, New York 100033; and Albert Einstein College of Medicine, Bronx, New York 104614 Received 25 June 1990/Accepted 15 January 1991 The mec gene of a number of clinical methicillin-resistant Staphylococcus aureus isolates exhibiting a variety of heterogeneous expression modes was selectively inactivated by allelic replacement mutagenesis. While the resistance level of each of the transformants was reduced, the methicillin MIC for these transformants was well above the MIC for susceptible laboratory strains of S. aureus and was similar to the methicillin MIC for many contemporary clinical isolates which did not react with the mec-specific DNA probe but which showed a low or borderline level of resistance to methicillin. A number of those strains had no detectable I8-lactamase, and for about half of the isolates that did carry plasmid-borne j8-lactamase, elimination of the plasmid caused only partial reduction of the methicillin MIC or no reduction at all. The findings suggest that many contemporary strains of staphylococci harbor a combination of at least three distinct I8-lactam resistance mechanisms: (i) the mechanism related to the acquisition of the foreign mec gene and (ii) a,b-lactamase-dependent and (iii) a I3-lactamase-independent mechanism, each one of which can provide a certain degree of resistance against penicillinase-resistant Il-lactam antibiotics. Methicillin-resistant clinical isolates of Staphylococcus aureus (MRSA) carry a complex, as yet only poorly understood resistance mechanism. All MRSA isolates examined so far have contained the mec gene, a 2,130-bp segment of foreign DNA coding for a low-affinity penicillin-binding protein (PBP 2A) (3, 19, 20). Despite the ubiquitous presence of this gene, MRSA isolates show tremendous variation in the MICs for the majority of the cells, and cultures of MRSA isolates are also heterogeneous: they contain a variable number of subpopulations for which the MICs range from very low to very high. Recently, it was shown that these complex modes of phenotypic expression are strain specific and appear to be under genetic control (37). In extreme cases, the methicillin MIC for the majority of bacteria (>99.99% of cells) may be as low as 3 p.g/ml (a MIC for susceptibility is 0.5 to 1.0 p.g/ml), despite the presence of the mec gene and its gene product (PBP 2A) in every cell. Similar, moderately increased MICs (4 to 8 p.g/ml) were also detected recently in some isolates which did not carry the mec gene, whether or not they produced f-lactamase (6, 36), the overproduction of which has been proposed as one mechanism that causes a moderate (borderline) elevation of the MIC of typically penicillinase-resistant antibiotics (6, 22, 23) Ȯne purpose of this study was to determine the relative contribution(s) of these mechanisms to the MIC for some selected MRSA isolates. Of particular interest was testing of the possibility that these distinct mechanisms may coexist in single isolates. An additional purpose of this study was to test to what extent these mechanistic complexities contribute to the * Corresponding author. t Present address: Centro de Tecnologia Quimica e Biol6gica, Oeiras, and Universidade Nova de Lisboa, Lisbon, Portugal. 632 problem of identification of these potentially dangerous pathogens in clinical specimens (10, 34). We compared a frequently used identification technique (Kirby-Bauer [KB] method) with a method that is not based on the phenotypes of the cells but, rather, on the recognition of mec-specific DNA sequences in the bacteria. (A preliminary form of this report has been presented previously [8].) MATERIALS AND METHODS Strains. S. aureus isolates were obtained through the courtesy of colleagues at the following institutions: Clinical Microbiology Laboratory, Booth Memorial Medical Center, Queens, N.Y.; Department of Microbiology, New York University School of Medicine, New York; New York Hospital-Cornell University Medical Center, New York (Lawrence Senterfit); Centers for Disease Control, Atlanta, Ga. (Richard Facklam); American Type Culture Collection, Rockville, Md.; and Instituto Nacional de Saude Dr. Ricardo Jorge (INSRJ), Lisbon, Portugal (Maria V. Vaz Pato). Preparation of stocks. Clinical isolates were received on agar slants or in lyophilized form and were grown in tryptic soy broth (TSB; Difco, Detroit, Mich.) and plated onto tryptic soy agar (TSA). A single colony was picked from the TSA plate, grown to the stationary phase in a few milliliters of TSB and then supplemented with sterile glycerol (final concentration, 10%), frozen in small aliquots on dry ice, and stored at -70 C. Stock cultures were also retained on TSA at 4 C. Purification of isolates from clinical sources by single-colony isolation was extremely important, since we often found that such cultures were contaminated with highly methicillin-resistant (both coagulase-positive and -negative) staphylococci. These contaminants, presumably, originated from the same nosocomial sources (equipment,

VOL. 35, 1991 MULTIPLE MECHANISMS OF METHICILLIN RESISTANCE 633 carriers), the importance of which has been well recognized in the epidemiology of MRSA infections. Culture growth. Overnight cultures (10 ml of TSB in standard 18-mm-diameter test tubes) were initiated by inoculation of cultures either with a single colony picked from TSA or with 10 to 50,ul of the frozen stock culture and were incubated at 37 C in a horizontal rotating device that ensured vigorous aeration and a high bacterial yield (about 1010 CFU/ml). Testing for methicillin resistance. (i) Preliminary agar screen. Before undertaking determination of the precise MIC for an isolate, the following preliminary tests were performed. The overnight cultures were plated at appropriate dilutions onto TSA (to determine the precise cell concentration) and undiluted 100-,ul portions were spread onto three kinds of TSA plates, each of which contained 4,ug of clavulanic acid per ml plus methicillin at concentrations of 5, 25, and 100,ug/ml. The plates were evaluated after incubation at 37 C for 24 h. Identical results were obtained without clavulanic acid, so its addition is not necessary for this test. (ii) MIC determination with screen-negative isolates. The methicillin MICs for staphylococcal isolates that showed no colonies on any of the three test plates in the preliminary agar screen were determined by agar dilution in the following manner. Portions of the overnight cultures were plated onto TSA containing 0, 0.5, 1.0, 2, and 4,ug of methicillin per ml at two cell concentrations: one without predilution (25,ul of an overnight culture directly spread onto the agar) and another after an appropriate (106- to 107-fold) predilution that produced countable numbers of colonies (a few hundred to 1,000) on the antibiotic-free TSA plate. Plating of undiluted cultures allowed detection of cells for which the MICs were somewhat elevated and that may have been present at a low frequency, while the prediluted samples allowed an accurate estimation of the MIC for the predominant (majority) cell population, which we defined as the drug concentration that caused at least a 99% loss in the number of CFU. In practice, the decrease in CFU at the MIC for the majority cell population almost always involved 99.9% or more of the culture. Repeated determinations by this method of the MICs for the same strains gave excellent reproducibility. (iii) MIC determination with screen-positive isolates. Detailed population analysis was used to determine the methicillin MICs for staphylococcal isolates that showed growth (from confluent to various numbers of colonies) on any of the test plates in the preliminary agar screen. The overnight cultures were plated at several dilutions (10-1, 10-3, 10-5, and 10-7) onto a series of TSA plates containing twofold dilutions of methicillin ranging in concentration from 0 and 0.75 to 800,ug/ml. Dot blot hybridization. Whole-cell lysates were prepared from 0.5 ml of the overnight cultures that were grown as described above. Cells were centrifuged and resuspended in 1 ml of TE (18), and cell walls were digested by the addition of 10,ul of lysostaphin (10 mg/ml; Sigma, St. Louis, Mo.) and 5,lI of muramidase (10 mg/ml; Sigma). The protoplasts were then lysed with 10,ul of sodium dodecyl sulfate (10%) and 2,lI of proteinase K (10 mg/ml; Boehringer Mannheim Biochemicals, Indianapolis, Ind.) at 37 C for 1 h. The lysate was then treated with phenol, the water phase was separated by centrifugation, and the DNA (water phase) was denatured at alkaline ph by the addition of 10 [l of 3 M NaOH and incubation at room temperature for 10 min. The samples were then chilled at 0 C, and the ph was neutralized by the addition of 110 pal of 2 M ammonium acetate. Lysates were kept on ice, and serial dilutions were made in TE at 0 C. Samples of 25 pi were then loaded into dot blot wells. A Bio-Rad dot blot apparatus and Gene Screen membranes were used (36). A radiolabeled, mec-specific DNA probe (see below) was added to Gene Screen membranes after prehybridization and hybridization at 60 C by standard methods (18). DNA probe. The 32P-labeled mec DNA probe used in this study was a PstI-XbaI fragment (MF13) from the mec gene of the Australian type strain ANS 46 (20) cloned in ptz219. This fragment is totally internal to the PBP 2A open reading frame, running from nucleotide positions 773 to 1929 in the mec gene sequence of Song et al (31). A DNA preparation of the plasmid was obtained by established techniques (18). A radiolabeled probe was prepared by using a random priming kit and [32P]dATP (Amersham Corp., Arlington Heights, Ill.), as described previously (36). Disk diffusion test of susceptibility to antibiotics. One specific group of isolates (108 strains obtained from Booth Memorial Medical Center) was used to compare the KB disk susceptibility testing method with the mec DNA probe. The KB tests were performed at the Clinical Microbiology Laboratory of Booth Memorial Medical Center by the 1986 approved methodology recommended by the National Committee for Clinical Laboratory Standards (25). Bacteria were plated onto Mueller-Hinton agar (no salt added) with oxacillin disks (1 pug/ml), and the plates were evaluated after incubation at 35 C for 18 to 24 h. In the latest (1990) methodology (26), the incubation time is not more than 24 h. Strains demonstrating inhibition zones of -10 mm in diameter were considered resistant, and those yielding inhibition zones of.13 mm in diameter were considered susceptible. Strains producing zone diameters of 11 to 12 mm or those with detectable growth within a zone of inhibition were reincubated for 24 h at room temperature. All strains were reported ultimately as either susceptible or resistant. The 108 strains used in this study were picked at random from among the isolates that were identified as resistant or susceptible on the basis of such KB test results. P-Lactamase activity. P-Lactamase activity was determined by depositing 5 RI of nitrocefin (10-mg/ml solution, Glaxo Laboratories) on young (less than 24 h) bacterial colonies grown on TSA and on TSA plus a sub-mic (0.5 jig/ml) of methicillin. Curing of the P-lactamase plasmid. Curing of the,-lactamase plasmid was done by growing the staphylococcal strain (inoculum, 105 cells per ml) at 42 C in TSB containing one-half the MIC of ethidium bromide (2 to 4,ug/ml). When a culture became turbid, the bacteria were plated onto TSA and colonies were tested for 1-lactamase activity by the nitrocefin method. Genetic transformation. Transforming DNA was prepared by a previously described method (36) from a TnS5I mutant of a homogeneously resistant strain in which the insert was inside the mec gene (21). Recipient cells were made competent by treatment with helper phage 455 and 0.1 M CaCl2 (33). Transformants were scored by plating them onto TSA containing 10 jig of erythromycin per ml. RESULTS Contribution of the mec gene to the MICs for MRSA isolates. Despite the presence of the mec gene and its gene product PBP 2A in all MRSA isolates, individual isolates differed widely in the MICs for their cell majorities and also in the frequency with which they generated highly resistant cells during growth in culture. Thus, the actual, quantitative

634 DE LENCASTRE ET AL. ANTIMICROB. AGENTS CHEMOTHER. DNA Methicillin MIC MIC and frequency probfg (jlg/mi) for of most resistant (mec) maiority of cells jubpopulations EPand/sion clasm PBP 2A and/or mechgnism SELF PROBE susceptible -. BM 57 (Heterogeneous) BM 73 (Heterogeneous) BM 72 (Borderline resistant) BM 79 (Heterogeneous) BM 52 (Homogeneous) 1.0 1.5 6.0 8.0 200 800 - no > 200 (10-7) yes class 1 >400 (10-5) yes class 2 - no MOD 800 (10-3) yes class 3 800 (1) yes class 4 FIG. 1. Properties of representative strains of MRSA. A methicillin-susceptible laboratory strain of S. aureus, a borderline-resistant strain (BM 72), and four MRSA isolates representing different modes of phenotypic expression of methicillin resistance (classes 1 through 4 of expression of resistance [35]) were assayed for a number of relevant properties by the techniques described in the text. Each culture lysate was assayed with the mec-specific DNA probe at three cell equivalent concentrations (from left to right): 107, 106, and 105. MOD, See reference 36. contribution of the mec gene to the resistance phenotype of a given strain is not clear. An experimental test of this was made possible by the recent isolation of a Tn551 mutant in which the transposon was unequivocally localized within the mec gene (21, 24). Transforming DNA prepared from this transposon mutant was used to selectively inactivate the mec gene in clinical isolates. It has been shown that in such crosses the transforming DNA carrying the transposon marker (erythromycin resistance) replaces the active mec gene of the recipient with the transposon-inactivated gene by homologous recombination (24). Three clinical MRSA isolates, each of which was free of 1-lactamase activity, were chosen as recipients for the crosses. Each strain carried the mec gene (DNA probe positive) and contained comparable amounts of PBP 2A. On the other hand, the strains differed vastly in their phenotypic expressions of resistance (Fig. 1). Erythromycin-resistant transformants and the corresponding recipient strains were compared for their population analysis profiles (Fig. 2A to 2C). In each case, inactivation of the mec marker led to a dramatic reduction in the MIC. Nevertheless, the MICs for the transformants (2 to 4,ug/ml) were consistently higher than the MICs for the susceptible laboratory strains (0.5 to 1 jig/ml). These residual MICs were within the range of values that are considered susceptible and/or borderline by criteria of clinical microbiology, and as such, they are of limited clinical significance (10). Nevertheless, they are of considerable mechanistic interest, since they suggest the presence of a second kind of intrinsic resistance in the background of the mec carrier strains. Methicillin MICs among susceptible (mec probe-negative) isolates of S. aureus. In order to assess the significance and possible origin of the somewhat elevated residual methicillin MICs for MRSA strains in which the mec gene was specifically inactivated, we undertook a survey of the methicillin MICs for a large number of methicillin-susceptible S. aureus isolates from a variety of sources. We defined as methicillin susceptible all isolates that gave negative results in the preliminary agar screening test described in Materials and Methods. Thus, none of the 268 isolates tested produced detectable colonies (<10-9) on TSA containing 5, 25, or 100,ug of methicillin per ml plus 4 jig of clavulanic acid per ml. One hundred of these isolates were also tested with the mec DNA probe, each with negative results. After these preliminary tests, the precise methicillin MICs for the strains were determined by using two inocula (about 2 x 108 and about 1 x 103 CFU per plate) and a series of methicillin concentrations (0.5, 1.0, 2.0, 4.0, and 8.0 jig/ml), as described in Materials and Methods. The MIC was taken as the lowest drug concentration causing the loss of 99% or more of the colonies on the plate with the lower (i.e., about 103) inoculum. Figure 3 shows the appearance of the agar assay for a select group of strains. Each strain was also tested for 1-lactamase (constitutive or inducible) production, and penicillinase-negative strains were also tested for their penicillin MICs by the same agar dilution methods, except that methicillin was replaced with benzylpenicillin in the agar plates at the following concentrations: 0.02, 0.05, 0.1, and 0.2 jlg/ml. The results of these tests are summarized in Table 1. The data in Table 1 indicate that early (1928 to 1945) staphylococcal isolates were all free of penicillinase; the methicillin MICs for these isolates were low (0.5 to 1,ug/ml). Such staphylococci, with or without penicillinase, have become a rarity among the recent (1989 and 1990) clinical isolates (5 of 122) and colonizing strains (7 of 93). While most strains for which the methicillin MIC was 2 or 4 jig/ml carried penicillinase, this was not always the case, since the methicillin MIC for 32 of 122 of the clinical strains and 7 of 93 of the colonizing strains was 2,ug/ml and there was no detectable penicillinase activity. The penicillin MICs for these strains were 0.05 jlg/ml with the low inoculum and 0.1 to 0.2 jig/ml with the high inoculum. In these bacteria, the moderately elevated MIC of methicillin cannot be attributed to 3-lactamase activity. In order to test further the contribution of penicillinase to the methicillin MIC, 11 penicillinase-positive strains for which the MICs were 2 or 4,ug/ml were cured of the penicillinase plasmid (by growth in ethidium bromide, as described in Materials and Methods). Only for one strain (SA43) did curing decrease the methicillin MIC from 2 to 1 jig/ml, i.e., to that for truly susceptible strains; for five of the rest of the strains, the MIC dropped from 4 to 2 jig/ml, and

VOL. 35, 1991 MULTIPLE MECHANISMS OF METHICILLIN RESISTANCE 635 109t 18 107 16 Class 1 0 1 0 D 5 U. 10 0 103 1010 1 09 108 V 107 V 106 D 105 0 10 i 1o04 1o03 102V 101i 100 Li II I I I II I III I III I III I II I III I III Methicillin (jg/mi) a A A5 mec::tn551 I -llr-i I 1 I 0.75 1.5 6.0 25 Methicillin (jg/mi) I C:lass 4 0.75 1.5 6.0 25 100' 400'1600 Methicillin (ig/ml) FIG. 2. Decrease in the methicillin MICs after transposon inactivation of the mec gene by genetic transformation in three MRSA strains belonging to expression classes 1 (A), 2 (B), and 4 (C). Population analysis profiles are shown for each of the original MRSA strains (x), for their transposon-inactivated transformants (mec::tn551 [0]), and for a standard methicillin-susceptible strain (OI and dashed lines). bacteria. We determined the clavulanic acid MIC for several strains and found that it varied considerably, from about 8,ug/ml (in the laboratory susceptible strains) to as high as 64 p,g/ml or more (for strains CDC 6 [36] and BM 72, for which the methicillin MICs, 4 and 8 ptg/ml, respectively, were also the highest). It appears, therefore, that 4,ug of clavulanate per ml may lower the methicillin MIC for some strains through synergistic antibacterial action, similar to the case noted for sulbactam (16) and confirming the findings of Sierra-Madero and colleagues (30). Comparison of the KB method and a mec-specific DNA probe for the identification of methicillin-resistant staphylococci. The methicillin MIC for the majority of bacteria present in heteroresistant MRSA cultures may be as low as 3 to 6,ug/ml (Fig. 1), which is quite close to the MIC for many of the contemporary susceptible and/or borderlineresistant S. aureus isolates. The latter group of organisms, in 100 400 1600 contrast to the MRSA strains, do not seem to present chemotherapeutic problems in the most common types of staphylococcal infections. Therefore, accurate identification for five other strains there was no detectalble change in the of these two types of staphylococci in clinical specimens is MIC (2 jig/ml). important for choosing the appropriate kind of chemother- acid. We also apy. We compared the KB method and use of the mec- Methicillin MICs with and without clavulaanic tried to use the determination of methicilliin MICs with and specific DNA probe for their accuracies in identifying MRSA without the inclusion of 4,ug of clavulanilc acid per ml (to strains among 108 isolates obtained from the microbiology suppress,-lactamase activity) in order to i4dentify strains for laboratory of Booth Memorial Medical Center. All disk which the somewhat elevated methicillin M[IC was due to the diffusion assays were performed at the hospital laboratory, production of P-lactamase. However, these tests gave incon- while the tests with the DNA probe were done at The clusive results. While 4,ug/ml was below ti he clavulanic acid Rockefeller University. MIC for all strains, the addition of clavulanate resulted in a Of the 49 strains identified as methicillin susceptible by the reduction (by a factor of 2) in the methicilllin MIC for most KB method, 11 strains gave a positive signal with the mec-negative strains, including even,-lalctamase-negative mec-specific DNA probe.

636 DE LENCASTRE ET AL. FIG. 3. Low-level (intrinsic) methicillin resistance in staphylococcal strains that did not react with the mec-specific DNA probe. A highly methicillin-susceptible laboratory strain, 27s, and four other mec probe-negative clinical isolates (BM 25, BM 36, BM 2, and BM 37) were assayed for their methicillin MICs by a combination of an agar screen and agar dilution method. Each strain was tested at two different cell concentrations corresponding to approximately 2 x 108 and 2 x 102 to 1 x 103 CFU per plate. Numbers on the left indicate the concentration of antibiotic in the plates; numbers on the top identify the strains. For strain 27s cells, the MICs were uniform (1.0,ug/ml); strains BM 25 and BM 36 were free of,-lactamase; methicillin MICs for these strains were 2,ug/ml. Strains BM 2 and BM 37 produced P-lactamase; methicillin MICs for these strains were 4,ug/ml. Of the 59 strains classified as methicillin resistant by the KB method, 10 strains showed no detectable mec-specific DNA sequences. Thus, the two methods disagreed in about 18 to 20% of the cases, in both directions. In order to test further these conflicting findings, we undertook a more detailed analysis of the 21 problematic cultures by two additional methods: (i) the preliminary agar test screen described in Materials and Methods and (ii) an enrichment screen, in which a large number of bacteria (108 CFU) was used to inoculate 10 ml of TSB containing 25 plg of methicillin per ml with or without 4 [Lg of clavulanic acid per ml. Each of the 11 strains that gave a positive reaction with the mec DNA probe (even though the KB method classified them as methicillin susceptible) gave unambiguous positive reactions both in the agar screen and in the enrichment screen as well. On the other hand, none of the 10 mec probe-negative strains (identified as methicillin resistant by the KB method) showed any colonies on the agar screen, and no strain produced detectable turbidity in the enrichment test, even after 5 days of incubation at 37 C. A detailed population analysis performed on the 11 problem strains showed that they were all MRSA strains belonging to expression classes 1 or 2 (37); i.e., they were strains which expressed the highly resistant phenotypes with very low frequencies only (Fig. 1 and 2). When the population analysis was performed on all the 60 mec probe-positive strains from the Booth Memorial Medical Center, more than 70% (43 of 60) were shown to belong to expression classes 1 or 2. Figure 4 shows a comparison of the results of disk diffusion assays performed at The Rockefeller University with the following strains: (i) four MRSA strains representing different expression classes (as defined by the population ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Some properties of methicillin-susceptible strains of S. aureus MIC (,uglml)' Source No. of Penicil- (collection dates)a strains linase Methi- Penicillin cillin ATCC (1928 to 1945) 14-0.5-1 -s0.02 CDC (1952 to 1962) 9-0.5-1 '<0.02 11-2 0.05 19 + 2 New York Hospital (1990) 2-1 <0.02 3 + 1 16-2 0.05 48 + 2 5 + 4 Booth Memorial Medical 5-2 0.05 Center (1989) 38 + 2-5 + 4 New York University School 3-1.0.02 of Medicine (1990) 1-2 0.05 29 + 2 INSRJ, Lisbon (1989) 3-1 <0.02 1 + 1 6-2 0.05 49 + 2 1 + 4 a Strains from the American Type Culture Collection (ATCC), the Centers for Disease Control (CDC), New York Hospital-Cornell University Medical Center, and Booth Memorial Medical Center were clinical isolates; strains from New York University Medical Center and INSRJ were from patients with nasal carriage of the organism. b MICs were determined as described in the text.-, Penicillin MICs for penicillinase-producing strains were all >200,ug/ml with the high bacterial inocula. analyses in Fig. 4A), (ii) a susceptible strain (MIC, 1 plg/ml), and (iii) a borderline-resistant strain (MIC, 4 p.g/ml). The conditions of the assay were those suggested by the National Committee for Clinical Laboratory Standards (25, 26), except that a whole series of bacterial inoculum sizes (from 109 to 105 CFU per plate) was also used and incubation was done at 37 C. However, exactly the same results were obtained at 35 C (data not shown). Four MRSA strains, each representing a different expression class (Fig. 4A), were plated at a series of cell concentrations (from 109 to 105 CFU per plate) (Fig. 4B). Two features of Fig. 4B stand out. (i) The respective inhibition zone sizes of the susceptible strain (26 mm) and the borderline-resistant strain (14 mm) were independent of the inoculum size. (ii) In contrast, the diameter of the zone of inhibition changed markedly for the class 1 and class 2 heteroresistant MRSA strains (but not for class 3 or 4 strains; Fig. 4C) as an inverse function of the inoculum size (Fig. 4B and D). By using a bacterial inoculum of 107 cells per plate (corresponding to the inoculum recommended by the National Committee for Clinical Laboratory Standards [25, 26]), the borderline-resistant and heteroresistant strains could not be reliably distinguished. The identification of the two MRSA isolates belonging to expression classes 3 and 4 (homogeneous) posed no problems with the disk method. It is less clear why 10 of 59 of the mec probe-negative strains were misidentified as MRSA. For these isolates, the

VOL. 35, 1991 MULTIPLE MECHANISMS OF METHICILLIN RESISTANCE 637 A 10 2 103 100 B Class Class Class Class 4 3 2 FIG. 4. Population analysis and antibiotic disk susceptibility tests for the characterization of MRSA strains with different modes of phenotypic expression of methicillin resistance. Four MRSA strains exhibiting the particular methicillin resistance phenotypes (A) were plated at five different cell concentrations (109, 108, 107, 106, and 105 CFU per plate) on Mueller-Hinton agar and tested with oxacillin disks (1,ug) for their respective inhibition zones (B) by the KB method and the recommendation of the National Committee for Clinical Laboratory Standards (25, 26). (C) Results of the KB method for the same strains with a single concentration of the bacteria (107 CFU per plate) recommended by the National Committee for Clinical Laboratory Standards (25, 26). (D) Two MRSA isolates belonging to expression classes 1 and 2 were plated along with a susceptible laboratory strain (MIC, 1 jig/ml) and a 3-lactamase-negative, mec probe-negative, borderline-resistant strain (MIC, 4,ug/ml). MOD, See reference 36. methicillin MIC was 2,ug/ml, and for the rest of the (correctly identified) mec probe-negative strains, the MICs were 2 or 4,ug/ml. All the strains had P-lactamase activity. It is conceivable that the original cultures of the 10 problem strains were contaminated with low numbers of MRSA. These contaminants would then have been eliminated by the single colony picking that preceded all work at The Rockefeller University so that the strains tested as probe negative. DISCUSSION The purposes of this study were twofold: (i) to evaluate the frequencies of occurrence of the various mechanisms that have been proposed to contribute to the increased resistance against penicillinase-resistant,-lactam antibiotics in clinical isolates of staphylococci, and (ii) to compare the effectiveness of a mechanism-based test (DNA probe) with that of a traditional clinical technique for the identification of methicillin-resistant staphylococcal isolates. We first discuss the problem of MRSA detection. The rationale for using a mec-specific DNA probe for the detection of MRSA is well supported by data in the literature indicating the ubiquitous presence of these DNA sequences in all MRSA isolates, independent of the wide variations individual strains exhibit in the phenotypic expression of resistance (1, 3, 12, 19, 20, 21). The application of the mec-specific DNA probe for the detection of MRSA gave results that conflicted with the results of a test (KB) frequently used in clinical microbiology laboratories: 11 of 49 strains classified as methicillin susceptible by the KB method gave a positive signal with the DNA probe. The results obtained with the DNA probe were fully confirmed by an agar screen and an enrichment screen, in both of which large bacterial inocula were used. The identity of isolates obtained from results of the mec probe and these two phenotypic screens indicates that the KB method used to identify this particular collection of isolates was in error in 18 to 20% of the cases. A likely reason for the lack of detection of MRSA strains by the KB method was that these belonged to phenotypic expression class 1 or 2, in which the frequency of highly resistant cells in the cultures was extremely low (10-7 to 10-6). The results of the study described here indicate that multiple mechanisms of methicillin resistance coexist in many contemporary strains of staphylococci. Selective inactivation of the mec gene by the genetic techniques described above resulted in the reduction of the methicillin MICs for each one of the three MRSA isolates tested: a highly and homogeneously resistant (expression class 4) strain and two additional strains belonging to expression classes 1 and 2, respectively (37). Interestingly, however, the residual methicillin MICs for these inactivated MRSAs were significantly higher than the MIC expected for a truly susceptible strain, suggesting the presence of a low-level intrinsic resistance mechanism in the background of MRSA (i.e., mec carrier) strains. Extensive published surveys in the early literature (9, 27, 28, 32) indicate that for the majority of S. aureus isolates in the era preceding the spread of the penicillinase plasmid, penicillin MICs were 0.02,ug/ml or less (9), which corresponds to a methicillin MIC of 0.5 to 1 ig/ml (28). We identified the same MICs for isolates from early collections obtained from the American Type Culture Collection and the Centers for Disease Control (Table 1). The data in Table 1 suggest that such highly methicillin-susceptible staphylococci have become a rarity among contemporary strains, and the majority of contemporary isolates (both from clinical and colonizing specimens) have significantly higher MICs. This finding is likely to be representative of the situation beyond the two medical centers in New York, since the samples collected from nasal carriage came from newly enrolled medical students at New York University Medical Center and ambulatory patients at the National Institute of Health, INSRJ, Lisbon, Portugal. The majority of contemporary strains had penicillinase activity. Yet, a significant portion of strains for which the MIC was increased had no detectable penicillinase. This was also true for the MRSA strains with an inactivated mec gene. Furthermore, curing of the penicillinase activity caused a decrease to the truly

638 DE LENCASTRE ET AL. susceptible level in only 1 of the 11 strains tested. For half of the rest of the strains, the elimination of P-lactamase caused a partial reduction in the methicillin MIC or no reduction at all Ṫhese findings suggest that at least two distinct mechanisms may be responsible, singly or in combination, for the low but significant elevation of methicillin MICs that has taken place for most of the contemporary methicillin-susceptible isolates of S. aureus between the late 1950s and now: (i) production of r-lactamase, as suggested by Thornsberry and McDougal (34), and (ii) another, P-lactamase-independent, mechanism which we propose should be called "low-level intrinsic resistance." The biochemical basis of the,3-lactamase-independent intrinsic resistance is not known. An attractive candidate would be the type of mechanism described in step mutants of staphylococci (2, 14, 15) and pneumococci (11) isolated in the laboratory in which the increased 1-lactam MICs were related to the altered PBPs in the mutants (5, 38). Indeed, we found that S. aureus mutants for which the methicillin MIC was 2 p.g/ml can be isolated easily if a sufficiently high number of susceptible (MIC, 1.Lg/ml) bacteria are plated onto agar containing 1 plg of methicillin per ml (as is the case with the higher [108] inocula used in our agar MIC assay). Stable mutants for which the MIC was 2 p.g/ml appeared in cultures of strain RN2677 with a frequency of 3 x 10-7. High-density cultures of the mutant for which the MIC was 2 1Lg/ml yielded bacteria for which the MIC was 4 p.g/ml at a frequency of about 5 x 10-7. Experimental evidence for the presence of PBPs with decreased penicillin affinity was described in some borderline-resistant (and 3-lactamasenegative) clinical strains of staphylococci (30, 36). We suggest that the intense antibiotic pressure in the clinical environment of staphylococci since the late 1940s resulted in a modest but significant upward shift in the methicillin MICs for a substantial portion of clinical strains, at least in part through an intrinsic mechanism that involved mutational alterations in PBP genes. A similar, global shift upward to a low level of intrinsic resistance may also have occurred among clinical isolates of pneumococci (11). Such intrinsically resistant strains may then have served as hosts to acquired (exogenous) mechanisms, such as,-lactamaseor mec-related resistance. Thus, in contemporary clinical isolates, these three distinct mechanisms, i.e., a low degree of intrinsic resistance and 1-lactamase-associated and mecrelated resistance, may cohabitate in the same staphylococcal cell. In addition, these mechanisms may even become interactive with each other, as shown by the inducibility of PBP 2A production in MRSA strains that also carry,b-lactamase (apparently through the sharing of some control elements with the,b-lactamase regulon [5, 7, 29, 39]). The coexistence of mec and the intrinsic resistance in MRSA raises the intriguing possibility that some of the genes responsible for the 3-lactamase-independent (intrinsic) form of resistance may be related to the auxiliary genes postulated to control the mode of phenotypic expression of resistance in MRSA (4, 12, 17, 35). ACKNOWLEDGMENTS This investigation received partial support from the National Institutes of Health, U.S. Public Health Service. A. M. 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