ANTIMICROBIAL AGENT8 AND CHEMWFERAPY, Apr. 1977, Copyright © 1977 American Society for Microbiology
p. 661-668
Vol. 11, No. 4 Printed in U.S.A.
Macrolide Resistance in Staphylococcus aureus: Induction of Macrolide-Resistant Protein Synthesis NORRIS E. ALLEN The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46206 Received for publication 12 October 1976
Induction of resistance to macrolide-, lincosamide-, and streptogramin B-type antibiotics in Staphylococcus aureus was studied by monitoring the appearance of erythromycin A (EM)-resistant [14C]leucine incorporation. Examination of the induction process revealed saturation kinetics and a time course much like that reported for penicillinase in gram-positive bacteria. Induction kinetics in exponentially growing cells were sigmoidal and appeared to reach a maximum and constant rate when growth reached stationary phase. Since the induction of EM-resistant colony-forming ability was complete within 60 min, ribosome modification cannot be limited to a fraction of the population and must occur in essentially every cell. However, EM-resistant growth was expressed in cells where less than half the ['4C]leucine-incorporating activity was resistant to EM. This suggests that resistance requires that only a threshold level of ribosome modification be exceeded and that, once exceeded, resistance is dominant to sensitivity.
Inducible resistance to erythromycin A (EM) has been described for certain clinically isolated strains of Staphylococcus aureus (2,3; B. Weisblum, in Microbiology -1974, p. 199-206, American Society for Microbiology, Washington, D.C.). Exposure to subinhibitory concentrations of EM induces resistance to high concentrations of EM plus other macrolide-, lincosamide-, and streptogramin B-type (MLS) antibiotics (2, 3, 21), all of which bind specifically to the 50S ribosomal subunit and inhibit ribosome function (14, 20). EM does not induce resistance to nonmacrolide 50S ribosome inhibitors other than those cited or to 30S inhibitors (21). Inducible resistance to macrolides and lincosamides also has been reported in clinically isolated strains of Streptococcus pyogenes (4, 24). Macrolide resistance (i.e., resistance to MLS antibiotics) in S. aureus is due to an alteration of the 50S ribosomal subunit (20). Specifically, N6,N6-dimethyl adenine has been detected in 23S ribosomal ribonucleic acid (RNA) of inducible strains grown under inducing conditions and in constitutively resistant mutants grown in the absence of any inducer (6, 7). Ribosomes from resistant cells show a decreased binding affinity for EM (7, 22), and ribosome reconstitution experiments have established that methylation ofribosomal RNA is a cause rather than a result of resistance (8). Although methylation of ribosomal RNA provides an attractive explanation for the mode of macrolide resistance, little information is 661
available to explain the specific mechanism of the induction process. Protein and RNA synthesis are required for induction, but synthesis of deoxyribonucleic acid is not (19, 22). Moreover, induced resistance is reversible and is lost when resistant cells are transferred and grown in an inducer-free medium (19, 22). These findings suggest classical induction, but the possibility that macrolide resistance is induced via a direct interaction between EM and the ribosome cannot be ruled out. In this paper, the measurement of induced EM-resistant protein synthesis is described. The method requires measuring rates of EMresistant [14C]leucine incorporation after exposure to subinhibitory concentrations of EM. Unlike measuring EM-resistant growth, this technique reflects directly the functional change due to the induced ribosomal alteration. In an accompanying paper (1), this method is used to survey MLS antibiotics, including EM derivatives, for inducer activity. MATERIALS AND METHODS Strains. S. aureus 1206, kindly supplied by B. Weisblum, has been described previously (Weisblum, Microbiology-1974, p. 199-206). This strain demonstrates a partial requirement for leucine (unpublished findings, this laboratory). S. aureus 1206-C4 is a generalized constitutively macrolide-resistant mutant according to the nomenclature of Weisblum and Demohn (21). The mutant was isolated in this laboratory from S. aureus 1206 as a resistant clone growing on agar supplemented
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with 10 ,ug of niddamycin per ml. Constitutive methylating activity was not tested. Media. Synthetic growth medium contained (in grams per liter): K2HPO4, 7; KH2PO4, 2; MgSO4. 7H20, 0.1; (NH4)2SO4, 1; and sodium citrate, 0.5. It was supplemented after autoclaving with appropriate stock solutions to give (final concentration): glucose, 2 g/liter; calcium pantothenate, (0.2 mg/liter; pyrodoxine-hydrochloride, 0.2 mg/liter; niacinamide, 0.2 mg/liter; inositol, 0.2 mg/liter; thiamine-hydrochloride, 0.2 mg/liter; para-aminobenzoic acid, 0.2 mg/liter; riboflavin, 0.05 mg/liter; biotin, 0.002 mg/liter; folic acid, 0.02 mg/liter; adenine, 1.5 mg/liter; guanine, 1.5 mg/liter; xanthine, 1.5 mg/liter; hypoxanthine, 1.5 mg/liter; thymine 1.5 mg/liter; uracil, 1.5 mg/liter; cytosine, 1.5 mg/ liter; and each of 20 amino acids (50 mg/liter). Antibiotic medium no. 1 (Difco) or Trypticase agar (BBL) was used for antibiotic disk diffusion assays. Antibacterial activity. Antibiotic sensitivity was indicated by a zone of inhibition around antibioticcontaining disks (20 jig/disk) placed on seeded agar and incubated for 18 h at 37°C. Inducible resistance to an antibiotic was indicated by a distorted zone of inhibition when placed adjacent to EM (21). Constitutive resistance was indicated by the absence of a zone of inhibition. Induction of resistance. Both log-phase and stationary-phase cells were used to measure induction of EM resistance. The methods used in each experiment are given in the appropriate figure legends. Incorporation of [14C]leucine. The relative capacity of cells to synthesize protein in the presence or absence of EM was determined by measuring the incorporation of [14C]leucine, using cells washed once and resuspended in phosphate-buffered saline (0.05 M potassium phosphate in 0.15 M sodium chloride, pH 6.9) to a density of 0.06 to 0.12 mg of wholecell protein per ml. Reactions contained, in a total volume of 1.0 ml: 0.05 M potassium phosphate (pH 6.9); 0.15 M sodium chloride; 0.001 M glucose; 0.05 ug of 19 amino acids not including leucine; 0.05 ,uCi of [14C]leucine (280 mCi/mmol); and 0.06 to 0.12 mg of cell protein. After a preincubation period of 5 min at 370C, reactions were initiated with the addition of [14C]leucine. Unless otherwise indicated, reactions were run in duplicate and incubated for 1 min in a 370C water bath. EM-resistant ['4C]leucine incorporation was measured by adding EM to the reaction mixture at a final concentration of 140 ,uM (100 Ag/ml). These conditions provide a rapid assay of EM-resistant incorporation unaffected by changes due to cell growth. Reactions were stopped by adding ice-cold 10% trichloroacetic acid supplemented with 10 mM (cold) leucine. Tubes were heated at 90°C for 15 min, and the precipitates were collected by filtration on Whatman GF/C glass-fiber filters. The filters were washed three times with 5% trichloroacetic acid containing (cold) leucine, dried, and counted by liquid scintillation spectrometry. EM and other antibiotics were prepared as 1- to 5-
ANTIMICROB. AGENTS CHEMOTHER. mg/ml methanol-water stock solutions. The maximum level of 0.2% methanol in the reaction mixture had no measurable effect on rates of [14C]leucine incorporation. Uptake of [4C]ileucine. Cells and reaction mixtures were exactly as described for measuring acidinsoluble incorporation of [14C]leucine except that each reaction contained 200 ,ug of chloramphenicol per ml to stop protein synthesis. After 1 min of incubation at 370C, cells were rapidly collected on membrane filters (0.45 nm, type HA; Millipore Corp.) at room temperature, immediately washed three times with 5-ml portions of phosphate-buffered saline, dried, and counted. Protein determination. Whole-cell protein was determined on cells grown in synthetic broth, washed, and resuspended in phosphate-buffered saline. Protein was extracted with trichloroacetic acid and solubilized according to the procedures of Winshell and Shaw (23). Solubilized protein was measured by the method of Lowry et al. (10). Antibiotics. Antibiotics used in this study and their sources were: methymycin, vernamycin Ba
(Squibb); oleandomycin, triacetyloleandomycin, carbomycin (Pfizer); narbomycin (Ciba-Geigy); lankamycin, desacetyllankamycin (Taisho); pikromycin (Farbenfabriken-Bayer); megalomycin, rosamycin (Schering); niddamycin (Farbwerke Hoechst AG); spiramycin (Rhone-Poulenc); chalcomycin, viridogrisein (Parke-Davis); shincomycin A (Tohoku); cirramycin A and B (Bristol); josamycin (Yamanouchi); propionylmaridomycin (Takeda); relomycin (Lederle); mydecamycin (Meiji Sheika Kaisha); leucomycin (Toyo Jozo); lincomycin, clindamycin, celesticetin (Upjohn); griseomycin (University of Louvain, Louvain, Belgium); chloramphenicol (Calbiochem); erythromycin, tylosin, desmycosin, benzylpenicillin G (Eli Lilly).
RESULTS Antibiotic sensitivity. When the 30 MLS antibiotics listed in Table 1 were tested against S. TABLE 1. MLS antibiotics 16-membered-ring macrol-
12-membered-ring macrolides Methymycin
14-membered-ring macrolides Erythromycin A Oleandomycin Triacetyloleandomycin Narbomycin Lankamycin Desacetyllankamycin Pikromycin Griseomycin Megalomycin Lincosamides
ides Carbomycin Niddamycin Spiramycin Tylosin Desmycosin Chalcomycin Shincomycin A Cirramycin A Cirramycin B Josamycin
Propionylmaridomycin
Relomycin Mydecamycin Rosamycin Leucomycin
Lincomycin Clindamycin Celesticetin
Streptogramin-B types Vernamycin Ba Viridogrisein
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INDUCTION OF MACROLIDE RESISTANCE
1206, a zone of inhibition was observed around each antibiotic disk except those containing EM, desacetyllankamycin, megalomycin, and celesticetin. With the exception of the latter four antibiotics, placing drug-containing disks adjacent to a disk containing EM (21) resulted in distorted zones of inhibition. Desacetyllankamycin, megalomycin, and celesticetin behaved like EM and caused distorted zones of inhibition when disks containing these drugs were placed adjacent to lincomycin. These results suggest that at least three antibiotics in addition to EM are capable of inducing resistance and that induced S. aureus 1206 is cross-resistant to a variety of MLS antibiotics (Table 1). This pattern of resistance supports and extends a previous report (21) showing that S. aureus 1206 is inducibly resistant to at least 10 MLS antibiotics. S. aureus 1206-C4 was isolated as a niddamycin-resistant mutant and demonstrated constitutive resistance to the 30 antibiotics in Table 1, but remained sensitive to nonmacrolide antibiotics. Inhibition of protein synthesis. Incorporation of ['4C]leucine by S. aureus 1206 grown without prior exposure to inducing levels of EM is illustrated in Fig. 1. Incorporation was sensitive to the same minimal levels of EM (0.14 to 1.4 uM) required to inhibit growth of this strain (22). Maximum but incomplete inhibition was obtained with 140 /,M EM. EM may be unable to completely inhibit incorporation because it has little effect on ribosomes actively synthesizing peptides (18). Since the time course of incorporation in the presence or absence of EM was linear during the first 2 min of the reaction (Fig. 1), initial rates of incorporation in the presence of EM were used to estimate EM resistance in subsequent experiments. [14C]leucine incorporation by S. aureus 1206 was sensitive to other MLS antibiotics providing that cells were grown in the absence of EM (Table 2). In contrast, incorporation by the constitutively resistant mutant was essentially insensitive to these drugs. Induction of EM resistance in dividing cells. Although S. aureus 1206 was sensitive to EM and other MLS antibiotics when grown under noninducing conditions, rates of [14C]leucine incorporation in the presence of 140 psM EM (EM-resistant incorporation) increased after exposing mid-log-phase cells to 0.014 ,uM EM. A differential plot where rates of EMresistant incorporati2n are expressed relative to the increase in cell mass is illustrated in Fig. 2. The maximum differential rate occurred as the culture approached higher turbidities. At a turbidity of 1.4 the cells were entering stationary phase, so that maximum induction apaureus
2
-
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c
E E
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0
10 5 MINUTES
15
FIG. 1. Sensitivity of protein synthesis in uninduced S. aureus 1206 to EM. Cells were grown to early stationary phase in synthetic broth (approximately 0.15 mg ofprotein per ml), harvested, washed once, and resuspended in phosphate-buffered saline. [14C]leucine incorporation (see Materials and Methods) was measured in reactions containing: 0 (0), 0.14 (0), 1.4 (A), and 140 or 700 (0) pM EM.
TABLz 2. Antibiotic sensitivity of protein synthesis in inducible and constitutive S. aureUSa
stnc
Induibl
Inducible strain
Antibioticb
Constitutive stai straind
[14C]leu- % of ['4C]leu- % of cine incor- con- cine incorporation trol poration control
261 None ........... 251 100 100 58 23 258 99 Erythromycin . . 21 8 235 90 Megalomycin... 33 Tylosin ........ 13 245 94 22 188 72 9 Carbomycin .... 32 250 96 13 Lincomycin .... a Cells were grown under noninducing conditions, and [14C]leucine incorporation was measured as described in the legend to Fig. 1. b Each antibiotic was tested at 100 lAg/ml. c S. aureus 1206. Incorporation is expressed as counts/minute per milligram of protein x 10-3. d S. aureus 1206 C4. Incorporation is expressed as in footnote c.
peared to coincide with a diminution in growth rate. Without added inducer, the rate of EMresistant incorporation remained unchanged. Identical kinetics were observed in a separate
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ANTIMICROB. AGENTS CHEMOTHER.
ing or resting cells. Figure 4 illustrates the induction of EM resistance using stationaryphase cells. Both the rate and maximum level of EM-resistant incorporation increased relative to the uninhibited rates during the 2-h ina duction period. Initial rates of incorporation (Table 3) indicated that EM-resistant incorporation in< creased more than eightfold, whereas control (uninhibited) incorporation increased only twoL x fold. (EM resistance is expressed as a percent-J age of the uninhibited rate in order to adjust for Coc =: changes in rates of protein synthesis occurring during induction.) In repeated experiments (data not shown), the level of EM-resistant incorporation by cell suspensions incubated for E 120 min without inducer remained between 10 and 20% of the uninhibited rate. _n E In Fig. 5, rates of EM-resistant incorporation 20are plotted against time of induction. The data :ifor this figure were taken from two separate '" experiments and include the data in Table 3. Induction kinetics under these conditions apA peared linear. Changes in amino acid transport. Induction ofEM-resistant [14C]leucine incorporation is as1.6 0.8 1.2 sumed to reflect a ribosomal alteration. Similar changes in rates of incorporation might occur if TURBIDITY (600nm) induced resistance led to an alteration that reFIG. 2. Differential plot of induction of EM-resist- sulted in increased transport of the radioactive ant incorporation. Synthetic broth was inoculated amino acid. The experiment summarized in Tawith an overnight culture of S. aureus 1206 and ble 4 indicated that the uptake of [14C]leucine grown at 37°C to mid-exponential phase. EM was added to the growing culture to give a final concen- was slightly reduced in the presence of EM. tration of 0.014 and incubation was continued. However, the extent ofthis reduction as well as A companion culture received no EM. Samples were the level of leucine uptake remained essentially taken periodically over a 2-h period for measuring unchanged during the 120-min induction period turbidity and macrolide-resistant protein synthesis. and cannot account for the increases in EM['4C]leucine incorporation was measured on washed resistant ['4C]leucine incorporation. I
I
I
60F
40F
M,
cells as described in Materials and Methods, except that glucose was omitted from the mixture and reactions were incubated for 2 min. Symbols: (a)Rates of EM-resistant ['4C]leucine incorporation after addition of inducer; (A) rate of EM-resistant ['4C]leucine incorporation after 2 h of incubation without inducer.
I
z
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o) CLX
20
oZ
IF
z
CD
experiment where inducer was added to earlylog-phase cells (data not shown). The effect of inducer concentration on EMresistant incorporation is shown in Fig. 3. The concentration of EM required for maximum induction was between 0.03 and 0.06 ,uM, with higher concentrations being less effective due to inhibition of protein synthesis and induction (see Fig. 1 and references 19 and 22). Weisblum et al. (22) reported similar concentration optima for induction of colony-forming ability on EM-supplemented agar. Induction of EM resistance in slowly grow-
z
0X
cn
E CD = Lu LU
0
0.04
0.08
0.12
EM CONCENTRATION ({M) FIG. 3. Effect of inducer concentration on induction of EM-resistant incorporation. EM was added at the concentrations shown to exponentially growing S. aureus 1206 as described in Fig. 2, and incubation was continued for 2 h. EM-resistant [14C]leucine in-
corporation was measured on washed cells scribed in Materials and Methods.
as
de-
VOL. 11, 1977
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x
2
Ir00 0
~~ 60 =
INDUCTION OF MACROLIDE RESISTANCE
665
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0
400
z
20
-E IN
15 10 15 0 5 10 5 MINUTES FIG. 4. Induction of EM-resistant incorporation in slowly growing or resting cells. S. aureus 1206 was grown to early stationary phase in synthetic broth, at which time cells were washed once and resuspended in fresh broth to a cell density of0.24 mg ofprotein per ml. The cell suspension was preincubated for 30 min, EM (0.04 MM) was added as inducer, and incubation was continued. Samples were taken before, 60 min after, and 120 min after adding EM. There was less than a 35% increase in cell mass during the 2-h incubation. Measurement of ["4C]leucine incorporation was as described in Materials and Methods. (A) Incorporation by uninduced cells; (B) incorporation after 60 min of induction; (C) incorporation after 120 min of induction. Symbols: (0) Uninhibited ['4C]leucine incorporation; (@) l4C]leucine incorporation in the presence of140 MM EM.
0
5
10
15
0
TABLE 3. Effects of induction on rates ofuninhibited and EM-resistant incorporationa Induction
[14C]leucine incorporation"
time (min)
-EM
+EM
0 60
162 207 275
23.3 100
120
198
% EM-resistant incorpora-
14.4 48.3 72.0
Data were taken from the experiment illustrated in Fig. 4. b Initial rates were calculated from the linear portions of the curves in Fig. 4. Incorporation is expressed as in Table 2. c EM resistance is expressed as EM-resistant incorporation calculated as a percentage of the uninhibited rate. a
EM-resistant translation of functional protein. Cells induced for resistance grow and divide in EM-containing media (19, 22), indicating indirectly that the induced change allows synthesis of functional protein in the presence of antibiotic. The experiment illustrated in Fig. 6 further rules out the possibility that EMresistant incorporation of [14C]leucine might represent synthesis of nonfunctional protein. It is readily apparent that penicillinase was syn-
thesized in the presence of 140 ,uM EM by cells preinduced for EM resistance but not by uninduced cells. In the absence of EM, induced and uninduced cells synthesized equal amounts of enzyme. EM-resistant colony formation. Induction of EM-resistant colony-forming ability was compared with the induction of EM-resistant [14C]leucine incorporation. Induction of resistant colony-forming ability was complete within 60 min, yet resistant incorporation was less than 40% of the uninhibited rate in these cells (Table 5). Although these findings imply that ribosome modification must occur in all cells, the actual rate of modification may not be reflected by the apparent rate of induced EMresistant colony formation. DISCUSSION Our current understanding of the induction of macrolide resistance in S. aureus has come from studies that, for the most part, were based on the ability of induced cells to grow in media containing high concentrations of EM (20). This methodology, especially the disk method (21), has been effectively used to establish that EM induces resistance to all MLS antibiotics. How-
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ANTIMICROB. AGENTS CHEMOTHER.
80 60 LU
0m
p. 661-668
Vol. 11, No. 4 Printed in U.S.A.
Macrolide Resistance in Staphylococcus aureus: Induction of Macrolide-Resistant Protein Synthesis NORRIS E. ALLEN The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46206 Received for publication 12 October 1976
Induction of resistance to macrolide-, lincosamide-, and streptogramin B-type antibiotics in Staphylococcus aureus was studied by monitoring the appearance of erythromycin A (EM)-resistant [14C]leucine incorporation. Examination of the induction process revealed saturation kinetics and a time course much like that reported for penicillinase in gram-positive bacteria. Induction kinetics in exponentially growing cells were sigmoidal and appeared to reach a maximum and constant rate when growth reached stationary phase. Since the induction of EM-resistant colony-forming ability was complete within 60 min, ribosome modification cannot be limited to a fraction of the population and must occur in essentially every cell. However, EM-resistant growth was expressed in cells where less than half the ['4C]leucine-incorporating activity was resistant to EM. This suggests that resistance requires that only a threshold level of ribosome modification be exceeded and that, once exceeded, resistance is dominant to sensitivity.
Inducible resistance to erythromycin A (EM) has been described for certain clinically isolated strains of Staphylococcus aureus (2,3; B. Weisblum, in Microbiology -1974, p. 199-206, American Society for Microbiology, Washington, D.C.). Exposure to subinhibitory concentrations of EM induces resistance to high concentrations of EM plus other macrolide-, lincosamide-, and streptogramin B-type (MLS) antibiotics (2, 3, 21), all of which bind specifically to the 50S ribosomal subunit and inhibit ribosome function (14, 20). EM does not induce resistance to nonmacrolide 50S ribosome inhibitors other than those cited or to 30S inhibitors (21). Inducible resistance to macrolides and lincosamides also has been reported in clinically isolated strains of Streptococcus pyogenes (4, 24). Macrolide resistance (i.e., resistance to MLS antibiotics) in S. aureus is due to an alteration of the 50S ribosomal subunit (20). Specifically, N6,N6-dimethyl adenine has been detected in 23S ribosomal ribonucleic acid (RNA) of inducible strains grown under inducing conditions and in constitutively resistant mutants grown in the absence of any inducer (6, 7). Ribosomes from resistant cells show a decreased binding affinity for EM (7, 22), and ribosome reconstitution experiments have established that methylation ofribosomal RNA is a cause rather than a result of resistance (8). Although methylation of ribosomal RNA provides an attractive explanation for the mode of macrolide resistance, little information is 661
available to explain the specific mechanism of the induction process. Protein and RNA synthesis are required for induction, but synthesis of deoxyribonucleic acid is not (19, 22). Moreover, induced resistance is reversible and is lost when resistant cells are transferred and grown in an inducer-free medium (19, 22). These findings suggest classical induction, but the possibility that macrolide resistance is induced via a direct interaction between EM and the ribosome cannot be ruled out. In this paper, the measurement of induced EM-resistant protein synthesis is described. The method requires measuring rates of EMresistant [14C]leucine incorporation after exposure to subinhibitory concentrations of EM. Unlike measuring EM-resistant growth, this technique reflects directly the functional change due to the induced ribosomal alteration. In an accompanying paper (1), this method is used to survey MLS antibiotics, including EM derivatives, for inducer activity. MATERIALS AND METHODS Strains. S. aureus 1206, kindly supplied by B. Weisblum, has been described previously (Weisblum, Microbiology-1974, p. 199-206). This strain demonstrates a partial requirement for leucine (unpublished findings, this laboratory). S. aureus 1206-C4 is a generalized constitutively macrolide-resistant mutant according to the nomenclature of Weisblum and Demohn (21). The mutant was isolated in this laboratory from S. aureus 1206 as a resistant clone growing on agar supplemented
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with 10 ,ug of niddamycin per ml. Constitutive methylating activity was not tested. Media. Synthetic growth medium contained (in grams per liter): K2HPO4, 7; KH2PO4, 2; MgSO4. 7H20, 0.1; (NH4)2SO4, 1; and sodium citrate, 0.5. It was supplemented after autoclaving with appropriate stock solutions to give (final concentration): glucose, 2 g/liter; calcium pantothenate, (0.2 mg/liter; pyrodoxine-hydrochloride, 0.2 mg/liter; niacinamide, 0.2 mg/liter; inositol, 0.2 mg/liter; thiamine-hydrochloride, 0.2 mg/liter; para-aminobenzoic acid, 0.2 mg/liter; riboflavin, 0.05 mg/liter; biotin, 0.002 mg/liter; folic acid, 0.02 mg/liter; adenine, 1.5 mg/liter; guanine, 1.5 mg/liter; xanthine, 1.5 mg/liter; hypoxanthine, 1.5 mg/liter; thymine 1.5 mg/liter; uracil, 1.5 mg/liter; cytosine, 1.5 mg/ liter; and each of 20 amino acids (50 mg/liter). Antibiotic medium no. 1 (Difco) or Trypticase agar (BBL) was used for antibiotic disk diffusion assays. Antibacterial activity. Antibiotic sensitivity was indicated by a zone of inhibition around antibioticcontaining disks (20 jig/disk) placed on seeded agar and incubated for 18 h at 37°C. Inducible resistance to an antibiotic was indicated by a distorted zone of inhibition when placed adjacent to EM (21). Constitutive resistance was indicated by the absence of a zone of inhibition. Induction of resistance. Both log-phase and stationary-phase cells were used to measure induction of EM resistance. The methods used in each experiment are given in the appropriate figure legends. Incorporation of [14C]leucine. The relative capacity of cells to synthesize protein in the presence or absence of EM was determined by measuring the incorporation of [14C]leucine, using cells washed once and resuspended in phosphate-buffered saline (0.05 M potassium phosphate in 0.15 M sodium chloride, pH 6.9) to a density of 0.06 to 0.12 mg of wholecell protein per ml. Reactions contained, in a total volume of 1.0 ml: 0.05 M potassium phosphate (pH 6.9); 0.15 M sodium chloride; 0.001 M glucose; 0.05 ug of 19 amino acids not including leucine; 0.05 ,uCi of [14C]leucine (280 mCi/mmol); and 0.06 to 0.12 mg of cell protein. After a preincubation period of 5 min at 370C, reactions were initiated with the addition of [14C]leucine. Unless otherwise indicated, reactions were run in duplicate and incubated for 1 min in a 370C water bath. EM-resistant ['4C]leucine incorporation was measured by adding EM to the reaction mixture at a final concentration of 140 ,uM (100 Ag/ml). These conditions provide a rapid assay of EM-resistant incorporation unaffected by changes due to cell growth. Reactions were stopped by adding ice-cold 10% trichloroacetic acid supplemented with 10 mM (cold) leucine. Tubes were heated at 90°C for 15 min, and the precipitates were collected by filtration on Whatman GF/C glass-fiber filters. The filters were washed three times with 5% trichloroacetic acid containing (cold) leucine, dried, and counted by liquid scintillation spectrometry. EM and other antibiotics were prepared as 1- to 5-
ANTIMICROB. AGENTS CHEMOTHER. mg/ml methanol-water stock solutions. The maximum level of 0.2% methanol in the reaction mixture had no measurable effect on rates of [14C]leucine incorporation. Uptake of [4C]ileucine. Cells and reaction mixtures were exactly as described for measuring acidinsoluble incorporation of [14C]leucine except that each reaction contained 200 ,ug of chloramphenicol per ml to stop protein synthesis. After 1 min of incubation at 370C, cells were rapidly collected on membrane filters (0.45 nm, type HA; Millipore Corp.) at room temperature, immediately washed three times with 5-ml portions of phosphate-buffered saline, dried, and counted. Protein determination. Whole-cell protein was determined on cells grown in synthetic broth, washed, and resuspended in phosphate-buffered saline. Protein was extracted with trichloroacetic acid and solubilized according to the procedures of Winshell and Shaw (23). Solubilized protein was measured by the method of Lowry et al. (10). Antibiotics. Antibiotics used in this study and their sources were: methymycin, vernamycin Ba
(Squibb); oleandomycin, triacetyloleandomycin, carbomycin (Pfizer); narbomycin (Ciba-Geigy); lankamycin, desacetyllankamycin (Taisho); pikromycin (Farbenfabriken-Bayer); megalomycin, rosamycin (Schering); niddamycin (Farbwerke Hoechst AG); spiramycin (Rhone-Poulenc); chalcomycin, viridogrisein (Parke-Davis); shincomycin A (Tohoku); cirramycin A and B (Bristol); josamycin (Yamanouchi); propionylmaridomycin (Takeda); relomycin (Lederle); mydecamycin (Meiji Sheika Kaisha); leucomycin (Toyo Jozo); lincomycin, clindamycin, celesticetin (Upjohn); griseomycin (University of Louvain, Louvain, Belgium); chloramphenicol (Calbiochem); erythromycin, tylosin, desmycosin, benzylpenicillin G (Eli Lilly).
RESULTS Antibiotic sensitivity. When the 30 MLS antibiotics listed in Table 1 were tested against S. TABLE 1. MLS antibiotics 16-membered-ring macrol-
12-membered-ring macrolides Methymycin
14-membered-ring macrolides Erythromycin A Oleandomycin Triacetyloleandomycin Narbomycin Lankamycin Desacetyllankamycin Pikromycin Griseomycin Megalomycin Lincosamides
ides Carbomycin Niddamycin Spiramycin Tylosin Desmycosin Chalcomycin Shincomycin A Cirramycin A Cirramycin B Josamycin
Propionylmaridomycin
Relomycin Mydecamycin Rosamycin Leucomycin
Lincomycin Clindamycin Celesticetin
Streptogramin-B types Vernamycin Ba Viridogrisein
VOL. 11, 1977
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INDUCTION OF MACROLIDE RESISTANCE
1206, a zone of inhibition was observed around each antibiotic disk except those containing EM, desacetyllankamycin, megalomycin, and celesticetin. With the exception of the latter four antibiotics, placing drug-containing disks adjacent to a disk containing EM (21) resulted in distorted zones of inhibition. Desacetyllankamycin, megalomycin, and celesticetin behaved like EM and caused distorted zones of inhibition when disks containing these drugs were placed adjacent to lincomycin. These results suggest that at least three antibiotics in addition to EM are capable of inducing resistance and that induced S. aureus 1206 is cross-resistant to a variety of MLS antibiotics (Table 1). This pattern of resistance supports and extends a previous report (21) showing that S. aureus 1206 is inducibly resistant to at least 10 MLS antibiotics. S. aureus 1206-C4 was isolated as a niddamycin-resistant mutant and demonstrated constitutive resistance to the 30 antibiotics in Table 1, but remained sensitive to nonmacrolide antibiotics. Inhibition of protein synthesis. Incorporation of ['4C]leucine by S. aureus 1206 grown without prior exposure to inducing levels of EM is illustrated in Fig. 1. Incorporation was sensitive to the same minimal levels of EM (0.14 to 1.4 uM) required to inhibit growth of this strain (22). Maximum but incomplete inhibition was obtained with 140 /,M EM. EM may be unable to completely inhibit incorporation because it has little effect on ribosomes actively synthesizing peptides (18). Since the time course of incorporation in the presence or absence of EM was linear during the first 2 min of the reaction (Fig. 1), initial rates of incorporation in the presence of EM were used to estimate EM resistance in subsequent experiments. [14C]leucine incorporation by S. aureus 1206 was sensitive to other MLS antibiotics providing that cells were grown in the absence of EM (Table 2). In contrast, incorporation by the constitutively resistant mutant was essentially insensitive to these drugs. Induction of EM resistance in dividing cells. Although S. aureus 1206 was sensitive to EM and other MLS antibiotics when grown under noninducing conditions, rates of [14C]leucine incorporation in the presence of 140 psM EM (EM-resistant incorporation) increased after exposing mid-log-phase cells to 0.014 ,uM EM. A differential plot where rates of EMresistant incorporati2n are expressed relative to the increase in cell mass is illustrated in Fig. 2. The maximum differential rate occurred as the culture approached higher turbidities. At a turbidity of 1.4 the cells were entering stationary phase, so that maximum induction apaureus
2
-
z o
x
M
CZ) a) C.3 Z
c
E E
C.)
0
10 5 MINUTES
15
FIG. 1. Sensitivity of protein synthesis in uninduced S. aureus 1206 to EM. Cells were grown to early stationary phase in synthetic broth (approximately 0.15 mg ofprotein per ml), harvested, washed once, and resuspended in phosphate-buffered saline. [14C]leucine incorporation (see Materials and Methods) was measured in reactions containing: 0 (0), 0.14 (0), 1.4 (A), and 140 or 700 (0) pM EM.
TABLz 2. Antibiotic sensitivity of protein synthesis in inducible and constitutive S. aureUSa
stnc
Induibl
Inducible strain
Antibioticb
Constitutive stai straind
[14C]leu- % of ['4C]leu- % of cine incor- con- cine incorporation trol poration control
261 None ........... 251 100 100 58 23 258 99 Erythromycin . . 21 8 235 90 Megalomycin... 33 Tylosin ........ 13 245 94 22 188 72 9 Carbomycin .... 32 250 96 13 Lincomycin .... a Cells were grown under noninducing conditions, and [14C]leucine incorporation was measured as described in the legend to Fig. 1. b Each antibiotic was tested at 100 lAg/ml. c S. aureus 1206. Incorporation is expressed as counts/minute per milligram of protein x 10-3. d S. aureus 1206 C4. Incorporation is expressed as in footnote c.
peared to coincide with a diminution in growth rate. Without added inducer, the rate of EMresistant incorporation remained unchanged. Identical kinetics were observed in a separate
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ANTIMICROB. AGENTS CHEMOTHER.
ing or resting cells. Figure 4 illustrates the induction of EM resistance using stationaryphase cells. Both the rate and maximum level of EM-resistant incorporation increased relative to the uninhibited rates during the 2-h ina duction period. Initial rates of incorporation (Table 3) indicated that EM-resistant incorporation in< creased more than eightfold, whereas control (uninhibited) incorporation increased only twoL x fold. (EM resistance is expressed as a percent-J age of the uninhibited rate in order to adjust for Coc =: changes in rates of protein synthesis occurring during induction.) In repeated experiments (data not shown), the level of EM-resistant incorporation by cell suspensions incubated for E 120 min without inducer remained between 10 and 20% of the uninhibited rate. _n E In Fig. 5, rates of EM-resistant incorporation 20are plotted against time of induction. The data :ifor this figure were taken from two separate '" experiments and include the data in Table 3. Induction kinetics under these conditions apA peared linear. Changes in amino acid transport. Induction ofEM-resistant [14C]leucine incorporation is as1.6 0.8 1.2 sumed to reflect a ribosomal alteration. Similar changes in rates of incorporation might occur if TURBIDITY (600nm) induced resistance led to an alteration that reFIG. 2. Differential plot of induction of EM-resist- sulted in increased transport of the radioactive ant incorporation. Synthetic broth was inoculated amino acid. The experiment summarized in Tawith an overnight culture of S. aureus 1206 and ble 4 indicated that the uptake of [14C]leucine grown at 37°C to mid-exponential phase. EM was added to the growing culture to give a final concen- was slightly reduced in the presence of EM. tration of 0.014 and incubation was continued. However, the extent ofthis reduction as well as A companion culture received no EM. Samples were the level of leucine uptake remained essentially taken periodically over a 2-h period for measuring unchanged during the 120-min induction period turbidity and macrolide-resistant protein synthesis. and cannot account for the increases in EM['4C]leucine incorporation was measured on washed resistant ['4C]leucine incorporation. I
I
I
60F
40F
M,
cells as described in Materials and Methods, except that glucose was omitted from the mixture and reactions were incubated for 2 min. Symbols: (a)Rates of EM-resistant ['4C]leucine incorporation after addition of inducer; (A) rate of EM-resistant ['4C]leucine incorporation after 2 h of incubation without inducer.
I
z
< -
o) CLX
20
oZ
IF
z
CD
experiment where inducer was added to earlylog-phase cells (data not shown). The effect of inducer concentration on EMresistant incorporation is shown in Fig. 3. The concentration of EM required for maximum induction was between 0.03 and 0.06 ,uM, with higher concentrations being less effective due to inhibition of protein synthesis and induction (see Fig. 1 and references 19 and 22). Weisblum et al. (22) reported similar concentration optima for induction of colony-forming ability on EM-supplemented agar. Induction of EM resistance in slowly grow-
z
0X
cn
E CD = Lu LU
0
0.04
0.08
0.12
EM CONCENTRATION ({M) FIG. 3. Effect of inducer concentration on induction of EM-resistant incorporation. EM was added at the concentrations shown to exponentially growing S. aureus 1206 as described in Fig. 2, and incubation was continued for 2 h. EM-resistant [14C]leucine in-
corporation was measured on washed cells scribed in Materials and Methods.
as
de-
VOL. 11, 1977
0
x
2
Ir00 0
~~ 60 =
INDUCTION OF MACROLIDE RESISTANCE
665
-
0
400
z
20
-E IN
15 10 15 0 5 10 5 MINUTES FIG. 4. Induction of EM-resistant incorporation in slowly growing or resting cells. S. aureus 1206 was grown to early stationary phase in synthetic broth, at which time cells were washed once and resuspended in fresh broth to a cell density of0.24 mg ofprotein per ml. The cell suspension was preincubated for 30 min, EM (0.04 MM) was added as inducer, and incubation was continued. Samples were taken before, 60 min after, and 120 min after adding EM. There was less than a 35% increase in cell mass during the 2-h incubation. Measurement of ["4C]leucine incorporation was as described in Materials and Methods. (A) Incorporation by uninduced cells; (B) incorporation after 60 min of induction; (C) incorporation after 120 min of induction. Symbols: (0) Uninhibited ['4C]leucine incorporation; (@) l4C]leucine incorporation in the presence of140 MM EM.
0
5
10
15
0
TABLE 3. Effects of induction on rates ofuninhibited and EM-resistant incorporationa Induction
[14C]leucine incorporation"
time (min)
-EM
+EM
0 60
162 207 275
23.3 100
120
198
% EM-resistant incorpora-
14.4 48.3 72.0
Data were taken from the experiment illustrated in Fig. 4. b Initial rates were calculated from the linear portions of the curves in Fig. 4. Incorporation is expressed as in Table 2. c EM resistance is expressed as EM-resistant incorporation calculated as a percentage of the uninhibited rate. a
EM-resistant translation of functional protein. Cells induced for resistance grow and divide in EM-containing media (19, 22), indicating indirectly that the induced change allows synthesis of functional protein in the presence of antibiotic. The experiment illustrated in Fig. 6 further rules out the possibility that EMresistant incorporation of [14C]leucine might represent synthesis of nonfunctional protein. It is readily apparent that penicillinase was syn-
thesized in the presence of 140 ,uM EM by cells preinduced for EM resistance but not by uninduced cells. In the absence of EM, induced and uninduced cells synthesized equal amounts of enzyme. EM-resistant colony formation. Induction of EM-resistant colony-forming ability was compared with the induction of EM-resistant [14C]leucine incorporation. Induction of resistant colony-forming ability was complete within 60 min, yet resistant incorporation was less than 40% of the uninhibited rate in these cells (Table 5). Although these findings imply that ribosome modification must occur in all cells, the actual rate of modification may not be reflected by the apparent rate of induced EMresistant colony formation. DISCUSSION Our current understanding of the induction of macrolide resistance in S. aureus has come from studies that, for the most part, were based on the ability of induced cells to grow in media containing high concentrations of EM (20). This methodology, especially the disk method (21), has been effectively used to establish that EM induces resistance to all MLS antibiotics. How-
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80 60 LU
0m
