of water. Each strain was cultured in 2 liters of ... C and for 15 min with deoxyribonuclease at a final .... c Counts per minute of the "C-component of 8-14C-ATP.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1973, p. 1-5 Copyright © 1973 American Society for Microbiology
Vol. 4, No. 1
Printed in U.S.A.
Lividomycin Resistance in Staphylococci by Enzymatic Phosphorylation FUJIO KOBAYASHI, TOMOYUKI KOSHI, JUNJI EDA, YOKO YOSHIMURA,
AND
SUSUMU
MITSUHASHI
Tokyo Research Laboratories, Kowa Co., Ltd., Higashimurayama, Tokyo, and Department of Microbiology, School of Medicine, Gunma University, Maebashi, Japan Received for publication 13 February 1973
Enzymatic inactivation of lividomycin (LV) was attempted with nine clinical isolates of staphylococci including LV-susceptible and -resistant strains. LV inactivation and the incorporation into LV of 32P from y-32P-adenosine triphosphate were demonstrated in the presence of cell-free extracts from LV-resistant strains but not from LV-susceptible ones. The enzyme was purified approximately 82-fold from a resistant Staphylococcus aureus strain by means of ammonium sulfate fractionation and column chromatography. Some properties of the partially purified LV-phosphorylating enzyme were quite similar to those of an enzyme from Escherichia coli carrying an R factor conferring LV resistance, and the phosphorylated product of the drug was also found to be identical with that produced by E. coli carrying an R factor, i.e., 5"-phosphoryl-LV. Many enzymes which inactivate aminoglycosidic antibiotics have been reported in drugresistant strains of bacteria of several species (1, 12). We reported that the lividomycin (LV)inactivating enzyme capable of phosphorylating the drug was demonstrated in resistant strains of Pseudomonas aeruginosa (9) and of Escherichia coli carrying an R factor (19). Further studies indicated that the inactivated LV was a monophosphorylated product, in which the Dribose moiety of LV was phosphorylated (10, 20). In a survey of Staphylococcus aureus, LVresistant strains whose growth was not inhibited by 50 qig of LV/ml were isolated at a frequency of 23.4% (6). This paper deals with some properties of the LV-inactivating enzyme and the relation between LV resistance and the enzyme activity. MATERIALS AND METHODS Bacterial strains. The nine clinical isolates used included three LV-resistant and three LV-susceptible strains of S. aureus and three LV-resistant strains of S. epidermidis. These strains were selected at random from our stocks. Preparation of the S-105 fraction. Glucose-peptone broth (pH 7.2) consisted of 10 g of peptone, 5 g of NaCl, 3 g of glucose, 4 g of yeast extract, and 1,000 ml of water. Each strain was cultured in 2 liters of glucose-peptone broth at 35 C for 5 h with shaking, after 18 h of precultivation in the same broth at 37 C
without shaking. The cells were harvested by centrifugation at 4,500 x g for 10 min and were washed with TMK solution (0.06 M KCI, 0.01 M magnesium acetate, and 0.006 M 2-mercaptoethanol in 0.1 M tris [hydroxymethyl Jaminomethane-hydrochloride buffer, pH 7.8). Washed cells were suspended in 20 ml of TMK solution and disrupted by passage twice through a French press under a pressure of 600 kg/cm2. The suspension of disrupted cells was centrifuged at 105,000 x g for 60 min after digestion at 37 C and for 15 min with deoxyribonuclease at a final concentration of 4 gg/ml. The supernatant fluid was designated as the S-105 fraction. Protein concentration in each S-105 fraction was determined by the method of Lowry et al. (11) and was adjusted to 5.2 mg of protein/ml. Inactivation reaction. The inactivation reaction was performed by the same procedure described previously (19). The reaction mixture, consisting of 0.5 ml of TMK solution, 0.1 ml of 40 mM adenosine triphosphate (ATP), 0.1 ml of 1.0 mM LV, and 0.3 ml of enzyme solution (S-105 fraction), was incubated at 30 C for 60 min, and the reaction was then stopped by heating at 80 C for 5 min. Residual antibiotic activity in the reaction mixture was determined by a paperdisk method with Bacillus subtilis as the test organism. Radioisotopic assay. The incorporation into LV of 32p from -y-32P-ATP or of 14C from 8-'4C-ATP by inactivating enzyme was carried out by a modification of the method described by Ozanne et al. (15). The reaction mixture consisted of 30 Aliters of the S-105 fraction, 10 uliters of 1.0 mM LV, 10 gliters of 40 mM ATP solution containing 1 1ACi of -Y_32P-ATP (or 0.33
2
ANTIMICROB. AG. CHEMOTHER.
KOBAYASHI ET AL.
pCi of 8-14C-ATP), and 50 uliters of 'I'MK solution. The reaction mixture was incubated at 30 C for 60 min; 10 gliters of the reaction mixture was then spotted on 1 cm2 of phosphocellulose paper (Whatman P-81), washed several times with hot distilled water (80 C), and dried. Radioactivity on the paper was counted in toluene-based scintillator with the use of a liquid scintillation counter (Packard Instrument Co., Inc.). Isolation of inactivated LV. LV inactivation was carried out in a reaction mixture containing 30 ml of the S-105 fraction (20 mg of protein/ml) from S. aureus KW-2, 510 mg of ATP, and 170 mg of LV sulfate. The reaction mixture was incubated at 30 C for 5 h, and the reaction was stopped by heating at 80 C for 5 min. LV was completely inactivated by this reaction. The inactivated product was isolated by the procedure described previously (9); 120 mg of the inactivated product was obtained. Preparation of 32P-labeled phosphoryl-LV. The inactivation reaction was carried out at 30 C for 20 h in a total volume of 0.5 ml containing 0.1 gmol of LV, 8.4 mg of protein of the S-105 fraction from S. aureus KW-2, and 0.4 jumol of ATP containing 18 pCi of 'y-32P-ATP. The supematant fluid of the reaction mixture was absorbed onto phosphocellulose paper after heating at 80 C for 5 min and was washed with large volumes of water. The 32P-labeled compound absorbed on phosphocellulose paper was extracted with 0.4 N NH4OH solution and dried. Thin-layer chromatography. The 32P-labeled compound and the inactivated product of LV were developed with the following solvent system on a thin layer of silica gel H (Merck): chloroformmethanol-17% NH4OH (2:1:1). The spot on a chromatogram was detected with an Aloka thin-layer chromatogram scanner (model TLC-2BR) following the ninhydrin reaction. Antibiotics and chemicals. LV sulfate was prepared in this laboratory. Deoxyribonuclease (from E. coli) and alkaline phosphatase (from calf intestinal mucosa, type I) were purchased from Worthington Biochemical Corp. and Sigma Chemical Co., respectively. The -y32P-ATP (1,200 mCi/mmol) and 8-14CATP (50 mCi/mmol) were supplied by the Radiochemical Centre (Amersham, England) and by International Chemical and Nuclear Corp., respectively. 5"-Phosphoryl-LV was prepared by inactivation of LV by E. coli ML1410 Rms.+ as reported previously (19). RESULTS
Detection of LV-phosphorylating enzyme. LV inactivation was attempted with six LVresistant strains. As shown in Table 1, the incorporation of 32p from -y2P-ATP into LV occurred in the presence of the S-105 fraction from LV-resistant strains but not from LV-susceptible strains. Incorporation of 14C from 8"4C-ATP could not be demonstrated, indicating that LV-resistant strains inactivated LV by phosphorylation of the drug. LV-phosphorylating enzyme. LV-resistant
TABLE 1. Distribution of lividomycin (LV)-phosphorylating enzyme in susceptible and resistant strains Incorporation Strain
MICa (pAg/Mi)
of
Enzymed
32pb
14Cc
>800 >800 >800
7,152 7,839 5,028
75 68 70
+ + +
> 800 > 800 > 800
8,770 7,487 6,423
89 66 84
+ + +
511 571 284
71 87 76
_ _ -
542
101
S. aureus KW-1 ............ KW-2 ............ KW-3 ............
S. epidermidis KW-11 ........... KW-12 ........... KW-13 ...........
S. aureus KW-4 ............ KW-5 ............ KW-6 ............ Reaction system minus enzyme ..
0.78 0.39 0.39
inhibitory LV concentration. Counts per minute of the 32p of -y-"P-ATP incorporated into LV. c Counts per minute of the "C-component of 8-14C-ATP incorporated into LV. d Detection of LV-phosphorylating enzyme. a Minimal "
S. aureus strain KW-2 was selected, and the LV-phosphorylating enzyme in the S-105 fraction was partially purified. S. aureus KW-2 was cultured in 20 liters of glucose-peptone broth at 35 C for 5 h with shaking. The cells at logarithmic phase were harvested by centrifugation, yielding approximately 90 g (wet weight) of cell pellet. The cells were suspended in TMK solution, and 70 ml of the S-105 fraction was obtained by the procedure described in Materials and Methods. LV-phosphorylating enzyme in the S-105 fraction was precipitated by ammonium sulfate at 66 to 99% saturation. The precipitate was dissolved in 8 ml of TMK solution. After dialysis against a large volume of TMK solution, the solution was absorbed onto a diethylaminoethyl-cellulose column (25 by 550 mm) and eluted by a linear gradient of NaCl from 0 to 0.5 M. The enzyme was eluted at a concentration of 0.25 M NaCl. Gel filtration of the enzyme solution was carried out by use of TMK solution with Sephadex G-100 (37 by 600 mm) and Sephadex G-75 (26 by 550 mm) columns. Each purification step is summarized in Table 2. LV-phosphorylating enzyme was purified approximately 82-fold from the S-105 fraction by these procedures with a recovery of 18.4%. The optimal pH for the LV-inactivation reaction, as can be seen in Fig. 1, was nearly 6.5, and the enzyme required both ATP and Mg2+ for the inactivation reaction. Dialysis in TMK solution
LIVIDOMYCIN RESISTANCE IN STAPHYLOCOCCI
VOL. 4, 1973
3
TABLE 2. Partial purification of the lividomycin (LV)-phosphorylating enzyme from S. aureus KW-2
|
Step Step
Total SpecifiCa activity | ~~protein (mg) activity
activityb enzyme
Total
Recovery (%
21,756.0
100.0 94.5
1.0 5.3
53.6 49.8 18.4
28.9 46.6
S-105 fraction .......................... 66-99% saturation with (NH4)2SO4
14.8 77.9
1,470.0 264.0
Diethylaminoethyl-cellulose ....... ...... Sephadex G-100 ........................ Sephadex G-75 .........................
428.3 690.0
27.2 15.7
1,212.1
3.3
.......
20,565.6 11,649.8 10,833.0 3,999.9
Prfcto urfiato
81.9
Specific activity is expressed as units of activity per milligram of protein. h One unit of enzyme activity is equal to 1 nmol of LV inactivated per minute.
a
1001
3
4
5
6
7
8
9
10 11
pH FIG. 1. Effect of pH on lividomycin (LV) inactivation. The reaction mixture consisted of 0.1 ml of partially purified enzyme (28 ug/ml), 0.1 ml of 40 mM ATP, 0.1 ml of 1 mM LV, and 0.7 ml of buffer solution. The reaction was carried out at 30 C for 15 min. Symbols: *, 0.1 M Veronal-HCI buffer; 0, 0.1 M glycine-NaOH buffer.
was demonstrated on thin-layer chromatography (CHCl-methanol-17% NH,OH, 2: 1: 1). The antimicrobial activity of the product was restored by incubation at 37 C for 3 h with alkaline phosphatase. Elemental analysis of the phosphorylated product was as follows. Calculated for C29H,,N518.*PO(OH)2 2H2O: C, 39.67; H, 6.90; N, 7.97; P, 3.52. Found: C, 39.92; H, 6.54; N, 8,08; P, 3.50. As shown in Fig. 3, the radioactive spot of 32P-labeled phosphoryl-LV which was produced by the S-105 fraction from S. aureus KW-2 coincided completely with the ninhydrin-positive spots of the purified phosphoryl product of LV from the reaction mixture and with 5"-phosphoryl-LV which was produced by the enzyme from E. coli ML1410 Rm8i+. Furthermore, the infrared spectrum of the inactivated product showed a specific band at 965 cm-l, and was identical with the phos-
100 Z-
80
E/
without Mg2+ caused an irreversible loss of the enzyme activity. This fact suggests that Mg2+ is necessary not only for the activity of the enzyme but also for its stability. The stability of the enzyme against heat treatment for 5 min was -X 640 20 tested. Losses of enzyme activity of 24.9% at 40 C, 65.2% at 60 C, and 100% at 80 C were found, 0 although the activity was not affected at 30 C. The time course of LV inactivation is shown in Fig. 2. After 60 min of incubation, 100 nmol of LV was completely inactivated by 2.8 ug of protein of a partially purified enzyme. 5 10 15 30 60 Identification of the phosphorylated prodTime ( min uct. LV enzymatically inactivated by S. aureus KW-2 was isolated and purified by the procecourse FIG. 2. Time of lividomycin (LV) inactivadure described in a previous paper (9). Inactiva- tion. The reaction mixture consisted of 0.3 ml of ted LV showed positive tests for ninhydrin, partially purified enzyme (9 Ag/ml), 0.1 ml of 40 mM anthrone, and Hanes reactions, and one spot ATP, 0.1 ml of 1 mMLV, and 0.5 ml of TMK solution.
4
KOBAYASHI ET AL.
ANTIMICROB. AG. CHEMOTHER.
Rmsl+, i.e., 5"-phosphoryl-LV. These studies showed LV-phosphorylating enzyme to be responsible for aminoglycoside resistance in staphylococci, in addition to streptomycinadenylating (5) and kanamycin-phosphorylating (2) enzymes, and these enzymes have also been demonstrated in aminoglycoside-resistant x strains of gram-negative bacteria with or withE out R factor (7-9, 16-18). Since macrolide resistance in staphylococci was irreversively eliminated by treatment with acridine dyes, Mitsuhashi (13) concluded that tthe determinant governing macrolide resistance 31 *A ~~~~~~~~Z was located on a plasmid. Similarly, the deterfiB GTZ 0fi 31 minant governing penicillin resistance in staphC Q= a60 ylococci was found to be located on a plasmid by genetic studies (14) and curing experiments (3). FIG. 3. Thin-layer chromatogram of phosphoryl- IRecent studies by curing experiments and lividomycin (LV) enzymatically produced by S. au- ttransduction to a rec- (4) recipient have shown reus KW-2. The solvent system used was: chloroform, tthat the determinants goveming kanamycin methanol, and 17%o NH4OH (2:1:1). The spots on the and LV resistance in staphylococci are also chromatogram were detected with an Aloka thin-layer located on a plasmid (S. Mitsuhashi and M. chromatogram scanner following the ninhydrin reaction. A: phosphoryl-LV enzymatically produced by S. Inoue, U.S.-Japan Scientific Seminar on Bacteaureus KW-2. B: 5"-phosphoryl-LV enzymatically rial Plasmid, Hawaii, 1973, in press). It should produced by E. coli ML1410Rmsi+. C: LV.1be noted that most of the determinants are located on R factors or staphylococcal plasmid. fact that LV has not been clinically used The . E. coli ML1410 from phorylated product Rm.,& Both phosphoryl-LVs showed the same Rm val- 3yet but resistant strains producing LV-phosues (Rm, 1.7) on high-voltage paper electropho- Iphorylating enzyme are widely distributed resis at 3,000 V with a solvent containing formic Iarouse interest in the origin of the LV-resistant acid, acetic acid, and water (22:75:900). These determinant. The origin of the determinant results indicated that LV-phosphorylating en- Egoverning LV phosphorylation should be studzyme detected in a resistant strain of S. aureus iied. was similar to that from E. coli ML1410 Rm.,+, ACKNOWLEDGMENTS and that the phosphoryl-LV produced by S. We are indebted to H. Mori, the director of Tokyo greatly aureus KW-2 was also identical with that pro- Research Laboratories, Kowa Co., for his encouragement. duced by E. coli ML1410 Rm8l+. Thanks also go to the members of the analytical section for m
DISCUSSION The LV-phosphorylating enzyme was detected in LV-resistant strains of staphylococci, indicating that the enzyme is widely distributed in not only gram-negative but also gram-positive bacteria. The LV-resistant determinant in E. coli was found to be located on an R factor and conjugally transmissible to a susceptible strain (19). LV-resistant strains of staphylococci were found capable of phosphorylating the drug. Therefore, the LV resistance in E. coli, P. aeruginosa, and S. aureus is accounted for by the presence of an enzyme capable of phosphorylating the drug. Biochemical studies of the LV-phosphorylating enzyme purified from S. aureus KW-2 disclosed that the enzyme was quite similar to that from E. coli ML1410 Rmsn+ (20), and the phosphorylated product of LV was found to be identical with that produced by E. coli ML1410
their devoted assistance in performing elemental analysis and determining the infrared spectra.
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LIVIDOMYCIN RESISTANCE IN STAPHYLOCOCCI
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