Vol. 7, No. 1 Printed in U.S.A.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1975, P. 50-54 Copyright i 1975 American Society for Microbiology
Acetylation of Amikacin, a New Semisynthetic Antibiotic, by Pseudomonas aeruginosa Carrying an R Factor HARUHIDE KAWABE,* TAKAYUKI NAITO, AND SUSUMU MITSUHASHI Department of Microbiology, School of Medicine, Gunma University, Maebashi,* and Bristol-Banyu Research
Institute Ltd., Tokyo, Japan Received for publication 31 July 1974
A clinical isolate Pseudomonas aeruginosa GN315 resistant to amikacin (AK), semisynthetic antibiotic, inactivated AK by acetylation. The acetylating enzyme was purified approximately 146-fold from a crude extract of GN315 by affinity chromatography. Fractionated samples obtained by affinity chromatography showed almost the same inactivation curves toward 3',4'-dideoxykanamycin B (DKB) and AK. Partially purified AK-acetylating enzyme inactivated DKB and kanamycin A but could not inactivate gentamicin C1 The optimal pH for their inactivation was 6.0 to 7.0, and the pH curves for the inactivation of both drugs were almost the same. These facts indicate that AK and DKB are inactivated by the same aminoglycoside-acetylating enzyme. Through elemental analysis, the inactivated AK was found to be a monoacetylated product of AK. A sample of inactivated AK was purified and compared with a synthetic 6'-N-acetyl AK by thin-layer chromatography, and the results indicated that AK was inactivated by acetylation of the 6'-NH2 group. The ultraviolet, infrared, and nuclear magnetic resonance spectra of the inactivated AK showed that AK was inactivated by the enzyme through acetylation of the amino group of 6'-amino-6'-deoxy-D-glucose moiety of AK. This enzyme, mediated by R factor, is capable of conferring resistance to AK, DKB, kanamycin, gentamicin, and sulfanilamide. a new
.
A 6'-N-acetylating enzyme that inactivates aminoglycosidic antibiotics has been found in drug-resistant strains of Escherichia coli (1, 9, 11) and Pseudomonas aeruginosa (4, 8, 14, 15). We reported that an enzyme capable of acetylating 3',4'-dideoxykanamycin B (DKB) was demonstrated in a resistant strain of P. aeruginosa cape 15, and the inactivated product appeared to be 6'-N-acetyl DKB (4). Yagisawa et al. (14) reported that the inactivation of DKB by P. aeruginosa GN315 was due to the formation of DKB 6'-N-acetate. Furthermore, Yamamoto et al. (15) reported the inactivation of kanamycin (KM) A and ribostamycin by the same enzyme solution of GN315, producing KM 6'-N-acetate and ribostamycin 6'-N-acetate. This paper deals with some properties of the 6'-N-acetylating enzyme from GN315 and the chemical structure of inactivated amikacin (AK). MATERIALS AND METHODS Bacterial strains. Ten strains of P. aeruginosa were isolated from clinical specimens and stocked in this laboratory. Their resistance patterns to KMA, DKB, gentamicin Cl (GM-Cl), and AK are shown in Table 1. P. aeruginosa ML4561 (leu-arg-ilv-his50
rifampin-resistant) and P. aeruginosa ML4344 (leu-trp-) were used as recipients of R factor. Drugs. A semisynthetic aminoglycoside antibiotic, DKB, was produced from KM B (12) and was kindly supplied by H. Umezawa, Institute of Microbial Chemistry, Tokyo. AK was produced by acylation of the 1-NH2 group of KM A with L(-)-y-amino-ahydroxybutyric acid (5) and was provided by H. Kawaguchi, Bristol-Banyu Research Institute, Ltd., Tokyo. GM-Cl was kindly supplied by the Schering Corp., Bl6oomfield, N.J. Media. Nutrient broth containing 0.25% KNO, was used for liquid culture. Minimal medium was used for the transfer of R factor from exconjugant ML4561 to ML4344, and consisted of 8.0 g of Na,PHO4, 2.0 g of KH2PO4, 1 g of (NH4)2SO4, 13 g of agar, 80 mg of bromothymol blue, 10 g of glucose, 0.1 g of MgSO4, and 1,000 ml of distilled water. Preparation of S-105 fraction and inactivation reaction. Cell-free extract was prepared as described previously (3). The reaction mixture consisted of 0.2 ml of acetate buffer (pH 6.0), 0.05 ml of 20 mM disodium adenosine triphosphate (ATP), 0.05 ml of 0.5 mM coenzyme A (CoA) 0.05 ml of 0.02 M magnesium acetate, 0.05 ml of 0.5 mM drug, and 0.1 ml of S-105 fraction. After incubation at 37 C for 30 min, the reaction was stopped by heating in boiling water for 3 min. Residual antibiotic activity in the reaction mixture was determined by bioassay using Bacillus subtilis PCI 219.
AMIKACIN RESISTANCE IN P. AERUGINOSA
VOL. 7, 1975
TABLE 1. Minimal inhibitory concentrations of aminoglycoside antibiotics against P. aeruginosa Minimal inhibitory concn (ug/ml)
Strain
GN237 GN269 GN315 GN362 GN4475 GN4489 GN4491 GN4493 GN4494 GN4495 ML4344a ML4561a
KM
DKB
GM-Cl
AK
100 100 100 100 25 25 25 25 25 25 12.5 25
50 50 100 100 1.6 0.8 1.6 0.8 1.6 1.6 0.4 0.4
100 3.1 3.1 3.1 100 100 100 50 50 50 1.5 1.5
25 50 25 25 1.6 3.1 3.1 3.1 1.6 0.8 0.8 0.8
aTwo strains of P. aeruginosa recipient of R factor.
were
used
as
the
Conjugal transfer of resistance. From each donor and recipient culture in nutrient broth at 37 C for 18 h, 0.5 ml was removed and inoculated into 5 ml of fresh nutrient broth and shaken at 37 C. After 2 h of incubation, the cultures (about 2 x 109 cells/ml) reached the late logarithmic phase of growth and were used for the conjugal transfer of resistance. One part of donor culture was mixed with four parts of recipient culture, and the mixture was incubated at 37 C with gentle shaking. After 90 min of incubation, a 0.1-ml sample of the mixed culture was plated on a selective plate containing rifampin (200 Ag/ml) and either DKB (3.1 lAg/ml) or AK (3.1 ;ig/ml). The colonies that developed on selective plates after 18 h of incubation at 37 C were picked and purified by three successive single-colony isolations, and their drug resistance was determined. For the transfer of resistance from exconjugant ML4561 to ML4344, the minimal medium containing DKB (or AK), leucine (50 ug/ml), and tryptophan (50 jug/ml) was used for the selection of exoconjugant ML4344. The transfer frequency was expressed as the ratio of the number of donor to recipient cells that acquired drug resistance by conjugation. Preparation of the column. Aminoglycoside acetyltransferase was purified by affinity chromatography (6, 13). KM A-Sepharose 4B was prepared as follows. Cyanogen bromide-activated Sepharose 4B (Pharmacia Fine Chemicals AB, Upsala, Sweden) was washed with 1,000 ml of 1 mM HCl, 500 ml of cold water, and 25 ml of 0.1 M NaHCOa-0.5 M NaCl, successively, and was mixed with 0.5 g of KM in 45 ml of 0.1 M NaHCO,-0.5 M NaCl. The mixture was stirred at 4 C for 24 h. KM A-Sepharose 4B thus prepared was alternately washed several times with 50 ml of 0.1 M acetate buffer-1.0 M NaCl (pH 4.0) and then with 50 ml of 0.1 M borate buffer-1.0 M NaCl (pH 8.0). Then KM A-Sepharose 4B was washed with distilled water and with 20 mM acetate buffer (pH 6.0)-20% glycerin containing 5 mM magnesium acetate and 10 mM 2-mercaptoethanol. KM A-Sepharose 4B was packed in a column (1 by 12 cm), and 4 ml of S-105 fraction (60 mg of protein per ml) was passed
51
through the column at a flow rate of 25 ml/h. The eluted solution was collected at 5 ml per tube. Isolation of the inactivated AK. AK inactivation was carried out in the reaction mixture containing 58 ml of the S-105 fraction (78 mg of protein per ml), 612 mg of disodium ATP, 39 mg of trilithium CoA, 958 mg of magnesium acetate, 100 mg of AK, and 15 ml of 1 M acetate buffer (pH 6.0). The total volume of the reaction mixture was brought to 108 ml with distilled water and incubated at 37 C. After 3 h of incubation, the mixture was diluted with 200 ml of distilled water and the reaction was stopped by heating in boiling water for 10 min. The supernatant obtained by centrifugation at 30,000 x g for 30 min was passed through a column of Amberlite CG-50 (NH4+ form, 50 ml). After the column was washed with 2,000 ml of distilled water, the inactivated AK was eluted with 0.5 N NH4OH. The eluate giving a positive ninhydrin reaction was collected and concentrated to dryness, and the powder was applied to thin-layer chromatography (TLC). The spot on the chromatogram was raked up, washed with distilled water, and then extracted with 0.5 N NH4OH and dried. This powder was further subjected to chromatography with Amberlite CG-50(NH4+ form, 15 ml). TLC. TLC was carried out by using the following solvent systems on a thin layer of silica gel G (Tokyo Kasei Co., Tokyo): S-110; chloroform-methanol-28% NH4OH-water (1:4:2:1); S-117; and chloroformmethanol-2$% NH4OH (1:2:1). The spot on the chromatogram was detected by ninhydrin reaction.
RESULTS Resistance to new aminoglycoside antibiotics. We could select 10 strains of P. aeruginosa from our stock cultures that were resistant to either DKB, GM-C1, or AK, or to various combinations of these drugs. The included three GM-C18 DKBrAKr strains, six GM-C,rDKB'AKs strains, and one GM-C1rDKBrAKr strain (Table 1). Among 10 strains examined, 4 of which were resistant to KM, DKB and AK were selected and the mechanisms of resistance to these drugs were examined. Three strains could inactivate KM, DKB, and AK but not GM-C1 in the presence of ATP, CoA, and magnesium acetate (Table 2). An inactivation reaction did not take place without either CoA or ATP, or without both, in the reaction mixture. But ATP, CoA, and magnesium acetate could be replaced by acetyl CoA, and [14C]acetic acid was incorporated into KM, DKB, and AK by the enzymatic inactivation of the drugs, indicating that these drugs were inactivated by acetylation. GN237 could inactivate KM by KM 3'-0-phosphorylation and was resistant to AK, DKB, KM, and GM-C1. However, GN237 could not inactivate AK, DKB, and GM-C1 by phosphorylation, acetylation, and adenylylation. The detailed mechanism of GN237 resistance to these drugs is now being studied and will be described elsewhere.
ANTIMICROB. AGENTsCHEMOTHER.
KAWABE, NAITO, AND MITSUHASHI
52
Conjugal transfer of drug resistance. Conjugal transfer of drug resistance was examined by using four strains. Three strains could transfer resistance to KM, DKB, AK, GM, and sulfanilamide. The exconjugants showed a low level of resistance to the GM-C complex (minimal inhibitory concentration, 3.1 gg/ml) and could inactivate both GM-C18 and GM-C2 by 6'-N-acetylation (Table 3). Purification and properties of 6'-Nacetyltransferase. The S-105 fraction from GN315 was passed through a KM A-Sepharose 4B column and was eluted by a linear gradient elution with NaCl from 0 to 0.5 M in 20 mM acetate buffer (pH 6.0) -20% glycerin containing 5 mM magnesium acetate and 10 mM 2-mercaptoethanol. The enzyme that inactivated KM, AK, and DKB appeared in the eluate with 0.3 M NaCl, and the inactivation curves of two drugs using eluted fractions were almost the same (Fig. 1). An AK acetyltransferTABLE 2. Inactivation of KM, DKB, AK, and GM-C1 by resistant strains of P. aeruginosaa Inactivation of:
Strain GN237 GN269 GN315 ML4561 (KM.DKB' AK.GM.SA ML4344 (KM.DKBb AK.GM.SA)
KM
DKB
AK
GM-Cl
+
+ + +
_ + + +
_ + + +
-
+
+
+
-
_
a The S-105 fractions were prepared from each strain as described in Materials and Methods. After 30 min of incubation at 37 C, residual antibiotic activity in the reaction mixture was determined by bioassay. b Two strains acquired drug resistance by conjugation in the experiment shown in Table 3.
ase was purified approximately 146-fold from the S-105 fraction. The optimal pH for AK and DKB inactivation, was 6.0 to 7.0, and the pH curves for inactivation of two drugs were almost the same (Fig. 2). These results indicated that an aminoglycoside acetyltransferase from GN315 could inactivate KM, AK, and DKB. Identification of the acetylated product. AK inactivation was carried out as described in Materials and Methods, and AK was found to be completely inactivated by this reaction. Inactivated AK (73 mg) was purified and obtained as a white powder. The inactivated AK and the synthetic sample of 6'-N-acetyl AK melted at 167 to 170 C and 168 to 173 C, respectively. Specific rotations of the inacti-
vated AK and synthetic sample were [a]23 + 69
(c 0.5, H20) and [a]123 + 72 (c 0.5, H2O), respectively. Elemental analysis of the inactivated AK was as follows. Calculated for C24N4,N50.3/2 H2CO,,; C, 42.50; H, 6.71; N, 9.71. Found: C, 42.35; H, 6.82; N, 10.08. Elemental analysis of the synthetic sample was as follows. Found: C, 42.30; H, 6.97; N, 9.54. The infrared spectra were very similar. Each sample was developed by TLC and examined by ninhydrin reagent. R, values of the inactivated AK and the synthetic sample were 0.24 by solvent S-110 and 0.16 by solvent S-117, respectively. Both samples were subjected- to a mild alkaline hydrolysis with 0.4 N NaOH at 80 C for 1 h. The hydrolysates of the inactivated product showed five ninhydrin-positive spots on the two TLC plates, the R, value of each spot being identical with that of the hydrolysate of synthetic sample (Fig. 3). Two bioactive zones revealed by bioautography of the TLC plates were identified as those of AK and KM, respectively. The nuclear magnetic resonance spectrum of the
TABLE 3. Conjugal transfer of drug resistance in P. aeruginosa Dono R n
GN315
ML4561
ML4561a (AK.DKB.KM.GM.SA)
ML4344
GN269
ML4561
ML4561a (AK.DKB.KM.GM.SA)
ML4344
GN362
ML4561
ML4561a (AK.DKB.KM.GM.SA)
ML4344
Selective drug
Transfer frequency
Resistance pattern of exconjugants
AK DKB AK DKB
1.5 x 4.4 x 2.5 x 3.1 x
10-5
(AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA)
AK DKB AK DKB
9.5 x 10-6 9.7 x 10-i 3.8 x 10-6 6.5 x 10-6
(AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA)
AK DKB AK DKB
4.0 x 8.5 x 4.3 x 6.6 x
(AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA)
10- 6 10-5
10-'
10-5 10-5
10-6
10-6
An exconjugant ML4561 that had acquired (AK.DKB.KM.GM.SA) resistance from GN315, GN269, and GN362 by conjugation was used as the donor of resistance. a
AMIKACIN RESISTANCE IN P. AERUGINOSA
VOL. 7, 1975
L'J
.9
-8'5
20
10
40 30 Fraction number
50
FIG. 1. Purification of aminoglycoside acetyltransferase of P. aeruginosa GN315. The reaction mixture consisted of 0.1 ml of each fraction, 0.05 ml of 0.5 mM drug, 0.025 ml of 0.5 mM CoA, 0.025 ml of 20 mM ATP, 0.05 ml of S-105 fraction of E. coli K-12 ML1410 (5 mg of protein/ml), 0.05 ml of 0.02 M magnesium acetate, and 0.2 ml of 0.2 M acetate buffer (pH 6.0).
100I I
90 Zn 0.)
80
E N
a
Lu
70 lo
53
streptomycin, and neomycin. However, the GM-C complex and semisynthetic aminoglycoside antibiotics such as DKB and AK were found to be effective against P. aeruginosa strains isolated from clinical specimens. Recent studies have indicated, however, that GMresistant P. aeruginosa strains are isolated from clinical specimens and that their isolation frequency is now increasing owing to the worldwide use of the drug. Based on the studies of biochemical mechanisms of aminoglycoside resistance, DKB (12) and AK (5) were produced from kanamycins B and A, respectively, and they were found to be effective against KM-resistant strains of bacteria, P. aeruginosa (7), and even against P. aeruginosa strains resistant to GM-Cla and GM-C2 . According to epidemiological studies of aminoglycoside resistance in P. aeruginosa, we could demonstrate the strains resistant to DKB (S. Mitsuhashi and H. Kawabe, 2nd Int. Symp. Antibiot. Resistance, June, 1974, Czechoslovakia) or resistant to both DKB and AK even before the practical use of the drugs. AK was produced by acylation of the 1-NH2 group of KM A with y-amino-a-hydroxybutyric acid but still had the hydroxy groups at the C-3' and C-2" positions. The 3'-OH and 2"-OH groups of KM A are known to be phosphorylated or adenylylated by enzymes from resistant strains (2, 10), but almost all strains capable of
la
h.-
ol 5.0
--
6.0 pH
7.0
0.64 0.59
FIG. 2. Effect of pH on AK and DKB inactivation. The reaction mixture consisted of 0.1 ml of enzyme purified by affinity chromatography (150 usg of protein/ml), 0.05 ml of 0.5 mM of DKB (or AK), 0.05 ml of 0.5 mM acetyl CoA, 0.2 ml of 0.2 M buffer, and 0.1 ml of distilled water. Symbols: 0, DKB; 0, AK.
0.41
0 0 0
0.22 inactivated AK in D2O showed one N-acetyl 0.15 signal at or 2.43. Furthermore, the nuclear magnetic resonance spectra of the inactivated AK and the synthetic sample were found to be similar (Fig. 4). In view of the above experimenA B C D E F G H tal results, the inactivated AK was concluded to FIG. 3. Thin-sayer chromatography of KM, AK, and be 6'-N-acetyl AK. their acetylated products hydrolysates using Silica Gel. The solvent system was S-110. (A) hydrolysate of the synthetic 6'-N-acetyl AK; (B) hydrolysate of DISCUSSION inactivated AK; (C) AK; (D) KM; (E) 6'-N-acetyl Epidemiological studies disclosed that almost KM; (F) synthetic sample of 6'-N-acetyl AK; (G) all P. aeruginosa strains were resistant to known sample of the inactivated AK; (H) L(-)--y-amino-aaminoglycoside antibiotics, including KM, hydroxybutyric acid.
54
KAWABE, NAITO, AND MITSUHASHI
ANTIMICROB. AGENTS CHEMOTHER.
tection of the inactivated position of the drugs by chemical modification of some position different from the inactivated one. ACKNOWLEDGMENTS
I
11
1 6 4 2 3 5 FIG. 4. Nuclear magnetic resonance spectra of the inactivated products of AK and the synthetic 6'-Nacetyl AK. (I) Synthetic sample of 6'-N-acetyl AK; (II) an inactivated product of AK by GN315.
inactivating the drugs by 3'-phosphorylatransferase or 2"-adenylyltransferase are found to be susceptible to AK and cannot inactivate the drug (unpublished observation). Furthermore, AK has the amino groups at the C-6' and C-3 positions, but the strains having 3-Nacetyltransferase are susceptible to AK. We have also recently isolated P. aeruginosa strains that can inactivate KM A and B by the enzyme through acetylation of the 6'-NH2 group but are susceptible to AK. These results strongly suggest that the acylation of the 1-NH2 group of KM A protects the inactivated position of AK, resulting in resistance of AK to known aminoglycoside acetyltransferases, adenylyltransferase or phosphotransferase. Thus there are two methods to tailor the known chemotherapeutic agents to become more effective against resistant bacteria; (i) chemical modification of the inactivated position of the drugs; and (ii) pro-
We are greatly indebted to H. Umezawa and S. Kondo, Institute of Microbial Chemistry, Tokyo, and to H. Kawaguchi, Bristol-Banyu Research Institute, Ltd., Tokyo, for their technical advice and chemical analysis of the inactivated products.
LITERATURE CITED 1. Benveniste, R., and J. Davies. 1971. Enzymatic acetylation of aminoglycoside antibiotics by Escherichia coli carrying an R factor. Biochemistry 10:1787-1796. 2. Benveniste, R., and J. Davies. 1971. R-factor mediated gentamicin resistance: a new enzyme which modified aminoglycoside antibiotics. FEBS Lett. 14:293-296. 3. Kawabe, H., M. Inoue, and S. Mitsuhashi. 1974. Inactivation of dihydrostreptomycin and spectinomycin by Staphylococcus aureus. Antimicrob. Agents Chemother. 5:553-557. 4. Kawaba, H., and S. Mitsuhashi. 1972. Acetylation of dideoxykanamycin B by Pseudomonas aeruginosa. Jap. J. Microbiol. 16:436-437. 5. Kawaguchi, H., T. Naito, S. Nakagawa, and K. Fujisawa. 1972. BB-K8, a new semisynthetic aminoglycoside antibiotic. J. Antibiot. 25:695-708. 6. Le Goffic, F., and N. Moreau. 1973. Purification by affinity chromatography of an enzyme involved in gentamicin inactivation. FEBS Lett. 29:289-291. 7. Mitsuhashi, S., F. Kobayashi, M. Yamaguchi, K. O'hara, and M. Kono. 1972. Enzymatic inactivation of aminoglycoside antibiotics by resistant strains of bacteria, p. 561-564. In M. Hejzlar (ed.), Advances in antimicirobiol and antineoplasmic chemotherapy (Proc. VIIth Int. Congr. Chemother.). University Park Press, Baltimore. 8. O'hara, K., M. Kono, and S. Mitsuhashi. 1974. Structure of enzymatically acetylated sisomicin by Pseudomonas aerguinosa. J. Antibiot. 27:349-351. 9. Okanishi, M., S. Kondo, Y. Suzuki, S. Okamoto, and H. Umezawa. 1967. Studies on inactivation of kanamycin and resistances of E. coli. J. Antibiot. 20:132-135. 10. Umezawa, H., M. Okanishi, S. Kondo, K. Hamana, R. Utahara, K. Maeda, and S. Mitsuhashi. 1967. Phosphorylative inactivation of aminoglycosidic antibiotics by Escherichia coli carrying R factor. Science 157:1559-1561. 11. Umezawa, H., M. Okanishi, R. Utahara, K. Maeda, and S. Kondo. 1967. Isolation and structure of kanamycin inactivated by a cell free system of kanamycin-resistant E. coli. J. Antibiot. 20:136-141. 12. Umezawa, H., S. Umezawa, T. Tsuchiya, and Y. Okazaki, 1971. 3',4'-Dideoxykanamycin B active against kanamycin-resistant Escherichia coli and Pseudomonas aeruginosa. J. Antibiot. 24:485-487. 13. Umezawa, H., H. Yamamoto, M. Yagisawa, S. Kondo, T. Takeuchi, and Y. A. Chabbert. 1973. Kanamycin phosphotransferase I. Mechanism of cross resistance between kanamycin and lividomycin. J. Antibiot. 26:407-410. 14. Yagisawa, M., H. Naganawa, S. Kondo, T. Takeuchi, and H. Umezawa. 1972. 6'-N-acetylation of 3',4'dideoxykanamycin B by an enzyme in a resistant strain of Pseudomonas aeruginosa. J. Antibiot. 25:495-496. 15. Yamamoto, H., M. Yagisawa, H. Naganawa, S. Kondo, T. Takeuchi, and H. Umezawa. 1972. Kanamycin 6'-acetate and ribostamycin 6'-acetate, enzymatically inactivated products by Pseudomonas aeruginosa. J. Antibiot. 25:746-747.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1975, P. 50-54 Copyright i 1975 American Society for Microbiology
Acetylation of Amikacin, a New Semisynthetic Antibiotic, by Pseudomonas aeruginosa Carrying an R Factor HARUHIDE KAWABE,* TAKAYUKI NAITO, AND SUSUMU MITSUHASHI Department of Microbiology, School of Medicine, Gunma University, Maebashi,* and Bristol-Banyu Research
Institute Ltd., Tokyo, Japan Received for publication 31 July 1974
A clinical isolate Pseudomonas aeruginosa GN315 resistant to amikacin (AK), semisynthetic antibiotic, inactivated AK by acetylation. The acetylating enzyme was purified approximately 146-fold from a crude extract of GN315 by affinity chromatography. Fractionated samples obtained by affinity chromatography showed almost the same inactivation curves toward 3',4'-dideoxykanamycin B (DKB) and AK. Partially purified AK-acetylating enzyme inactivated DKB and kanamycin A but could not inactivate gentamicin C1 The optimal pH for their inactivation was 6.0 to 7.0, and the pH curves for the inactivation of both drugs were almost the same. These facts indicate that AK and DKB are inactivated by the same aminoglycoside-acetylating enzyme. Through elemental analysis, the inactivated AK was found to be a monoacetylated product of AK. A sample of inactivated AK was purified and compared with a synthetic 6'-N-acetyl AK by thin-layer chromatography, and the results indicated that AK was inactivated by acetylation of the 6'-NH2 group. The ultraviolet, infrared, and nuclear magnetic resonance spectra of the inactivated AK showed that AK was inactivated by the enzyme through acetylation of the amino group of 6'-amino-6'-deoxy-D-glucose moiety of AK. This enzyme, mediated by R factor, is capable of conferring resistance to AK, DKB, kanamycin, gentamicin, and sulfanilamide. a new
.
A 6'-N-acetylating enzyme that inactivates aminoglycosidic antibiotics has been found in drug-resistant strains of Escherichia coli (1, 9, 11) and Pseudomonas aeruginosa (4, 8, 14, 15). We reported that an enzyme capable of acetylating 3',4'-dideoxykanamycin B (DKB) was demonstrated in a resistant strain of P. aeruginosa cape 15, and the inactivated product appeared to be 6'-N-acetyl DKB (4). Yagisawa et al. (14) reported that the inactivation of DKB by P. aeruginosa GN315 was due to the formation of DKB 6'-N-acetate. Furthermore, Yamamoto et al. (15) reported the inactivation of kanamycin (KM) A and ribostamycin by the same enzyme solution of GN315, producing KM 6'-N-acetate and ribostamycin 6'-N-acetate. This paper deals with some properties of the 6'-N-acetylating enzyme from GN315 and the chemical structure of inactivated amikacin (AK). MATERIALS AND METHODS Bacterial strains. Ten strains of P. aeruginosa were isolated from clinical specimens and stocked in this laboratory. Their resistance patterns to KMA, DKB, gentamicin Cl (GM-Cl), and AK are shown in Table 1. P. aeruginosa ML4561 (leu-arg-ilv-his50
rifampin-resistant) and P. aeruginosa ML4344 (leu-trp-) were used as recipients of R factor. Drugs. A semisynthetic aminoglycoside antibiotic, DKB, was produced from KM B (12) and was kindly supplied by H. Umezawa, Institute of Microbial Chemistry, Tokyo. AK was produced by acylation of the 1-NH2 group of KM A with L(-)-y-amino-ahydroxybutyric acid (5) and was provided by H. Kawaguchi, Bristol-Banyu Research Institute, Ltd., Tokyo. GM-Cl was kindly supplied by the Schering Corp., Bl6oomfield, N.J. Media. Nutrient broth containing 0.25% KNO, was used for liquid culture. Minimal medium was used for the transfer of R factor from exconjugant ML4561 to ML4344, and consisted of 8.0 g of Na,PHO4, 2.0 g of KH2PO4, 1 g of (NH4)2SO4, 13 g of agar, 80 mg of bromothymol blue, 10 g of glucose, 0.1 g of MgSO4, and 1,000 ml of distilled water. Preparation of S-105 fraction and inactivation reaction. Cell-free extract was prepared as described previously (3). The reaction mixture consisted of 0.2 ml of acetate buffer (pH 6.0), 0.05 ml of 20 mM disodium adenosine triphosphate (ATP), 0.05 ml of 0.5 mM coenzyme A (CoA) 0.05 ml of 0.02 M magnesium acetate, 0.05 ml of 0.5 mM drug, and 0.1 ml of S-105 fraction. After incubation at 37 C for 30 min, the reaction was stopped by heating in boiling water for 3 min. Residual antibiotic activity in the reaction mixture was determined by bioassay using Bacillus subtilis PCI 219.
AMIKACIN RESISTANCE IN P. AERUGINOSA
VOL. 7, 1975
TABLE 1. Minimal inhibitory concentrations of aminoglycoside antibiotics against P. aeruginosa Minimal inhibitory concn (ug/ml)
Strain
GN237 GN269 GN315 GN362 GN4475 GN4489 GN4491 GN4493 GN4494 GN4495 ML4344a ML4561a
KM
DKB
GM-Cl
AK
100 100 100 100 25 25 25 25 25 25 12.5 25
50 50 100 100 1.6 0.8 1.6 0.8 1.6 1.6 0.4 0.4
100 3.1 3.1 3.1 100 100 100 50 50 50 1.5 1.5
25 50 25 25 1.6 3.1 3.1 3.1 1.6 0.8 0.8 0.8
aTwo strains of P. aeruginosa recipient of R factor.
were
used
as
the
Conjugal transfer of resistance. From each donor and recipient culture in nutrient broth at 37 C for 18 h, 0.5 ml was removed and inoculated into 5 ml of fresh nutrient broth and shaken at 37 C. After 2 h of incubation, the cultures (about 2 x 109 cells/ml) reached the late logarithmic phase of growth and were used for the conjugal transfer of resistance. One part of donor culture was mixed with four parts of recipient culture, and the mixture was incubated at 37 C with gentle shaking. After 90 min of incubation, a 0.1-ml sample of the mixed culture was plated on a selective plate containing rifampin (200 Ag/ml) and either DKB (3.1 lAg/ml) or AK (3.1 ;ig/ml). The colonies that developed on selective plates after 18 h of incubation at 37 C were picked and purified by three successive single-colony isolations, and their drug resistance was determined. For the transfer of resistance from exconjugant ML4561 to ML4344, the minimal medium containing DKB (or AK), leucine (50 ug/ml), and tryptophan (50 jug/ml) was used for the selection of exoconjugant ML4344. The transfer frequency was expressed as the ratio of the number of donor to recipient cells that acquired drug resistance by conjugation. Preparation of the column. Aminoglycoside acetyltransferase was purified by affinity chromatography (6, 13). KM A-Sepharose 4B was prepared as follows. Cyanogen bromide-activated Sepharose 4B (Pharmacia Fine Chemicals AB, Upsala, Sweden) was washed with 1,000 ml of 1 mM HCl, 500 ml of cold water, and 25 ml of 0.1 M NaHCOa-0.5 M NaCl, successively, and was mixed with 0.5 g of KM in 45 ml of 0.1 M NaHCO,-0.5 M NaCl. The mixture was stirred at 4 C for 24 h. KM A-Sepharose 4B thus prepared was alternately washed several times with 50 ml of 0.1 M acetate buffer-1.0 M NaCl (pH 4.0) and then with 50 ml of 0.1 M borate buffer-1.0 M NaCl (pH 8.0). Then KM A-Sepharose 4B was washed with distilled water and with 20 mM acetate buffer (pH 6.0)-20% glycerin containing 5 mM magnesium acetate and 10 mM 2-mercaptoethanol. KM A-Sepharose 4B was packed in a column (1 by 12 cm), and 4 ml of S-105 fraction (60 mg of protein per ml) was passed
51
through the column at a flow rate of 25 ml/h. The eluted solution was collected at 5 ml per tube. Isolation of the inactivated AK. AK inactivation was carried out in the reaction mixture containing 58 ml of the S-105 fraction (78 mg of protein per ml), 612 mg of disodium ATP, 39 mg of trilithium CoA, 958 mg of magnesium acetate, 100 mg of AK, and 15 ml of 1 M acetate buffer (pH 6.0). The total volume of the reaction mixture was brought to 108 ml with distilled water and incubated at 37 C. After 3 h of incubation, the mixture was diluted with 200 ml of distilled water and the reaction was stopped by heating in boiling water for 10 min. The supernatant obtained by centrifugation at 30,000 x g for 30 min was passed through a column of Amberlite CG-50 (NH4+ form, 50 ml). After the column was washed with 2,000 ml of distilled water, the inactivated AK was eluted with 0.5 N NH4OH. The eluate giving a positive ninhydrin reaction was collected and concentrated to dryness, and the powder was applied to thin-layer chromatography (TLC). The spot on the chromatogram was raked up, washed with distilled water, and then extracted with 0.5 N NH4OH and dried. This powder was further subjected to chromatography with Amberlite CG-50(NH4+ form, 15 ml). TLC. TLC was carried out by using the following solvent systems on a thin layer of silica gel G (Tokyo Kasei Co., Tokyo): S-110; chloroform-methanol-28% NH4OH-water (1:4:2:1); S-117; and chloroformmethanol-2$% NH4OH (1:2:1). The spot on the chromatogram was detected by ninhydrin reaction.
RESULTS Resistance to new aminoglycoside antibiotics. We could select 10 strains of P. aeruginosa from our stock cultures that were resistant to either DKB, GM-C1, or AK, or to various combinations of these drugs. The included three GM-C18 DKBrAKr strains, six GM-C,rDKB'AKs strains, and one GM-C1rDKBrAKr strain (Table 1). Among 10 strains examined, 4 of which were resistant to KM, DKB and AK were selected and the mechanisms of resistance to these drugs were examined. Three strains could inactivate KM, DKB, and AK but not GM-C1 in the presence of ATP, CoA, and magnesium acetate (Table 2). An inactivation reaction did not take place without either CoA or ATP, or without both, in the reaction mixture. But ATP, CoA, and magnesium acetate could be replaced by acetyl CoA, and [14C]acetic acid was incorporated into KM, DKB, and AK by the enzymatic inactivation of the drugs, indicating that these drugs were inactivated by acetylation. GN237 could inactivate KM by KM 3'-0-phosphorylation and was resistant to AK, DKB, KM, and GM-C1. However, GN237 could not inactivate AK, DKB, and GM-C1 by phosphorylation, acetylation, and adenylylation. The detailed mechanism of GN237 resistance to these drugs is now being studied and will be described elsewhere.
ANTIMICROB. AGENTsCHEMOTHER.
KAWABE, NAITO, AND MITSUHASHI
52
Conjugal transfer of drug resistance. Conjugal transfer of drug resistance was examined by using four strains. Three strains could transfer resistance to KM, DKB, AK, GM, and sulfanilamide. The exconjugants showed a low level of resistance to the GM-C complex (minimal inhibitory concentration, 3.1 gg/ml) and could inactivate both GM-C18 and GM-C2 by 6'-N-acetylation (Table 3). Purification and properties of 6'-Nacetyltransferase. The S-105 fraction from GN315 was passed through a KM A-Sepharose 4B column and was eluted by a linear gradient elution with NaCl from 0 to 0.5 M in 20 mM acetate buffer (pH 6.0) -20% glycerin containing 5 mM magnesium acetate and 10 mM 2-mercaptoethanol. The enzyme that inactivated KM, AK, and DKB appeared in the eluate with 0.3 M NaCl, and the inactivation curves of two drugs using eluted fractions were almost the same (Fig. 1). An AK acetyltransferTABLE 2. Inactivation of KM, DKB, AK, and GM-C1 by resistant strains of P. aeruginosaa Inactivation of:
Strain GN237 GN269 GN315 ML4561 (KM.DKB' AK.GM.SA ML4344 (KM.DKBb AK.GM.SA)
KM
DKB
AK
GM-Cl
+
+ + +
_ + + +
_ + + +
-
+
+
+
-
_
a The S-105 fractions were prepared from each strain as described in Materials and Methods. After 30 min of incubation at 37 C, residual antibiotic activity in the reaction mixture was determined by bioassay. b Two strains acquired drug resistance by conjugation in the experiment shown in Table 3.
ase was purified approximately 146-fold from the S-105 fraction. The optimal pH for AK and DKB inactivation, was 6.0 to 7.0, and the pH curves for inactivation of two drugs were almost the same (Fig. 2). These results indicated that an aminoglycoside acetyltransferase from GN315 could inactivate KM, AK, and DKB. Identification of the acetylated product. AK inactivation was carried out as described in Materials and Methods, and AK was found to be completely inactivated by this reaction. Inactivated AK (73 mg) was purified and obtained as a white powder. The inactivated AK and the synthetic sample of 6'-N-acetyl AK melted at 167 to 170 C and 168 to 173 C, respectively. Specific rotations of the inacti-
vated AK and synthetic sample were [a]23 + 69
(c 0.5, H20) and [a]123 + 72 (c 0.5, H2O), respectively. Elemental analysis of the inactivated AK was as follows. Calculated for C24N4,N50.3/2 H2CO,,; C, 42.50; H, 6.71; N, 9.71. Found: C, 42.35; H, 6.82; N, 10.08. Elemental analysis of the synthetic sample was as follows. Found: C, 42.30; H, 6.97; N, 9.54. The infrared spectra were very similar. Each sample was developed by TLC and examined by ninhydrin reagent. R, values of the inactivated AK and the synthetic sample were 0.24 by solvent S-110 and 0.16 by solvent S-117, respectively. Both samples were subjected- to a mild alkaline hydrolysis with 0.4 N NaOH at 80 C for 1 h. The hydrolysates of the inactivated product showed five ninhydrin-positive spots on the two TLC plates, the R, value of each spot being identical with that of the hydrolysate of synthetic sample (Fig. 3). Two bioactive zones revealed by bioautography of the TLC plates were identified as those of AK and KM, respectively. The nuclear magnetic resonance spectrum of the
TABLE 3. Conjugal transfer of drug resistance in P. aeruginosa Dono R n
GN315
ML4561
ML4561a (AK.DKB.KM.GM.SA)
ML4344
GN269
ML4561
ML4561a (AK.DKB.KM.GM.SA)
ML4344
GN362
ML4561
ML4561a (AK.DKB.KM.GM.SA)
ML4344
Selective drug
Transfer frequency
Resistance pattern of exconjugants
AK DKB AK DKB
1.5 x 4.4 x 2.5 x 3.1 x
10-5
(AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA)
AK DKB AK DKB
9.5 x 10-6 9.7 x 10-i 3.8 x 10-6 6.5 x 10-6
(AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA)
AK DKB AK DKB
4.0 x 8.5 x 4.3 x 6.6 x
(AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA) (AK.DKB.KM.GM.SA)
10- 6 10-5
10-'
10-5 10-5
10-6
10-6
An exconjugant ML4561 that had acquired (AK.DKB.KM.GM.SA) resistance from GN315, GN269, and GN362 by conjugation was used as the donor of resistance. a
AMIKACIN RESISTANCE IN P. AERUGINOSA
VOL. 7, 1975
L'J
.9
-8'5
20
10
40 30 Fraction number
50
FIG. 1. Purification of aminoglycoside acetyltransferase of P. aeruginosa GN315. The reaction mixture consisted of 0.1 ml of each fraction, 0.05 ml of 0.5 mM drug, 0.025 ml of 0.5 mM CoA, 0.025 ml of 20 mM ATP, 0.05 ml of S-105 fraction of E. coli K-12 ML1410 (5 mg of protein/ml), 0.05 ml of 0.02 M magnesium acetate, and 0.2 ml of 0.2 M acetate buffer (pH 6.0).
100I I
90 Zn 0.)
80
E N
a
Lu
70 lo
53
streptomycin, and neomycin. However, the GM-C complex and semisynthetic aminoglycoside antibiotics such as DKB and AK were found to be effective against P. aeruginosa strains isolated from clinical specimens. Recent studies have indicated, however, that GMresistant P. aeruginosa strains are isolated from clinical specimens and that their isolation frequency is now increasing owing to the worldwide use of the drug. Based on the studies of biochemical mechanisms of aminoglycoside resistance, DKB (12) and AK (5) were produced from kanamycins B and A, respectively, and they were found to be effective against KM-resistant strains of bacteria, P. aeruginosa (7), and even against P. aeruginosa strains resistant to GM-Cla and GM-C2 . According to epidemiological studies of aminoglycoside resistance in P. aeruginosa, we could demonstrate the strains resistant to DKB (S. Mitsuhashi and H. Kawabe, 2nd Int. Symp. Antibiot. Resistance, June, 1974, Czechoslovakia) or resistant to both DKB and AK even before the practical use of the drugs. AK was produced by acylation of the 1-NH2 group of KM A with y-amino-a-hydroxybutyric acid but still had the hydroxy groups at the C-3' and C-2" positions. The 3'-OH and 2"-OH groups of KM A are known to be phosphorylated or adenylylated by enzymes from resistant strains (2, 10), but almost all strains capable of
la
h.-
ol 5.0
--
6.0 pH
7.0
0.64 0.59
FIG. 2. Effect of pH on AK and DKB inactivation. The reaction mixture consisted of 0.1 ml of enzyme purified by affinity chromatography (150 usg of protein/ml), 0.05 ml of 0.5 mM of DKB (or AK), 0.05 ml of 0.5 mM acetyl CoA, 0.2 ml of 0.2 M buffer, and 0.1 ml of distilled water. Symbols: 0, DKB; 0, AK.
0.41
0 0 0
0.22 inactivated AK in D2O showed one N-acetyl 0.15 signal at or 2.43. Furthermore, the nuclear magnetic resonance spectra of the inactivated AK and the synthetic sample were found to be similar (Fig. 4). In view of the above experimenA B C D E F G H tal results, the inactivated AK was concluded to FIG. 3. Thin-sayer chromatography of KM, AK, and be 6'-N-acetyl AK. their acetylated products hydrolysates using Silica Gel. The solvent system was S-110. (A) hydrolysate of the synthetic 6'-N-acetyl AK; (B) hydrolysate of DISCUSSION inactivated AK; (C) AK; (D) KM; (E) 6'-N-acetyl Epidemiological studies disclosed that almost KM; (F) synthetic sample of 6'-N-acetyl AK; (G) all P. aeruginosa strains were resistant to known sample of the inactivated AK; (H) L(-)--y-amino-aaminoglycoside antibiotics, including KM, hydroxybutyric acid.
54
KAWABE, NAITO, AND MITSUHASHI
ANTIMICROB. AGENTS CHEMOTHER.
tection of the inactivated position of the drugs by chemical modification of some position different from the inactivated one. ACKNOWLEDGMENTS
I
11
1 6 4 2 3 5 FIG. 4. Nuclear magnetic resonance spectra of the inactivated products of AK and the synthetic 6'-Nacetyl AK. (I) Synthetic sample of 6'-N-acetyl AK; (II) an inactivated product of AK by GN315.
inactivating the drugs by 3'-phosphorylatransferase or 2"-adenylyltransferase are found to be susceptible to AK and cannot inactivate the drug (unpublished observation). Furthermore, AK has the amino groups at the C-6' and C-3 positions, but the strains having 3-Nacetyltransferase are susceptible to AK. We have also recently isolated P. aeruginosa strains that can inactivate KM A and B by the enzyme through acetylation of the 6'-NH2 group but are susceptible to AK. These results strongly suggest that the acylation of the 1-NH2 group of KM A protects the inactivated position of AK, resulting in resistance of AK to known aminoglycoside acetyltransferases, adenylyltransferase or phosphotransferase. Thus there are two methods to tailor the known chemotherapeutic agents to become more effective against resistant bacteria; (i) chemical modification of the inactivated position of the drugs; and (ii) pro-
We are greatly indebted to H. Umezawa and S. Kondo, Institute of Microbial Chemistry, Tokyo, and to H. Kawaguchi, Bristol-Banyu Research Institute, Ltd., Tokyo, for their technical advice and chemical analysis of the inactivated products.
LITERATURE CITED 1. Benveniste, R., and J. Davies. 1971. Enzymatic acetylation of aminoglycoside antibiotics by Escherichia coli carrying an R factor. Biochemistry 10:1787-1796. 2. Benveniste, R., and J. Davies. 1971. R-factor mediated gentamicin resistance: a new enzyme which modified aminoglycoside antibiotics. FEBS Lett. 14:293-296. 3. Kawabe, H., M. Inoue, and S. Mitsuhashi. 1974. Inactivation of dihydrostreptomycin and spectinomycin by Staphylococcus aureus. Antimicrob. Agents Chemother. 5:553-557. 4. Kawaba, H., and S. Mitsuhashi. 1972. Acetylation of dideoxykanamycin B by Pseudomonas aeruginosa. Jap. J. Microbiol. 16:436-437. 5. Kawaguchi, H., T. Naito, S. Nakagawa, and K. Fujisawa. 1972. BB-K8, a new semisynthetic aminoglycoside antibiotic. J. Antibiot. 25:695-708. 6. Le Goffic, F., and N. Moreau. 1973. Purification by affinity chromatography of an enzyme involved in gentamicin inactivation. FEBS Lett. 29:289-291. 7. Mitsuhashi, S., F. Kobayashi, M. Yamaguchi, K. O'hara, and M. Kono. 1972. Enzymatic inactivation of aminoglycoside antibiotics by resistant strains of bacteria, p. 561-564. In M. Hejzlar (ed.), Advances in antimicirobiol and antineoplasmic chemotherapy (Proc. VIIth Int. Congr. Chemother.). University Park Press, Baltimore. 8. O'hara, K., M. Kono, and S. Mitsuhashi. 1974. Structure of enzymatically acetylated sisomicin by Pseudomonas aerguinosa. J. Antibiot. 27:349-351. 9. Okanishi, M., S. Kondo, Y. Suzuki, S. Okamoto, and H. Umezawa. 1967. Studies on inactivation of kanamycin and resistances of E. coli. J. Antibiot. 20:132-135. 10. Umezawa, H., M. Okanishi, S. Kondo, K. Hamana, R. Utahara, K. Maeda, and S. Mitsuhashi. 1967. Phosphorylative inactivation of aminoglycosidic antibiotics by Escherichia coli carrying R factor. Science 157:1559-1561. 11. Umezawa, H., M. Okanishi, R. Utahara, K. Maeda, and S. Kondo. 1967. Isolation and structure of kanamycin inactivated by a cell free system of kanamycin-resistant E. coli. J. Antibiot. 20:136-141. 12. Umezawa, H., S. Umezawa, T. Tsuchiya, and Y. Okazaki, 1971. 3',4'-Dideoxykanamycin B active against kanamycin-resistant Escherichia coli and Pseudomonas aeruginosa. J. Antibiot. 24:485-487. 13. Umezawa, H., H. Yamamoto, M. Yagisawa, S. Kondo, T. Takeuchi, and Y. A. Chabbert. 1973. Kanamycin phosphotransferase I. Mechanism of cross resistance between kanamycin and lividomycin. J. Antibiot. 26:407-410. 14. Yagisawa, M., H. Naganawa, S. Kondo, T. Takeuchi, and H. Umezawa. 1972. 6'-N-acetylation of 3',4'dideoxykanamycin B by an enzyme in a resistant strain of Pseudomonas aeruginosa. J. Antibiot. 25:495-496. 15. Yamamoto, H., M. Yagisawa, H. Naganawa, S. Kondo, T. Takeuchi, and H. Umezawa. 1972. Kanamycin 6'-acetate and ribostamycin 6'-acetate, enzymatically inactivated products by Pseudomonas aeruginosa. J. Antibiot. 25:746-747.
