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ENZYMATIC MODIFICATION OF to this drug but the results were negative (J. D. HYGROMYCIN B IN unpublished observations). Since no resistance STREPTOMYCES HYGROSCOPICUS mechanism had been detected to date, we have examined extracts of S. hygroscopicus to see if an enzymatic modification exists for hygromycin J. LEBOUL* and J. DAVIES* in the producing organism. Dept. of Biochemistry, University of Streptomyces hygroscopicus (NRRL 2387) was Wisconsin, U.S.A. grown in trypticase soy broth and the cells were harvested, washed and resuspended in buffer. (Received for publication October 19, 1981) The cells were broken by passing the suspension through a French pressure cell (Aminco) at 1,400 Antibiotic producing-organisms generally ex- kg/cm2 and the resulting extract centrifuged at hibit resistance to the antibiotics that they pro- 30,000 xg for 30 minutes to remove the cell de- duce1). Such resistance mechanisms may take bris. Samples of the extract were tested for ace- the form of target site modification, as is the case tyltransferase, adenylyltransferase and phospho- with erythromycin2) and thiostrepton3), or enzy- transferase activity, using a variety of substrates. matic modification of the antibiotic itself, as has A phosphotransferase activity was detected with been shown for a number of aminocyclitols4) hygromycin B and structurally related antibiotics The existence of these resistance mechanisms in as substrates (Table 1). Note that weak acetyl- producing organisms is thought to be the means transferase activity against sisomicin was de- by which the organism avoids growth inhibition tected; the enzyme aminoglycoside 3-acetyl- by its own antibiotic product although other pos- transferase appears to be widely distributed in sibilities cannot be ruled out. For example, in Streptomycetes6). Since previous experience the case of the aminocyclitols the enzymatic modi- with phosphotransferases had shown that detec- fications may be concerned with specific steps in tion was often made difficult because of the pre- antibiotic biosynthesis or transport and the fact sence of potent endogenous ATPase activity, the that the antibiotics are inactivated may be S. hygroscopicus extract was passed through a coincidental. It has been suggested that these column of Bio-Gel P-100; the protein eluting in genes may be evolutionary precursors of the re- the void volume was discarded and assay of ma- sistance determinants of R-plasmids in clinical terial retarded by gel filtration chromatography isolates of bacteria5). Regardless of the role or demonstrated potent hygromycin phosphotrans- evolution of antibiotic modifications in producing organisms, the fact that they are biochemically Table 1. Modification of aminocyclitol antibiotics homologous to the determinants of R-plasmids catalyzed by extracts* of St•eptomyces Ingroscopi- provides a useful means of predicting possible cus. resistance mechanisms that may be encountered Substrate cpm in the therapeutic use of antibiotics. Chemical transferred" modifications of antibiotics to block these enzy- Acetylation Hygromycin B M8 matic modifications could be undertaken in anti- Sisomicin 1,048 cipation of the appearance of resistant bacterial Adenylylation Tobramycin 0 strains. Spectinomycin 0 The hygromycin group of antibiotics have not Hygromycin B 178 been used extensively, apart from veterinary ap- plications. Hygromycin B, produced by Stre- Phosphorylation Neomycin 1,708 ptomyces hygroscopicus, has a good range of anti- Sisomicin 2,018 bacterial activity; a limited number of R-plasmids Streptomycin 1,630 have been screened for resistant determinants Hygromycin B 68,797 * Present addresses: J * In the case of phosphorylation . DAVIES, Biogen S. A., , a crude extract 3, rte de Troinex 1227, Carouge, Switzerland and J. was partially purified by gel-filtration on Bio- LEBOUL, Rhone-Poulenc, Centre de Recherches Vitry Gel P-100. 13, quai Jules Guesde B. P. 24, F-94 400 Vitry-sur- ** Activity transferred in phosphocelIulose paper Seine, France. binding assay4). 528 THE JOURNAL OF ANTIBIOTICS APR. 1982 ferase activity. A number of aminocyclitol References antibiotics were tested as substrates for the pho- 1) DEMAIN, A. L.: How do antibiotic producing sphotransferase but none were active; the enzyme organisms avoid suicide ? Ann. N. Y. Acad. demonstrates high specificity for the destomycin- Sci. 235: 601-612, 1974 hygromycirt antibiotics. 2) FUJISAWA,Y. & B. WEISBLUM: A family of r- This finding extends the notion that enzymatic determinants in Streptomyces spp. that specifies inactivation of antibiotics is common among pro- inducible resistance to macrolide, lincosamide, ducing organisms and we suggest that should R- and streptogramin B type antibiotics. J. Bac- plasmid delermined resistance to the hygromycin teriol. 146: 621 - 632, 1981 antibiotics appear, phosphorylation is likely to 3) CUNDLIFFE, E.: Mechanism of resistance to be an important biochemical mechanism. It thiostrepton in the producing-organism Strepto- has been demonstrated, recently, that phosphoryl- myces azureus. Nature 272: 792-.795, 1978 4) DOWDING, J. & J. DAVIES: Mechanisms and ation is the major mechanism of self-protection origins of plasmid-determined resistance. Micro- for a number of aminoglycoside-producing or- biology 1974: 179186, 1974 ganisms7). It would be profitable to study the na- 5) BENVENISTE,R. & J. DAVIES: Aminoglycoside ture of phosphorylated hygromycin B in the event antibiotic-inactivating enzymes in Actinomycetes that extensive clinical use of this antibiotic (or similar to those present in clinical isolates of related compounds) is anticipated. antibiotic resistant bacteria. Proc. Nat. Acad. In addition, since hygromycin B is a potent Sci. (U.S.) 70: 22762280, 1973 inhibitor of protein synthesis in a wide range of 6) DAVIES, J.; C. HOUK, M. YAGISAWA& T. J. pro- and eakaryotes8) this drug, in combination WHITE: Occurrence and function of amino- with the resistance gene, might be a useful selec- glycoside-modifying enzymes. Genetics of In- dustrial Microorganisms, pp. 166-169, 1979 tion system in gene transfer experiments, as has 7) HOTTA,K.; H. YAMAMOTO,Y. OKAMI& H. UME- been shown previously for the aminoglycoside zAwA: Resistance mechanisms of kanamycin-, antibiotic G4189). neomycin- and streptomycin-producing strepto- mycetes to aminoglycoside antibiotics. J. Anti- Acknowledgements biotics 34: 1175 - 1182, 1981 8) SCHINDLER,D.: Ph. D. thesis, University of This study was supported generously by Rhone- Wisconsin, 1976 Poulenc and also by funds from the National Science 9) DAVIES,J. & A. JIMENEZ: A new selective agent Foundation. We wish to thank Dr. STEPHENHAR- for eukaryotic cloning vectors. Am. J. Trop. FORDfor his interest and helpful suggestions. Hyg. 29 Suppl.: 1089- 1092, 1980