Resistance Factor-Mediated Spectinomycin Resistance
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INFECTION AND IMMUNITY, Jan. 1970, p. 120-127 Vol. 1, No. 1 Copyright ( 1970 American Society for Microbiology Priinted inz U.S.A. Resistance Factor-Mediated Spectinomycin Resistance DAVID H. SMITH, J. A. JANJIGIAN, NAOMI PRESCOTT, AND PORTER W. ANDERSON Department ofPediatrics, Harvard Medical School anid Chlildreni's Hospital Medical Cenlter, Boston, Massachusetts 02115 Received for publication 22 September 1969 Of 100 natural isolates of drug-resistant enteric bacteria, 51 were resistant to spec- tinomycin (Spc) and 46 contained transferable R factors mediating Spc resistance. All SpcR R factors mediated streptomycin and bluensomycin resistance and were fi+ type. Extracts of R-SpcR strains adenylated Spc, dihydrospectinomycin, actinamine streptomycin, and bluensomycin in vitro in the presence of adenosine triphosphate and Mg++. Results of genetic and biochemical studies support the hypothesis that these reactions are mediated by a single enzyme. Spectinomycin (Spc), a dibasic aminoglycoside and the Massachusetts General Hospital were the antibiotic produced by Streptomyces spectabilis, is source of most of the R factors studied. RE130 bacteriostatic for a wide variety of clinically (fi+ SpcR StrR BluR ChlR TetR MerR SulR; 11), important bacteria (16). Spc selectively inhibits the R factor studied in many of the experiments, was in and carried originally by a strain of E. coli. Bacteria used protein synthesis Escherichia coli, strains as to a chromosomal recipients for the R factors included E. coli B resistant to Spc owing muta- and E. coli K-12 strains AB1932-1 (Arg- Met- Lac- tion have 30S ribosomal subunits that are re- NalR; 27) and FW 13A (Thr- Leu- Met- StrR, 19). sistant to the drug (6). Chromosomal Spc re- Klebsiella UC-59, obtained from George Whitfield sistance arises by high-level, one-step mutation of The Upjohn Co., Kalamazoo, Mich., was used and is recessive in diploid strains with SpcS and in the bioassay of Spc. E. coli K-12 strain A 352 SpcR loci (2, 24). (lacdel/F-lac), obtained from S. Luria, was used as During a recent survey of the functions medi- the donor of the F factor. ated by R factors, resistance to Spc was fre- Bacteriological methods. Bacteria were grown in quently found to be carried by the R factors of Trypticase Soy Broth (BBL) unless otherwise noted. enteric bacteria isolated in local from Resistance to each of several antibacterial agents hospitals was determined by inoculating 5 X 104 to 105 bac- 1967 to 1969 (21). Subsequently, some of the teria in a diameter of 4 mm on the surface of selective R factors first described in Japan (22) and an media with a replicative device (25) and assessing R factor carried by an E. coli isolated in the growth after 48 hr of incubation at 37 C. Strains United States in 1947 (20) were also found to were defined as resistant if they formed confluent mediate resistance to Spc. These results were patches of growth. To test bacterial growth, we used somewhat surprising because Spc was isolated Levine Lactose Eosin Methylene Blue (EMB; BBL) only in 1961 (16) and has not yet been intro- to which the following drugs were added to result duced commercially. This apparent paradox in the indicated concentrations per milliliter: Spc, prompted us to continue our studies of R factor- 50,g; Str, 20,ug; bluensomycin (Blu), 100,g; kana- mycin (Kan), 50 mg; chloramphenicol (Chi), 50,pg; mediated Spc resistance in the hope that they tetracycline (Tet), 20 Mg; ampicillin (Amp), 50,g. might provide insight into the problem of the We also used minimal medium A (7) supplemented origin of R factor genes and the mechanisms with 0.2% glucose, 0.1 ,ug of thiamine per ml, and responsible for their selection. Our findings 0.2% Casamino Acids (Difco) in 1.5% agar (Difco) indicate that a locus mediating the synthesis of containing sulfadiazine (Sul) at 900 an which ,ug/nd and Trypti- adenylate transferase inactivates Spc case soy agar containing mercuric chloride (Mer) and, in addition, streptomycin (Str) is found at a concentration of 10-4 M. frequently on fi+ R factors. Some of the pre- The methods of this have been used for bacterial conjugation (28) liminary findings study pre- and for transduction with phage Plkc (13) were sented elsewhere (D. H. Smith, Int Symp. described earlier. Infec. in Multiple Drug Resistance, press; 23). The fi type of the R factors (29) was determined Some of these results have been independently by testing F+ R+ bacteria obtained Benveniste et al. for susceptibility to MS-2 by (4). phage. R factors were transferred to E. coli AB1932-1 MATERIALS AND METHODS by conjugation; the recipient bacteria, after purifica- Bacterial strains. Enteric bacteria isolated in the tion, were mated with E. coli A 352 and selected for diagnostic bacteriology laboratories of this hospital infection with both episomes on EMB agar contain- 120 VOL. l, 1970 SPECTINOMYCIN RESISTANCE 121 ing mrultiple antibiotics. After purification, the method of Lowry et al. (15), with bovine serum al- doubly infected bacteria were tested for susceptibility bumin (Calbiochem, Los Angeles, Calif.) as standard. to MS-2 phage using the media and techniques de The assay for inactivation of the antimicrobial scribed earlier (8). activity of Spc was a modification of the method of To obtain R factors mutated in Spc or Str re- Harwood and Smith (11). Complete incubation sistance, E. coli B/RE130 was exposed to N-methyl- mixtures contained, per milliliter, 2 to 8 mg of cell N'-nitrosoguanidine (NTG) by the method of extract protein in 500 ,liters of extraction buffer, Adelberg et al. (1), incubated overnight in broth at 0.4 Amole of Spc (a gift of G. Whitfield, The Upjohn 37 C, and diluted and plated for individual colonies Co.), 3.5 p&moles of adenosine triphosphate (ATP; on EMB agar. After incubation overnight at 37 C, Sigma Chemical Co., St. Louis, Mo.), plus 8.3 ,umoles the colonies were replicated onto plates of EMB of MgCl2 and 40 ,moles of Tris-hydrochloride agar with or without Spc (200,g/ml) or Str (20,pg/ml), (pH 8.6); control mixtures containing no extract, or which were incubated further at 37 C; colonies that that of E. coli B, were incubated in parallel. Samples appeared to be altered in Spc or Str resistance were (200 ,liters) were removed after intervals of incu- purified and tested for resistance to Spc, Str, Chl, bation at 37 C, heated for 5 min at 70 C to destroy Tet, and Mer (to establish that the R factor was still enzymatic activity, and centrifuged for 15 min at present and altered only in Spc or Str resistance). 3,000 X g in 12-ml conical tubes. Samples (100 pliters) These mutant colonies were initially tested for of the supernatant fluids were assayed for antimicro- Spc and Str resistances by velveteen replication or bial activity as described above. inoculation of 105 bacteria (see above) onto EMB The assay for adenylation of aminoglycoside drugs agar with or without Spc (200 ,ug/ml) or Str (20 by cell extract (actually aminoglycoside-dependent Ag/ml). Growth on the EMB-antibiotic medium was conversion of radioactive label from ATP into a usually either not visible or confluent and thus easily form retained by phosphocellulose paper) was a interpreted; in many instances, however, only a few modification of the method of Yamada et al. (31). colonies appeared at the site of inoculation, making Complete incubation mixtures (100,liters) contained it difficult to interpret the resistance of the strain. 0.3 to 1.5 mg of cell extract protein in 70 pliters of Therefore resistance was determined subsequently by extraction buffer, 50 pmoles of aminoglycoside, 20 plating approximately 200 viable cells on a series of Aliters of a buffer (R buffer) containing 0.4 M NH4CI, EMB plates with increasing concentrations of Spc or 0.04 M MgCl2, 0.66 M Tris-hydrochloride (pH 8.0), and Str, counting colonies after an overnight incubation 0.1 M 13-mercaptoethanol, and 150 pmoles of labeled at appropriate temperatures, and calculating the ATP (uniformly 14C-labeled, New England Nuclear percentage of cells surviving at each of the drug con- Corp., Boston, Mass., 1.33 ,uc/,umole; a- or 7y32p- centrations. The results with this method were easily labeled, International Chemical and Nuclear Corp., interpreted and indicated that replication of colonies Irvine, Calif., 0.04 plc//umole). Control mixtures lack- or inoculation of large numbers of cells onto media ing aminoglycoside drug or containing extract of containing Spc (200 pg/ml) or Str (20 pg/ml) did not E. coli B were incubated in parallel. After various accurately assess the Spc and Str resistances of the periods of incubation at 37 C, 10-,Aliter samples were mutant strains with "partial" resistance (see below). pipetted onto 7-mm squares of Whatman P81 phos- Microbiological assay of Spc. Bioassay of Spc was phocellulose paper (Reeve Angell, Clifton, N. J.). The performed by a modification of the method of Hanka squares were dried in air for 15 to 30 sec, shaken vigor- et al. (10). Klebsiella UC-59 was diluted to approxi- ously at 37 C in 5 mm Tris-HCl (pH 7.1) for 2 min, mately 106 per ml in Trypticase soy agar, and 25 briefly rinsed twice in distilled water at room tempera- ml of this suspension was poured into each of several ture, dried under an infrared lamp, and counted in petri dishes. Upon solidification, wells 7 mm in 10 ml of Bray's solution (5). The complex formed diameter were made with a sterile cork borer, and between Spc and ATP appears to be more easily wash- 0.1-ml samples of test solutions were added.