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 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 , 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 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. After the able from phosphocellulose paper than Str adenylate, plates were incubated overnight at room tempera- for which the original assay method was designed (31); ture and for 4 to 8 hr at 37 C, the zones of inhibition the conditions employed for washing the paper were were measured. The diameters of the inhibition zones designed for best removal of the excess unreacted were a linear function of the logarithm of the Spc ATP with minimal loss of the labeled Spc complex. concentration. Experiments with cell extracts. Cell extracts RESULTS were prepared from overnight cultures in Trypticase Soy Broth. Cells from 40 mi of culture were washed Distribution and characteristics of R factors twice in 15 ml and resuspended in 5 ml of a buffer mediating Spc resistance. Of the 100 natural containing 10 mM tris(hydroxymethyl)aminomethane- isolates of enteric bacteria found at routine (Tris)-hydrochloride (pH 7.1), 33 mM NaCl, and testing to be resistant to any of the nine anti- 1 mM MgCl2, and were broken with a Branson bacterial agents tested, 51 were resistant to Spc. Sonifier (Branson Instruments Inc., Melville, N.Y.), (Agents to which resistance was tested included by using an ice-water bath for cooling and six 15-sec Spc, Str, Blu, Kan, Chl, Tet, Sul, Mer, Amp.) bursts at an energy setting of 60, each followed by a 15-sec cooling period. The sonic extracts were clarified Forty-six of the SpcR strains (90%) contained bv centrifugation for 10 min at 18,000 X g; the transferable R factors that mediated the Spc re- resulting clear supernatant fluids were stored at sistance. The R factors mediating Spc resistance at -20 C. Protein content was measured by the were found in isolates of E. coli, Klebsiella, 122 SMITH ET AL. INFEC. IMMUN.

TABLE 1. Transfer of R factor-mediated spectinzo- TABLE 4. Levels of speclinioinycini resistance mycin resistance by conljugatiolna mediated by R factorsa No. of recipient clones acquir- Spc resistance No. of R ing indicated properties of (,ug/ml) factors donor bacteria No. of Drug on which >50 <100 1 bacteria recipient recipient clones >100 <250 5 selected tested SpcRStrR TetR Arg+ Met+ Lac+ >250 < 500 8 ChIlR >500 <750 5 MlerE >750 <1,000 1 >1,000 <2,000 6 Spectinomyin. . 713 713 0 0 0 >2,000 7 Tetracycline ..... 546 546 0 0 0 a The Spc resistance of E. coli AB1932-1, in- a E. coli B/RE130 was mated with E. coli AB fected with each of 33 R factors, was determined 1932-1 (Lac7 Arg- Met- NalR); recipient bacteria by inoculating bacteria onto plates of EMB agar were selected on EMB agar containing Nal (100 with increasing concentrations of Spc as de- ,ug/ml) and either Spc (50,4g/ml) or Tet (30 ,ug/ml) scribed. and were tested for the indicated genetic proper- ties as described. Salmonella, Shigella, Aerobacter, and Proteus. That the Spc resistance was mediated by an TABLE 2. Transfer of R factor-mediated spectinio- R factor was inferred from the following findings. mycin resistance by transductioni with phage (i) The Spc resistance could be transferred by Plkca conjugation to sensitive E. coli under conditions Drug on which No. of No. of SpcR StrR in which several known R factor genes, but none transductants transductants TetR ChIR MerR of the three tested chromosomal genes, were also selected tested transductants transferred (Table 1). (ii) Such recipient bacteria, in turn, could transfer the Spc resistance to other Spectinomycin .. 497 497 sensitive E. coli by conjugation. (iii) The Spc Tetracycline ..... 212 212 resistance could be cotransduced with other R a Plkc, prepared on E. coli FW 13A/RE130, was known factor genes by phage Plkc (Table 2). used to transduce loci mediating drug resistance (iv) SpcR R+ bacteria lost Spc resistance together into E. coli AB 1932-1 (NalR); transductant clones with known R factor genes as a consequence of were selected on EMB agar containing Nal (100 incubation in either 10% sodium dodecyl sulfate ,Lg/ml) and either Spc (50 Ag/ml) or Tet (20,ug/ml) or 235 ,ug of ethidium bromide per ml (J. Pitt and and were tested for resistance to the antibacterial D. H. Smith, in press). agents indicated as described. Some of the genetic characteristics of R factors mediating Spc resistance are reported in Table 3. TABLE 3. Some genetic expressions of R factors All mediated resistance to Str and Blu, and the 10 mediating spectiniomycin resistancea ested R factors also mediated resistance to Patterns of resistance and No. of R dihydrospectinomycin; all also inhibited the fi type mediated factors biological expression of the F factor, i.e., they have the fi+ genotype (29). And, conversely, 46 fi+ Spc Str Blu Tet Chl Sul Mer Amp Kan ....3 of the 49 fi+ R factors studied during these experi- fi+ Spc Str Blu Tet Chl Sul Mer Kan...... 11 ments mediated Spc resistance. fi+ Spc Str Blu Tet Chl Sul Mer Amp...... 8 fi+ Spc Str Blu Tet Chl Sul Amp ...... 3 The levels of Spc resistance mediated by R fi+ Spc Str Blu Tet Chl Sul Mer...... 8 factors depend on the test medium, the concen- fi+ Spc Str Blu Tet Chi Sul...... 1 tration of bacteria used in the assay, the host fi+ Spc Str Blu Tet Sul Mer Amp...... 5 bacterium, and the R factor (21). Therefore, fi+ Spc Str Blu Tet Sul Mer Kan...... 1 to compare the levels of Spc resistance mediated fi+ Spc Str Blu Tet Sul Mer ...... 2 by individual R factors, certain of them were fi+ Spc Str Blu Tet Sul...... 1 1 transferred by conjugation to the same host fi+ Spc Str Blu Chl Sul Amp ...... strain, E. coli AB1932-1, and the resulting re- fi+ Spr Str Blu Sul Mer ...... 2 sistant substrains were tested under identical a The patterns of drug resistance and the fi type conditions. The 33 tested R factors conferred mediated by the R factors were determined as various levels of resistance: 18 mediated levels of described. The order of presentation of the > 100 <750 ,ug/ml, whereas 13 mediated > 1,000 individual genetic expressions does not indicate ,qg/ml (Table 4). genetic linkage groups. Mechanism for the R factor-mediated Spc VOL. 1, 1970 SPECIINOMYCIN RESISTANCE 123

KINETICS OF PHENOTYPIC EXPRESSION E. co/i F co/i B OF R FACTOR MEDIATED SpcR 12- B/RE /30 104- Chi 1000 _ 0 8 K 6-

4 z 800 - 2 A -0-O-Q-- -- -!" en Q@ 600 ,/Spc 0 20 40 60 80 0 20 40 60 80 0 7 600 MINUTES OF INCUBA TION FIG. 2. Adenylation of Spc, dihydrospectinomycin, and actinamine by extracts of E. coli B/RE130 and o 400 0 E. coli B. The assayfor adenylation of aminoglycosides was performed with 14C-ATP as described. The "con- trol" incubation contained no drug. Substrates used: 0, none; 0, Spc; A, dihydrospectinomycin; O, actinamine. 2 200 /

I occur through enzymatic acetylation (18). These C findings led us to test directly for Spc inactiva- 10 20 30 40 tion during a recent study (11) on inactivation of Str by an R-SpcR StrR strain. Extracts of this MINUTES strain were also found to inactivate Spc if (as FIG. 1. Kinetics of expression of R factor-mediated with Str) ATP and Mg++ were added. Subse- Spc and Chl resistance. E. coli AB1932 was incubated quent studies have confirmed that Spc is inac- in Lennox broth, centrifuged, washed twice, and re- tivated at a rate of approximately 62 ,ug per suspended in 0.9% NaCI containing 10-3 M CaCl2, mg of protein per hr by ATP-Mg++-supple- and incubated for 15 min at 37 C, after which equal mented extracts of R_SpcR strains but not by concentrations (2 X 108/ml) of bacteria and phage Plkc, prepared on E. coli AB1932-4/RE130, were those of R-SpcS, SpcR (chromosomal), or SpcS mixed. This suspension was incubated for 15 min at strains. 37 C and then chilled and centrifuged in the cold, after Because the cofactor requirement observed which the pellet was resuspended in prewarmed Lennox is the same as for the inactivation of Str (and broth and incubated at 37 C. Samples of 0.1 ml were Blu) by adenylation (11), the possibility of Spc removed at intervals (zero time being the time of resus- adenylation was explored more directly. Portions pension in Lennox broth) and spread on EMB agar of incubation mixtures containing uniformly containing either Spc (50 ,ug/ml), or Chl (50 ,Ag/ml). labeled 14C-ATP, Mg+, Spc, and extract of Symbols: 0, SpcR transductants; 0, ChlR trans- R_SpcR bacteria were removed at intervals to ductants. phosphocellulose paper (to which Spc binds by virtue of its two cationic groups). The amount resistance. When the resistance of R-SpcR of radioactive label retained by the paper in- strains was tested by the tube dilution assay, creased linearly with the time of incubation (Fig. the minimal inhibitory concentration of Spc 2) and with the concentration of extract; control was found to increase substantially with the experiments showed that Mg++, Spc, and extract number of bacteria used as inoculum (21). were all necessary for transfer of label from ATP Such a relationship has been observed when to the paper. Transfer of label was also observed drug resistance is mediated through enzymatic when a-32P-ATP, but not y-32P-ATP, was sub- inactivation, e.g., penicillin resistance due to stituted for '4C-ATP or when Str or Blu, but not j3-lactamase (3). Likewise consistent with inac- Kan, was substituted for Spc (Table 5). Thus, the tivation was the very rapid expression of Spc extract forms a complex that retains the cationic resistance after introduction of the locus into a properties of Spc and that includes some 14C_ sensitive cell. Figure 1 shows that, after trans- labeled moiety of and the a-phosphate of ATP. duction of the two R factor loci by phage Plkc, This reaction, like the inactivation of the anti- Spc resistance was expresssed even slightly more microbial activity of the drug, is specific to ex- rapidly than Chl resistance, which is known to tracts of R-SpcR strains and is not observed in 124 SMITH ET AL. INFEC. IMMUN.

TABLE 5. Aminoglycoside-dependent transfer of adenylating Spc and Str were examined as radioactivity from ATP, variously labeled, follows. Extract of E. coli B/RE130 was applied to phosphocellulose by an extract of to a column of Sephadex 200 and eluted with R E. coli B/REI30a buffer; the elution profile of Spc-adenylating Aminoglycoside 14C-ATP a-32P-ATP S-32P-ATP activity was found to be identical with that of the Str-adenylating activity. Extract of E. coli was heated at 48 C, and samples were Streptomycin ...... 2,043 1,236 58 B/RE130 Spectinomycin .....981 507 54 removed at intervals and assayed for Spc and Kanamycin ...... 165 61 60 Str adenylating activities. Fig. 4 indicates that None ...... 180 59 59 both activities were inactivated coordinately: 50% of both activities were lost after 3 min of exposure a Adenylation of aminoglycoside drugs was of extracts to this temperature. These results assayed as described. Reaction mixtures were are consistent with the proposal that one en- incubated for 60 min; results are expressed as zyme adenylates Spc and Str, and they prompted counts per minute. the following studies of substrains of E. coli B/RE130 mutated in Spc or Str resistance. SPEC TINOMYC IN Of 2,399 colonies of E. coli B/RE130 examined for mutations to Spcs or Strs after exposure to NTG, 11 had lost their R factor (R-), 4 were HOH STREPTOMYCIN H3C-N 0 0 CH3 (Bl uensomyci n) Spc5 StrS ChlR TetR MerR SulR, and 8 were interpreted by the replication method to be SpcR H H StrS ChlR TetR MerR SulR (see below); thus HO 0H H3C-N OH NH 0 0.5 % of the clones carried R factors mutated in in CH3 0 0 C-OH Spc or Str resistance, or both. / R3 When one of the Spcs Str5 clones was examined further, it was found to have the same in vivo sensitivity to Spc and Str (and Blu, not shown) as ACTINAMINE 0 CH3 0 did the isogenic R- strain (Fig. 5) and to transfer OH Spcs StrS ChlR TetR MerR SulR by conjugation H H3C- N OH to sensitive E. coli. Extracts of E. coli B carrying HO OH this R factor, RE130-17, did not detectably HO OH RI adenylate either Spc or Str in vitro (Fig. 5). NH When E. coli B/RE130-17 was spread on EMB CH3 containing Spc (200 Ag/ml), colonies were ob- tained at an approximate frequency of 1 in FIG. 3. Molecular structures of spectinomycin, actinamine, streptomycin, and bluensomycin. The structures have been depicted to indicate the structural I001 similarity of the drugs at the proposed site of attack of the adenylate synthetase; no stereospecificity is 80 intended. For Str, R1 and R2 = -N(H)-C(NH2) = 60 _ NH, and R3 = -CHO;,for Blu, the Rt and R2 posi- tions are occupied by -NH2and -N(H) -C (NH2) = A NH, but which group is at R1 and which at R2 is not 4 known; R3 = CH20H. extracts of R-Spc5, SpcS, or SpcR (chromosomal) a 20 strains; the reaction has been confirmed in eight additional R-SpcR StrR BluR strains tested. When equimolar concentrations of dihydrospectino- mycin or actinamine (30), the 1,3-dibasic 2 4 6 8 glycoside component of Spc (Fig. 3), were pres- ent in place of Spc, transfer of label proceeded MINUTES AT 48° C at similar rates (Fig. 2). FIG. 4. Inactivation at 48 C of thle Spc- and Studies on identity of Spc- and Str-adenylating Str-adenylating activities of E. coli B/RE130 extract. activities. Several lines of evidence suggest that Extract, at a concentration of I mg/ml, was heated one adenylate transferase mediates Spc and Str at 48 C; samples were removed at intervals, incu- resistance (see below). To examine this proposal, bated withcompletereaction mixtures,for30 min, and the size and heat stability of the enzyme(s) assayed as described. Substrates used: 0, Spc; A, str. VOL. l, 1970 SPECTINOMYCIN RESISTANCE 125 A A * Streptomycin 100 0 Spectinomycin 100 Spectinomycin Streptomycin

751 S 75 0

at 50~ 50 0

25 25-

i h 2200 4004 600 Ilo 20 200 400 600 0 20 DRUG CONCENTRATION f/g/r) DRUG CONCENTRA77ON,ug/m/) B B 15Spectinomycin 25Streptomycin 12- 20-

9- 151-

6- 10-

3h 5

20 40 20 40 40 INCUBATIONI(min) INCUBATION (min) FIG. 5. Correlation of in vivo resistance to and in FIG. 6. Correlation of in vivo resistance to and in vitro adenylation of Spc and Str by certain sub- vitro adenylation of Spc and Str by certain substrains strains of E. coli B. The resistance of bacteria (A) ofE. coli B. The resistance ofbacteria (A) andadenylat- and adenylating activity of extracts (B) were tested ing activity of extracts (B) were tested as described. as described. Adenylating activities were normalized Adenylating activities were normalized for concentra- for concentrations of extracts. Symbols: 0, E. coli tions of extracts. Symbols: 0, E. coli B (SpcS Str8); B (SpcS StrS); 0, E. coli B/RE130 (SpcR StrR); *, E. coli B/RE130 (SpcR StrR); U, E. coli B/RE130- 1, E. coli B/RE130-17 (SpcS StrS); E, E. coli 4 [Spc (partial R) Str (partial R)]. B/RE130-17-11 (SpcR StrR). every 5.5 X 107 cells plated; of the 10 of these of the mutated R factor rather than of the host revertants to Spc resistance tested, all were also cell, since the patterns could be transferred by resistant to Str (20 ,ug/ml). The in vivo resistance conjugation to sensitive E. coli. Moreover, ex- to and the in vitro adenylation of Spc and Str tracts of E. coli B/RE130-4 had less of both Spc by extracts of one of these revertant substrains, and Str adenylating activities per unit of extrac. E. coli B/RE130-17-11, were equal to or slightly protein than did extracts of E. coli B/RE1301 greater than that of E. coli B infected with the Thus, upon careful analysis, all mutationat parent R factor, RE130 (Fig. 5). changes affecting one resistance have been found The eight colonies interpreted to be SpcR to affect the other coordinately. Strs ChlR Tet* MerR SulR (see above) were of great interest because their existence implied DISCUSSION that Spc and Str resistance could be dissociated The present survey supports and extends our by mutation; hence, their Spc and Str resistance preliminary observation that SpcR R factors are levels were thoroughly examined by quantitating frequent among drug-resistant enteric bacteria colony formation on a series of plates with vari- (21): of 100 resistant strains examined, 46 car- ous drug concentrations. Figure 6 depicts the ried a transferable R factor mediating Spc results obtained with one of the mutants, E. coli resistance. Many of the bacteria used as the B/RE130-4; the results with the others were es- source of the R factors studied were selected, sentially identical. Each of these mutant sub- however, from resistant strains isolated in diag- strains (i) was not Str6 but had a level of Str nostic laboratories (for other studies) because resistance that was greater than that of E. coli they were resistant to either Str or Chl; most of B but less than that of E. coli B/RE130 ("partial them were E. coli or Klebsiella. R factors that resistance") and (ii) was not SpcR but was "par- infect these two genera generally have the fi+ tially" resistant to Spc. These patterns of "partial" genotype (17; D. H. Smith, unpublished data), as Spc and Str resistance were shown to be properties do all those reported to date that mediate Chl 126 SMITH ET AL. INFEC. IMMUN. resistance (T. Watanabe and N. Datta, personal StrR, and extracts of this strain regained Spc and communication). Furthermore, one R factor locus Str adenylation activity. Thus, a single mutation mediating Str resistance presumably mediates of an R factor can affect in vivo resistance to, and Spc resistance, and, from our observations, it in vitro adenylation of, Spc and Str. Moreover, appears to be carried generally, if not exclusively, the Str adenylate transferase reportedly attacks by fi+ R factors. Thus, our survey was made on the N-methyl glucosamine moiety of Str (Fig. 3) a sample population fortuitously preselected to at the 3'-OH group (26, 31), which may be con- include Spc resistance. It would be unlikely, sidered a part of a D-threo methylamino alcohol therefore, that the population of resistant enteric moiety. The actinamine moiety of Spc (Fig. 3) bacteria in general would include as high a includes an OH group with the same conforma- percentage of SpcR R factors as found in our tional relation to a methylamino group, and it survey; it is likely, however, that most fi+ R has been suggested that the D-threo methylamino factors will be found to carry the locus mediating alcohol moiety is the basis for the recognition of Spc (and Str-Blu) resistance. the adenylate transferase for its several substrates We have not undertaken chemical studies (G. Whitfield, personal communication). Our find- necessary to prove that R factors inactivate ings that extracts of R-SpcR bacteria adenylate Spc by adenylation. However, the observations Spc and actinamine equally are consistent with that extracts of R-SpcR bacteria mediate a Spc- this proposal. Thus, the results of several bio- dependent finding of some '4C-labeled moiety of chemical studies support and extend the bio- and the a-phosphate of ATP to phosphocellulose logical and genetic observations that suggest that and that this aminoglycoside-dependent adenylate a single adenylate transferase inactivates both transfer activity by extracts is positively correlated Spc and Str. with resistance of R-SpcR bacteria are consistent According to the recommendations of Demerec with this mechanism. A strong argument for Spc et al. (9), loci mediating drug resistance should adenylation can also be made from the close be designated by three-letter, lower case symbols correspondence ofSpc and Str resistance in R-fac- suggesting the name of the drug. Designation of tor strains in which inactivation of Str by adenyla- the R factor locus mediating Spc adenylation, tion is well documented (11, 26, 31). however, reveals a difficulty in the use of this Several lines of evidence indicate that R factors guideline, since this locus mediates resistance to at mediate the synthesis of a single adenylate trans- least three aminoglycoside antibiotics, Spc, ferase that inactivates Spc and Str. All R-SpcR Str, Blu (and perhaps to others untested, although bacteria isolated from natural sources that have not to Kan, , , or genta- been studied to date are also StrR. [The converse mycin). Str and Blu are quite similar in structure is not true, however, because certain R factors and action but differ appreciably from Spc in inactivate Str, but not Spc, by phosphorylation both these respects. For this and other loci (D. H. Smith, J. A. Janjigian, and N. Prescott, mediating resistance by an enzymatic inactiva- unpublished data; B. Ozanne et al., in press).] tion of established mechanism to a number of The positive correlation between Spc and Str important drugs, it might be useful if the three- resistance is not limited to bacteria isolated from letter symbol suggested the biochemical activity clinical specimens, since mutant substrains involved. Should more than one drug inactivation (selected after exposure to NTG) that had locus mediate resistance by the same activity, "partial" R-Spc resistance had a coordinate the loci would be distinguished (per Demerec decrease in R-Str resistance. Adenylation of Spc et al.) by an italicized capital letter, possibly, and Str by extracts of these strains was positively though not necessarily, suggesting the drug inac- correlated with their levels of cellular resistance. tivated. We suggest, therefore, that the locus Furthermore, of the Str8 R factors recovered from mediating Spc, Str, and Blu adenylation be 191 clones of E. coli RC (an Arg- conditionally designated adn; that mediating Kan and neo- Str-dependent strain) /RE130 obtained by selec- mycin phosphorylation (12), phs K; that medi- tion on minimal medium containing 20 Atg of ating Str and gentamycin phosphorylation Str per ml [a cultural condition that permits (D. H. Smith, J. A. Janjigian, and N. Prescott, growth only of substrains that are Arg+ or unpublished data; Ozanne et al., in press), ph/ S; R-Strs (J. H. Harwood and D. H. Smith, Genet. and those loci mediating the synthesis of the Res., in press)], all were SpcB (J. H. Harwood, several j3-lactamases that produce resistance J. Janjigian, and D. H. Smith, Genet. Res., in not only to various penicillins but also to cepha- press). Finally, SpCR revertants of an NTG-in- losporins (M. H. Richmond, personal communi- duced R-Spc8 Strs mutant substrain were also cation), ltm A, B, C, etc. VOL. 1, 1970 SPECTINOMYCIN RESISTANCE 127

ACKNOWLEDGMENTS 13. Lennox, E. S. 1955. Transduction of linked genetic characters. We thank G. Whitfield, The Upjohn Co., for spectinomycin, of the host by bacteriophage P1. Virology 1:190-206. 14. Lewis, C., and H. W. Clapp. 1961. Actinospectacin, a new actinamine, and Klebsiella UC-59 and for his helpful comments, and J. Davies and co-workers for discussions of their experi- antibiotic. III. In vitro and in vivo evaluation. Antibiot. Chemotherapy 11:127-133. ments before publication of their manuscript. 15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. This investigation was supported by Public Health Service J. Randall. 1951. Protein measurement with Folin phenol grants AI-0836 from the National Institute of Allergy and Infec- reagent. J. Biol. Chem. 193:265-275. tious Diseases and GM-14119 from the National Institute of 16. Mason, D. J., A. Dietz, and R. M. Smith. 1961. Actinospecta- General Medical Sciences. D. H. S. is the recipient of Career cin, a new antibiotic. I. Discovery and biological properties. Development Award AI-20376 from the National Institute of Antibiot. Chemotherapy 11:118-122. and Infectious Diseases. Allergy 17. Meynell, E., G. G. Meynell, and N. Datta. 1968. Phylogenetic relationships of drug-resistance factors and other trans- LITERATURE CITED missible bacterial plasmids. Bacteriol. Rev. 32:55-83. 18. Shaw, W. V. 1967. The enzymatic acetylation of chloram- 1. Adelberg, E. A., M. and G. C. C. Chen. 1965. Mandel, Opti- phenicol by extract of R factor-resistant Escherichia coli. J. mal conditions for mutagenesis by N-methyl-N'-nitro-N- Biol. Chem. 242:687-693. nitrosoguanidine in Escherichia coli. Biochem. Biophys. 19. Smith, D. H. 1966. Transferable drug resistance in Salmonella. Res. Commun. 18:788-795. New Engl. J. Med. 275:625-630. 2. Anderson, P., Jr. 1969. Sensitivity and resistance to spectino- 20. Smith, D. H. 1967. R factor infection of Escherichia coli mycin in Escherichia coli. J. Bacteiiol. 100:939-947. lyophilized in 1946. J. Bacteriol. 94:2071-2072. 3. Barber, M. 1947. Coagulase-positive staphylococci resistant to 21. Smith, D. H. 1967. R factor-mediated resistance to new amino- penicillin. J. Pathol. Bacteriol. 59:373-384. glycoside antibiotics. Lancet 1:252-254. 4. Benveniste, R., T. Yamada, and J. Davies. 1970. Enzymatic 22. Smith, D. H. 1967. R factors mediate resistance to mercury, adenylylation of streptomycin and spectinomycin in Esche- nickel and cobalt. Science 156:1114-1116. richia coli carrying resistance factors. Infec. Immun. 1:109- 23. Smith, D. H. 1969. R factors for aminoglycoside antibiotics. J. 119. Infect. Dis. 119:378-380. 5. Bray, G. A. 1960. A simple efficient liquid scintillator for 24. Sparling, P. F., J. Modalell, Y. Takeda, and B. D. Davis. 1968. counting aqueous solutions in a liquid scintillation counter. Ribosomes from Escherichia coli merodiploids heterozygous Anal. Biochem. 1:279. for resistance to streptomycin and to spectinomycin. J. Mol. 6. Davies, J. E., P. W. Anderson, and B. D. Davis. 1965. Inhibi- Biol. 37(Suppl. 3):407-421. tion of protein synthesis by spectinomycin. Science 149: 25. Steers, E., E. L. Foltz, and B. S. Graves. 1959. Inocula repli- 1096-1098. cating apparatus for routine testing of bacterial susceptibil- 7. Davis, B. D., and E. S. Mingioli. 1950. Mutants of Fscherichia ity to antibiotics. Antibiot. Chemotherapy 9:307-311. coli requiring methionine or vitamin B12. J. Bacteriol. 26. Umezawa, H., S. Takasawa, M. Okinishi, and R. Utahara. 60:17-28. 1968. Adenylylstreptomycin, a product of streptomycin in- 8. Davis, J. E., and R. L. Sinsheimer. 1963. The replication of activated by E. coli carrying R factor. J. Antibiot. (Tokyo) bacteriophage MS2. I. Transfer of parental nucleic acid to Ser. A 21:81-82. progeny phage. J. Mol. Biol. 6:203-207. 27. Walton, J. R., and D. H. Smith. 1969. New hemolysin (-y) 9. Demerec, M., E. A. Adelberg, A. J. Clatk, and P. E. Hartman. produced by Fscherichia col. J. Bacteriol. 98:304-305. 1966. A proposal for a uniform nomenclature in bacterial 28. Watanabe, T. 1964. Selected methods of genetic study of genetics. Genetics 54:61-76. episome-mediated drug resistance in bacteria. Methods 10. Hanka, L. J., D. J. Mason, and W. T. Sokolski. 1961. Actino- Med. Res. 10:202-220. spectacin, a new antibiotic. II. Microbiological assay. 29. Watanabe, T., H. Nishida, C. Ogata, T. Arai, and S. Sato. Antibiot. Chemotherapy 11:123-126. 1964. Episome-mediated transfer of drug resistance in 11. Harwood, J. H., and D. H. Smith. 1969. Resistance factor- Enterobacteriaceae. VII. Two types of naturally occurring R mediated streptomycin resistance. J. Bacteriol. 97:1262- factors. J. Bacteriol. 88:716-726. 1271. 30. Wiley, P. F. 1962. The chemistry of actinospectacin. I. Actina- 12. Kondo, S., M. Okanishi, R. Utahara, K. Maeda, and H. mine. J. Amer. Chem. Soc. 84:1514. Umezawa. 1968. Isolation of kanamycin and paromamine 31. Yamada, T., D. Tipper, and J. Davies. 1968. Enzymatic in- inactivated by E. coli carrying R factor. J. Antibiot. activation of streptomycin by R factor-resistant Echerichia (Tokyo) Ser. A 21:22-29. coli. Nature (London) 219:288-291.