INFECTION AND IMMUNITY, Jan. 1970, p. 120-127Copyright ( 1970 American Society for Microbiology

Vol. 1, No. 1Priinted inz U.S.A.

Resistance Factor-Mediated Spectinomycin ResistanceDAVID H. SMITH, J. A. JANJIGIAN, NAOMI PRESCOTT, AND PORTER W. ANDERSON

Department of Pediatrics, 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, actinaminestreptomycin, and bluensomycin in vitro in the presence of adenosine triphosphateand Mg++. Results of genetic and biochemical studies support the hypothesis thatthese reactions are mediated by a single enzyme.

Spectinomycin (Spc), a dibasic aminoglycosideantibiotic produced by Streptomyces spectabilis, isbacteriostatic for a wide variety of clinicallyimportant bacteria (16). Spc selectively inhibitsprotein synthesis in Escherichia coli, and strainsresistant to Spc owing to a chromosomal muta-tion have 30S ribosomal subunits that are re-sistant to the drug (6). Chromosomal Spc re-sistance arises by high-level, one-step mutationand is recessive in diploid strains with SpcS andSpcR loci (2, 24).During a recent survey of the functions medi-

ated by R factors, resistance to Spc was fre-quently found to be carried by the R factors ofenteric bacteria isolated in local hospitals from1967 to 1969 (21). Subsequently, some of theR factors first described in Japan (22) and anR factor carried by an E. coli isolated in theUnited States in 1947 (20) were also found tomediate resistance to Spc. These results weresomewhat surprising because Spc was isolatedonly in 1961 (16) and has not yet been intro-duced commercially. This apparent paradoxprompted us to continue our studies of R factor-mediated Spc resistance in the hope that theymight provide insight into the problem of theorigin of R factor genes and the mechanismsresponsible for their selection. Our findingsindicate that a locus mediating the synthesis ofan adenylate transferase which inactivates Spcand, in addition, streptomycin (Str) is foundfrequently on fi+ R factors. Some of the pre-liminary findings of this study have been pre-sented elsewhere (D. H. Smith, Int Symp.Infec. Multiple Drug Resistance, in press; 23).Some of these results have been independentlyobtained by Benveniste et al. (4).

MATERIALS AND METHODSBacterial strains. Enteric bacteria isolated in the

diagnostic bacteriology laboratories of this hospital

and the Massachusetts General Hospital were thesource of most of the R factors studied. RE130(fi+ SpcR StrR BluR ChlR TetR MerR SulR; 11),the R factor studied in many of the experiments, wascarried originally by a strain of E. coli. Bacteria usedas recipients for the R factors included E. coli Band E. coli K-12 strains AB1932-1 (Arg- Met- Lac-NalR; 27) and FW 13A (Thr- Leu- Met- StrR, 19).Klebsiella UC-59, obtained from George Whitfieldof The Upjohn Co., Kalamazoo, Mich., was usedin the bioassay of Spc. E. coli K-12 strain A 352(lacdel/F-lac), obtained from S. Luria, was used asthe donor of the F factor.

Bacteriological methods. Bacteria were grown inTrypticase Soy Broth (BBL) unless otherwise noted.

Resistance to each of several antibacterial agentswas determined by inoculating 5 X 104 to 105 bac-teria in a diameter of 4 mm on the surface of selectivemedia with a replicative device (25) and assessinggrowth after 48 hr of incubation at 37 C. Strainswere defined as resistant if they formed confluentpatches of growth. To test bacterial growth, we usedLevine Lactose Eosin Methylene Blue (EMB; BBL)to which the following drugs were added to resultin the indicated concentrations per milliliter: Spc,50,g; Str, 20,ug; bluensomycin (Blu), 100,g; kana-mycin (Kan), 50 mg; chloramphenicol (Chi), 50,pg;tetracycline (Tet), 20 Mg; ampicillin (Amp), 50,g.We also used minimal medium A (7) supplementedwith 0.2% glucose, 0.1 ,ug of thiamine per ml, and0.2% Casamino Acids (Difco) in 1.5% agar (Difco)containing sulfadiazine (Sul) at 900 ,ug/nd and Trypti-case soy agar containing mercuric chloride (Mer)at a concentration of 10-4 M.The methods used for bacterial conjugation (28)

and for transduction with phage Plkc (13) weredescribed earlier.The fi type of the R factors (29) was determined

by testing F+ R+ bacteria for susceptibility to MS-2phage. R factors were transferred to E. coli AB1932-1by conjugation; the recipient bacteria, after purifica-tion, were mated with E. coli A 352 and selected forinfection with both episomes on EMB agar contain-

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ing mrultiple antibiotics. After purification, thedoubly infected bacteria were tested for susceptibilityto MS-2 phage using the media and techniques described earlier (8).To obtain R factors mutated in Spc or Str re-

sistance, E. coli B/RE130 was exposed to N-methyl-N'-nitrosoguanidine (NTG) by the method ofAdelberg et al. (1), incubated overnight in broth at37 C, and diluted and plated for individual colonieson EMB agar. After incubation overnight at 37 C,the colonies were replicated onto plates of EMBagar with or without Spc (200,g/ml) or Str (20,pg/ml),which were incubated further at 37 C; colonies thatappeared to be altered in Spc or Str resistance werepurified and tested for resistance to Spc, Str, Chl,Tet, and Mer (to establish that the R factor was stillpresent and altered only in Spc or Str resistance).

These mutant colonies were initially tested forSpc and Str resistances by velveteen replication orinoculation of 105 bacteria (see above) onto EMBagar with or without Spc (200 ,ug/ml) or Str (20Ag/ml). Growth on the EMB-antibiotic medium wasusually either not visible or confluent and thus easilyinterpreted; in many instances, however, only a fewcolonies appeared at the site of inoculation, makingit difficult to interpret the resistance of the strain.Therefore resistance was determined subsequently byplating approximately 200 viable cells on a series ofEMB plates with increasing concentrations of Spc orStr, counting colonies after an overnight incubationat appropriate temperatures, and calculating thepercentage of cells surviving at each of the drug con-centrations. The results with this method were easilyinterpreted and indicated that replication of coloniesor inoculation of large numbers of cells onto mediacontaining Spc (200 pg/ml) or Str (20 pg/ml) did notaccurately assess the Spc and Str resistances of themutant strains with "partial" resistance (see below).

Microbiological assay of Spc. Bioassay of Spc wasperformed by a modification of the method of Hankaet al. (10). Klebsiella UC-59 was diluted to approxi-mately 106 per ml in Trypticase soy agar, and 25ml of this suspension was poured into each of severalpetri dishes. Upon solidification, wells 7 mm indiameter were made with a sterile cork borer, and0.1-ml samples of test solutions were added. After theplates were incubated overnight at room tempera-ture and for 4 to 8 hr at 37 C, the zones of inhibitionwere measured. The diameters of the inhibition zoneswere a linear function of the logarithm of the Spcconcentration.

Experiments with cell extracts. Cell extractswere prepared from overnight cultures in TrypticaseSoy Broth. Cells from 40 mi of culture were washedtwice in 15 ml and resuspended in 5 ml of a buffercontaining 10 mM tris(hydroxymethyl)aminomethane-(Tris)-hydrochloride (pH 7.1), 33 mM NaCl, and1 mM MgCl2, and were broken with a BransonSonifier (Branson Instruments Inc., Melville, N.Y.),by using an ice-water bath for cooling and six 15-secbursts at an energy setting of 60, each followed by a15-sec cooling period. The sonic extracts were clarifiedbv centrifugation for 10 min at 18,000 X g; theresulting clear supernatant fluids were stored atat -20 C. Protein content was measured by the

method of Lowry et al. (15), with bovine serum al-bumin (Calbiochem, Los Angeles, Calif.) as standard.The assay for inactivation of the antimicrobial

activity of Spc was a modification of the method ofHarwood and Smith (11). Complete incubationmixtures contained, per milliliter, 2 to 8 mg of cellextract protein in 500 ,liters of extraction buffer,0.4 Amole of Spc (a gift of G. Whitfield, The UpjohnCo.), 3.5 p&moles of adenosine triphosphate (ATP;Sigma Chemical Co., St. Louis, Mo.), plus 8.3 ,umolesof MgCl2 and 40 ,moles of Tris-hydrochloride(pH 8.6); control mixtures containing no extract, orthat of E. coli B, were incubated in parallel. Samples(200 ,liters) were removed after intervals of incu-bation at 37 C, heated for 5 min at 70 C to destroyenzymatic activity, and centrifuged for 15 min at3,000 X g in 12-ml conical tubes. Samples (100 pliters)of the supernatant fluids were assayed for antimicro-bial activity as described above.The assay for adenylation of aminoglycoside drugs

by cell extract (actually aminoglycoside-dependentconversion of radioactive label from ATP into aform retained by phosphocellulose paper) was amodification of the method of Yamada et al. (31).Complete incubation mixtures (100,liters) contained0.3 to 1.5 mg of cell extract protein in 70 pliters ofextraction buffer, 50 pmoles of aminoglycoside, 20Aliters of a buffer (R buffer) containing 0.4 M NH4CI,0.04 M MgCl2, 0.66 M Tris-hydrochloride (pH 8.0), and0.1 M 13-mercaptoethanol, and 150 pmoles of labeledATP (uniformly 14C-labeled, New England NuclearCorp., Boston, Mass., 1.33 ,uc/,umole; a- or 7y32p-labeled, International Chemical and Nuclear Corp.,Irvine, Calif., 0.04 plc//umole). Control mixtures lack-ing aminoglycoside drug or containing extract ofE. coli B were incubated in parallel. After variousperiods of incubation at 37 C, 10-,Aliter samples werepipetted onto 7-mm squares of Whatman P81 phos-phocellulose paper (Reeve Angell, Clifton, N. J.). Thesquares were dried in air for 15 to 30 sec, shaken vigor-ously at 37 C in 5 mm Tris-HCl (pH 7.1) for 2 min,briefly rinsed twice in distilled water at room tempera-ture, dried under an infrared lamp, and counted in10 ml of Bray's solution (5). The complex formedbetween Spc and ATP appears to be more easily wash-able from phosphocellulose paper than Str adenylate,for which the original assay method was designed (31);the conditions employed for washing the paper weredesigned for best removal of the excess unreactedATP with minimal loss of the labeled Spc complex.

RESULTS

Distribution and characteristics of R factorsmediating Spc resistance. Of the 100 naturalisolates of enteric bacteria found at routinetesting to be resistant to any of the nine anti-bacterial agents tested, 51 were resistant to Spc.(Agents to which resistance was tested includedSpc, Str, Blu, Kan, Chl, Tet, Sul, Mer, Amp.)Forty-six of the SpcR strains (90%) containedtransferable R factors that mediated the Spc re-sistance. The R factors mediating Spc resistancewere found in isolates of E. coli, Klebsiella,

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TABLE 1. Transfer of R factor-mediated spectinzo-mycin resistance by conljugatiolna

No. of recipient clones acquir-ing indicated properties of

donor bacteriaDrug on which No. of

recipient bacteria recipientselected clones SpcRtested StrR

TetR Arg+ Met+ Lac+ChIlRMlerE

Spectinomyin. . 713 713 0 0 0Tetracycline ..... 546 546 0 0 0

a E. coli B/RE130 was mated with E. coli AB1932-1 (Lac7 Arg- Met- NalR); recipient bacteriawere selected on EMB agar containing Nal (100,ug/ml) and either Spc (50,4g/ml) or Tet (30 ,ug/ml)and were tested for the indicated genetic proper-ties as described.

TABLE 2. Transfer of R factor-mediated spectinio-mycin resistance by transductioni with phage

Plkca

Drug on which No. of No. of SpcR StrRtransductants transductants TetR ChIR MerR

selected tested transductants

Spectinomycin .. 497 497Tetracycline ..... 212 212

a Plkc, prepared on E. coli FW 13A/RE130, wasused to transduce loci mediating drug resistanceinto E. coli AB 1932-1 (NalR); transductant cloneswere selected on EMB agar containing Nal (100,Lg/ml) and either Spc (50 Ag/ml) or Tet (20,ug/ml)and were tested for resistance to the antibacterialagents indicated as described.

TABLE 3. Some genetic expressions of R factorsmediating spectiniomycin resistancea

Patterns of resistance and No. of Rfi type mediated factors

fi+ Spc Str Blu Tet Chl Sul Mer Amp Kan ....3fi+ Spc Str Blu Tet Chl Sul Mer Kan....... 11fi+ Spc Str Blu Tet Chl Sul Mer Amp........ 8fi+ Spc Str Blu Tet Chl Sul Amp ............ 3fi+ Spc Str Blu Tet Chl Sul Mer............. 8fi+ Spc Str Blu Tet Chi Sul.................. 1fi+ Spc Str Blu Tet Sul Mer Amp............ 5fi+ Spc Str Blu Tet Sul Mer Kan............. 1fi+ Spc Str Blu Tet Sul Mer .................2fi+ Spc Str Blu Tet Sul...................... 1fi+ Spc Str Blu Chl Sul Amp ................ 1fi+ Spr Str Blu Sul Mer .....................2

TABLE 4. Levels of speclinioinycini resistancemediated by R factorsa

Spc resistance No. of R(,ug/ml) factors

>50 <100 1>100 <250 5>250 < 500 8>500 <750 5>750 <1,000 1

>1,000 <2,000 6>2,000 7

a The Spc resistance of E. coli AB1932-1, in-fected with each of 33 R factors, was determinedby inoculating bacteria onto plates of EMB agarwith increasing concentrations of Spc as de-scribed.

Salmonella, Shigella, Aerobacter, and Proteus.That the Spc resistance was mediated by anR factor was inferred from the following findings.(i) The Spc resistance could be transferred byconjugation to sensitive E. coli under conditionsin which several known R factor genes, but noneof the three tested chromosomal genes, were alsotransferred (Table 1). (ii) Such recipient bacteria,in turn, could transfer the Spc resistance to othersensitive E. coli by conjugation. (iii) The Spcresistance could be cotransduced with otherknown R factor genes by phage Plkc (Table 2).(iv) SpcR R+ bacteria lost Spc resistance togetherwith known R factor genes as a consequence ofincubation in either 10% sodium dodecyl sulfateor 235 ,ug of ethidium bromide per ml (J. Pitt andD. H. Smith, in press).Some of the genetic characteristics of R factors

mediating Spc resistance are reported in Table 3.All mediated resistance to Str and Blu, and the 10ested R factors also mediated resistance todihydrospectinomycin; all also inhibited thebiological expression of the F factor, i.e., theyhave the fi+ genotype (29). And, conversely, 46of the 49 fi+ R factors studied during these experi-ments mediated Spc resistance.The levels of Spc resistance mediated by R

factors depend on the test medium, the concen-tration of bacteria used in the assay, the hostbacterium, and the R factor (21). Therefore,to compare the levels of Spc resistance mediatedby individual R factors, certain of them weretransferred by conjugation to the same hoststrain, E. coli AB1932-1, and the resulting re-sistant substrains were tested under identicalconditions. The 33 tested R factors conferredvarious levels of resistance: 18 mediated levels of> 100 <750 ,ug/ml, whereas 13 mediated > 1,000,qg/ml (Table 4).Mechanism for the R factor-mediated Spc

a The patterns of drug resistance and the fi typemediated by the R factors were determined asdescribed. The order of presentation of theindividual genetic expressions does not indicategenetic linkage groups.

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KINETICS OF PHENOTYPIC EXPRESSIONOF R FACTOR MEDIATED SpcR

Chi1000 _ 0

z 800 -

Q@ 600 ,/Spc600 0 7

o 400 0

2 200 /

IC

10 20 30 40

MINUTESFIG. 1. Kinetics of expression of R factor-mediated

Spc and Chl resistance. E. coli AB1932 was incubatedin Lennox broth, centrifuged, washed twice, and re-suspended in 0.9% NaCI containing 10-3 M CaCl2,and incubated for 15 min at 37 C, after which equalconcentrations (2 X 108/ml) of bacteria and phagePlkc, prepared on E. coli AB1932-4/RE130, weremixed. This suspension was incubated for 15 min at37 C and then chilled and centrifuged in the cold, afterwhich the pellet was resuspended in prewarmed Lennoxbroth and incubated at 37 C. Samples of 0.1 ml wereremoved at intervals (zero time being the time of resus-pension in Lennox broth) and spread on EMB agarcontaining either Spc (50 ,ug/ml), or Chl (50 ,Ag/ml).Symbols: 0, SpcR transductants; 0, ChlR trans-ductants.

resistance. When the resistance of R-SpcRstrains was tested by the tube dilution assay,the minimal inhibitory concentration of Spcwas found to increase substantially with thenumber of bacteria used as inoculum (21).Such a relationship has been observed whendrug resistance is mediated through enzymaticinactivation, e.g., penicillin resistance due toj3-lactamase (3). Likewise consistent with inac-tivation was the very rapid expression of Spcresistance after introduction of the locus into asensitive cell. Figure 1 shows that, after trans-duction of the two R factor loci by phage Plkc,Spc resistance was expresssed even slightly morerapidly than Chl resistance, which is known to

E. co/i12- B/RE /30

104-

8 K

6-

4

2-0-O-Q-- --

0 20 40 60 80

F co/i B

A-!" en0 20 40 60 80

MINUTES OF INCUBA TION

FIG. 2. Adenylation of Spc, dihydrospectinomycin,and actinamine by extracts of E. coli B/RE130 andE. coli B. The assayfor adenylation of aminoglycosideswas performed with 14C-ATP as described. The "con-trol" incubation contained no drug. Substrates used:0, none; 0, Spc; A, dihydrospectinomycin; O,actinamine.

occur through enzymatic acetylation (18). Thesefindings led us to test directly for Spc inactiva-tion during a recent study (11) on inactivationof Str by an R-SpcR StrR strain. Extracts of thisstrain were also found to inactivate Spc if (aswith Str) ATP and Mg++ were added. Subse-quent studies have confirmed that Spc is inac-tivated at a rate of approximately 62 ,ug permg of protein per hr by ATP-Mg++-supple-mented extracts of R_SpcR strains but not bythose of R-SpcS, SpcR (chromosomal), or SpcSstrains.

Because the cofactor requirement observedis the same as for the inactivation of Str (andBlu) by adenylation (11), the possibility of Spcadenylation was explored more directly. Portionsof incubation mixtures containing uniformlylabeled 14C-ATP, Mg+, Spc, and extract ofR_SpcR bacteria were removed at intervals tophosphocellulose paper (to which Spc binds byvirtue of its two cationic groups). The amountof radioactive label retained by the paper in-creased linearly with the time of incubation (Fig.2) and with the concentration of extract; controlexperiments showed that Mg++, Spc, and extractwere all necessary for transfer of label from ATPto the paper. Transfer of label was also observedwhen a-32P-ATP, but not y-32P-ATP, was sub-stituted for '4C-ATP or when Str or Blu, but notKan, was substituted for Spc (Table 5). Thus, theextract forms a complex that retains the cationicproperties of Spc and that includes some 14C_labeled moiety of and the a-phosphate of ATP.This reaction, like the inactivation of the anti-microbial activity of the drug, is specific to ex-tracts of R-SpcR strains and is not observed in

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TABLE 5. Aminoglycoside-dependent transfer ofradioactivity from ATP, variously labeled,

to phosphocellulose by an extract ofE. coli B/REI30a

Aminoglycoside 14C-ATP a-32P-ATP S-32P-ATP

Streptomycin ...... 2,043 1,236 58Spectinomycin .....981 507 54Kanamycin........ 165 61 60None............. 180 59 59

a Adenylation of aminoglycoside drugs wasassayed as described. Reaction mixtures wereincubated for 60 min; results are expressed ascounts per minute.

SPEC TINOMYC IN

HOHH3C-N 0 0 CH3

HO 0HNH 0CH3

ACTINAMINE

OHH

H3C- N OH

HO OHNH

CH3

STREPTOMYCIN(Bl uensomyci n)

H HH3C-N OH

0 0 C-OH/ R3

0 CH30

HO OH

RI

FIG. 3. Molecular structures of spectinomycin,actinamine, streptomycin, and bluensomycin. Thestructures have been depicted to indicate the structuralsimilarity of the drugs at the proposed site of attackof the adenylate synthetase; no stereospecificity isintended. For Str, R1 and R2 = -N(H)-C(NH2) =NH, and R3 = -CHO;,for Blu, the Rt and R2 posi-tions are occupied by -NH2and -N(H)-C (NH2) =NH, but which group is at R1 and which at R2 is notknown; R3 = CH20H.

extracts of R-Spc5, SpcS, or SpcR (chromosomal)strains; the reaction has been confirmed in eightadditional R-SpcR StrR BluR strains tested. Whenequimolar concentrations of dihydrospectino-mycin or actinamine (30), the 1,3-dibasicglycoside component of Spc (Fig. 3), were pres-ent in place of Spc, transfer of label proceededat similar rates (Fig. 2).

Studies on identity of Spc- and Str-adenylatingactivities. Several lines of evidence suggest thatone adenylate transferase mediates Spc and Strresistance (see below). To examine this proposal,the size and heat stability of the enzyme(s)

adenylating Spc and Str were examined asfollows. Extract of E. coli B/RE130 was appliedto a column of Sephadex 200 and eluted with Rbuffer; the elution profile of Spc-adenylatingactivity was found to be identical with that of theStr-adenylating activity. Extract of E. coliB/RE130 was heated at 48 C, and samples wereremoved at intervals and assayed for Spc andStr adenylating activities. Fig. 4 indicates thatboth activities were inactivated coordinately: 50%of both activities were lost after 3 min of exposureof extracts to this temperature. These resultsare consistent with the proposal that one en-zyme adenylates Spc and Str, and they promptedthe following studies of substrains of E. coliB/RE130 mutated in Spc or Str resistance.Of 2,399 colonies of E. coli B/RE130 examined

for mutations to Spcs or Strs after exposure toNTG, 11 had lost their R factor (R-), 4 wereSpc5 StrS ChlR TetR MerR SulR, and 8 wereinterpreted by the replication method to be SpcRStrS ChlR TetR MerR SulR (see below); thus0.5 % of the clones carried R factors mutated inSpc or Str resistance, or in both.When one of the Spcs Str5 clones was examined

further, it was found to have the same in vivosensitivity to Spc and Str (and Blu, not shown) asdid the isogenic R- strain (Fig. 5) and to transferSpcs StrS ChlR TetR MerR SulR by conjugationto sensitive E. coli. Extracts of E. coli B carryingthis R factor, RE130-17, did not detectablyadenylate either Spc or Str in vitro (Fig. 5).When E. coli B/RE130-17 was spread on EMBcontaining Spc (200 Ag/ml), colonies were ob-tained at an approximate frequency of 1 in

I00180

60 _

4 A

a 20

2 4 6 8

MINUTES AT 48° C

FIG. 4. Inactivation at 48 C of thle Spc- andStr-adenylating activities of E. coli B/RE130 extract.Extract, at a concentration of I mg/ml, was heatedat 48 C; samples were removed at intervals, incu-bated withcompletereaction mixtures,for30 min, andassayed as described. Substrates used: 0, Spc; A, str.

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A100

751

50~

25

0 Spectinomycin

i2 4 600200 400 600

* Streptomycin

hIlo 20

DRUG CONCENTRATION f/g/r)

B15Spectinomycin 25Streptomycin

12- 20-

9- 151-

6- 10-

3h 5

20 40 20 40

INCUBATIONI(min)FIG. 5. Correlation of in vivo resistance to and in

vitro adenylation of Spc and Str by certain sub-strains of E. coli B. The resistance of bacteria (A)and adenylating activity of extracts (B) were testedas described. Adenylating activities were normalizedfor concentrations of extracts. Symbols: 0, E. coliB (SpcS StrS); 0, E. coli B/RE130 (SpcR StrR);1, E. coli B/RE130-17 (SpcS StrS); E, E. coliB/RE130-17-11 (SpcR StrR).

every 5.5 X 107 cells plated; of the 10 of theserevertants to Spc resistance tested, all were alsoresistant to Str (20 ,ug/ml). The in vivo resistanceto and the in vitro adenylation of Spc and Strby extracts of one of these revertant substrains,E. coli B/RE130-17-11, were equal to or slightlygreater than that of E. coli B infected with theparent R factor, RE130 (Fig. 5).The eight colonies interpreted to be SpcR

Strs ChlR Tet* MerR SulR (see above) were ofgreat interest because their existence impliedthat Spc and Str resistance could be dissociatedby mutation; hence, their Spc and Str resistancelevels were thoroughly examined by quantitatingcolony formation on a series of plates with vari-ous drug concentrations. Figure 6 depicts theresults obtained with one of the mutants, E. coliB/RE130-4; the results with the others were es-sentially identical. Each of these mutant sub-strains (i) was not Str6 but had a level of Strresistance that was greater than that of E. coliB but less than that of E. coli B/RE130 ("partialresistance") and (ii) was not SpcR but was "par-tially" resistant to Spc. These patterns of "partial"Spc and Str resistance were shown to be properties

A100 Spectinomycin Streptomycin

S 75 0

50 0

25-

200 400 600 0 20

DRUG CONCENTRA77ON,ug/m/)

B

40

INCUBATION (min)

FIG. 6. Correlation of in vivo resistance to and invitro adenylation of Spc and Str by certain substrainsofE. coli B. The resistance ofbacteria (A) and adenylat-ing activity of extracts (B) were tested as described.Adenylating activities were normalized for concentra-tions of extracts. Symbols: 0, E. coli B (SpcS Str8);*, E. coli B/RE130 (SpcR StrR); U, E. coli B/RE130-4 [Spc (partial R) Str (partial R) ].

of the mutated R factor rather than of the hostcell, since the patterns could be transferred byconjugation to sensitive E. coli. Moreover, ex-tracts of E. coli B/RE130-4 had less of both Spcand Str adenylating activities per unit of extrac.protein than did extracts of E. coli B/RE1301Thus, upon careful analysis, all mutationatchanges affecting one resistance have been foundto affect the other coordinately.

DISCUSSIONThe present survey supports and extends our

preliminary observation that SpcR R factors arefrequent among drug-resistant enteric bacteria(21): of 100 resistant strains examined, 46 car-ried a transferable R factor mediating Spcresistance. Many of the bacteria used as thesource of the R factors studied were selected,however, from resistant strains isolated in diag-nostic laboratories (for other studies) becausethey were resistant to either Str or Chl; most ofthem were E. coli or Klebsiella. R factors thatinfect these two genera generally have the fi+genotype (17; D. H. Smith, unpublished data), asdo all those reported to date that mediate Chl

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resistance (T. Watanabe and N. Datta, personalcommunication). Furthermore, one R factor locusmediating Str resistance presumably mediatesSpc resistance, and, from our observations, itappears to be carried generally, if not exclusively,by fi+ R factors. Thus, our survey was made ona sample population fortuitously preselected toinclude Spc resistance. It would be unlikely,therefore, that the population of resistant entericbacteria in general would include as high apercentage of SpcR R factors as found in oursurvey; it is likely, however, that most fi+ Rfactors will be found to carry the locus mediatingSpc (and Str-Blu) resistance.We have not undertaken chemical studies

necessary to prove that R factors inactivateSpc by adenylation. However, the observationsthat extracts of R-SpcR bacteria mediate a Spc-dependent finding of some '4C-labeled moiety ofand the a-phosphate of ATP to phosphocelluloseand that this aminoglycoside-dependent adenylatetransfer activity by extracts is positively correlatedwith resistance of R-SpcR bacteria are consistentwith this mechanism. A strong argument for Spcadenylation can also be made from the closecorrespondence of Spc and Str resistance in R-fac-tor strains in which inactivation of Str by adenyla-tion is well documented (11, 26, 31).

Several lines of evidence indicate that R factorsmediate the synthesis of a single adenylate trans-ferase that inactivates Spc and Str. All R-SpcRbacteria isolated from natural sources that havebeen studied to date are also StrR. [The converseis not true, however, because certain R factorsinactivate Str, but not Spc, by phosphorylation(D. H. Smith, J. A. Janjigian, and N. Prescott,unpublished data; B. Ozanne et al., in press).]The positive correlation between Spc and Strresistance is not limited to bacteria isolated fromclinical specimens, since mutant substrains(selected after exposure to NTG) that had"partial" R-Spc resistance had a coordinatedecrease in R-Str resistance. Adenylation of Spcand Str by extracts of these strains was positivelycorrelated with their levels of cellular resistance.Furthermore, of the Str8 R factors recovered from191 clones of E. coli RC (an Arg- conditionallyStr-dependent strain) /RE130 obtained by selec-tion on minimal medium containing 20 Atg ofStr per ml [a cultural condition that permitsgrowth only of substrains that are Arg+ orR-Strs (J. H. Harwood and D. H. Smith, Genet.Res., in press)], all were SpcB (J. H. Harwood,J. Janjigian, and D. H. Smith, Genet. Res., inpress). Finally, SpCR revertants of an NTG-in-duced R-Spc8 Strs mutant substrain were also

StrR, and extracts of this strain regained Spc andStr adenylation activity. Thus, a single mutationof an R factor can affect in vivo resistance to, andin vitro adenylation of, Spc and Str. Moreover,the Str adenylate transferase reportedly attacksthe N-methyl glucosamine moiety of Str (Fig. 3)at the 3'-OH group (26, 31), which may be con-sidered a part of a D-threo methylamino alcoholmoiety. The actinamine moiety of Spc (Fig. 3)includes an OH group with the same conforma-tional relation to a methylamino group, and ithas been suggested that the D-threo methylaminoalcohol moiety is the basis for the recognition ofthe adenylate transferase for its several substrates(G. Whitfield, personal communication). Our find-ings that extracts of R-SpcR bacteria adenylateSpc and actinamine equally are consistent withthis proposal. Thus, the results of several bio-chemical studies support and extend the bio-logical and genetic observations that suggest thata single adenylate transferase inactivates bothSpc and Str.

According to the recommendations of Demerecet al. (9), loci mediating drug resistance shouldbe designated by three-letter, lower case symbolssuggesting the name of the drug. Designation ofthe R factor locus mediating Spc adenylation,however, reveals a difficulty in the use of thisguideline, since this locus mediates resistance to atleast three aminoglycoside antibiotics, Spc,Str, Blu (and perhaps to others untested, althoughnot to Kan, neomycin, paromomycin, or genta-mycin). Str and Blu are quite similar in structureand action but differ appreciably from Spc inboth these respects. For this and other locimediating resistance by an enzymatic inactiva-tion of established mechanism to a number ofimportant drugs, it might be useful if the three-letter symbol suggested the biochemical activityinvolved. Should more than one drug inactivationlocus mediate resistance by the same activity,the loci would be distinguished (per Demerecet al.) by an italicized capital letter, possibly,though not necessarily, suggesting the drug inac-tivated. We suggest, therefore, that the locusmediating Spc, Str, and Blu adenylation bedesignated adn; that mediating Kan and neo-mycin phosphorylation (12), phs K; that medi-ating Str and gentamycin phosphorylation(D. H. Smith, J. A. Janjigian, and N. Prescott,unpublished data; Ozanne et al., in press), ph/ S;and those loci mediating the synthesis of theseveral j3-lactamases that produce resistancenot only to various penicillins but also to cepha-losporins (M. H. Richmond, personal communi-cation), ltm A, B, C, etc.

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SPECTINOMYCIN RESISTANCE

ACKNOWLEDGMENTS

We thank G. Whitfield, The Upjohn Co., for spectinomycin,actinamine, and Klebsiella UC-59 and for his helpful comments,and J. Davies and co-workers for discussions of their experi-ments before publication of their manuscript.

This investigation was supported by Public Health Servicegrants AI-0836 from the National Institute of Allergy and Infec-tious Diseases and GM-14119 from the National Institute ofGeneral Medical Sciences. D. H. S. is the recipient of CareerDevelopment Award AI-20376 from the National Institute ofAllergy and Infectious Diseases.

LITERATURE CITED

1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. Opti-mal conditions for mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine in Escherichia coli. Biochem. Biophys.Res. Commun. 18:788-795.

2. Anderson, P., Jr. 1969. Sensitivity and resistance to spectino-mycin in Escherichia coli. J. Bacteiiol. 100:939-947.

3. Barber, M. 1947. Coagulase-positive staphylococci resistant topenicillin. J. Pathol. Bacteriol. 59:373-384.

4. Benveniste, R., T. Yamada, and J. Davies. 1970. Enzymaticadenylylation of streptomycin and spectinomycin in Esche-richia coli carrying resistance factors. Infec. Immun. 1:109-119.

5. Bray, G. A. 1960. A simple efficient liquid scintillator forcounting aqueous solutions in a liquid scintillation counter.Anal. Biochem. 1:279.

6. Davies, J. E., P. W. Anderson, and B. D. Davis. 1965. Inhibi-tion of protein synthesis by spectinomycin. Science 149:1096-1098.

7. Davis, B. D., and E. S. Mingioli. 1950. Mutants of Fscherichiacoli requiring methionine or vitamin B12. J. Bacteriol.60:17-28.

8. Davis, J. E., and R. L. Sinsheimer. 1963. The replication ofbacteriophage MS2. I. Transfer of parental nucleic acid toprogeny phage. J. Mol. Biol. 6:203-207.

9. Demerec, M., E. A. Adelberg, A. J. Clatk, and P. E. Hartman.1966. A proposal for a uniform nomenclature in bacterialgenetics. Genetics 54:61-76.

10. Hanka, L. J., D. J. Mason, and W. T. Sokolski. 1961. Actino-spectacin, a new antibiotic. II. Microbiological assay.Antibiot. Chemotherapy 11:123-126.

11. Harwood, J. H., and D. H. Smith. 1969. Resistance factor-mediated streptomycin resistance. J. Bacteriol. 97:1262-

1271.12. Kondo, S., M. Okanishi, R. Utahara, K. Maeda, and H.

Umezawa. 1968. Isolation of kanamycin and paromamineinactivated by E. coli carrying R factor. J. Antibiot.(Tokyo) Ser. A 21:22-29.

13. Lennox, E. S. 1955. Transduction of linked genetic characters.of the host by bacteriophage P1. Virology 1:190-206.

14. Lewis, C., and H. W. Clapp. 1961. Actinospectacin, a new

antibiotic. III. In vitro and in vivo evaluation. Antibiot.Chemotherapy 11:127-133.

15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R.J. Randall. 1951. Protein measurement with Folin phenolreagent. J. Biol. Chem. 193:265-275.

16. Mason, D. J., A. Dietz, and R. M. Smith. 1961. Actinospecta-cin, a new antibiotic. I. Discovery and biological properties.Antibiot. Chemotherapy 11:118-122.

17. Meynell, E., G. G. Meynell, and N. Datta. 1968. Phylogeneticrelationships of drug-resistance factors and other trans-

missible bacterial plasmids. Bacteriol. Rev. 32:55-83.18. Shaw, W. V. 1967. The enzymatic acetylation of chloram-

phenicol by extract of R factor-resistant Escherichia coli. J.Biol. Chem. 242:687-693.

19. Smith, D. H. 1966. Transferable drug resistance in Salmonella.New Engl. J. Med. 275:625-630.

20. Smith, D. H. 1967. R factor infection of Escherichia colilyophilized in 1946. J. Bacteriol. 94:2071-2072.

21. Smith, D. H. 1967. R factor-mediated resistance to new amino-glycoside antibiotics. Lancet 1:252-254.

22. Smith, D. H. 1967. R factors mediate resistance to mercury,

nickel and cobalt. Science 156:1114-1116.23. Smith, D. H. 1969. R factors for aminoglycoside antibiotics. J.

Infect. Dis. 119:378-380.24. Sparling, P. F., J. Modalell, Y. Takeda, and B. D. Davis. 1968.

Ribosomes from Escherichia coli merodiploids heterozygousfor resistance to streptomycin and to spectinomycin. J. Mol.Biol. 37(Suppl. 3):407-421.

25. Steers, E., E. L. Foltz, and B. S. Graves. 1959. Inocula repli-cating apparatus for routine testing of bacterial susceptibil-ity to antibiotics. Antibiot. Chemotherapy 9:307-311.

26. Umezawa, H., S. Takasawa, M. Okinishi, and R. Utahara.1968. Adenylylstreptomycin, a product of streptomycin in-activated by E. coli carrying R factor. J. Antibiot. (Tokyo)Ser. A 21:81-82.

27. Walton, J. R., and D. H. Smith. 1969. New hemolysin (-y)produced by Fscherichia col. J. Bacteriol. 98:304-305.

28. Watanabe, T. 1964. Selected methods of genetic study ofepisome-mediated drug resistance in bacteria. MethodsMed. Res. 10:202-220.

29. Watanabe, T., H. Nishida, C. Ogata, T. Arai, and S. Sato.1964. Episome-mediated transfer of drug resistance inEnterobacteriaceae. VII. Two types of naturally occurring Rfactors. J. Bacteriol. 88:716-726.

30. Wiley, P. F. 1962. The chemistry of actinospectacin. I. Actina-mine. J. Amer. Chem. Soc. 84:1514.

31. Yamada, T., D. Tipper, and J. Davies. 1968. Enzymatic in-activation of streptomycin by R factor-resistant Echerichiacoli. Nature (London) 219:288-291.

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