JOURNAL OF BACTERIOLOGY, July 1975, p. 329-335Copyright 0 1975 American Society for Microbiology

Vol. 123, No. 1Printed in U.S.A.

Genetic Properties of an R Factor Carrying Resistance toAminoglycoside Antibiotics

KATSUMI HASUDA,* VLADIMIR KRCMERY, SHIZUKO IYOBE, AND SUSUMU MITSUHASHI

Department of Microbiology, School ofMedicine, Gunma University, Maebashi, Japan* and Research Instituteof Hygiene, Bratislava, Czechoslovakia

Received for publication 17 January 1975

R factor Rmsl51 is an fi+ R factor and belongs to a incompatibility group FIT. Itcarries the genes governing resistance to various aminoglycoside antibiotics, i.e.,kanamycin (KM), lividomycin (LV), gentamicin C complex (GM), and 3',4'-dideoxykanamycin B (DKB), in addition to those governing to tetracycline (TC),chloramphenicol (CM), sulfanilamide (SA), and ampicillin (APC). Electronmicroscopy observation disclosed that the Rmsl51 deoxyribonucleic acid was a

circular form with length of 32.2 Mm. A probable circular genetic map of Rmsl51was proposed by genetic and biochemical studies, the genes being in the order of-tet-tra-amp-aad-sul-aph-cml-, in which aad and aph confer resist-ance to KM.GM.DKB by adenylyltransferase or resistance to KM.LV byphosphotransferase, respectively.

The gentamicin C complex (GM) has beenused as an effective aminoglycoside antibioticagainst infections with various gram-negativeenteric bacteria, especially kanamycin (KM)-resistant strains. Recent surveys of bacterialresistance has disclosed, however, that the fre-quency of isolation of GM-resistant strains isnow increasing, owing to the world-wide use ofthe drug (10, 20). We recently demonstrated anR factor carrying resistance to various amino-glycoside antibiotics including GM and 3',4'-dideoxykanamycin B (DKB) from an Esche-richia coli strain isolated from a clinical speci-men in Frankfurt (16). This paper deals withthe genetic studies of this R factor and itsbiochemical mechanisms of resistance to ami-noglycoside antibiotics.

MATERIALS AND METHODS

Bacterial strains. E. coli F-027 was used as thedonor of the R factor and was isolated from a clinicalspecimen by H. Knothe in Frankfurt (16). For therecipient strains, E. coli K-12 ML1410 met- nalr(resistant to nalidixic acid) and E. coli K-12W3630(malh) were used. E. coli K-12 ML3674 (HfrCmet- nalr) was used as the R factor host strain to testthe fi character. Fifteen kinds of R factors belongingto different incompatibility groups were used todetermine the incompatibility type of our R factor.These were kindly supplied by N. Datta (RoyalPostgraduate Medical School, London). Incom-patilibity tests were carried out by the method ofDatta and Hedges (2, 3).

Drugs. Tetracycline (TC), chloramphenicol (CM),

streptomycin (SM), and sulfanilamide (SA) werekindly supplied by the Japan Lederly Co., SankyoSeiyaku Co., Kyowa Hakko Co., and the DainihonSeiyaku Co., respectively. KM, DKB (18), and am-picillin (APC) were given by the Meiji Seika Co.Amikacin (BB-K8) (9) and lividomycin (LV) weresupplied by the Bristol Banyu Co. and Kowa Co.,respectively. GM, its Cl component(GM-C l), andnalidixic acid were the gifts from the Shionogi Co.,Schering Co., and the Daiich Seiyaku Co., respec-tively.

Media. Brain heart infusion broth (Difco) was usedroutinely. For the determination of drug resistance,heart infusion agar (Eiken, Tokyo) and peptone waterwere used. Peptone water consisted of 1,000 ml ofdistilled water, 5 g of NaCl, and 10 g of peptone. Forthe assay of SA resistance, a semi-synthetic mediumwas used and consisted of 1,000 ml of medium A (12),2 g of Casamino Acids, 10 mg of tryptophan, 2 mg ofnicotinic acid, 10 mg of thiamine, and 2 g of glucose.Eosin methylene blue-maltose agar (12), containingboth 50 vg of NA per ml and one of the drugs toselect drug-resistance markers on the R factor, wasused for the selection plate for R factor transfer fromeither F-027 R+ or W3630 R+ to ML1410. Maltoseagar consisted of medium A (12) without citrate,0.2% bromothymol blue, 1.5% agar and 1.0% maltosein distilled water. L-broth and L-agar (13) were usedfor phage propagation and transduction experiments.

Conjugal transfer of R factor. Two milliliters ofrecipient and 0.5 ml of donor cultures were mixed atearly stationary phase of growth. The mixture wasshaken gently at 37 C for 30 min and 0.1 ml of themixture was put into 9.9 ml of cold saline to interruptthe conjugation by blender treatment at 2,200 rpm for2 min. Then the appropriate dilutions of the mixturewere spread on selective plates. Transfer frequency

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330 HASUDA ET AL.

was expressed by the number of exconjugants per thatof donor cells.

Transduction. Transduction was carried out bythe method of Lennox (13). The recipient was kept at37 C for 30 min in fresh broth before spreading onselective plate for phenotypic expression of drugresistance. The number of input phage and recipientcells were 109 plaque-forming units per ml and 5 x 109cells/ml, respectively. Transduction frequency wasexpressed by the number of transductants per that ofinput phages.

Preparation of the S-105 fraction and inacti-vation reaction. The S-105 fraction from E. coli wasprepared by the procedure described previously (8).To determine the inactivation by phosphorylationor adenylylation, disodium adenosine triphosphate(ATP) was used in the reaction mixture called the Psystem. The reaction mixture consisted of 0.2 ml oftris(hydroxymethyl)aminomethane (Tris)-hydrochlo-ride buffer (pH 7.5), 0.05 ml of 20 mM ATP, 0.05 ml of0.02 M magnesium chloride, 0.05 ml of 1 mM drug,0.05 ml of distilled water, and 0.1 ml of S-105fraction (15 mg of protein/ml). After incubation at37 C for 60 min, the reaction was then stopped byheating at 80 C for 3 min. Residual antibiotic activityin the reaction mixture was determined by bioassayusing Bacillus subtilis PCI 219. To determine the in-activation by acetylation, acetyl coenzyme A wasadded to a reaction mixture called the A system.This reaction mixture 'consisted of 0.2 ml of Tris-hydrochloride buffer (pH 7.5), 0.05 ml of 1 mM drug,0.05 ml of 1 mM acetyl coenzyme A, 0.05 ml ofdistilled water, and 0.1 ml of S-105 fraction (15 mg ofprotein/ml).

Radioisotope assay. The incorporation into antibi-otics of [14C]ATP and y- [3:P]ATP by drug-inactivat-ing enzymes were carried out by the procedure de-scribed previously (7). The reaction mixture consistedof 30 Ml of S-105 fraction (15 mg of protein/ml), 10 Ml of0.02 M magnesium chloride, 10 Ml of 0.5 mM drug, 40Ml of 0.2 M Tris buffer (pH 7.5), and 10 ul of-y [32P]ATP (12 MCi/ml) or [8-14C]ATP (50 MCi/ml).The reaction was carried out at 37 C for 2 h.

Preparation of radioactive deoxyribonucleicacid (DNA) of Rmsl51 by ethidium bromide CsClgradient centrifugation. E. coli K-12 W3630Rmsl51 + was grown at 37 C with aeration. When thenumber of bacterial cells reached 2 x 108/ml, deoxy-adenosine and [3H lthymidine were added to theculture in final concentrations of 250 ug/ml and 5MCi/ml, respectively, and incubation was continueduntil 8 x 108 cells/ml were obtained. Then the cellswere sedimented and washed twice in 0.02 M Tris-sodium chloride buffer (pH 8.0). The bacterial cellswhich were collected by centrifugation from 350 ml ofanother nonlabeled culture (109 cells/ml) were addedto the labeled cells to increase the number of bacterialcells. The cells were lysed by lysozyme (0.4 mg/ml) in6 ml of 0.02 M Tris-sodium chloride buffer (pH 8.0)containing 0.02 M disodium ethylenediaminetetra-acetate and 0.5% sodium dodecyl sulfate in water.The DNA of the cell lysate thus obtained was purifiedby phenol treatment and precipitation by isopropylalcohol. The purified DNA was dissolved in 2 ml of

0.02 M Tris-sodium chloride buffer. One milliliter ofthe DNA solution was mixed with 6 ml of water thatcontained 1.4 mg of ethidium bromide and 6.55 g ofCsCl and was centrifuged at 44,000 rpm for 40 h at20 C with a Hitachi RT65T angle roter in a Hitachi 65P preparative ultracentrifuge. After centrifugation,each 0.1-ml fraction was obtained serially from thebottom of the tube. Three fractions corresponding tothe covalently closed circular DNA were pooled aftercounting the radioactivity of each fraction. Ethidiumbromide was extracted by n-octanol from the fraction.The covalently closed circular DNA solution wasdialysed against SSC (0.15 M NaCl plus 0.015 Msodium citrate) and stored at 2 C.

Electron microscopy. The R factor DNA in SSCwas mixed with an equal volume of a 0.04% solution ofcytochrome c and with twice the volume of 4 Mammonium acetate. About 0.01 ml of the solution wasspread on the surface of distilled water. The mono-layer of cytochrome c including the concerned DNAwas transferred to a specimen grid coated with carbonand shadowed with platinum-palladium at a distanceof 4 cm by angle of 6°. The preparations wereexamined by a Nihondenshi electron microscope,JEOL type 7. Contour lengths were measured by en-larging the photographs of the circular DNA mole-cules. Molecular weights were calculated as 2.07 x106 per Mm contour length (1, 11).

RESULTSIsolation of an R factor carrying gentami-

cin resistance. E. coli F-027 was found to beresistant to various aminoglycoside antibioticsincluding KM, LV, GM, and DKB. In addition,it was found to be resistant to TC, CM, SA, andAPC but was sensitive to SM. We examined theconjugal transferability of its resistance usingML1410 as a recipient. As shown in Table 1, theresistances were transferred at a frequency of10-5 to 10-4, when selection was made for eachof TC, CM, KM, SA, GM, or APC resistance.After examination for unselected markers inexconjugants, we found that almost all of theexconjugants carried resistances to the eightdrugs mentioned above, and the resistances ofall exconjugants were transferable again to thesecond recipient W3630. Two types of the ex-conjugants which did not receive all of eightresistances at the same time were obtained. Onetype carried only TC resistance and anothertype carried resistances to the remaining sevendrugs. The R factors possessing TC resistanceand the R factor carrying seven-drug resistancesderived from each of the exconjugants wereincompatible with each other when transferredinto the same cell, indicating that they weresegregants of the R factor carrying eight resist-ances. It was concluded that F-027 possessed anR factor, called Rmsl51, carrying resistances toeight drugs. The levels of drug resistances

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AMINOGLYCOSIDE RESISTANCE R FACTOR 331

conferred by Rmsl51 in each of three hoststrains are shown in Table 2.Transduction of Rmsl51. The transduction

experiment was carried out using ML1410Rms151+ as a donor strain. Transductants were

obtained by selection for TC, CM, or KMresistance, and resistance patterns and conjugaltransferability in transductants were examined(Table 3). We could obtain transductants carry-

ing transferable eight-drug resistance, even

when selected for TC, CM, or KM resistance,indicating that the genes governing resistancesto eight drugs were located on an Rmsl51 factor.The transductants carrying various resistancepatterns were isolated and some of them were

conjugally nontransferable. As for resistances toaminoglycoside antibiotics, two nonparentaltypes of transductants were obtained, one ofwhich carried only resistances to KM and LV in

addition to TC and CM, and another carriedresistances to KM, LV, GM, and DKB inaddition to CM, SA, and APC. Their resist-ances were transferable by conjugation and we

called these R factors carrying resistances toTC, CM, KM, and LV, or resistances to CM,KM, LV, SA, GM, DKB, and APC, Rmsl51-1and Rmsl51-2, respectively.Biochemical mechanisms of resistance to

aminoglycoside antibiotics. To investigate thebiochemical mechanisms of aminoglycoside re-

sistance conferred by the Rmsl51 factor, we

examined the inactivation of KM, LV, GM-C1,and DKB using F-027 and two types of strainsderived by transduction of Rmsl51, W3630Rmsl51-1(TC.CM.KM.LV), and W3630Rmsl51- 2(CM . KM. LV. SA.GM .DKB. APC).Two inactivation systems were applied, the Psystem and the A system described above. Both

TABLE 1. Transfer of drug resistance from E. coli F-027a

[ ExconjugantsbSelection (,ug/ml) Transfer frequency No. of exconju-Selecion|Trasferrygants obtained |Drug resistance pattern

TC(25) 9.3 x 10-5 45 TC.CM.KM.LV.SA.GM.DKB.APC5 TC

CM(12.5) 9.6 x 10-5 50 TC.CM.KM.LV.SA.GM.DKB.APC

KM(50) 9.9 x 10-5 50 TC.CM.KM.LV.SA.GM.DKB.APC

SA(200) 5.6 x 10-5 50 TC.CM.KM.LV.SA.GM.DKB.APC

GM(3.2) 5.6 x 10-5 49 TC.CM.KM.LV.SA.GM.DKB.APC1 CM.KM.LV.SA.GM.DKB.APC

APC(50) 6.9 x 10 - 50 TC.CM.KM.LV.SA.GM.DKB.APC

aDonor, E. coli F-027; recipient, E. coli K-12 ML1410 nair. For conjugation experiment see Materials andMethods.

b Fifty exconjugants were picked at random from each selective plate and purified by successive inoculationon the same selective plate.

TABLE 2. Resistance of E. coli strains with or without the Rmsl51 factora

Host Rmsl5I TC CM KM LV SA GM-C| DKB APC SM BB-K8

027 +b > 50 > 25 > 400 > 100 > 200 12.5 25 > 50 < 1.56 0.8c < 1.56 1.56 <6.25 6.25 25 <0.8 <3.12 3.12 < 1.56 0.8

W3630 +d > 50 > 25 > 400 > 100 > 200 6.25 12.5 > 50 < 1.56 0.8- < 1.56 1.56 <6.25 1.56 < 12.5 <0.8 <3.12 1.56 < 1.56 0.8

ML1410 +d > 50 > 25 > 400 . 100 > 200 12.5 12.5 > 50 < 1.56 0.8_- <1.56 1.56 <6.25 1.56 <12.5 <0.8 <3.12 <1.56 <1.56 0.8

a For transfer of R factor see Materials and Methods.b Original donor of R factor.c F-027 derivatives which had lost the R factor during storage.d Exconjugants which acquired the R factor from E. coli F-027 Rms151+.

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TABLE 3. Segregation of Rmsl51 factor markers after transduction with bacteriophage pla

Genetic properties of transductants

Selection Transduction No. of(,Ug/ml) frequency transductants Resistance pattern Transferability

obtained (%) of resistance

TC(25) 4.0 x 10-7 148 (94.9) TC +6 (3.8) TC.CM.APC +2 (1.3) TC.CM.KM.LV.SA.GM.DKB.APC +

CM(12.5) 4.8 x 10-7 40 (44.4) TC.CM.APC +26 (28.9) CM.KM.LV.SA.GM.DKB.APC1 +11 (12.2) TC.CM.KM.LV.SA.GM.DKB.APC +6 (6.7) CM.KM.LV.SA.GM.DKB.APC _6 (6.7) TC.CM.KM.LV5 +1 (1.1) TC.CM.KM.LV.APC +

KM(50) 3.0 x 10-8 13 (32.5) CM.KM.LV.SA.GM.DKB.APC _11(27.5) CM.KM.LV.SA.GM.DKB.APC +1(27.5) TC.CM.KM.LV +3 (7.5) TC.CM.KM.LV.SA.GM.DKB.APC +2 (5.0) KM.LV.SA.GM.DKB.APC

a Donor of resistance was ML1410 Rmsl51+. It was obtained in the experiment shown in Table 1. Recipientwas W3630. For transduction see Materials and Methods.

b Transductants were used for the experimentation of biochemical mechanisms of aminoglycoside resistanceand their R factors were named Rmsl51-1 (TC.CM.KM.LV) and Rmsl51-2 (CM.KM.LV.SA.GM.DKB.APC).

F-027 and one transductant carrying resistancesto KM, LV, GM, and DKB could inactivate allof the four drugs by the P system, and anothertransductant carrying only KM and LV inacti-vated KM and LV by the same system (Table4). But none of these three strains could inacti-vate the drugs in the presence of acetyl coen-

zyme A instead of ATP(A system), indicatingthat they could not inactivate the aminoglyco-side antibiotics by acetylation. To knowwhether inactivation occurred by adenylylationor phosphorylation, we examined the incorpora-tion of [14C]ATP or y-[32P]ATP into KM or

GM-C,. When a cell-free extract from a straincarrying KM and LV resistance was used, a

high level of 32p incorporation from y- [3t2P]ATPinto KM-A was observed, indicating inactiva-tion by phosphorylation (Table 5). On the otherhand, the extract from a strain resistant to KM,LV, GM, and DKB could phosphorylate KM-Aand adenylylate both KM-A and GM-C1, indi-cating that this strain possessed two enzymes,KM phosphotransferase and GM adenylyl-transferase. From these results, it was con-cluded that the Rmsl51 carried two genes, aphand aad, which phosphorylate the 3'-OH groupof KM and LV by aminoglycoside phospho-transferase (3')-1 and adenylylate the 2"-OHgroup of certain aminoglycosides by adenylyl-transferase(2"), respectively (15).

TABLE 4. Inactivation ofKM, L V, GM-C1, and DKBby resistant strains of W3630

Inactivation of:Strain

KM LV GM-C, DKB

F-027a + + +W3630 Rms151-1(TC.CM. + +KM.LV)b

W3630 Rmsl5l-2(CM.KM. + + + +LV.SA.GM.DKB.APC)b

W3630

a F-027 was an original strain isolated in Frankfurt.Inactivation of drugs was carried out by the P system.For details see text.

b Transductant shown in Table 3.

TABLE 5. Incorporation of [14CJATP or y [32PIATPinto aminoglycoside antibiotics by cell-free extracta

W360 5 (C.W3630 Rms151-2(CM.W363ORms151-1(TC. KM.LV.SA.GM.DKB.Drug CMK.VbAPC)b

3"p "14C3fp 14C

KM 10,120 56 8,672 379GM-C1 ND ND 17 1,064

a Figures express the radioactivity incorporatedinto aminoglycoside antibiotics (counts per minute).ND, Not detected.

bSee Table 4.

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AMINOGLYCOSIDE RESISTANCE R FACTOR 333

Genetic properties of Rmsl51. We examined tetsome genetic and molecular properties ofRmsl51. Rmsl51 inhibited F function when / \introduced into an Hfr strain, ML3674. WhenRmsl51 was introduced to each one of bacterialstrains carrying each R factor belonging to the15 different incompatibility groups, it was aphfound that Rmsl51 was incompatible with only trathe FII type of R factor, R100. These results aad sulindicated that Rmsl51 was fi+ in fi character iadand belonged to the compatibility type FII.The molecular nature of Rmsl51 was exam- amp

ined by electron microscopy observation. Asshown in Fig. 1, we could observe the circularform of Rmsl51 DNA. The contour length of the FIG. 2. A probable genetic map of the Rmsl51DNA was 31.4 to 32.7 um(average 32.2 Mm), factor from E. coli F-027. The distances between eachwhen 38 molecules of DNA were estimated. The marker are drawn arbitrarily. Genetic symbols of eachaverage molecular weight calculated from the marker: tet, TC resistance; cml, CM resistance; aph,length was 66.7 x 106. phosphorylation ofKM and LVat 3'-OH residue; aad,

lengthc

w asp6. Xf106. adenylylation ofGM and DKB at 2"-OH residue; sul,Genetic map of Rmsl5l. According to the SA resistance; amp, APC resistance; tra, conjugal

results obtained by biochemical and genetic transferability.

FIG. 1. DNA ofRmsl5l factor. Bar represents 1 im.

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334 HASUDA ET AL.

studies, we could propose a probable geneticmap of Rmsl51 (Fig. 2). Although conclusiveexperiments were not done, this map satisfiedall of the gene sequences on various segre-gants obtained by transduction. The genes con-cerning KM.LV and KM.GM.DKB resistancewere expressed as aph and aad, respectively.

DISCUSSIONBiochemical and genetic studies disclosed

that resistance of E. coli F-027 to aminoglyco-side antibiotics was due to the presence of an Rfactor denoted Rmsl51, which carried two genesconcerning resistance to aminoglycoside antibi-otics, aad and aph. The former gene couldconfer resistance to KM, GM, and DKB in itshost by adenylylation of the drugs at the 2"-OHgroup on the presence of ATP. The latter genecould mediate the formation of aminoglycosidephosphotransferase 1, which inactivated KMand LV by phosphorylation at the 3'-OH and5"-OH group, respectively (19). Therefore, theresistance of E. coli F-027 to aminoglycosideantibiotics was due to two genes on Rmsl51factor, i.e., aad and aph, and we could separatethe gene aph from the gene aad by transductionwith phage P1.The Rmsl51 factor is an fi+ R factor and

belongs to the incompatibility group FII andthis type of R factor is isolated frequently frombacterial strains which belong to the familyEnterobacteriaceae. The R factors carryingTC.CM.SM.SA, CM.SM.SA, TC.SM.SA, andSM.SA resistance are isolated frequently andthe genes governing SM resistance are com-monly located on R factors (14, 17). Rmsl51,however, does not possess SM resistance butconfers resistance to other aminoglycoside an-tibiotics and APC resistance, although thisfactor is phylogenetically related to fi+ R100(TC.CM.SM.SA) of the FII incompatibilitygroup. The tet.tra genes of R100 that govern,respectively, TC resistance and conjugaltransferability could be separated from threeother resistance genes, i.e., cml.str.sul, bytransduction of R100 factor with bacteriophageP1(5). The tet and tra genes on R factors of R100type were segregated from other genes by conju-gal transfer to Salmonella strains (5). Similarlywe could obtain segregants carrying the tet.traregion from Rmsl51 by conjugal transfer or bytransduction with P1. This result also shows thesimilarity between R100 and Rmsl51 in itsgenetical constitution.We obtained various types of segregant from

Rmsl51 by transduction with P1. According to

the type of segregation among the genes govern-ing tra and drug resistance, we proposed agenetic map of Rmsl51. It is phylogeneticallyinteresting to note that the order of genes onRmsl51, i.e., -tet.tra-, -cml.tet.tra-, -tet.-tra.amp-, is common to many R factors (4, 6),and that the isolation frequency of R factorscarrying single CM resistance or resistance toonly aminoglycoside antibiotics is rather low(14, 17).

ACKNOWLEDGMENTSWe are greatly indebted to H. Kawabe, The Institute of

Microbial Chemistry, Tokyo, for the assay of biochemicalmechanisms of drug resistance.

LITERATURE CITED1. Clowes, R. C. 1972. Molecular structure of bacterial

plasmids. Bacteriol. Rev. 36:361-405.2. Datta, N., and R. W. Hedges. 1971. Compatibility groups

among fi- R factors. Nature (London) 234:222-223.3. Datta, N., and R. W. Hedges. 1972. Trimethoprim

resistance conferred by W plasmids in Enterobacteria-ceae. J. Gen. Microbiol. 72:349-356.

4. Hashimoto, H., and S. Mitsuhashi. 1966. Drug resistanceof enteric bacteria. VI. Recombination of R factor withtetracycline-sensitive mutants. J. Bacteriol.92:1351-1356.

5. Hashimoto, H., and S. Mitsuhashi. 1971. Segregation ofR factors, p. 545-551. In H. Umezawa (ed.), Progressin antimicrobial and anticancer chemotherapy. Int.Congr. Chemother. Proc. VI, University of TokyoPress, Tokyo.

6. Hashimoto, H., T. Tanaka, and S. Mitsuhashi. 1973.Genetic structure of an R factor conferring ampicillinresistance. Jpn. J. Microbiol. 17:331-337.

7. Kawabe, H., M. Inoue, and S. Mitsuhashi. 1974. Inacti-vation of dihydrostreptomycin and spectinomycin byStaphylococcus aureus. Antimicrob. Agents Chemo-ther. 5:553-557.

8. Kawabe, H., and S. Mitsuhashi. 1972. Acetylation ofdideoxykanamycin B by Pseudomonas aeruginosa.Jpn. J. Microbiol. 16:436-437.

9. Kawaguchi, H., T. Naito, S. Nakagawa, and K. Fujisawa.1972. BB-K8, a new semisynthetic aminoglycosideantibiotic. J. Antibiot. 25:695-708.

10. Knothe, H., V. Krcmery, W. Sietzen, and J. Borst. 1973.Transfer of gentamicin resistance from Pseudomonasaeruginosa strains highly resistance to gentamicin andcarbenicillin. Chemotherapy 18:229-234.

11. Lang, D. 1970. Molecular weights of coliphage andcoliphage DNA. III. Contour length and molecularweight ofDNA from bacteriophage T4, T5 and T7, andfrom bovine papilloma virus. J. Mol. Biol. 54:557-565.

12. Lederberg, J. 1950. Isolation and characterization ofbiochemical mutants of bacteria. Methods Med. Res.3:5-22.

13. Lennox, E. S. 1955. Transduction linked genetic charac-ters of the host by bacteriophage P1. Virology1:190-206.

14. Mitsuhashi, S. 1971. Epidemiology and genetics of Rfactors. Ann. N.Y. Acad. Sci. 185:141-152.

15. Mitsuhashi, S. 1974. Contribution of bacterial genetics tochemotherapy. In L. Rosival (ed.), Second internationalsymposium on antibiotic resistance: drug-inactivatingenzymes and other problems of resistant bacteria,

J. BACTERIOL.

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http://jb.asm.org/

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AMINOGLYCOSIDE RESISTANCE R FACTOR 335

in press. Avicenum, Czechoslovakia.16. Sietzen, W., H. Knothe, and V. Krcmery. 1973. Simulta-

neous transfer of gentamicin from an urine strain of E.coli. Z. Allg. Mikrobiol. 13:277-278.

17. Tanaka, T., A. Kobayashi, K. Ikemura, H. Hashimoto,and S. Mitsuhashi. 1974. Drug resistance and distribu-tion of R factors among Escherichia coli strains. Jpn. J.Microbiol. 18:343-347.

18. Umezawa, H., S. Umezawa, T. Tsuchiya, and Y. Oka-zaki. 1971. 3',4'-Dideoxykanamycin B active against

kanamycin-resistant Escherichia coli and Pseudomo-nas aeruginosa. J. Antibiot. 24:485-487.

19. Umezawa, H., H. Yamamoto, M. Yagisawa, S. Kondo, T.Takeuchi, and Y. A. Chabbert. 1973. Kanamycinphosphotransferase I: mechanism of cross resistancebetween kanamycin and lividomycin. J. Antibiot.25:407-411.

20. Witchitz, J. L., and Y. A. Chabbert. 1971. High leveltransferable resistance to gentamicin. J. Antibiot.24:137-139.

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