JOURNAL OF BACTERIOLOGYVol. 88, No. 2, p. 322-328 August, 1964Copyright © 1964 American Society for Microbiology

Printed in U.S.A.

TRANSFER OF DRUG RESISTANCE BETWEEN ENTERICBACTERIA INDUCED IN THE MOUSE INTESTINE

MORIMASA KASUYADepartment of Bacteriology, Nagoya University School of Medicine, Nagoya, Japan

Received for publication 10 March 1964

ABSTRACT

KASUYA, MORIMASA (Nagoya University Schoolof Medicine, Nagoya, Japan). Transfer of drugresistance between enteric bacteria induced in themouse intestine. J. Bacteriol. 88:322-328. 1964.-Transfer of multiple drug resistance in the in-testines of germ-free and conventional mice wasstudied with strains of Shigella, Escherichia, andKlebsiella. The transfer experiment was carriedout under antibiotic-free conditions to eliminatethe production of drug-resistant bacteria by anti-biotics. All resistance factors (chloramphenicol,streptomycin, tetracycline, and sulfathiazole)were transferred with ease in the intestinal tractsof mice, when donors and recipients multipliedfreely, and acquired resistance was further trans-ferred to other sensitive enteric bacteria in theintestinal tract. Bacteria to which resistance fac-tors were transferred showed, in most of the ex-periments, exactly the same level and pattern ofresistance as the donors. Based on the above, ahypothesis that the same process may possiblyoccur in the human intestine is presented.

In Japan, information on the transfer of drugresistance in enteric bacteria has accumulatedsince Ochiai et al. (1959) and Akiba et al. (1960)reported investigations of the possible transfer ofmultiple drug resistance by mixed cultivation.Most of these studies were carried out in vitro,

with the exception of in vivo experiments doneby Kagiwada et al. (1960) with human volun-teers and by Akiba et al. (1961) with a volunteerand animals. In both cases, however, the effectsof administered antibiotics upon the origin orselection of drug-resistant bacteria were not con-sidered at all.

In studies of this nature, in vivo difficultiesusually arise in eliminating the occurrence ofbacterial resistance to the antibiotics admin-istered, and in conducting experiments withonly the "infecting strains" without contamina-tion by other bacteria. In this study, therefore,attention was directed to the above, resulting in

satisfactory infection of mice by oral administra-tion of Shigella, Klebsiella, and Escherichia underantibiotic-free conditions. A germ-free mouse,which should theoretically be most suited forsuch an experiment, was fortunately available,and was tested in parallel.The present communication deals with a study

on the transfer of drug resistance in vivo amongthe above-mentioned bacteria. A preliminaryreport on some phases of this study was alreadypublished (Kasuya, 1962).

MATERIALS AND METHODS

Animals. One germ-free female mouse weighing18 g (supplied by M. Miyagawa, originally fromthe University of Notre Dame) and 11 conven-tional male mice weighing approximately 25 gwere used. Six series of experiments were carriedout with two animals each.

Organisms. S. flexneri 3a strain Kurano (sup-plied by K. Ochiai) and E. coli strain LNH (iso-lated from a mouse) were used as originallyresistant donors. Both are resistant to chlor-amphenicol, streptomycin, tetracycline, andsulfathiazole. Strain Kurano is a erythromycin-resistant mutant selected from the parent strainby the gradient broth method. As sensitive re-cipients, K. pneumoniae strain Kasuya (isolatedfrom a human being), E. coli strain Ml (isolatedfrom a mouse), S. flexneri 2a strain K (fromstock culture), and S. flexneri 2a strain Yamada(supplied by S. Naito) were used.Drugs and media. Chloramphenicol powder

(Parke, Davis & Co., Detroit, Mich.), dihydro-streptomycin sulfate (Takeda Chemical In-dustries, Ltd., Osaka, Japan), tetracycline hy-drochloride powder (Lederle Laboratories, PearlRiver, N.Y.), sulfathiazole (Takeda), and eryth-romycin lactobionate (Abbott Laboratories,North Chicago, Ill.) were used. For selection ofdrug-resistant colonies (strains), SS Agar andDesoxycholate Agar (Nihoneiyokagaku Co.,Ltd., Tokyo, Japan) containing 12.5 ,ug of chlor-

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IN VIVO TRANSMISSION OF DRUG RESISTANCE

amphenicol, 12.5 ,ug of streptomycin, 12.5 ,ug oftetracycline, or 1 mg of sulfathiazole per ml, as

well as the four drugs combined, were preparedand used. Multiplication of the administeredbacteria in intestinal tracts and contaminationof mice with other bacteria were determined byfecal cultures with SS Agar, Desoxycholate Agar,EMB Agar (Nihoneiyokagaku Co., Ltd.), En-terococcus Confirmatory Agar and Broth (Difco),and beef extract-peptone-agar.

Infection in mice. These procedures were car-

ried out by a modification of the technique ofFreter (1956), Cooper (1959), and Cooper andPillow (1959). With two polyvinyl chloridestomach tubes, a germ-free mouse was fed 0.1ml of a 24-hr broth culture of the donor and laterof the recipient without preliminary treatmentwith antibiotics. In conventional mice, 2 daysbefore administration of the donor, 10 mg ofstreptomycin and 2 mg of erythromycin in 0.3ml of water were first given by a stomach tube;later, tap water containing 1.5 mg of streptomycinand 0.025 mg of erythromycin per ml was givenad libitum. At the end of the 2 days, mice were

again given 0.3 ml of a solution containing 10mg of streptomycin and 2 mg of erythromycinby a stomach tube, and with another stomachtube the donor was then given to each animal.The mice were returned to their respective cages,

and given food and water containing strepto-mycin and erythromycin ad libitum, up to 48 hrbefore administration of the recipient. For some

days afterward, fecal cultures of the mice were

carried out every other day, to determine thatthe donors had multiplied and that no contami-nation had occurred. The animals were thentransferred to new cages, and were fed withsterilized food and boiled tap water free fromantibiotics. After 2 days, the recipients were

given, and fecal cultures were carried out every

day for the succeeding 10 days.Fecal culture. A stool pellet obtained directly

from each animal was emulsified with a pestlein a mortar containing 1 ml of sterile saline, and0.1 ml of the emulsion was spread evenly over

the surface of drug-containing and drug-free agarplates by means of bent glass rods. The plateswere then incubated at 37 C for 24 hr, and were

examined for counts of colonies of the recipients.Five colonies from each plate containing thedrugs were then selected at random, and were

isolated and examined for resistance to drugs bythe disk method (Eiken). When the colonies

were fewer than five in number, all were examinedfor their drug resistance. The identities of thecolonies examined were determined by biochemi-cal reactions and slide agglutinations with specificantisera.When the recipient was a Shigella, SS Agar

was used as the drug-containing medium; in thecase of Escherichia or Klebsiella, DesoxycholateAgar was used.

Breeding environment. The animals were bredas germ-free as possible to prevent contamina-tion. For drinking water they were given tapwater sterilized in an autoclave at 121 C for 15min, and for food, germ-free solid food (suppliedby Germfree Life Research Laboratory, NagoyaUniversity School of Medicine) or solid foodsterilized in a hot-air oven for 2 hr. The cages,water bottles, etc., were also sterilized, at 121 Cfor 15 min or at 80 to 100 C for 1 hr. The micewere kept isolated in their own cages with a wire-mesh bottom, under conditions that could mini-mize, if not prevent, coprophagy, and the cageswere placed in a box with commercial lampswhich radiated rays of 2,537 A.

RESULTSIn preliminary experiments, a loopful of a

24-hr broth culture of the recipient was streakedon four plates, each containing a different typeof drug, and on one containing all four drugs.When the plates were incubated at 37 C for 24hr, the recipients did not grow. In addition, aloopful of a 1:1 mixture of a 24-hr broth cultureof donor and recipient was streaked similarly onthe five drug-containing plates. When the plateswere incubated, the donor grew abundantly, butthe recipient did not, as was expected. From theabove, it was concluded that the recipients weresensitive to the drugs, and transfer of drug re-sistance between the experimented strains didnot take place on the plates.

Experiment 1. In experiment 1, transfer ofresistance from S. flexneri 3a to K. pneumoniaewas determined (Table 1). (In this experiment,a germ-free mouse was used.) Throughout theexperiment, both donor and recipient grewabundantly on SS and Desoxycholate Agar freefrom antibiotics. Two or three colonies of therecipient were first found to grow on Desoxy-cholate Agar plates containing chloramphenicol,tetracycline, sulfathiazole, or the four drugs, onthe second day after administration of the re-cipient. Thereafter, there was a tendency for

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TABLE 1. Number of colonies of recipient and donor developing on drug-containing and drug-free mediaafter oral administration to a germ-free mouse of resistant Shigella flexneri Sa strain Kurano

and sensitive Klebsiella pneumoniae strain Kasuya*

Day after challengeOrganism Medium Drugt

1 2 3 4 5 6 7 8 9 10

S. flexneri Sa SS Agar Free: +3 +3 +3 +3 +3 +3 +3 +3 +3 +3

K. pneumoniae Desoxycholate Free$ +3 +4 +4 +4 +3 +4 +4 +3 +3 +3Agar

CM 0 3 16 20 1 77 95 16 13 9TC 0 3 31 37 0 78 72 19 10 12SM 0 0 17 31 0 61 61 15 8 14ST 0 2 22 45 1 72 39 19 1 9CM, TC, 0 3 16 39 0 74 59 7 13 13SM, andST

* Number of visible colonies developing on a plate spread with 0.1 ml of an emulsified stool afterincubation at 37 C for 24 hr.

t CM = chloramphenicol; TC = tetracycline; SM = streptomycin; ST = sulfathiazole. Amount ofCM, TC, and SM in the medium was 12.5 ,Ag/ml, and that of ST was 1 mg/ml.

t The number of visible colonies developing on drug-free media is graded +1 to +4; +1 = <200;+2 = >200; +3 = >+2 plus adherent colonies; +4 = >+3 with most colonies adherent.

TABLE 2. Number of colonies of recipient and donor developing on drug-containing and drug-free mediaafter oral administration to a conventional mouse of resistant Shigella flexneri Sa strain Kurano

and sensitive Escherichia coli strain Ml*

Day after challengeOrganism Medium Drug -

1 2 3 4 5 6 7 8 9 10

S. flexneri 3a SS Agar Free +3 +3 0 +2 +2 +1 +1 +1 +1 +1

E. coli Desoxycholate Free +3 +3 +3 +3 +3 +3 +3 +3 +3 +3Agar

CM 0 0 0 1 4 98 81 222 103 227TC 0 0 0 0 12 96 56 218 103 237SM 0 0 0 0 9 96 81 212 103 226ST 0 0 0 0 5 88 73 209 103 199CM, TC, 0 0 0 0 5 98 83 182 103 248SM, andST

* See footnotes to Table 1.

increase in number with prolongation of culture,but a decrease in number was also seen on cer-tain days of the 8-day period of observation; thenumbers of the colonies on the five drug-contain-ing plates of a definite day were approximatelyequal. The five colonies selected at random fromeach plate were examined for their sensitivitiesto the drugs, and it was found that all colonies

were resistant to the four drugs at an equal leveland in the same pattern.A control experiment, with administration of

K. pneumoniae alone and no donor, failed toshow the growth of resistant colonies.

Experiment 2. Transfer of resistance from S.flexneri 3a to E. coli was determined in experi-ment 2. (In experiments 2 to 6, conventional

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TABLE 3. Number of colonies of recipient and donor developing on drug-containing and drug-free mediaafter oral administration to a conventional mouse of resistant Escherichia coli strain LNH

and sensitive Klebsiella pneumoniae strain Kasuya*

Medium

SS and Desoxy-cholate Agar

DesoxycholateAgar

* See footnotes to Table 1.

Drug

Free

Free

CM

TCSM

STCM, TC,SM, andST

Day after challenge

1

+3

+3

0

0

10

0

mice were used.) As shown in Table 2, largeamounts of both donor and recipient were con-

tinuously excreted. One colony of the recipientwas first found to grow on the DesoxycholateAgar plate containing chloramphenicol on thefourth day after challenge with the recipient.Thereafter, the colonies of the recipient increasedin number, and colonies developing on the fivedrug-containing plates were also found to bealmost equal in number. Examination of theirsensitivities to the drugs revealed all the coloniesto be resistant to the four drugs to an equal level.When E. coli Ml alone was given, no resistant

colony was found to develop.Experiment 3. Transfer of resistance from E.

coli to K. pneumoniae was determined in experi-ment 3 (Table 3). Lactose-nonfermenting E. coliLNH was used as donor. Excretion of the donorand recipient continued, but recipient coloniesdeveloping on the drug-containing plates num-

bered only two during the 10 days of observation,probably owing to the transfer of resistance fac-tors being less frequent here than in the precedingcases. One of the two colonies was resistant tothe four drugs, and the other was resistant onlyto three (streptomycin, tetracycline, and sulfa-thiazole).

In the case of administration of the recipientalone, no resistant colony was found to develop.

Experiment 4. Retransfer of resistance from K.pneumoniae to S. flexneri 2a was attempted inexperiment 4. A resistant K. pneumoniae strain

2

+3

+3

0

0

0

0

0

3

+3

+3

0

0

0

0

0

4

+3

+3

0

0

0

0

0

5

+3

+3

0

0

0

10

6

+3

+3

0

0

0

0

0

7

+3

+2

0

0

0

0

0

8

+3

+2

0

0

0

0

0

9

+3

+1

0

0

0

0

0

10

+3

0

0

0

0

0

0

Kasuya 4R6-1, isolated in experiment 1 from theDesoxycholate Agar plate containing the fourdrugs on the sixth day after the administrationof recipient, was used as donor, and further trans-fer of the acquired resistance was attempted.The recipient used was S. Jlexneri 2a strain K.

Excretion of the donor occurred abundantly,but on a drug-free SS Agar plate the recipientnumbered only about 200. The colonies of therecipient developing on the plates containingdrugs totaled only two, one developing on the10th day and the other on the 15th day afteradministration of the recipient. These resultsindicate probably that the transmission fre-quency between the two strains is less than 102per recipient cell. The two colonies were foundto be resistant to all four drugs.

In the case of administration of the recipientalone, no resistant colony of the recipient was

found to develop during the 15 days after ad-ministration of the recipient.Experiment 5. Retransfer of resistance from E.

coli to S. flexneri 2a was attempted in experiment5. A resistant E. coli strain M1 4R5-1 Ar, iso-lated in experiment 2 from the DesoxycholateAgar plate containing the four drugs on the fifthday after the administration of recipient, was

used as donor. The recipient used was S. flexneri2a strain K.

Excretion of the donor occurred abundantly,but the recipient was excreted poorly and rapidlydisappeared from the feces. Therefore, the re-

Organism

E. coli

K. pneumoniae

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TABLE 4. Number of colonies of recipient and donor developing on drug-containing and drug-free mediaafter oral administration to a conventional mouse of resistant Escherichia coli strain Ml

4R5-l A r and sensitive Shigella flexneri 2a strain Yamada*

Day after challengeOrganism Medium Drug

1 2 3 4 5 6 7 8 9 10

E. coli Desoxycholate Free +2 +2 +1 +3 +2 +2 +3 +3 +3 +3Agar

S. flexneri 2a SS Agar Free +2 +2 +2 +3 +2 +3 +3 +2 +3 +2CM 0 0 0 0 0 0 0 0 2 5TC 0 0 0 0 0 0 0 0 2 3SM 10 500 160 464 103 103 103 103 103 872ST 105 400 300 103 103 10' 103 103 103 103CM, TC, 0 0 0 0 0 0 0 0 0 0SM, andST

* See footnotes to Table 1.

cipient was administered again to the same mouseon the fifth day after the first administration ofrecipient. However, excretion of the recipientwas poor, and the colonies of the recipient failedto develop on SS Agar plates containing drugsduring the 6 days of observation after reinfection.The two colonies of the recipient recovered on

a drug-free S3 Agar plate on the sixth day afterreinfection were therefore isolated and examinedfor their sensitivities to drugs; both were foundto be resistant to streptomycin and sulfathiazole.When the recipient alone was given, no re-

sistant colony was found to develop.Experiment 6. Retransfer of resistance from E.

coli to S. Jlexneri 2a was attempted in experiment6. Excretion of S. flexneri 2a strain K was foundto be poor in experiments 4 and 5; hence, fivesensitive strains of S. flxneri (2a, 2a, 2b, 2b, and3a) were examined for possible multiplication inthe mouse intestine. Two of the five strains, 2aand 2b, were abundantly recovered from thefeces of infected animals pretreated with anti-biotics. One of these two strains, S. flexneri 2astrain Yamada, was therefore used as recipientin this experiment. As donor, E. coli strain Ml4R5-1 Ar was again used.

Large amounts of both donor and recipientwere excreted constantly during the course ofthe experiment, and a large number of resistantcolonies of the recipient were recovered (Table4). Most of these resistant colonies were repeat-edly found to grow on SS Agar containing strep-

tomycin or sulfathiazole, and some were foundto develop first on SS Agar containing chloram-phenicol or tetracycline on the ninth and tenthdays after administration of the recipient. Theformer group of colonies was resistant to twodrugs (streptomycin and sulfathiazole), and thelatter, to the four drugs.When the recipient alone was given, no re-

sistant colony was found to grow.

DISCUSSIONThe preliminary finding that drug resistance

cannot be transferred on SS and DesoxycholateAgar plates was confirmed. It was also proventhat resistant recipients obtained in the presentexperiments were neither spontaneous mutantsnor contaminants, from the control experimentson administration of recipients alone.

In the experiment with a germ-free mouse, theeffect of antibiotics in producing drug-resistantbacteria may be ignored, as no drugs were given.Therefore, the fact that generation of resistantrecipients in the intestinal tract occurred afteradministration of a resistant donor and a sensi-tive recipient indicates clearly the possible trans-fer of drug resistance in vivo between a previouslyexisting donor and a recipient entering subse-quently. In experiments with conventional mice,an antibiotic-free state was produced 2 daysbefore administration of the recipient. Therefore,the results obtained from these cases also indicatethe possible transfer of drug resistance in vivo.

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IN VIVO TRANSMISSION OF DRUG RESISTANCE

TABLE 5. Patterns of acquired resistance*

Donor RecipientExpt no.

Organism Resistant to Organism Resistance acquired

1 Shigella flexneri 3a CM, TC, SM, ST K. pneumoniae strain CM, TC, SM, STstrain Kurano Kasuya

2 S. flexneri 3a strain CM, TC, SM, ST E. coli strain Ml CM, TC, SM, STKurano

3 Escherichia coli strain CM, TC, SM, ST K. pneumoniae strain (1) TC, SM, STLNH Kasuya (2) CM, TC, SM, ST

4 Klebsiella pneumo- CM, TC, SM, ST S.flexneri 2a strain K CM, TC, SM, STniae strain Kasuya4R6-1

5 E. coli strain Ml CM, TC, SM, ST S.flexneri 2a strain K SM, ST4R5-1 Ar

6 E. coli strain Ml CM, TC, SM, ST S. flexneri 2a strain (1) SM, ST4R5-1 Ar Yamada (2) CM, TC, SM, ST

* CM = chloramphenicol; TC = tetracycline; SM = streptomycin; ST = sulfathiazole.

The germ-free cages used were structurally notperfect, but were believed to prevent bacterialcontamination of mice. Mice exposed to ultra-violet rays and poorly nourished were attackedwith alopecia, dermatitis, and ophthalmia, anddid not increase in body weight, but did not dieduring the course of experiments.

Experiments 4 and 5 showed failure of multi-plication of Shigella in the intestines of mice.When the susceptibilities of mice to enteric in-fections were examined with five strains of Shi-gella, it was found that two strains, 2a and 2b,could continue to be recovered in large numbersfrom the feces, but the remaining three strains,2a, 2b, and 3a, disappeared from the feces within2 or 3 days. Hence, the existence of a relation-ship between possible multiplication of Shigellain the intestine of mice and their serotypes wasnot noted. These findings suggest that selectionof a strain which may multiply in the intestinaltract of an experimental animal is necessary forthe success of this type of investigation. In theother four experiments, a considerable number ofrecipient colonies grew on the plates and resistantcolonies of the recipients could be selected andisolated, so technical difficulties did not arise.

All the results obtained are briefly summarizedin Table 5. Resistance factors were transferred inall cases, and acquired resistance was furthertransferred in experiments 4, 5, and 6. In four ofthe six experiments, bacteria to which resistancehad been transferred showed single patterns and

levels of resistance common to each group. Inthe remaining two, the patterns of resistance wereof two types but the levels of resistance to specificdrugs were equal. It was thus found that transferof drug resistance in vivo occurs according to adefinite mechanism, and it is believed that themechanism is cell-to-cell contact. In experiments1 and 2, and 5 and 6, the patterns of acquiredresistance were found to be exactly the same,when resistance factors were passed from thesame donor to recipients of a different genus.From experiments 1 and 3, and 4 and 5, if trans-fer in vivo occurred between donors of a differentgenus and the same recipient, the patterns ofacquired resistance might differ, or be the same.It seems, therefore, that the pattern of acquiredresistance is determined more by the donor thanby the recipient.Watanabe and Fukasawa (1961a, b) reported

that the level of the resistance transferred bymixed cultivation might differ from recipient torecipient. In the present study, however, thelevel did not differ from recipient to recipient, apoint that needs more detailed investigation.

In two experiments, bacteria to which resist-ance had been transferred showed resistance totwo drugs only, streptomycin and sulfathiazole.These results support the suggestion by Ochiai,Yamanaka, and Kimura (1961) that resistancefactors of streptomycin and sulfathiazole aretransferred together.The numbers of colonies of the recipients which

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J. BACTERIOL.

grew on plates containing drugs were comparedwith those recovered from the same feces spreadon drug-free plates on a definite day, and theirratios were obtained with the latter as denomi-nator. The results were 10-3 to 10-5 per recipientcell, but in experiment 6 the ratio was 10-2.Harada et al. (1961a, b, c) reported that thisratio is 10-4-5 to 10-6 per recipient cell in vitro,so transfer of resistance in vivo may occur atthe same frequencies as in vitro.From the results obtained above, it may be

considered that in the human intestines too, ifdonor and recipient multiply together, resistancefactors may be transferred also. Thus, generationof drug-resistant enteric bacteria in the humanintestine may occur owing to the transfer of re-sistance from one previously existing entericbacteria to a newcomer, or vice versa, from anewly arriving resistant bacteria to a previouslyexisting enteric bacteria.

ACKNOWLEDGMENTS

The author wishes to express his sincere ap-preciation to K. Ogasawara of the Departmentof Bacteriology, Nagoya University School ofMedicine, and to K. Ochiai of the Nagoya Mu-nicipal Higashi Shimin Hospital, for their encour-agement and helpful discussions in conductingthe present study; to M. Miyagawa of the Germ-free Life Research Laboratory, Nagoya Univer-sity School of Medicine, for supplying germ-freemouse and food; and to S. Naito of the NagoyaMunicipal Higashi Shimin Hospital for supplyingShigella strains.

LITERATURE CITEDAKIBA, T., T. KOYAMA, Y. ISSHIKI, S. KIMURA,

AND T. FUKIuSHIMA. 1960. Studies on the mech-anism of development of multiple drug-re-sistant Shigella strains. Nihon Iji Shimpo1866:45-50.

AKIBA, T., K. KOYAMA, S. KIMURA, AND T. FUKIu-SHIMA. 1961. Studies on the mechanism oftransfer of drug-resistance in bacteria. 4. Ex-periments on the transfer of drug-resistancein vivo. Med. Biol. (Tokyo) 59:185-188.

COOPER, G. N. 1959. Experimental shigellosis inmice. 1. Chronic infection with Shigella dysen-teriae type 2. Australian J. Exptl. Biol. Med.Sci. 37:193-200.

COOPER, G. N., AND J. A. PILLOW. 1959. Experi-

mental shigellosis in mice. 2. Immunologicalresponses to Shigella dysenteriae type 2 in-fection. Australian J. Exptl. Biol. Med. Sci.37:201-209.

FRETER, R. 1956. Experimental enteric Shigellaand Vibrio infections in mice and guinea pigs.J. Exptl. Med. 104:411-418.

HARADA, K., M. KAMEDA, M. SUZUKI, AND S.MITSUHASHI. 1961a. On the drug-resistance ofenteric bacteria between Shigella and E. coli.Japan J. Bacteriol. 16:6-16.

HARADA, K., M. KAMEDA, M. SUZUKI, H. HASHI-MOTO, AND S. MITSUHASHI. 1961b. On the drug-resistance of enteric bacteria. 1. Effect ofprotease, deoxyribonuclease, and ribonucleaseon the transmission of drug-resistance bymixed cultivation. Japan J. Bacteriol. 16:823-826.

HARADA, K., M. KAMEDA, M. SUZUKI, R. EGAWA,AND S. MITSUHASHI. 1961c. Studies on thedrug-resistance of enteric bacteria. 10. Rela-tion between transmissible drug-resistance(R) factor and fertility (F) factor. Japan. J.Exptl. Med. 31:291-299.

KAGIWADA, S., S. KATO, M. ROKUGO, J. HOSHINO,AND K. NISHIYAMA. 1960. Studies on the mech-anism of generation of drug-resistant Shigellain intestines. On the possibility of the trans-mission of drug-resistance in intestines fromresistant E. coli to sensitive Shigella. NihonIji Shimpo 1886:5-9.

KASUYA, M. 1962. The transmission of the drugresistance of enteric bacteria in the intestineof germ-free and conventional mice. Japan.J. Bacteriol. 17:687-694.

OCHIAI, K., T. YAMANAKA, K. KIMURA, AND 0.SAWADA. 1959. Studies on inheritance of drug-resistance between Shigella strains and Esch-erichia coli strains. Nihon Iji Shimpo 1861:34-46.

OCHIAI, K., T. YAMANAKA, AND K. KIMURA. 1961.Combination of drug-resistance betweenenteric bacteria by mixed culture. Nihon IjiShimpo 1935:38-42.

WATANABE, T., AND T. FUKASAWA. 1961a. Epi-some-mediated transfer of drug resistance inEnterobacteriaceae. I. Transfer of resistancefactors by conjugation. J. Bacteriol. 81:669-678.

WATANABE, T., AND T. FUKASAWA. 1961b. Epi-some-mediated transfer of drug resistance inEnterobacteiaceae. II. Elimination of resist-ance factors with acridine dyes. J. Bacteriol.81:679-683.

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