INFREQUENT APPEARANCE OF DRUG RESISTANT STRAINS OF TUBERCLE BACILLI IN EXPERIMENTAL ANIMALS TREATED WITH STREPTOMYCIN AND p-AMINO-SALICYLIC ACID1' 2 EMANUEL WOLINSKY AND WILLIAM STEEN:KEN, JR. The Trudeau Laboratory of The Trudeau Foundation for the Clinical and Experimental Study of Pulmonary Disease, Trudeau, New York Received for publication February 23, 1953 The emergence of drug resistant strains of tubercle bacilli has proved to be one of the main drawbacks to the successful use of antimicrobial therapy in pulmonary tuberculosis. Resistant strain also may be obtained readily by in vitro methods. Nevertheless, it has proven difficult to study this phenomenon experimentally because of the inability to induce regularly a high degree of drug resistance in cultures subjected to ex- posure to drugs in the animal body. This report summarizes the results of drug susceptibility determinations of several different strains of tubercle bacilli recovered from guinea pigs, mice, and rabbits after prolonged treatment. MATERIALS AND METHODS Mice. For recovering tubercle bacilli from mice the following method was used: One-half of the spleen and one entire lung were ground in a sterile mortar with a small amount of sterile physiological saline to produce a thick paste. After a little further dilution, approximately 1.0 ml of this suspension was injected sub- cutaneously in the inguinal region of each of 2 guinea pigs. Cultures were made directly from this suspension by planting a double loopful on solid medium. The material then was digested with 3 per cent sodium hydroxide, concen- trated, and again planted on tubes of solid medium. Rabbits and guinea pigs. Caseous material when available was removed aseptically with a platinum spade, emulsified in a small amount of sterile physiological saline, and planted directly on tubes of solid medium before and after sodium 1 This study was aided by grants from the Division of Research Grants and Fellowships of the National Institutes of Health, Public Health Service. ' Presented in part at the 52nd annual meeting of the Society of American Bacteriologists, Bos- ton, April, 1952. hydroxide digestion. Other lesions from various organs were ground in a mortar and handled in the same manner as described under mice. Material for guinea pig inoculation always was removed before any sodium hydroxide was added. The egg-yolk, potato, glycerin medium recom- mended by the American Trudeau Society (1946) was used. Direct drug sensitivity tests were made at the time of the original planting by distributing equal portions of the material on tubes of medium containing graded concentrations of the drug as well as on control tubes without drug. Sufficient streptomycin was added to the medium before inspissation to give concentrations of 10, 50, and 500 pg per ml Chemical analysis of the final medium indicates that the concentrations of available streptomycin are then approximately 3.5, 15, and 200 ug per ml, respectively. The p-amino-salicylic acid concentrations used were 1.0, 10, and 100 pg per ml. Comparison was made of the lag period and the final amount of growth in each of the drug containing tubes with that in the control tubes. The amount of growth of tubercle bacilli was graded according to the following scale: Actual count of colonies if 50 or less; 1+ for over 50 to 200 colonies; 2+ for several hundred discrete colonies; 3+ for innumerable colonies covering the inoculated surface; 4+ for confluent growth covering the entire inoculated surface. Indirect or subculture sensitivity tests were made by subculturing into liquid sorbitan mono- oleate albumin medium a representative sample of the growth occurring on the nondrug contain- ing tubes and then testing the culture so obtained. In the case of streptomycin the testing was carried out in a series of tubes containing graded concentrations of the drug in the same liquid medium. Incubation was carried on for 14 days at 37.5 C. For p-amino-salicylic acid the tests were done on solid Herrold's egg-yolk agar medium with an incubation time of 35 days. 229 on December 20, 2018 by guest http://jb.asm.org/ Downloaded from
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INFREQUENT APPEARANCE OF DRUG RESISTANT STRAINS OF TUBERCLEBACILLI IN EXPERIMENTAL ANIMALS TREATED WITHSTREPTOMYCIN AND p-AMINO-SALICYLIC ACID1' 2
EMANUEL WOLINSKY AND WILLIAM STEEN:KEN, JR.The Trudeau Laboratory of The Trudeau Foundation for the Clinical and Experimental
Study of Pulmonary Disease, Trudeau, New York
Received for publication February 23, 1953
The emergence of drug resistant strains oftubercle bacilli has proved to be one of the maindrawbacks to the successful use of antimicrobialtherapy in pulmonary tuberculosis. Resistantstrain also may be obtained readily by in vitromethods. Nevertheless, it has proven difficult tostudy this phenomenon experimentally becauseof the inability to induce regularly a high degreeof drug resistance in cultures subjected to ex-posure to drugs in the animal body. This reportsummarizes the results of drug susceptibilitydeterminations of several different strains oftubercle bacilli recovered from guinea pigs, mice,and rabbits after prolonged treatment.
MATERIALS AND METHODS
Mice. For recovering tubercle bacilli from micethe following method was used: One-half of thespleen and one entire lung were ground in asterile mortar with a small amount of sterilephysiological saline to produce a thick paste.After a little further dilution, approximately1.0 ml of this suspension was injected sub-cutaneously in the inguinal region of each of 2guinea pigs. Cultures were made directly fromthis suspension by planting a double loopful onsolid medium. The material then was digestedwith 3 per cent sodium hydroxide, concen-trated, and again planted on tubes of solidmedium.
Rabbits and guinea pigs. Caseous materialwhen available was removed aseptically with aplatinum spade, emulsified in a small amount ofsterile physiological saline, and planted directlyon tubes of solid medium before and after sodium
1 This study was aided by grants from theDivision of Research Grants and Fellowships ofthe National Institutes of Health, Public HealthService.
' Presented in part at the 52nd annual meetingof the Society of American Bacteriologists, Bos-ton, April, 1952.
hydroxide digestion. Other lesions from variousorgans were ground in a mortar and handled inthe same manner as described under mice.Material for guinea pig inoculation always wasremoved before any sodium hydroxide was added.The egg-yolk, potato, glycerin medium recom-
mended by the American Trudeau Society (1946)was used. Direct drug sensitivity tests were madeat the time of the original planting by distributingequal portions of the material on tubes of mediumcontaining graded concentrations of the drug aswell as on control tubes without drug. Sufficientstreptomycin was added to the medium beforeinspissation to give concentrations of 10, 50,and 500 pg per ml Chemical analysis of thefinal medium indicates that the concentrationsof available streptomycin are then approximately3.5, 15, and 200 ug per ml, respectively. Thep-amino-salicylic acid concentrations used were1.0, 10, and 100 pg per ml.Comparison was made of the lag period and
the final amount of growth in each of the drugcontaining tubes with that in the control tubes.The amount of growth of tubercle bacilli wasgraded according to the following scale: Actualcount of colonies if 50 or less; 1+ for over 50to 200 colonies; 2+ for several hundred discretecolonies; 3+ for innumerable colonies coveringthe inoculated surface; 4+ for confluent growthcovering the entire inoculated surface.
Indirect or subculture sensitivity tests weremade by subculturing into liquid sorbitan mono-oleate albumin medium a representative sampleof the growth occurring on the nondrug contain-ing tubes and then testing the culture so obtained.In the case of streptomycin the testing wascarried out in a series of tubes containing gradedconcentrations of the drug in the same liquidmedium. Incubation was carried on for 14 daysat 37.5 C. For p-amino-salicylic acid the testswere done on solid Herrold's egg-yolk agarmedium with an incubation time of 35 days.
All of the human strains of Mycobacterium tu-berculosis used in these experiments were in-hibited by 0.5 Mg of streptomycin or 1.0 ,ug ofp-amino-salicylic acid per ml of medium whentested in the manner described.The degrees of resistance to streptomycin were
defined as follows:Sensitie: Growth in the control tubes only in a
direct test, or inhibition of growth by 1.0 ug perml in liquid medium.
Slight loss of susceptibility or borderline re-sistance: Appreciable growth in the first tubeonly (containing 3.5 Mug of streptomycin per ml)in the direct test, or resistance to 1.0 but growthinhibition by 5.0 Mug per ml in the subculturetest.
Moderately resistant: In the direct test, com-parable growth in the control tubes and the tubescontaining 3.5 and 15 Mug of streptomycin perml, and resistant to at least 5.0 MAg per ml, butstill susceptible to 30 ,g per ml in thesubculturetest.
Highly resistant: Comparable growth in thecontrol tubes and all the streptomycin containingtubes in the direct test, and growth in 30 or moreMug per ml in liquid medium.
EXPERIMENTAL RESULTS
Guinea pigs. In the first guinea pig experimenttreatment with streptomycin was started 6weeks after subcutaneous infection with thehuman virulent strain, H37Rv. The streptomy-cin was given intramuscularly in doses varyingfrom 7.5 to 30 mg per day, but since there was nosignificant variation in the results obtained withthe different doses, the treated animals areconsidered as a single group. Certain animalswere killed at various intervals after strepto-mycin was begun; a few died of causes otherthan tuberculosis. The survivors were sacrificedafter 575 consecutive days of treatment. Theresults of the streptomycin sensitivity tests of 85strains recovered from 79 guinea pigs (in sixinstances 2 cultures were obtained from oneanimal) are shown in table 1.There were 43 such strains recovered from
animals which had been treated for 90 days orless; 41 of these were still completely sensitiveand 2 showed a borderline resistance. Of theanimals treated longer than 135 days, 3 out of42 cultures showed a definite resistance of eithermoderate or high degree; 22 exhibited a border-
line resistance or only slight loss of susceptibility;and 17 were still completely sensitive. Thus,although the animals treated for longer periodsof time tended to yield more strains exhibitingslight loss of susceptibility, the growth of virtu-ally all of the cultures still could be inhibited by5.0 ,ug of streptomycin per ml in liqulid medium.
In another experiment a deliberate attemptwas made to produce discrete, chronic, abscess-like lesions which would harbor many multiplyingtubercle bacilli for a long period of time. Theselesions were produced by subcutaneous infectionwith a virulent human strain (H37Rv) of guineapigs previously vaccinated with the avirulent
bacilli recovered from guinea pigs with acute tu-berculosis treated with streptomycin for variousperiods of time. Results of subculture tests inliquid sorbitan monooleate medium.
O.NO. RE- NO. 32-
DAYS OCI NO. OP SNO SIS1TAT SITAN NO.8M-S=roxMcn TU._w o 1, Tro 5, SISTA?4TRETMNT TOS 1 SENSO SENSFE To 30
strain, H37Ra, as described previously (Steenkenet al., 1952a). Ninety-seven days after infectionstreptomycin was started in dosage of 15 mgonce daily intramuscularly and continued for 300consecutive days. Table 2 shows the results ofstreptomycin sensitivity tests of the strains oftubercle bacilli recovered from 5 chronic abscess-like lesions in 3 guinea pigs after 300 days ofstreptomycin treatment.
It may be seen that all 5 strains were stillcompletely sensitive to streptomycin. Theamount of growth obtained in the control tubesfrom 4 of these lesions was moderately heavy,indicating that large numbers of viable tuberclebacilli were present in the caseous material fromthese lesions.
Mice. The finding of drug resistant strains oftubercle bacilli in mice treated with streptomycin
A92 Liver abscess 2+ 2+ 0 0 0 Not testedSpleen 10 15 0 0 0 Resistant to 0.5,
sensitive to 11-g/m
A95 Liver abscess 2+ 3+ 0 0 0 Not tested
TABLE 3Drug sensitivity of strains of HS7Rv tubercle bacilli recovered from dba mice treated with streptomycin or
p-amino-salicylic acid
DAYS 01 NO. OLP NO.TREAMNTCULTREDPOSITIVE DEUG SENSITIVIT
p-Amino-salicylic acid subcutane- 42 5 5 All sensitive to p-amino-salicylicously 5 mg once daily acid
Streptomycin subcutaneously 1 mg 42 5 5 All sensitive to streptomycintwice daily
Streptomycin subcutaneously 1.5 70 2 2 Both sensitive to streptomycinmg once daily
Streptomycin subcutaneously 1.5 198 8 6 All completely sensitive to strep-mg once daily tomycin by direct test, inhibited
by 0.5 pg/ml in liquid mediumsubculture test
p-Amino-salicylic acid in water or 198 8 8 All sensitive to p-amino-salicylicdiet acid
attempt to confirm these findings the two follow-ing experiments were carried out:
First, a subacute infection was produced indba mice (Rockland Farms) by intravenousinfection with 0.1 mg (dry weight) of H37Rvmicroorganisms. Treatment was started im-mediately after infection with either 1.0 mg ofstreptomycin subcutaneously twice daily, 1.5mg of streptomycin once daily subcutaneously,
in the amount of 0.7 per cent in the drinkingwater. After 42 days only 10 per cent of thecontrol untreated mice had died. At this time5 animals treated with p-amino-salicylic acidand 5 treated with streptomycin were sacrificed.Tubercle bacilli obtained from each of these 10mice were still completely sensitive to p-amino-salicylic acid or streptomycin. None of the 16cultures isolated from mice treated for 70 or 198
days showed any resistance to streptomycin orp-amino-salicylic acid. These results are shownin table 3.In a second experiment a total of 81 C57 black
mice were infected intravenously, 27 with 0.25mg (dry weight) of each of the following humanvirulent strains of tubercle bacilli: H37Rv,Campbell, and Erdman. Treatment was startedimmediately after infection with either strepto-mycin alone in dosage of 1.5 mg once a day
tissues of the treated mice, and the streptomycinsusceptibility (subculture method) of each re-covered strain.
It may be seen that tubercle bacilli werecultured from all but one of the 54 treated mice.For each of the infecting strains there was onlyone mouse from which moderately or highlyresistant organisms were obtained. Of the 53positive cultures, only 3 (6 per cent) were moder-ately or highly resistant; 32 (60 per cent) ex-
TABLE 4C-57 black mice infected intravenously with 3 strains of human virulent tubercle bacilli, killed qfter 154 to
160 days of streptomycin or streptomycin-p-amino-salicylic acid treatment. Results of subculturestreptomycin susceptibility tests of recovered strains
SUBCULTURE STREPTOMYCIN SENSITIVITY,
NO. OF LIQUID MDI
ORGANISN TREATMENT CL POSI- Resistant ResistantTURED TIVE Sensitive to 1, to 5 Resistant
to 1 jg/ml sensitive sensitive to 30 jg/mlto S g/ml to 30 pg/ml
* Difference not significant by Chi square and standard error tests.
subcutaneously, or a combination of the sameamount of streptomycin plus p-amino-salicylicacid in the amount of 0.7 per cent in the drinkingwater (approximately 1,500 mg of p-amino-salicylic acid per kg per day). In this experiment80 per cent of the control untreated animalsdied within 87 days, the mean survival time foreach group being 69 days for the H37Rv-infectedanimals, 68 days for those infected with theCampbell strain, and 58 days for those infectedwith the Erdman strain. Ninety per cent of thetreated animals remained alive when the experi-ment was terminated after 160 days. Table 4shows the results of the cultures made from the
hibited a slight loss of susceptibility but werestill inhibited by 5.0 pg of strepomycin per ml;and 18 (34 per cent) were still completely sensi-tive to streptomycin. About half of the testswere repeated in modified Proskauer and Beck'sliquid synthetic medium, and none demonstratedany more drug resistance by this method. Theresults of the direct tests on solid medium werenot significantly different, only 4 cultures (7.5per cent) showing a moderate or high degree ofresistance. All of the cultures remained sensitiveto p-amino-salicylic acid.The difference in the streptomycin suscepti-
bility of strains recovered from mice treated with
19531 APPEARANCE OF DRUG RESISTANT STRAINS OF TUBERCLE BACILLI
streptomycin alone as compared with those now been recovered from rabbits treated fortreated with the combination of streptomycin prolonged periods with antituberculous agentsand p-amino-salicylic acid was not striking. Of after acute miliary tuberculosis had been es-
TABLE 5Streptomycin seMitivity of tubercle bacilli recovered from rabbits infected intravenously with the virulent
bovine Ravenel strain. Treatment started after miliary pulmonary disease established
STTOYYCIN SENSITIVrTY
Direct, solid medium Subcultuep,RABBIT TATM Uul
liquid mediumNO.
Control tubes gg of strepto- Resis- Sensi-Control__tubes _mycm per mltant to tive to
virulent bovine strain, Ravenel. The infectingdose was 0.01 mg, dry weight. Treatment wasdelayed until advanced pulmonary disease ofthe miliary type was evident in the chest roent-genograms (Steenken et al., 1952b), usually2 to 4 weeks after infection. Therapy consistedof intramuscular injections of 75 or 150 mg of
cultures of tubercle bacilli recovered from 10different rabbits, 9 having been treated withoutinterruption for at least 1 year, none was moder-ately or highly resistant to streptomycin. Nineof the cultures, however, showed a slight loss ofsusceptibility or borderline resistance. The caseof rabbit no. 128 is especially interesting. After
TABLE 6Streptomycin sen8itivity of tubercle bacilli recovered from rabbits infected by inhalation with the virulent
bovine Ravenel strain. Treatment started after advanced caseous and cavitary lesions demonstratedby chest roentgenography
STRPTOYMYI SEIrNSIIVT
Direct, solid medium Subculture,RABBITZTIN ULut liquid mediumNO.
Control tubes pgo tet-Resist- Sensi-_________________ my_m__per_ml ant to tive toNo. 1 No. 2 3.5 15 200 _9g/ml pg/mI
9259-496 Streptomycin 150 mg once Lung, ten- 1+ 1+ 0 0 0 0.5 1.0daily 61 days sion cavity
* Tubercle bacilli recovered by guinea pig subinoculation.
streptomycin once daily, or the combination of75 mg of streptomycin plus 300 mg of viomycinintramuscularly once daily. Although the miliarylesions in the lungs generally responded verywell, several rabbits developed caseous abscessesin the eyes and in the testicles while under treat-ment. In one (no. 837) a residual pulnonarytuberculous cavity remained after 1 year oftreatment. Others yielded cultures positive fortubercle bacilli from small residual necroticlesions in the lungs.From table 5 it may be seen that of 18 positive
1 year of streptomycin therapy, positive cultureswere obtained from residual lesions in the lungsand from caseous abscesses in both eyes and inboth testicles. All 5 cultures showed a slight lossof susceptibility, requiring 2.5 ug of streptomycinper ml of liquid medium to inhibit growth. Rab-bit no. 391 also was treated with streptomycinfor 1 year, after which positive cultures wereobtained from the lung, from an abscess in theleft testicle, and from a caseous lesion in the lefteye. All 3 of these cultures were still sensitive tostreptomycin.
1953] APPEARANCE OF DRUG RESISTANT STRAINS OF TUBERCLE BACILLI
Other rabbits have been infected by the in-halation of aerosolized suspensions of the Rave-nel strain of bovine tubercle bacilli and treat-ment delayed until chronic caseous pneumonicand cavitary lesions were evident in the chestroentgenograms. The results of such treatmentwill be described elsewhere (Steenken et al.,1953). Residual cavitary lesions remained insome animals after 1 year of antimicrobialtherapy. Table 6 shows the results of culturesmade from these lesions and the drug sensitivityof the recovered tubercle bacilli.
It may be seen that from one rabbit (no. 492),which had a large persistent pulmonary cavityafter 8 months of streptomycin treatment,the recovered tubercle bacilli were moderatelyresistant to streptomycin. Of the other 5 posi-tive cultures, however, 3 still retained sensitivityto streptomycin, and 2 showed only a slight lossof susceptibility.
DISCSSION
It is evident from these results that insofaras the emergence to predominance of drug re-sistant populations is concerned, tubercle bacillibehave differently in experimental animals thanin human beings with pulmonary tuberculosis.Using the same techniques as those describedhere, it was found that the majority of patientswith pulmonary tuberculosis who still had posi-tive cultures after 4 months of treatment withstreptomycin alone yielded strains of tuberclebacilli with a moderate or high degree of resist-ance to the drug (Bernstein et al., 1948).
In guinea pigs with an acute tuberculous in-fection, prolonged treatment with streptomycindoes not favor the production of highly drugresistant strains of tubercle bacilli in any but avery small percentage of animals. Realizing thatthe type of lesion from which streptomycinresistant organisms are recovered most rapidlyand most frequently is the chronic pulmonarycavity, it was thought that perhaps a chronicabscess-like lesion in the guinea pig might yieldresistant organisms. Accordingly, the experimentwas set up whereby chronic caseous lesions wereproduced in the organs of guinea pigs whoseresistance to infection had been increased byprevious vaccination. Even after prolongedstreptomycin treatment of guinea pigs withsuch lesions, however, and despite the fact thatlarge numbers of tubercle bacilli were recovered
from these chronic lesions at the end of treatment,the recovered organisms still retained theirsensitivity to the drug.
It is difficult to explain the discrepancy in theresults reported herein and those obtained byYoumans and co-workers (Youmans et al., 1949;Williston and Youmans, 1950), who describedthe finding of streptomycin resistant strains in alarge percentage of mice treated with strepto-mycin for 155 days. Their sensitivity tests wereperformed by subculture method, using eithermodified Proskauer and Beck's liquid mediumwith serum or solid egg-yolk agar medium. Al-though the results obtained by these methodswould not be expected exactly to duplicate thosefound with the technique described in this paper,it has been the experience of this laboratory thatthe differences would not be great enough toaccount for the over-all discrepancy. One pointof definite variance was the severity of theinfection as indicated by the mean survival timeof the untreated control mice: 14.6 days in You-mans' first experiment; 44.4 in his second; and58 to 69 in the second experiment reported here.It is possible that larger infecting inocula willbe associated with a higher incidence of recover-able drug resistant populations of tubercle bacilli.At any rate, the combined administration of
streptomycin and p-amino-salicylic acid doesnot have the same effect on the emergence ofdrug resistant organisms in mice as it does inhuman beings. In the latter it is well establishedthat the combined treatment delays the emer-gence to predominance of drug resistant tuberclebacilli, whereas in mice it has been demonstratedby Williston and Youmans (1950), and confirmedin the experiments reported above, that theaddition of p-amino-salicylic acid has very littleif any effect on the emergence of drug resistance.
It is not within the scope of this paper to dis-cuss the mechanisms of drug resistance. It seemsclear, however, the the generally accepted theoryof spontaneous mutation and natural selectionis not adequate by itself to explain all thesefindings and that certain host factors must beplaying an important role. It is also clear thatinsofar as the development of drug resistantstrains is concerned, the experience gained fromthe described treatment of experimental tuber-culosis in animals may not be transferred tohuman beings.
caseous and cavitary pulmonary lesions resem-bling those in human beings and treated forprolonged periods with streptomycin would yielddrug resistant tubercle bacilli frequently fromthe residual cavities. The results so far have notbeen convincing, in that only one rabbit hasyielded a strain with definite resistance tostreptomycin, but these experiments have beenextended and are continuing. It would certainlybe a great help in the study of this problem ifdrug resistant cultures could be produced withsome degee of regularity in the experimentalanimal.
ACKNOWLEDGMENT
It is a pleasure to acknowledge the technicalaistance of William J. Costigan and MarjorieM. Smith.
Streptomycin, viomycin, and p-amino-salicylicacid were supplied by Chas. Pfizer & Co.,Brooklyn, New York.
SUMMLARY
Moderately or highly drug resistant strains oftubercle bacilli were found only rarely in mice,guinea pigs, and rabbits treated for prolongedperiods.
Guinea pigs. Guinea pigs with acute experi-mental tuberculosis produced by subcutaneousinfection with the H37Rv strain were treatedwith streptomycin for various periods up to 575days starting 6 weeks after infection. Of 43positive cultures obtained from animals treatedfor 35 to 90 days, all but 2 were completely sensi-tive to streptomycin, the 2 showing only a slightloss of susceptibility. Of 42 cultures obtainedfrom guinea pigs treated for 135 to 575 days, 3showed moderate or high resistance to strepto-mycin, 22 showed a slight loss of susceptibility,and 17 were still completely sensitive to strepto-mycin. Five positive cultures of tubercle bacillirecovered from residual chronic abscesses in 3guinea pigs after 300 days of streptomycin treat-ment were all sensitive to streptomycin.
Mice. In a subacute infection of dba mice,no drug resistant cultures were found after 198days of streptomycin or p-amino-salicylic acidtreatment. Groups of C57 black mice wereinfected with 3 different strains of tubercle bacilliand treated with streptomycin alone or thecombination of streptomycin and p-amino-
salicylic acid for 160 days, after which viablecultures were obtained from all but one of 54treated mice. Only 3 showed a moderate or highdegree of streptomycin resistance, and 32 a slightloss of susceptibility. The ministration of p-amino-salicylic acid apparently had no significanteffect on the streptomycin resistance.
Rabbits. Eighteen positive cultures were ob-tained from 10 rabbits infected intravenouslywith virulent bovine tubercle bacilli of theRavenel strain and treated for periods up to 16months with streptomycin or the combinationof streptomycin and viomycin. None of thesecultures proved to be moderately or highlyresistant to streptomycin; 9 showed a slight lossof susceptibility or borderline resistance. Inanother group of rabbits with chronic caseousand cavitary lung lesions, resistant tuberclebacilli were obtained from one after 8 months ofstreptomycin treatment, and sensitive or onlyslightly resistant cultures from 4 others aftertreatment for as long as one year.Moderately or highly resistant strains of
tubercle bacilli have been obtained from humanbeings with pulmonary tuberculosis treated withstreptomycin alone in a high percentage of cases,using the same methods as those described here.It is thus apparent that there are certain factorsoperating in experimental tuberculosis of ani-mals, as contrasted with the disease in man,which tend to suppress the emergence of a highdegree of bacterial resistance during drug treat-ment. Further investigation of these factors maybroaden our understanding of the whole phe-nomenon of drug resistance.
REFERENCESAMERICAN TRIUDEAU SOCETY 1946 Report of
the committee on evaluation of laboratoryprocedures. Am. Rev. Tuberc., 64, 428-432.
BERNSTEIN, S., D'EsoPo, N. D., AND STEENKEN,W., JR. 1948 Streptomycin resistant tuber-cle bacilli. Incidence in patients treated withstreptomycin. Am. Rev. Tuberc., 68, 344-352.
LENERT, T. F., AND HOBBY, G. L. 1949 Strepto-mycin-dependent strains of Mycobacteriumtuberculosi8. Am. Rev. Tuberc., 59, 219-220.
STEENKEN, W., JR., WOLINSKY, E., PRATr, P. C.,AND SNTH, M. M. 1952a Streptomycin inguinea pigs with discrete chronic tuberculouslesions. Am. Rev. Tuberc., 66, 194-212.
19531 APPEARANCE OF DRUG RESISTANT STRAINS OF TUBERCLE BACILLI
STEENKEN, W., JR., WOLINSKY, E., BRISTOL, L. J.,AND COSTIGAN,W. J. 1952b Use of the rabbitin experimental tuberculosis; a visual methodof evaluation of antituberculosis agents byserial chest roentgenograms. Trans. of theEleventh Conf. on the Chemotherapy of Tu-berculosis, Veterans Administration-Army-Navy, St. Louis. 266-268.
STEENKEN, W., JR., WOLINSKY, E., AND PRATT,P. C. 1953 To be published.
WILLISTON, E. H., AND YOUMANS, G. P. 1950The inability of p-aminosalicylic acid to de-lay the emergence of streptomycin-resistanttubercle bacilli in mice. Am. Rev. Tuberc.,62, 156-159.
YOUMANS, G. P., WILLISTON, E. H., AND OSBORNE,R. R. 1949 Occurrence of streptomycin re-
sistant tubercle bacilli in mice treated withstreptomycin. Proc. Soc. Exptl. Biol. Med.,70, 36-37.
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