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COMBINEDDRUGTREATMENTOF TUBERCULOSIS. I. PRE-VENTION OF EMERGENCEOF MUTANTPOPULATIONS

OF TUBERCLEBACILLI RESISTANT TO BOTHSTREPTOMYCINAND ISONIAZID

IN VITRO

By MAURICEL. COHN, GARDNERMIDDLEBROOKANDWILLIAM F. RUSSELL, JR.

(Front the Department of Research and Laboratories, National Jewish Hospital at Denver, andthe Departments of Medicine and Microbiology, University of Colorado School

of Medicine, Denver, Colo.)

(Submitted for publication September 10, 1958; accepted February 23, 1959)

Treatment of tuberculosis with single drug regi-mens employing streptomycin, para-aminosali-cylic acid, or isoniazid has proved to be clinicallyinferior to the use of combinations of these drugs(1-3). The superiority of combined drug regi-mens has been manifest both in the percentage ofpatients rendered bacteriologically negative andin the lower incidence of the emergence of drugresistance.

Ehrlich (4) first conceived the generalizationthat chemotherapy combining two or more effectiveagents with independent actions would prove su-perior to single drug treatment by tending toprevent the parasite from developing resistance tothe action of either of the drugs. This principlewas applied to antimicrobial attack in vitro onMycobacterium tuberculosis by Middlebrook andYegian (5), who also outlined the theoretical basisfor prevention of the emergence of drug-resistantmutants of tubercle bacilli by combined drug treat-ment. Demerec (6) employed more refinedgenetic principles to elaborate upon this theory.Then Karlson and co-workers (7) demonstratedthe suppressive effect of combined drugs (strepto-mycin, para-aminosalicylic acid and promin) uponthe emergence of streptomycin-resistant tuberclebacilli during the chemotherapy of tuberculosis inman. Since that time many studies pertaining tocombined drug therapy have been reported bothfrom the laboratory and from clinical investigation.

Thus, although it has been impossible to provideclinical evidence for superiority of combined drugtreatment in many other infectious diseases, thereseems to be no doubt of its superiority over theuse of single drugs in cavitary pulmonary or renaltuberculosis. Nevertheless, in spite of this estab-

lished superiority of combined drug regimens, thesputa of 10 to 20 per cent of previously untreatedpatients fail to become bacteriologically negative,on conventional double-drug programs, withoutancillary collapse procedures or surgical interven-tion (8-10). Many of these chemotherapeuticfailures are the result of the emergence of mutantforms 1 of tubercle bacilli which are resistant toone or more of the antimicrobial agents employed(12, 13).

Clinical investigation and laboratory studieshave established the fact that the action of a singleantimicrobial agent in adequate concentration invitro will usually select a drug-resistant popula-tion of tubercle bacilli if, initially, the number oforganisms is sufficiently large, and if they multi-ply to yield a detectable colony. Such investiga-tions depend upon laboratory determination of theprevalence of drug-resistant mutants in large pop-ulations of the parasites. Most attempts to esti-mate the prevalence of drug-resistant mutantshave been performed with cultures containing rela-tively small numbers of organisms, in liquid media,and with techniques requiring repeated subculture.

1 The advisability of employing the term mtutantts todescribe typical tubercle bacilli which are found to be re-sistant to the antimicrobial action of a drug may be ques-tioned. This usage has been justified on the followinggrounds: 1) Such organisms immediately proliferate frompreviously unexposed populations of organisms just asthough they were present in such populations prior toselection by the suppressive action of the drug on the sus-ceptible organisms. 2) All other peculiarities of theirproperties are just as immediately apparent. 3) In thecase of one strain of tubercle bacilli this has been provedby replica plating techniques which allow for preciseestimation of the drug susceptibility of clones before anydrug action (11).

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MAURICE L. COHN, GARDNERMIDDLEBROOKAND WILLIAM F. RUSSELL, JR.

Such techniques are not adequate for a real estima-tion of the prevalence of drug-resistant mutants;only a few studies (14, 15) have been performedwhich provide the quantitative information needed.

In order to provide a rational bacterial geneticbasis for the superiority of combined drug regi-mens and, perhaps, also for the not uncommonfailure of this approach to chemotherapy of tu-berculosis with conventional drug dosages, quan-titative information is required on the prevalenceof mutants resistant to two drugs (double mu-tants) in wild, previously unexposed populationsof tubercle bacilli. Whether or not, and, if so,how many mutant forms simultaneously resistantto both streptomycin and isoniazid can be selectedin vitro from extremely large populations oftubercle bacilli exposed to both of these agents incombination obviously has important clinical impli-cations. The purpose of this report (the first of aseries) is to present the results of a study of thisproblem.

MATERIALS ANDMETHODS

1. Culture medium. The experiments were performedwith base medium 7H-10,2 the composition and preparationof which follows:

KH2PO4(anhydrous)Na2HP04 (anhyldrous)L-glutamic acidAmmonium sulfateGlycerol (reagent grade)Tri-sodium citrate 2H20Ferric ammonium citrateMgSO4-7H20CaCl2 * 2H20ZnSO4 7H20CuSO4* 5H20Pyridoxine hydrochlorideBiotin

1.5 Gm.1.5 Gm.0.5 Gm.0.5 Gm.5.0 ml.0.4 Gm.0.04 Gm.0.05 Gm.0.0005 Gm.0.001 Gm.0.001 Gm.0.001 Gm.0.005 Gm.

Dissolve the above in 1,000 ml. of distilled water, andadjust to pH 6.6. For growth on solid medium, add agar 3in a final concentration of 1.5 per cent and add 0.25 mg.malachite green (oxalate salt) per ml. before autoclaving.After autoclaving, add oleic acid-albumin complex (16)in a final concentration of 0.5 per cent. For growth inliquid ST (standard transfer) medium, before autoclav-ing add Tween®9 80 in a final concentration of 0.05 percent, and add after autoclaving albumin instead of oleicacid-albumin complex, and omit agar. In either case,after autoclaving, add 0.2 per cent final concentration ofglucose (from a concentrated solution previously auto-claved with 0.005 M citric acid), and catalase4 in final

2A similar medium, 7H-9, is available from DifcoLaboratories, Detroit, Mich.

3 Baltimore Biological Laboratories, Inc., Baltimore,Md.

4 Nutritional Biochemical Corp., Cleveland, Ohio(listed as catalase "crude").

concentration of 3 ,ug. per ml. from a stock solution, 1.0mg. per ml. in 0.85 per cent saline, sterilized by passagethrough a sintered-glass filter.

2. Drugs. Sterile streptomycin sulfate powder in rub-ber-stopper bottles was dissolved aseptically in steriledistilled water to achieve a final concentration of 1,000 ,ug.of the base per ml. This stock solution was stored at- 100 C. Isoniazid was dissolved in sterile distilled wa-ter to a final concentration of 1,000 ,ug. per ml. Thissolution was then autoclaved for 10 minutes at 15 poundspressure (17), and stored at 40 C.

Petri dishes (100 mm. diameter) were prepared withthe oleic acid-albumin-agar medium, designated 7H-10.The drugs were added aseptically to this sterile mediumjust prior to pouring. In the first experiment, fourplates were prepared with 1.0 lsg. of isoniazid per ml. ofsolid culture medium, four plates contained 2.0 ,Ag. ofstreptomycin per ml., and 100 plates were poured withboth 2.0 ,ug. of streptomycin and 1.0 ug. of isoniazid perml. For the second experiment two plates were preparedcontaining 1.0 ,tg. of isoniazid per ml., two plates wereprepared containing 1.0 ,ug. of streptomycin per ml., and100 plates containing both 1.0 ,ug. of streptomycin and1.0 ,ug. of isoniazid per ml. were poured. The thirdexperiment was the same as the first except that 0.2 ,ug.of isoniazid instead of 1.0 ,ug. per ml. was used both inthe plates with isoniazid alone and in the 100 platestesting the combined action of streptomycin and isoniazid.The fourth experiment was carried out in liquid medium(ST); it was designed to reveal the sterilizing ratherthan the bacteriostatic action of 2.0 Ag. of streptomycinand 0.5 jug. of isoniazid per ml. of liquid culture medium.These concentrations of drugs were selected because suchconcentrations have been achieved in the serum of patientsduring treatment.

3. Cultures. Fully grown laboratory strains of tu-bercle bacilli (H37Rv and Pearson I), cultivated inTween®-albumin liquid medium, 7H-4, were used (18).H37Rv was used in both the first and second experi-ments. However, the stock H37Rv strain has yieldedcatalase-positive,5 isoniazid-resistant mutants in our labor-atory only in very small numbers. This differs fromour experience with most "wild" strains of tubercle ba-cilli freshly recovered from patients, such as the PearsonI strain. Studies in vitro on "wild" strains have demon-strated (19) that, by and large, critically low concentra-tions of isoniazid select many weakly isoniazid-resistantmutants which retain significalnt degrees of catalase ac-tivity (called catalase-positive) while higher concentra-tions of isoniazid result in the selection of only catalase-negative, highly isoniazid-resistant forms. Thus, an ap-proximate estimate of the concentration of isoniazid inthe free active state can be made after isoniazid-resistant

5 Catalase testing as performed in our laboratory is acrude test. A drop or two of a cold aqueous solution 15per cent hydrogen peroxide and 5 per cent Tween® isplaced on the colony of tubercle bacilli. Catalase-negativeorganisms give little or no foaming. Catalase-positiveorganisms yield foaming promptly.

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DRUG-RESISTANTMUTANTPOPULATIONSOF TUBERCLEBACILLI

mutants have been selected; low concentrations sterilizeonly the susceptible parent organisms while higher con-

centrations sterilize all but the highly isoniazid-resistant,catalase-negative mutants. Because the standard labora-tory strain, H37Rv, fails to yield catalase-positive, iso-niazid-resistant mutants, and, therefore, does not behavein the usual fashion toward high and low antimicrobially-active concentrations of isoniazid, the Pearson I strainwrhich does yield both catalase-negative and catalase-positive mutants was employed in the third and fourthexperiments.

4. Procedures. The first three experiments were per-

formed in the same manner. Petri dishes containing onlyisoniazid were inoculated with 0.1 ml. of a 1 x 101 dilu-tion of a seven to 10 day old dispersed culture of tuberclebacilli (neither filtered nor ground). These stock cul-tures in liquid medium have been shown to contain ap-

proximately 5 x 10' viable bacterial units per ml. (20).Distribution of the organisms on each plate was ac-

complished by means of a spreader while the plate was

rotating on a turntable. Similarly, the plates containingstreptomycin alone were inoculated with 0.1 ml. of theundiluted suspension, and the plates which contained bothstreptomycin and isoniazid were inoculated each with 0.2ml. of the undiluted suspension. All of the plates were

incubated at 36° C. under an atmosphere of 2 to 5 per

cent carbon dioxide in air for at least 28 days. Dryingout of the cultures was prevented by enclosing the platesin polyethylene plastic bags.

The fourth experiment was performed in duplicate.Two 250 ml. flasks containing 20 ml. of the liquid medium(ST) were inoculated with 0.2 ml. of an undiluted sus-

pension of five day old vigorously growing Pearson Iorganisms. These flasks were incubated in the same way

as the plates; however, two 0.1 ml. samples were removedfrom each duplicate flask at zero time, one, two andthree weeks, and were plated on Petri dishes containingthe 7H-10 solid medium without added drugs. Theseplates were incubated as indicated above.

RESULTS

Experiment No. 1

Four plates were used to test 1.0 jug. of isoniazidper ml. alone; four plates for 2.0 Mug. of strepto-mycin alone; and 100 plates tested 2.0 ,ug. ofstreptomycin combined with 1.0 ,ug. of isoniazid(Table I). The 108 plates were incubated as de-scribed under Mlaterials and Methods, and thenwere examined. The isoniazid plates yielded 99,101, 106 and 107 colonies, an average of 103colonies, consisting of isoniazid-resistant mutants.It is assumed that each of these colonies arose froma single drug-resistant mutant because of the rela-tively small numbers of organisms in each clump.Since there were roughly 5 x 106 viable bacterialunits originally inoculated onto each isoniazid

TABLE I

Results of Experiment 1

Colonies PrevalenceNumber Inoculum* per plate of resistanceof plates per plate Drug conc. (actual) mutants

pg./ml.4 5 X 106 INHt 1.0 99, 101, 1 in 5 X 104

106, 1074 5 X 107 SMt 2.0 55, 59, 1 in I X 106

64, 70100 1 X 108 INH 1.0 Nogrowth <1 in 1 X 1010

SM 2.0

* Number of viable, bacterial units from a seven to 10day old vigorously, growing, well dispersed H37Rv culturein liquid medium (ST).

t Isoniazid.$ Streptomycin.

plate, it is evident that there was approximatelyone isoniazid-resistant mutant for every 5 x 104drug-susceptible bacterial units. This is in agree-ment with previous estimates of the prevalenceof isoniazid-resistant mutants in the H37Rv strain(15).

The streptomycin plates supported growth of55, 59, 64 and 70 colonies, an average of 62colonies, consisting of streptomycin-resistant mu-tants. This represents about one streptomycin-resistant mutant in 1 X 106.

The 100 plates testing the combined action ofstreptomycin and isoniazid yielded no growthwhatsoever. In other words, no mutant whichwas simultaneously resistant to both streptomycinand isoniazid, the so-called double mutant, waspresent in 1 x 1010 viable bacterial units of theH37Rv strain. This population is about one-tenth of the number theoretically necessary toyield one such double mutant.

Only isoniazid-resistant mutants were recoveredfrom the colonies present on those plates whichcontained isoniazid alone; and only streptomycin-resistant mutants were present in the colonieswhich grew on the plates containing 2.0 ug. ofstreptomycin per ml. of solid culture medium.The combination of 2.0 ug. of streptomycin and1.0 ,ug. of isoniazid per ml. of culture mediumcompletely prevented the appearance of anycolonies.

Experiment No. 2

This experiment was the same as the first ex-cept that only 1.0 ug. of streptomycin per ml. was

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TABLE II


Colonies PrevalenceNumber Inoculum* per plate of resistantof plates per plate Drug conc. (actual) mutants

pg./mi.2 2.5 X 106 INH 1.0 23 26 1 in I X 1052 2.5 X 107 SM 1.0 30, 30 1 in 8 X 105

100 5 X 107 INH 1.0 lOt 1 in 5 X 108SM 1.0

* Number of viable bacterial units from a seven to 10day old vigorously growing, well dispersed H37Rv culturein liquid medium (ST).

tOnly 10 colonies appeared on a total of 100 plates;all susceptible to SM (streptomycin), 0.5 ,ug. per ml.;resistant to INH (isoniazid), catalase-negative.

employed (Table II). After the 104 plates hadbeen incubated for 28 days, they were inspectedfor evidence of growth. The isoniazid plates re-vealed 23 and 26 large colonies, all of which provedto consist of isoniazid-resistant, catalase-negativeorganisms. There was one isoniazid-resistant mu-tant in 1 X 105 viable bacterial units. The strepto-mycin plates revealed a thin, diffuse spreadinggrowth of streptomycin-susceptible organisms overthe entire surface of each plate. These organ-isms had been prevented from proliferating duringonly part of the incubation period. A total of 60large colonies were recognizable on these plates.These represented organisms which were strepto-mycin-resistant. Roughly speaking, this showedthat among 8 x 105 drug-susceptible bacterial unitsthere was one streptomycin-resistant mutant.

The 100 plates testing the combined action ofstreptomycin and isoniazid revealed 10 small col-onies. These colonies proved on subculture to con-sist of pure populations of organisms which weresusceptible to 0.5 ,ug. of streptomycin per ml. of cul-ture medium and resistant to at least 0.5 Mug. ofisoniazid. Since approximately 5 x 109 viablebacterial units were seeded on these plates, onlyone isoniazid-resistant mutant in 5 x 108 drug-susceptible bacterial units was able to proliferateon medium containing the stated concentrationsof both drugs together.

This experiment emphasized the fact that popu-lation size significantly modifies the antimicrobialaction of streptomycin. A concentration of about0.2 ,ug. of streptomycin per ml. of solid culturemedium is usually inhibitory, if not bactericidal,for small numbers of tubercle bacilli of the H37Rvstrain. These results show that when large num-

bers of tubercle bacilli are used, even a fivefold in-crease to 1.0 ,g. of streptomycin per ml. is notcompletely inhibitory for all drug-susceptible or-ganisms.

The usual minimum antibacterial concentrationof isoniazid for small numbers of tubercle bacilliof the H37Rv strain is 0.05 to 0.075 Mug. per ml.No significant antagonistic effect attributable tolarge inocula of tubercle bacilli has been observedin the case of isoniazid. The almost, but not quite,adequate concentration of streptomycin was cer-tainly responsible for the marked reduction innumbers of isoniazid-resistant colonies which didgrow. These experiments provide evidence forcross-antibacterial activity between streptomycinand isoniazid. However, when combined drugactivity failed due to an inadequate concentrationof Drug "A" (in this case, streptomycin), mu-tants grew which were resistant to Drug "B"(isoniazid) which was present in more effectiveconcentration.

Experiment No. 3

The previous experiments demonstrated that aconcentration of 2.0 ug. of streptomycin was theminimum totally inhibitory concentration for sucha large population of tubercle bacilli. On theother hand, 1.0 ,ug. of isoniazid was greatly in ex-cess of the minimum antibacterial concentrationof isoniazid for small numbers of tubercle bacilliof this strain, H37Rv.

Catalase-positive, isoniazid-resistant mutants canbe derived from the H37Rv strain by antimicro-bially-active concentrations of isoniazid only withgreat difficulty, although catalase-negative, iso-niazid-resistant mutants are readily selected.When low concentrations of isoniazid were underinvestigation, an organism which could produceboth types of resistant mutants was necessary toestablish the fact that the concentration of isoniazidwas suboptimal, even though exceeding the mini-mum antibacterial concentration for the drug-susceptible parent stock. In such an experimenta strain which readily yields a significant numberof catalase-positive, isoniazid-resistant mutantswhen subjected to the action of critically low con-centrations of isoniazid might reveal significantlydifferent responses to the antimicrobial activity ofthe combination of both drugs. Therefore, be-

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cause it readily yields both types of mutants, thePearson I strain was used.

The Pearson I strain (streptomycin-suscepti-ble, isoniazid-susceptible, catalase-positive, patho-genic) was seeded on 108 plates using the samedilutions as in the preceding experiments (TableIII). After incubation as before, these plateswere read. The isoniazid plates revealed 34, 44,45 and 54 colonies, respectively, an average of 44colonies, composed of isoniazid-resistant mutants.In accordance with the previous assumption, thismeans that there was roughly one isoniazid-resist-ant mutant among 1 X 105 drug-susceptible bac-terial units. There were 39, 39, 40 and 44 colonieson the streptomycin plates, an average of 40colonies consisting of streptomycin-resistant mu-tants, or about one streptomycin-resistant mutantin 1 x 106 drug-susceptible bacterial units. The100 plates containing both 2.0 ug. of streptomycinand 0.2 ug. of isoniazid per ml. of solid mediumyielded no growth.

The colonies which grew on the plates contain-ing only isoniazid were investigated for catalaseactivity. On one plate all 54 colonies were sub-jected to catalase testing: 41 colonies gave anegative reaction, and 13 colonies gave a positivereaction. Six colonies were selected at randomfrom another of the isoniazid plates in order toinvestigate isoniazid resistance as well as catalaseactivity. One colony consisted of organisms resist-ant to 0.2 Mug. of isoniazid, but susceptible to 5.0Mug. These organisms were catalase-positive. Thefive remaining colonies proved to consist of pure

TABLE III


Colonies PrevalenceNumber Inoculum* per plate of resistantof plates per plate Drug conc. (actual) mutants

,ug./ml.4 5 X 10 INH 0.2 34, 44,t t in t X 105

45, 54t4 5 X 107 SM 2.0 39, 39, l in t X 10,

40, 44100 1 X 108 INH 0.2 Nogrowth <1 in 1 X 1010

SM 2.0

* Number of viable bacterial units from a seven to 10day old vigorously growing, well-dispersed Pearson Iculture in liquid medium (ST).

t Six colonies selected at random: one catalase-positive,R-0.2 INH (isoniazid), S-5.0 INH; five catalase-negative,R-0.2 and 5.0 ,ug. INH.

t Fifty-four colonies: 41 catalase-negative, 13 catalase-positive.

populations which were catalase-negative and re-sistant to both 0.2 and 5.0 ug. of isoniazid per ml.of medium. These same colonies were susceptibleto the antimicrobial action of 2.0 Mug. of strepto-mycin per ml.

The results of this experiment confirmed ourprevious observations that exposure of large num-bers of the Pearson I strain (total for the fourisoniazid plates was 2 X 107 viable bacterial units)to 0.2 ,ug. of isoniazid per ml. selected both catalase-positive mutants which were resistant to 0.2 Mtg.of isoniazid and susceptible to 5.0 ug. of isoniazidper ml. of culture medium, and catalase-negativemutants which were resistant to both 0.2 and 5.0,Mg. of isoniazid per ml.

Although this concentration of isoniazid wasable to manifest effective antibacterial activityagainst all drug-susceptible parent bacilli, it wasstill low enough to select both types of resistantmutants. It seemed possible that a differentialselection of the two types of resistant mutantsmight result under the influence of combined strep-tomycin and low isoniazid. No such differentialselection occurred.

Experiment No. 4The first three experiments demonstrated the

antibacterial effect of combined drug action on verylarge populations of tubercle bacilli; this fourthexperiment was designed to show whether thisantibacterial activity was bacteriostatic or steriliz-ing. Viable colony counts were made by platingout from liquid culture medium (ST) containingdrugs onto oleic acid-albumin-agar plates con-taining no drugs. Two duplicate flasks, each con-taining 0.5 Mug. of isoniazid and 2.0 Mug. of strepto-mycin per ml., were sampled in duplicate at zerotime, and the samplings (each 0.1 ml.) plated onPetri dishes. These four control plates revealedconfluent growth after 28 days' incubation. Theflasks were sampled in duplicate after seven daysof incubation. This time the plates revealed four,four, three and zero colonies. Sampling of theflasks at 14 and 21 days failed to yield any col-onies, indicating a marked sterilizing effect uponthe 2 x 108 viable bacterial units originally inocu-lated. This experiment confirms the observationsof Singh and. Mitchison (21) relating to themarked sterilizing activity of these two drugs formultiplying tubercle bacilli.

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DISCUSSION

Replica plating techniques have established thatisoniazid-resistant mutants of typical tuberclebacilli are present in large populations of iso-niazid-susceptible organisms prior to any exposure

to this drug (11). Presumably this is also truefor streptomycin-resistant mutants. Therefore,whether or not these drugs also stimulate more

rapid mutation to drug resistance is not a matterfor further consideration in this report.

Analysis of the bacteriologic records of patientstreated inadequately in the past with streptomycinand isoniazid revealed that the drug-resistantorganisms which appeared during treatment were

initially resistant to only one drug.6 Theoreti-cally, there is a definite but extremely low inci-dence of mutants simultaneously resistant to bothof these drugs in enormous populations. Ef-fective concentrations of both streptomycin andisoniazid should select such double mutants when

they are present. It is evident that in our fourexperiments the populations tested were not suf-ficiently large to provide evidence on this point.

These experiments do establish by quantitativemethods the phenomenon of cross-sterilization ofmultiplying single-drug-resistant mutants in vitrowhen streptomycin and isoniazid are employed,each in concentrations adequate to sterilize allmultiplying parent drug-susceptible bacterial cellsin "wild" populations of stock laboratory strainsof H37Rv and Pearson I. Although the specificmodes of action of these two drugs have not beenbiochemically defined, this evidence corroboratesother observations that they have independentand unrelated mechanisms of action against drug-susceptible parasites.

These experiments also show that a nearly, butnot quite, adequate concentration of streptomycincombined with relatively high concentrations ofisoniazid reduces to a remarkable degree the sur-

6 It is possible that such drug-resistant organisms can beacquired from another patient by so-called exogenous re-

infection. However, careful study of the records of theinadequately treated patients, referred to above, revealednot one case in which resistance appeared to a drug thatthe patient had not previously received. Therefore, theseresults are consistent with the generally accepted con-

cept that the resistant forms of tubercle bacilli whichemerge to predominance in the sputa of drug-treated pa.tients are derived in vivo from spontaneous mutation inparent drug-susceptible populations.

vival of mutants resistant to isoniazid. Similarly,with an adequate concentration of streptomycin, acritically low concentration of isoniazid, alone un-able to prevent the selection of weakly isoniazid-resistant, catalase-positive mutants, is quite suf-ficient to prevent the emergence of both isoniazid-and streptomycin-resistant mutants.

It should be emphasized that combined drugaction, such as was tested here, actually preventsthe emergence of drug resistance unless a spon-taneous double mutant is encountered. These ex-periments failed to reveal even one double mu-tant from 2.5 x 1010 viable bacterial units.

Clinical experience indicated in the past thatdrug resistance would emerge provided only thatthe sputum remained persistently positive andcombined drug therapy continued for a sufficientperiod of time. This led to the idea that combineddrug treatment merely delayed the emergence ofresistance. However, another interpretation ofthe emergence of drug resistance under these con-ditions (combined drug treatment and persistentlypositive sputum) is possible. This view is basedon the results of Experiment No. 2, where aninadequate concentration of streptomycin alloweda few isoniazid-resistant mutants to be selectedby fully adequate concentrations of isoniazid. Ac-cording to this hypothesis, the patients who event-ually excreted drug-resistant organisms had beenunable to achieve or maintain for a sufficient periodof time adequate concentrations of one of the twodrugs in those sites where the tubercle bacilliwere multiplying during the time that the otherdrug was being delivered in fully adequate con-centration.

Finally, the results of these experiments providea microbiologic explanation for the increased con-version rates in those clinical trials (8) whichhave employed lower dosage of isoniazid (3 to5 mg. per Kg. per day) combined with higherdosage of streptomycin (7.0 Gm. or more perweek). Five to 10 times higher concentrationsof isoniazid (1.0 jug. per ml.. as compared to 0.1,ug. per ml.) were required to prevent the in vitroemergence of catalase-positive, isoniazid-resist-ant mutants than were needed to exert a strikingantibacterial effect against only the parent drug-susceptible population. Thus, in clinical practice,combined drug treatment employing lower dosagesof isoniazid should be sufficient with adequate dos-

1354


age of streptomycin to prevent the multiplication ofall mutants resistant to either drug (except, ofcourse, double mutants).

SUMMARY

The antimicrobial activity of isoniazid combinedwith streptomycin was tested against large num-bers of multiplying tubercle bacilli of the H37Rvand Pearson I strains in order to investigate theprevention of the selection of mutants simultane-ously resistant to both drugs. Concentrations ofstreptomycin not quite sufficient alone to inhibitthe growth of all streptomycin-susceptible bac-terial cells were remarkably effective in combina-tion with 20 times the minimum inhibitory con-centration of isoniazid in reducing the emergenceof drug-resistant mutants. Concentrations of iso-niazid, whether only slightly or far in excess of theminimum inhibitory concentration, when com-bined with the minimum sterilizing concentrationof streptomycin prevented the emergence of anydrug-resistant mutants. Prevention of emer-gence of drug-resistant mutants was attributed tothe cross-sterilizing action of the two drugs onindividual mutants separately resistant to eachone. The implications of these observations forclinical chemotherapy were discussed.

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14. Cohn, M. L., Oda, U., Kovitz, C., and Middlebrook, G.Studies on isoniazid and tubercle bacilli. I. Theisolation of isoniazid-resistant mutants in vitro.Amer. Rev. Tuberc. 1954, 70, 465.

15. Middlebrook, G. Sterilization of tubercle bacilli byisonicotinic acid hydrazide and the incidence ofvariants resistant to the drug in vitro. Amer. Rev.Tuberc. 1952, 65, 765.

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21. Singh, B., and Mitchison, D. A. Bactericidal ac-tivity of streptomycin and isoniazid against tu-bercle bacilli. Brit. med. J. 1954, 1, 130.

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