ENZYMATIC STUDIES ON THE MECHANISM OF THE RESISTANCEOF PNEUMOCOCCUS TO DRUGS

II. THiE INHIBITION OF DEHYDROGENASE ACTIVITIES BY DRUGS; ANTAGONISTICEFFECTS OF RIBOFLAVIN TO INHIBITIONS'

M. G. SEVAG AND JOSEPH S. GOTS2Department of Bacteriology, School of Medicine, University of Pennsylvania,

Philadelphia 4, Pennsylvania

Received for publication July 24, 1948

In the preceding study (Gots and Sevag, 1948) it was shown that pneumococcimade resistant to various drugs frequently possessed diminished dehydrogenaseactivities. It was suggested that the decreased dehydrogenase activity of theparticular resistant organisms was a manifestation of a deficiency of susceptiblesites, i.e., the resistant organisms depended on nonsusceptible metabolic sitesfor the maintenance of their physiological activities. The effect of the drugs onthe dehydrogenase activity in the presence of the various substrates was thereforeinvestigated to obtain information concerning these questions and to characterizecertain of the enzymes involved in the production of the observed phenomena.

Inhibition of Dehydrogenase ActivityMethods and results. For the determination of the methylene-blue-reducing

ability of the organisms a refinement of the technique used by Schnabel (1920)was used. This was substituted for the conventional Thunberg method, previ-ously used, in order to facilitate the performance of simultaneous experimentswith several substrates and varying concentrations of drugs, organisms, and otheradditions. Since some experiments involved 100 or more combinations, the useof Thunberg tubes would have been prohibitive. The technique, in effect, is amicroadaption with some experiments being conducted with as little as 0.05 mgof organisms. The results obtained by this method are qualitatively, but notquantitatively, comparable to those obtained by the Thunberg technique.The bacterial suspension and the reagents used were prepared as previously

described (Gots and Sevag, 1948). Using small serological tubes (10-by-100-mm), the system consisted of 0.05 ml of methylene blue (0.001 M in M/20 phos-phate buffer at pH 7.6), 0.1 ml of substrate, 0.1 ml of drug, bacterial suspension(0.1 mg per 0.1 ml), and M/20 phosphate buffer (pH 7.6) to give a total of 1 ml.The bacterial suspension was added last, the contents were immediately mixed,and 1 ml of melted petrolatum was carefully layered over the mixture. Thetubes were then placed in a water bath at 37 C and the time in minutes requiredfor 100 per cent reduction of the methylene blue was recorded.

I The investigation was begun under a grant from the Josiah Macy, Jr. Foundation.This investigation was supported, in part, by a research grant from the Division of

Research Grants and Fellowships of the National Institute of Health, U. S. Public HealthService.

2 Abbott Fellow in the Department of Bacteriology, 1947-1948.723

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Inhibition of dehydrogenase activities of drug-susceptible parent organisms.Since all of the susceptible types were affected by the drugs in a similar manner,data are presented (table 1) for a single type. The results from typical experi-ments are given in table 1 for glucose, hexose diphosphate (HDP), and ethyl

TABLE 1The effect on dehydrogenase activity of pneumococcus, type I, of acriflavine, atabrine,

propamidine, optochin, and sulfonamide in the presence of glucose, hexosediphosphate (HDP), ethyl alcohol, glycerol, and lactate

GLUCOSE X ED ETHYL ALOCHOL GLYCEROL LACTATE(0.001 M) (0.003 M) (0.1 l) (0.1 m) (0.1 K)

DRUG

DRUG CONCEN- Mg of organisms usedTRATION ____________________________(XlO-4) 0.2 0.1 j 0.3 0.2 0.2 J 0.1 0.5 0.5

Reduction time in minutes

Acriflavine 1.0 17 85 21 95 cc O 39 540.5 14 50 21 95 cc 00 30 480.25 11 27 21 95 >120 >120 30 400.00 8 18 11 19 22 38 29 38

Atabrine 4.0 27 95 oo co2.0 24 oX 211 681 00 43 681.0 18 21 32 120 150 35 660.5 13 26 27 400.00 8 18 1 1 19 22 38 33 45

Propamidine 10.0 14 cc 10o 16 31 38 >120 755.0 i 11 28 101 17 43 38 82 352.5 10 21 10 16 37 38 66 320.00 9 19 11 19 22 38 40 34

Optochin 10.0 c ccX 00 c cc ccX5.0 cc cc 22 72 cc cc 45 372.5 27 cc 14 28 51 150 30 351.25 17 85 11 20 35' 28 320.625 14 34 28 290.00 9 20 1i1 19 22j 38 28 29

Sulfathiazole (Na 40.0 8 19 8 16 22 35 28 48salt)

Sulfapyridine 4.0 8 22 12 21 25 37 30 4810.00 8 20i 11 19 221 38 30 48

alcohol with two different weights of organisms. The conditions chosen forHDP and ethyl alcohol were those under which normal activities approachedthat of glucose. Glycerol and sodium lactate dehydrogenase activities wereconsistently low, and the results shown are those obtained with 0.5 mg of organ-isms. A weight of organisms greater than this was impractical because of in-creased endogenous activity.

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In all cases, the degree of inhibition of the methylene-blue-reducing ability bya given concentration of a drug was related to the weight of organisms used.Inhibition by the drugs occurred less readily in the presence of greater amountsof bacterial cells.

Glucose dehydrogenase was inhibited by all the drugs except the sulfonamides.Concentrations as high as 1: 250 of sodium sulfathiazole and 1: 2,500 of sulfa-pyridine did not inhibit the activity of any of the substrates. (Factors responsi-ble for this effect are discussed below.) The two acridines, acriflavine andatabrine, inhibited the glucose dehydrogenase in comparatively high dilutions.Optochin required slightly higher concentrations, and propamidine even more,for the inhibition of glucose dehydrogenase activity. The activity in thepresence of HDP is less inhibited than that in the presence of glucose, and it isnot at all affected by propamidine. Ethyl alcohol activity is also little affectedby propamidine, but acriflavine exerts its greatest inhibition in the presenceof this substrate. Glycerol and lactate activity are only slightly affected by thedrugs, with propamidine being the most inhibitory. Concentrations higher thanthose shown for propamidine, optochin, and sulfapyridine could not be obtainedbecause of insolubility in the phosphate medium. Higher acriflavine concen-trations resulted in the clumping of the organisms.The amount of drug required to inhibit dehydrogenase activity is much

greater than that required to inhibit growth, as given in the preceding paper.However, the number of organisms required (300 to 400 million) to give demon-strable dehydrogenase activity is far greater than the number of organisms (80to 90 million) present in the typical inocula of the growth systems. Since thedehydrogenase activity and its degree of inhibition by the drugs are dependentupon the nulmber of organisms present, this would explain the difference in theamount of agent required for the inhibition in the two systems. In experimentsconducted with 0.05 mg of organisms (150 million cocci) or in syst ems with aparticularly low activity, glucose dehydrogenase activity could be inhibited byatabrine concentrations as low as 1:160,000. This is of the same order of mag-nitude as the growth-inhibiting concentration of atabrine of 1: 400,000.With glucose, the substrate concentration is also a factor determining the

degree of inhibition obtained with at least atabrine, propamidine, and, to a lesserextent, acriflavine. In an experiment with the type III organism, the followingresults were obtained in 0.1 M and 0.001 M of glucose with 0.1 mg (300 to 400million) of bacterial cells (table 2). Thus, the inhibition is smaller in higher con-centrations of glucose. This could only be examined with glucose as substratesince the activities in the presence of the other substrates are dependent on anoptimal substrate concentration, and the degree of inhibition is dependent on theactivity manifested under optimal conditions.The failure of sulfonamides to inhibit the activity in the presence of any of the

substrates was confirmed by use of the Thunberg technique. Concentrations ashigh as 1:160 of sodium sulfathiazole were completely ineffective. As will benoted in the discussion, this lack of inhibition is due to the antagonism of methyl-ene blue.

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The ability of propamidine to inhibit glucose activity but not HDP activitymight indicate an interference with phosphorylation mechanisms. This wouldbe more decisive if the exact nature of the two dehydrogenase systems werebetter defined. It is yet to be determined whether glucose must first be phos-phorylated in order to serve as a hydrogen donator in these dehydrogenasesystems or whether it can be directly oxidized to, e.g., gluconic acid withoutphosphorylation. In Thunberg experiments, however, some inhibition of HDPactivity by propamidine could be obtained with 1:1,600 concentration. In-hibition by propamidine was found to be peculiarly dependent on the order ofaddition of various agents. When the drug was added with the substrate fromthe side arm of Thunberg tubes after 10 minutes' incubation, the inhibition ofglucose activity was less than when the drug was allowed to incubate in the

TABLE 2The effect of glucose concentration on the inhibition of glucose dehydrogenase of pneumococcuw,

type III, by acriflavine, atabrine, and propamidine

PROPAMIDINZ ACRInAVINNE ATABRIN CONTRO(X10>4) (X10-4) (X10^) IOTO

10 1 5 1 2.5 1.0 0.5 0.25 0.5 0.25 1 0.1251 0.00

Methylene blue reduction time (minutes)

Glucose 0.1M....... co 43 20 !o 50 45 |X 50 33 16Glucose 0.001m..... co C| 68 || 90 45 | cc || 16

presence of the organisms before substrate was added alone from the side arm.Since in the experiments using the Schnabel technique the organisms are addedlast, this may well explain the failure of HDP inhibition by propamidine whendetermined by this method.'At any rate, the inhibition of glucose activity in any system is greater than

that of HDP activity by propamidine, as well as by atabrine. The possibleaction of these drugs on the phosphorylation mechanisms will require furtherstudy. Atabrine has already been implicated in the interference with thesesystems (Speck and Evans, 1945; Bovarick, Lindsay, and Hellerman, 1946;Marshall, 1948a).

Effects of the drugs on the dehydrogenase activity of resistant organisms. In-hibition of the dehydrogenase activities of the resistant organisms was studiedin order to determine if the resistance to inhibition of growth was associated witha resistance to inhibition of dehydrogenase activity by the drugs.

Using the Schnabel technique, a comparison of the inhibitions of glucose

3Propamidine possesses another property that might add to its dehydrogenase-inhibitingaction. In Thunberg experiments, if high concentrations of propamidine are added fromthe side arm after the organisms have reduced the methylene blue in the presence of glucose,the leuco form is slowly reoxidized to the blue form. This would indicate a competitionbetween methylene blue and propamidine for the hydrogen mobilized from the substrate,which would explain the observed decrease in inhibition in the presence of increased sub-strate.

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dehydrogenase activity for susceptible and resistant organsms was made. Onlythe glucose dehydrogenase activity was studied in these experiments because thiswas the most readily inhibited and, Ms previously reported (Gots and Sevag,1948), the most frequently decreased in resistant organisms. This difference inthe glucose activity of the susceptible and resistant organisms in the absence ofthe drugs presented difficulties when the degrees of inhibition of the activities of

TABLE 3Inhibition of glucose dehydrogenase of pneumococci by acriflavine, atabrine, propamidine,

and optochin(A comparison of reduction time in minutes for susceptible and resistant organisms in

glucose, 0.001 M)

TYP I TYPE rl TYP|m

DRUGO CONNU Susceptible Resistant Susceptible Resistant Susceptible ResistantTRATION(X1O4) Mg of organisms used

0.2 0.1 0.2 0.1 0.2 0.1 1 0.2 J 0.1 0.2 1 0.1- 0.2 1 0.1

reduction time ix minutesAcriflavine 1.0 17 85 44 eo en en 21 115 25 oo 67 co

0.5 14 50 18 en 26 eo 18 52 18 45 28 eo0.25 11 27 14 76 22 65 16 43 15 28 18 700.00 8 18 11 27 10 29 12 38 8 16 16 44

Atabrine 2.0 24 co 94 co co co on on co co co1.0 18 co 77 co co en 73 co 27 ao 40 eo0.5 13 26 60 eo 35 co 24 co 21 eo 30 eo0.25 20 co 21 en 18 49 18 co0.00 8 18 38 9 28 20 52 81616 43

Propamidine 10.0 12 co 17 co 17 co 12 305.0 10 40 16 47 9 29 12 292.5 9 21 16 33 8 17 12 270.00 9 20 16 33 7 16 12 27

Optochin 5.0 en en en en2.5 27 en 50 en1.25 17 43 28 en0.625 14 34 26 780.00 8 24 22 50

the two organisms were compared. Since the degree of inhibition is related in aninverse manner to the ability of the bacterial suspensions to reduce methyleneblue, a decreased activity will naturally show greater susceptibility to inhibition.Therefore, in comparing the effect of the drugs on dehydrogenase activities, asimilar base line of control activity had to be made in most instances. Thiscould be accomplished by increasing the number of the resistant organisms in thecomparative test systems so that their control activities approached the activitiescharacteristically demonstrated by fewer susceptible organisms.

Table 3 presents data from typical experiments permitting this adjustment.

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For example, in type III resistant organisms the control activity of 0.2 mg cells

is approximately that of 0.1 mg of the susceptible organisms, and the inhibitionof activity by the respective drugs is much, greater for the susceptible than for theresistant organisms. The effect of an increase in the nuimber of organisms,irrespective of activity, on the degree of inhibition obtained may well be a sig-nificant factor. However, in several cases when, in control systems, the resistantcells do not show a greatly decreased activity, a comparison of activities obtainedwith the same number of susceptible and resistant cells can be made. Forexample, the inhibition of type II cells by acriflavine shows that, whether one

compares the activities of (a) 0.2 mg of susceptible cells with those of 0.2 mgresistant cells, or (b) 0.1 mg of susceptible cells with that of 0.2 mg resistant cells,or (c) 0.1 mg of susceptible cells with that of 0.1 mg of resistant cells, the dehydro-genase activities of the resistant cells are markedly more resistant than those ofthe susceptible cells. The results with type II atabrine cells show that 0.2mg ofresistant cells, with an activity slightly greater than that of 0.1 mg susceptiblecells, are more resistant. Two-tenths mg of type I atabrine-resistant organismsmanifest a greatly decreased methylene blue reduction time of 38 minutes. Incontrast, 0.1 mg of the respective susceptible cells required 18 minutes for the sameperformance. Despite this difference in activity, the resistant organisms wereinhibited only 60 per cent by a 1:5,000 concentration of atabrine, whereas thesusceptible organisms were completelyinhibited by a 1:10,000 concentration ofthe drug. This relationship is indicated also by types I and III propamidine-resistant cells. For example, 0.2 mg of propamidine-resistant type I cells havinga control activity comparable to that of 0.1 mg of susceptible cells show strongresistance to propamidine. This relationship can similarly be seen with type IIIpropamidine-resistant and type I optochin-resistant cells.With the possible exception of the type I acriflavine-resistant organism, all

of the resistant pneumococci are thus less subjected toinhibition of their glucosedehydrogenase activity by the respective drugs than are the correspondingsusceptible parent stains.

Acridine and diamidinecross resistance to inhibition of dehydrogenase activity.In the preceding study, the rationale for the inclusion of the aromatic diamidine,propamidine, was based upon published observations of a similarity of actionbetween this compound and the acridines. This similarity of action was ex-

tended in the observations (a) that a cross resistance to inhibition of growthbetween the two compounds existed; and (b) that loss of dehydrogenase activityby the pneumococci made resistant to these agents followed a similar pattern.Since the resistant organisms are also resistant to glucose dehydrogenase in-hibition, a study was made of the cross resistance in this respect.

Using the same methods, theinhibition of glucose dehydrogenase activity byall three drugs was determined for the parent cells which were resistant andsusceptible to acriflavine, atabrine, and propamidine. This comparison was

made with all types with similar results, but, because of the large variations ofcontrol activities, a comprehensive comparison was only possible with the typeIII organisms. With these organisms a common range of activity could beobtained with a twofold increase in the number of the resistant organisms.

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Table 4 presents the results compared on a common basis of a drug-free activityof 12 to 16 minutes methylene blue reduction time. The results are given inpercentages of inhibitions calculated from reduction times (e.g., with 12 minutesas the reduction time for the control, 24 minutes in the presence of drug wouldcorrespond to a 50 per cent inhibition). With the exception of the atabrine-resistant organisms in propamidine, all the resistant organisms are less inhibitedthan the parent susceptible organisms. Organisms made resistant to one drugexhibit a resistance to the inhibition of dehydrogenase activity not only by thatdrug but also by the drugs to which they never had been exposed. This crosresistance is manifested more between acriflavine and propamidine than betweenatabrine and the others. A similar relationship existed between the crossresistance to the inhibition of growth.

TABLE 4Inhibition of glucose dehydrogenases by atabrine, acriflavine, and propamidine of susceptible

type III and corresponding resistant pneumococci

ACRIRLAVINE- ATABRINE- PROPAKIDINI-DRUG COKCZX)(.1 MCEL)RSTANT RESISTANT RESISTANT

DRUG TRATION (0.2 MGCELL(0.(0.2 MGCESIS) (0.2 MC CELLs)(X 104) ..

Percentage of inhibition

Acriflavine 1.0 100 76 80 380.5 65 28 34 160.25 53 11 21 12

Atabrine 2.0 100 100 100 471.0 88 45 60 330.5 83 30 46 22

Propamidine 10.0 100 43 100 275.0 54 28 38 82.5 30 20 32 0

The activity of organisms in the absence of a drug was 12 to 16 minutes.

The Effects of RiboflavinIn order to determine the role of flavoprotein in the resistance phenomenon,

experiments were performed with riboflavin to study its effects on the inhibitionof growth and dehydrogenase activity by the drugs and on the impaired activityof the resistant strains.

Methods. The inhibitory effect of the drugs on growth systems was deter-mined in the partially defined medium of Adams and Roe (1945) containingvitamin-free casein hydrolyzate (SMACO) instead of the "casamino acids."Various batches of media were prepared containing varying concentrations ofriboflavin (Merck) added from stock solution in 0.02 N solution of acetic acid.The inhibition of growth was determined as previously described (Gots andSevag, 1948).

Dehydrogenase experiments were performed by the Schnabel method. Thevitamins, prepared freshly in phosphate buffer, were substituted for part of the

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phosphate buffer in the final system. All other reagents and preparationsremained as previously described.

Effect of riboflavin on the inhibition of growth. The minimal amounts of drugrequired to inhibit the organisms in the presence and absence of added riboflavinare given in table 5. The presence of riboflavin had no effect on the inhibition ofgrowth by sulfathiazole and only slight effect on inhibition by optochin andpropamidine. The inhibition by acriflavine and atabrine is affected more by thepresence of riboflavin, in that higher concentrations of these drugs are requiredfor inhibition of growth as the concentration of riboflavin is increased. Forexample, twice as much atabrine is required to inhibit type III organism in thepresence of 10 pg of riboflavin per ml, 4 times as much with 100 ,g, and 8 timesas much with 250 pg. The data given are based on 24-hour readings. Therelief of inhibition by riboflavin was more pronounced with prolonged readings.By turbidimetric readings with the Klett photoelectric colorimeter, it was foundthat the relieving of inhibition by riboflavin could not have been due to a stimu-latory action of the vitamin since its presence, per se, in the absence of drugs,had no such effect.

Effect of riboflain on the inhibition of glucose dehydrogenase activity by the drugs.The inhibition of glucose activity by propamidine, acriflavine, and atabrine isalso relieved by the presence of riboflavin. Table 6 gives the results for a typicalexperiment with 0.1 mg of susceptible type II organisms in 0.001 M glucose.The other types behaved similarly. The activity in the absence of the drug wasstimulated as riboflavin concentration was increased. However, the effect of theadded riboflavin on relieving the inhibition by propamidine and the acridines wasquite disproportionate to what might have been expected on a stimulatory basisalone. The effect of the vitamin on inhibition produced by optochin is, however,of the same order as the stimulation which it shows in the absence of the drug.In this case, there does not appear to be any true antagonism. Nicotinamideand thiamine in concentrations equimolar to that of riboflavin were also testedfor antagonistic effect in these systems and were found to be completely in-effective.

DISCUSSION AND CONCLUSIONS

Pneumococci possess dehydrogenase systems, particularly active on glucose assubstrate, which are susceptible to the action of the drugs; the dehydrogenaseactivities of the resistant organisms, though reduced, are more resistant to theaction of the drugs. This suggests that the susceptibility and resistance toinhibition of growth may well be a function of these systems. In the absence ofwell-defined information concerning the intermediary steps involved in normaldehydrogenase activity, as well as incomplete extension to other systems,peremptory conclusions cannot be drawn. However, since riboflavin is at leastinvolved as an antagonist to the inhibition of both growth and dehydrogenaseactivity by the acridines and propamidine, the flavoprotein is offered as a site ofaction of these compounds.A correlation of the pattern of decreased dehydrogenase activity of the resistant

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TABLE 5Antagonism by riboflavin to the inhibition of growth* of pneumococci by drugs

RIBOnLAVIN (g/ML)

TYPE DRUO None 10 100 250

Minimal inhibitory concentration of drug (g/ml)

I Propamidine 6.25 12.50 12.50 12.50Acriflavine 1.25 2.50 2.50 5.00Atabrine 2.50 5.00 5.00 10.00Optochin 0.62 0.62 0.62 1.25Sulfathiazole 31.20 31.20 31.20 31.20

II Propamidine 12.50 12.50 25.00Acriflavine 0.62 0.62 1.25 2.50Atabrine 5.00 5.00 - 40.00Optochin 2.50 2.50 2.50 2.50Sulfathiazole 15.60 15.60 15.60 7.80

III Propamidine 12.50 12.50 12.50 25.00Acriflavine 2.50 2.50 5.00 10.00Atabrine 2.50 5.00 10.00 20.00Optochin 5.00 5.00 10.00 10.00Sulfathiazole 31.20 31.20 31.20 31.20

* Twenty-four-hour growth at 37 C.

TABLE 6Effect of riboflavin on the inhibition of glucose dehydrogenase

(Type II susceptible pneumococcus, 0.1 mg.; glucose concentration = 0.001 M)

DRUG CONCEN- RIBOFLAVIN (ag/Ml)DDRUG TRATICONCN

None 100 200 400

pg1/mi reduction time in minutes

Control 16 13 11 10

Propamidine 1000 co cc cW 42500 cc 94 39 25250 24 20 18 12

Acriflavine 100 cc 00 56 3250 44 31 23 1625 16 14 13 12

Atabrine 400 c co cc cc200 cc 0c cc 106100 90' 50 32 24-

Optochin 250 cO o co c cO125 45 43 38 2862.5 36 35 30 18

No glucose - OOc cc co

Parallel tests with equimolar concentrations of nicotinamide and thiamine showed no

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organisms (Gots and Sevag, 1948) with the inhibition spectrum obtained in thepresence of various drugs and substrates is desirable. Atabrine-resistant organ-isms showed a marked decrease in glucose, HDP, and ethyl alcohol dehydro-genase activity; slight decrease in lactate; and no change in glycerol. The in-hibition of the corresponding dehydrogenase systems of the susceptible cells byatabrine corresponds to this pattern remarkably in that atabrine inhibits thedehydrogenation of glucose, HDP, and ethyl alcohol, but practically no effect onlactate and glycerol dehydrogenases was observed. Acriflavine and propami-dine, in general, also follow this correlation of impaired dehydrogenase activityof resistant organisms and the susceptibility of the susceptible cells to the in-activation of dehydrogenase activities by these drugs. The results with propami-dine in the inhibition experiments with HDP and ethyl alcohol present certainpeculiarities which have been fully discussed in the text. The activities of allthree of the susceptible organisms were similarly inhibited by optochin yet onlythe resistant type I organism showed any loss of activity. The present experi-mental data offer no explanation for this discrepancy.MacLeod (1939) reported sulfapyridine inhibition of glycerol, lactate, and

pyruvate dehydrogenase activities with type I pneumococcus. The dehydro-genase activities in all substrates examined by us were completely unaffected byhigh concentrations of the sulfonamides. Even though the glycerol activity ofthe parent strain was not inhibited by sulfonamides, the sulfonamide-resistantorganisms did show a decrease in glycerol activity. The observations of Cliftonand Loewinger (1943) may throw some light on this inconsistency. They foundthat the oxygen consumption and anaerobic glycolysis of Escherichia coli areinhibited by sulfonamides. However, the dehydrogenase activity measured bymethylene blue reduction was not interfered with even with saturated solutionsof sulfonamide. This observation was interpreted by Sevag (1946) to indicatethat methylene blue competes effectively with sulfonamide for flavoprotein andtherefore no inhibition of methylene blue reduction could be observed. Theseobservations can be analogously applied to the pneumococci in that respirationof type I pneumococci is inhibited by sulfathiazole up to 40 per cent (Sevag andShelburne, 1942; Sevag, Richardson, and Henry, 1945) and, as is found in thepresent study, the dehydrogenase activity in the presence of methylene blue isnot affected in the highest concentrations of sulfathiazole. Eyster (1942) foundthat sulfanilamide in comparison to other narcotics is extremely potent in itseffect on the adsorption of methylene blue by charcoal particles.

In a preliminary study, the antagonistic effect of methylene blue on the in-hibition of aerobic respiration by sulfonamide was determined in the Barcroft-Warburg apparatus. The system contained 1 mg of pneumococcus, type I, con-taining catalase, 0.03 M glucose, and M/20 phosphate buffer of pH 7.6. Thefollowing results were obtained: the control had a Q02 value of 250; with3.8 X 10- M sulfathiazole there was 61 per cent inhibition. This inhibition with1.67 X 10' M methylene blue was reduced to 6 per cent. Methylene blue alonealso showed 54 per cent inhibition.Although the interrelationship of the findings above is being further investi-

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gated, the results of similar experiments with the Thunberg technique onsulfonamide, methylene blue antagonism can be reported as conclusive. Inthree systems in which the order of the addition of various factors was varied,only the system in which methylene blue was added last from the side arm of theThunberg tube showed 57 per cent inhibition of the reduction of the dye bysulfathiazole. This inhibition indicates that sulfonamides and methylene bluecompete for the same enzyme site.On the basis of the antagonistic effect of riboflavin on the inhibition of both

growth and dehydrogenase activity, and the failure to produce these effects bythiamine and nicotinamide, there would seem to be a definite correlation betweenflavoprotein metabolism and resistance to acriflavine, atabrine, and propamidine.The action of optochin cannot be so correlated from the present findings, and, inview of methylene blue antagonism, the data with sulfonamides are as yet inade-quate and require further detailed study.The competition of the acridines with riboflavin can be postulated on the basis

of structural similarity. Propamidine has an action similar to that of theacridines, in many respects, yet bears no structural similarity to either theacridines or riboflavin. Conversely, the two acridines show points of dissimi-larity in behavior in spite of similar structure. It would appear, therefore, thatstructural similarity is not an absolutely essential factor for the accomplishmentof the interferences observed. Yet, irrespective of the nature of the competition,the fact that a dimensional similarity between an inhibitor and competingmetabolite does exist would naturally result in an enhancement of the interfer-ence. The interference by agents which are dissimilar in structure would dependon other properties which could still be associated with the competition. Thestructural analogues, in addition to the property of similar configuration, mayalso share these other properties. In the present study, the acridines andpropamidines show a common property of basicity. The cationic nature of thesecompounds has led to an explanation of their mode of action on the basis ofcompetition with hydrogen ions for vital enzyme positions (Browning, Gulbran-sen, and Kennaway, 1919; Elson, 1945; Albert et al., 1945; Massart et al., 1947b).On the basis of the observed inhibitions of respiration by acridines (Silverman,Ceithaml, Taliaferro, and Evans, 1944; Ferguson and Thorne, 1946; Massartet al., 1947a; Deley, Peeters, and Massart, 1947; Marshall, 1948a) and by diami-dines (Bernheim, 1943; Marshall, 1948b; Wien, Harrison, and Freeman, 1948), thenegatively charged enzyme protein for which the drug cation and hydrogen ionscompete is probably the dissociating acidic group of a respiratory enzyme.Quastel and Wheatley (1931) have suggested that the dehydrogenase system isprimarily of an acidic nature on the basis that basic dyes inhibited and theacidic dyes did not inhibit the dehydrogenase activity. This would be in agree-ment with the observations which have been presented. However, the compe-tition of an ionizable enzyme hydrogen with a cation of a drug, as discussedpreviously (Sevag, 1946), does not explain the lack of such a competition in aresistant organism.

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SUMMARY

The effects of acriflavine, atabrine, propamidine, optochin, and sulfonamideson the dehydrogenase activities of pneumococci (types I, II, and III) in thepresence of glucose, hexose diphosphate (HDP), glycerol, lactate, and ethylalcohol were investigated. The glucose dehydrogenase activities of the parentsusceptible pneumococci were inhibited by all the drugs except sulfonamides.The latter had no effect on dehydrogenase activity in the presence of any of thesubstrates investigated. This effect has been found to be due to a competitionbetween sulfathiazole and methylene blue. Dehydrogenase activity is lessinhibited in the presence of HDP than in the presence of glucose by acridines andoptochin, and it is not at all affected by propamidine. Ethyl alcohol activity isalso little affected by propamidine but is inhibited by the acridines and optochin.Glycerol and lactate activity are only slightly affected.The degrees of inhibition by drugs are related to the control activities of the

organisms, the number of organisms present in the systems, and, in the case ofglucose, the substrate concentration.The drug-resistant pneumococci are less subject to the inhibition of their

glucose dehydrogenase activity by the respective drugs than are the correspond-ing susceptible parent strains. A cross resistance to inhibition of glucosedehydrogenase exists between organisms made resistant to the acridines and topropamidine.

Riboflavin can counteract the inhibitory effect of acriflavine and atabrineon the growth of the susceptible organisms. It is only slightly antagonistic topropamidine and optochin in the same systems and not at all to sulfonamides.The inhibition of glucose dehydrogenase activity by the acridines and propami-

dine is also relieved by the presence of riboflavin, but not by nicotinamide orthiamine. The inhibition of the glucose dehydrogenase by optochin is unaffectedby any of the vitamins.The findings are analyzed and discussed in relation to (a) similarity of the

physical properties and action of structurally dissimilar acridines and propami-dine; (b) the effects of these drugs on dehydrogenases in relation to their modeof antibacterial action; and (c) the antagonism of riboflavin to inhibition bythe drugs.

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BOVARNICK, M. R., LINDSAY, A., AND HELLERMAN, L. 1946 Metabolism of the malarialparasite, with reference particularly to the action of antimalarial agents. II. Atabrine(quinacrine) inhibition of glucose oxidation in parasites initially depleted of substrate.Reversal by adenylic acid. J. Biol. Chem., 163, 535-551.

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RESISTANCE OF PNEUMOCOCCUS TO DRUGS

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