JOURNAL OF BACrERIOLOGY, Oct. 1971, p. 320-327Copyright 0 1971 American Society for Microbiology

Vol. 108, No. IPrinted in U.S.A.

Antibacterial Nitroacridine, Nitroakridin 3582:Effects on Bacterial Growth and Macromolecular

Biosynthesis In VivoALAN D. WOLFE,' THOMAS M. COOK, AND FRED E. HAHN

Department of Molecular Biology, Walter Reed Army Institute ofResearch, Washington, D.C. 20012, andDepartment of Microbiology, and University of Maryland, College Park, Maryland 20742

Received for publication 24 May 1971

The antibacterial drug Nitroakridin 3582 inhibited the growth of selected gram-positive bacteria more strongly than it inhibited the growth of gram-negative ba-cilli. Nitroakridin at concentrations of the order of 5 x 10-6 M induced lysis ofBacillus licheniformis and Micrococcus lysodeikticus. At concentrations less than10-4 M, Nitroakridin 3582 reduced the exponential growth rate of Escherichia coliC-2; at 10-4 M the drug was bacteriostatic, and, at concentrations greater than 10- 4

M, it was bactericidal. Prolonged bacteriostasis resulted in the formation of longfilaments by E. coli, Serratia marcescens, Shigella sonnei, and Proteus mirabilis.The reversible effects of Nitroakridin 3582 on the growth of E. coli correlated withpartial inhibitions of deoxyribonucleic acid biosynthesis; ribonucleic acid and pro-tein syntheses were inhibited less strongly. Nitroakridin 3582 at concentrationsgreater than 2 x 10-' M, which block deoxyribonucleic acid biosynthesis, producedan accelerated bactericidal action.

The investigation reported here was under-taken to contribute to the knowledge of a partic-ular antibacterial nitroacridine, Nitroakridin3582 (NA), and to determine its mode of action.Aminoacridines are known to inhibit the assem-blage and replication of bacterial viruses (10, 11),to act as frameshift mutagens in such viruses(6, 23) or as antimutagens in bacteria (28), toeliminate episomes and R-factors from bacteria(17, 32), to bind specifically to deoxyribonucleicacid (DNA) (19, 24), and to be useful clinicallyas topical antibacterial drugs. However, notmany studies on nitroacridines have been re-ported, despite early observations of Albert andhis associates (1, 2) that nitroacridines exceededother substituted acridines in antibacterial po-tency.NA is [1 -diethylamino-3-(2, 3-dimethoxy-6-

nitro-9-acridinyl) amino]propanol (Fig. 1). It is1 of 87 amino-nitroacridines synthesized morethan 40 years ago (27) in the hope of obtainingeffective trypanocides. NA has antitrypanosomalactivity (27), but it also suppresses streptococcal(26) and other bacterial infections (1, 26, 27).NA is useful as a veterinary drug (1), and it is

I This investigation was carried out in partial fulfillment ofthe requirements for graduation as a Ph.D. at the University ofMaryland.

effective against rickettsial infections in mice andin embryonated eggs (12, 30). NA inhibits theproliferation of influenza B virus (13, 25), but itis without influence on the multiplication of in-fluenza A virus (18). NA suppresses infectionswith the organisms of the lymphogranuloma andpsittacosis groups in chick embryos and in mice(8, 14, 18), but it is ineffective against certainneurotropic viruses. Conversely, another acri-dine, substituting a nitro group in position 7 andcarrying the same side chain as quinacrine, iseffective against neurotropic viruses but inactiveagainst organisms of the psittacosis group (14,18). Low concentrations of Nitroakridin 3663were reported to inhibit both phage assemblageat low concentrations and phage-DNA synthesisat higher concentrations. The phage assemblageto which Denes and Polgar refer (7) was theunion of DNA and protein, not of head and tail.

Generally, antimicrobial acridines, at low con-centrations, induce bacteriostasis and the forma-tion of long bacterial filaments; at higher concen-trations they block DNA biosynthesis and arebactericidal. This has been shown for proflavinein Bacillus lactis aerogenes (Enterobacter aero-genes) (4) and for quinacrine in Escherichia coli(21, 5). Proflavine also interferes with proteinbiosynthesis, apparently by inhibiting transcrip-

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ANTIBACTERIAL EFFECTS OF NITROAKRIDIN 3582

tion of messenger ribonucleic acid (RNA) (33)or, in a gram-positive thermophile, by inducingdegradation of messenger RNA (15). Quinacrineat bacteriostatic concentrations partly inhibitsbiosynthesis of both DNA and protein, but doesnot influence RNA biosynthesis. Conversely,quinacrine at bactericidal concentrations blocksDNA biosynthesis and affects biosynthesis ofboth protein and RNA (5). At the outset of ourinvestigation of NA, the only clue to its mode ofaction was the observation that NA inhibits theinduced synthesis of s-galactosidase in E. coli(16).We found that NA exhibited antibacterial ef-

fects typical of substituted acridines. It inhibitedthe growth of a variety of bacteria, induced bac-teriostasis and the formation of long filaments atlow concentrations, and was bactericidal athigher concentrations. Partial inhibition ofgrowth of E. coli correlated with inhibition ofDNA biosynthesis, and lesser inhibitions of pro-tein and RNA biosyntheses correlated with eachother. These correlations suggest an effect of NAon transcription of messenger RNA.

MATERIALS AND METHODSBacteria cited in the text and tables were strains

from the collections of the Department of MolecularBiology, Walter Reed Army Institute of Research, orfrom the Department of Microbiology, University ofMaryland. NA was the generous gift of E. von Wasi-liewski of the Farbwerke Hoechst, Frankfurt-Hoechst,Federal Republic of Germany. "4C-labeled thymidine,uracil, and isoleucine were purchased from New Eng-land Nuclear Corp., Boston, Mass.

Minimal inhibitory concentrations of NA were de-termined in standard serial dilution assays by inocu-lating 5-ml portions of either Brain Heart InfusionBroth or Trypticase Soy Broth (containing twofoldgraded concentrations of NA) with small inocula, incu-bating overnight, and observing whether culturalgrowth occurred.

Reductions in the exponential growth rates of bac-terial cultures were determined by inoculating liquidmedia with freshly grown bacteria to initial turbiditiesof 0.020 measured in a Beckman DU spectropho-tometer at 580 nm, incubating these cultures at 37 Cwith vigorous shaking until a turbidity of 0.125 wasattained, adding NA to the desired concentrations, andmeasuring turbidities periodically while continuing theincubation. Viability was determined by serial dilutionplate counting of portions of such cultures grown inBrain Heart Infusion Agar and incubated for 48 hr.

Experiments on the incorporation of thymidine, ura-cil, or isoleucine into bacterial DNA, RNA, or proteinwere carried out in cultures of E. coli C-2 growingexponentially at 37 C with vigorous shaking in a glu-cose-salts medium supplemented with 20 Ag/ml of L-phenylalanine. When the turbidity value reached 0.125(approximately 2 x 108 cells/ml) measured at 420 nm,the cultures were divided into 15-ml portions to which

NH-CH2.CH(OH).CH2NEt2

OsN)NNOMe

02N N~~OMeFIG. 1. Structure of Nitroakridin 3582 (1).

were added the desired quantities of NA and 2 umoles(specific activity, 0.13 uCi/,umole) of the respectivelabeled compounds. Incubation was resumed, and 1-mlsamples of cultures were withdrawn at intervals andcombined with I ml of cold 10% trichloroacetic acid.Samples containing "4C-thymidine or "4C-uracil werekept at 4 C for I hr; samples containing "4C-isoleucinewere heated at 70 C for 30 min. All samples then werefiltered through membrane filters (type HA, pore size0.45 um, Millipore Corp., Bedford, Mass.). The filterswere washed free of nonincorporated radioactive com-pounds with 5% cold trichloroacetic acid; the com-pounds were dissolved in a dioxane-based scintillationfluid [naphthalene, 380 g (Eastman Organic Chemicals,Rochester, N.Y.); 2, 5-diphenyloxazole, 22.8 g; 1,4-bis-2-(5-phenyloxazolyl)-benzene, 380 mg (Packard Instru-ment Co., Inc., Downers Grove, Ill.); and dioxane, 3.8liters (Fisher Scientific Co., Pittsburgh, Pa)] andcounted in a liquid scintillation counter (Nuclear Chi-cago Corp., Des Plaines, Ill.).

RESULTSAntibacterial effects of NA. Selected strains of

bacteria were screened for their sensitivity to NAby serial dilution assays either in Trypticase SoyBroth or in Brain Heart Infusion Broth (Tables Iand 2). Generally, gram-positive organisms weremore sensitive to inhibition by NA than weregram-negative bacteria. At the highest concen-trations of NA tested, the growth of Proteusmirabilis, Serratia marcescens, and Salmonellatyphimurium was not inhibited.

Determining the influence of NA on the expo-nential growth rates of bacteria revealed thatmost organisms exhibited systematic decreases intheir growth rates in response to increasing con-centrations of NA. These experiments were per-formed by periodic measurements of bacterialdensities at 580 nm of drug-free control culturesand of parallel cultures containing graded con-centrations of NA. B. licheniformis exhibited aninteresting departure from typical dosage re-sponse (Fig. 2). NA at concentrations above 2 x10- M evoked lysis of these bacteria after thelapse of slightly more than one doubling time ofthe drug-free control culture. NA at a concentra-tion of 5 x 10-5 M also produced lysis in culturesof Micrococcus lysodeikticus. (We find no refer-ence to comparable observations with other acri-dines.)Gram-negative enterobacteria did not lyse

under the influence of NA but grew into long fil-

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WOLFE, COOK, AND HAHN

TABLE 1. Sensitivity to Nitroakridin 3582 ofselectedbacteria growing in Trypticase Soy Broth

MinimalOrganism inhibitory

concn (M)

Leuconostoc citrovorum ............. 1 x 10-6Sarcina lutea .....1................. xX 10-6Bacillus licheniformis ............... 1I x 10-6Bacillus subtilis ............ ........ 2 x 10-6Micrococcus lysodeikticus ...... ..... 2 x 10-6Bacillus megaterium ........ ........ 2 x 10-5Staphylococcus aureus ....... ....... 2 x 10-5Shigella sonnei ............ ........ 4 x 10-'5Escherichia coli ............ ........ 8 x 10-'Pseudomonas fluorescens ...... ...... 4 x 1o- 4

Enterobacter aerogenes ....... ...... 4 x 1o- 4

Proteus mirabilis ........... ........ >4 x 1o- 4

Serratia marcescens ........ ........ >4 x 10- 4

TABLE 2. Sensitivity to Nitroakridin 3582 ofselectedbacteria growing in Brain Heart Infusion Broth

MinimalOrganism inhibitory

concn (M)

Sarcina lutea ...................... 2 x 10-'6Bacillus subtilis ............ ........ 2 x 10-'Micrococcus lysodeikticus ...... ..... 2 x 1o- 5

Bacillus licheniformis ........ ....... 2 x 10- 'Shigella sonnei ............ ........ 2 x 10-'Enterobacter aerogenes ....... ...... I x 10-4Staphylococcus aureus .............. 1 x 10-4Serratia marcescens ........ ........ >2 x 1o- 4

Salmonella typhimurium ...... ...... >2 x 1o- 4

Proteus mirabilis ........... ........ >2 x 10- 4

aments, as observed with other antibacterial acri-dines (4, 5). This probably results from suppres-sion of DNA biosynthesis. The filaments formedby E. coli C-2, exposed overnight to a partiallygrowth-inhibiting concentration of NA, are illus-trated in Fig. 3. Similar observations were madewith Enterobacter aerogenes, Serratia marces-cens, Shigella sonnei, and P. mirabilis.At concentrations above 10-4 M, NA was bac-

tericidal for E. coli C-2 (Fig. 4). NA at 10-4 Mwas bacteriostatic, and at lower concentration itreduced the rates of replication of the test orga-nism. In this type of experiment, the bacteriawere grown in a synthetic medium (9) supple-mented with 20 gg of phenylalanine per ml.Strain C-2 of E. coli is a phenylalanine auxo-troph. Use of this test strain allowed testing for abactericidal effect of NA in phenylalanine-freebacterial suspensions. In such suspensions, E.coli C-2 will fail to synthesize not only protein,but also RNA, the biosynthesis of which is under

stringent amino acid regulation in this organism.NA killed E. coli C-2 both in absence and in

presence of protein and RNA synthesis. NA at 3X 10- 4 M produced a marked decline in thenumber of viable bacteria in complete medium;the decline was even greater for the organisms inmineral-glucose medium without phenylalanine(Fig. 5). A slight decline in viability was ob-served as a result of phenylalanine starvationalone, beginning after approximately one dou-bling time of the control culture growing nor-mally in complete medium. Evidently, suppres-sion of protein and RNA synthesis did not pro-tect the bacteria from the lethal action of NA,but rather appeared to enhance it. Therefore, thebactericidal effect of NA is not one of inducingor tolerating some form of lethal biosynthesis.

Effects of NA on biosyntheses of proteins andnucleic adds. It was found in one of our labora-tories (16) that NA at the bacteriostatic concen-tration of 1.16 x 10-4 M inhibits the synthesis ofinduced enzymes in E. coli. This could resultfrom an inhibition of the formation of messengerRNA or from a direct effect on protein biosyn-thesis. In E. coli, a substituted 9-aminoacridine,quinacrine, inhibits DNA synthesis strongly, in-hibits protein synthesis partly, and inhibits RNA

1.o00

.800 Cant x 1O-6M

6 0 x10-5M

.400

.200-

0 .10S~~~~~~~~~~~~~~~~o5SxioM 4x0

TIME IN MINUTES

FIG. 2. Effect of graded concentrations of NA oncultures of Bacillus licheniformis growing in BrainHeart Infusion Broth. Growth or lysis was measuredturbidimetrically at 580 nm.

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ANTIBACTERIAL EFFECTS OF NITROAKRIDIN 3582

pe

01%

4%

J, =~.i*,~~~400,~~~~1

_-6_o

_ oo_Nt

FIG. 3. NA-induced filaments of Escherichia coli formed during overnight growth in the presence of 5 x 105M NA. Samples were heat-fixed, stained with Gram stain, and photographed with a Leitz A ristophot camera.Magnification: 2,200x.

biosynthesis least (5). It was logical to expectthat the antibacterial effects of NA reportedabove resulted from an interference with one or

several processes by which the vitally necessarypolymers of bacteria are synthesized. To test thishypothesis, the effect of NA on DNA, RNA,and protein biosynthesis was determined in E.coli C-2 by measuring the time courses of incor-poration of "4C-thymidine, "4C-uracil, and 14C-isoleucine into the polymers of exponentially re-

plicating cells of this bacterium in the presenceor absence of NA. Although it appeared logicalto employ 14C-phenylalanine to measure thecourse of protein biosynthesis in this phenylala-nine auxotroph, this was not done to avoid iso-topic dilution of this amino acid by the 20 ,g of12C-phenylalanine per ml which supplements theexperimental growth medium.

Figure 6 shows the time course of incorpora-tion of 14C-thymidine into exponentially growingE. coli C-2 at graded concentrations of NA.Concentrations of NA which inhibited growthpartially also inhibited incorporation partially; atthe bacteriostatic concentration of 10-4 M NA,radiothymidine was incorporated at a rate 10%that of the control, and no progressive incorpora-

tion was observed at the bacterial concentrationof 2 x 10-4 M NA. Hence, NA acted as a stronginhibitor of DNA biosynthesis in the test bacte-rium.

Figure 7 illustrates the effect of NA on thebacterial incorporation of "4C-uracil. Partiallygrowth-inhibiting concentrations of NA did notstrongly affect uracil incorporation. At the bac-teriostatic concentration, uracil incorporationwas inhibited 83%; at the bactericidal concentra-tion of 2 x 10-4 M, no uracil was incorporated.Clearly, NA had less effect on RNA biosynthesisthan on DNA biosynthesis.

Figure 8 shows the action of NA on the incor-poration of 14C-isoleucine into E. coli C-2. Theresults closely resemble those observed for theincorporation of radiouracil. At the bacterio-static concentration, NA inhibited the incorpora-tion by 77%; no isoleucine incorporation wasdetected at bactericidal concentrations of NA.The numerical results indicating partial inhibi-

tions of biosyntheses (Fig. 6-8) and thoseshowing reductions in the rate of bacterial prolif-eration (Fig. 4) could be correlated in a mannermeaningful to the interpretation of these data.All effects of NA were expressed as percentages

l/

I

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324 WOLFE, COOK, AND HAHN J. BACTERIOL.

1010 10o9 _

00

x2.xO5Mx \

K

-oJ

2 \ 0 NA+ -oI

J 108 F , IX10 M S *sNA-+10~~~~~~~~~~~~~~~~~~~~~~~~0

w~~~~~~~~~~~~~~~~~-J

- \ M30 60 90 120MINUTES

FIG. 5. Viability of E. coli C-2 incubated as in Fig.4 in presence or absence of phenylalanine, with or

*\ 4 x l0-4 without addition of3 x 10-4 M NA.

6 ,, 710 30 60 90 120

TIME IN MINUTES6o

FIG. 4. Effect of graded concentrations of NA onviability of Escherichia coli C-2 growing in glucose-salts medium supplemented with 20 pg of L-phenylala- J

nine per ml. a: ew6a: 5

of inhibition, and these numbers were repre-Xsented diagrammatically on semilogarithmic W -Controlprobability graph paper as functions of the loga-rithm of the molar concentration of NA (Fig. 9). /This procedure, first used in bacteriology to rep- 3wresent response of bacterial growth to graded aconcentrations of antibiotics (31), is a graphic 2probit transformation of desage response curves 24cto linearity, which facilitates arithmetic fitting of /

25_5Mthe best lines. The response lines of DNA bio-

_. xIC)

synthesis and of bacterial proliferation to the 5' O-5Mdosage of NA were superposable and showed a 12

50% effective dose (ED,.) of close to 3 x 10-' SW

M. The response lines of RNA and protein bio- IoIO4 Msyntheses were also superposable and showed anED,. of close to 6 x 10-' M. The slopes of the TIME IN MINUTEStwo pairs of dosage response lines differ, indicat- FIG. 6. Effect of NA on incorporation of "4C-thying, by extrapolation, that protein and RNA midine into Escherichia coli C-2, growing as in Fig. 4syntheses were not significantly affected at 2 x Cold trichloroacetic acid-soluble material was removec10-' M NA, but that DNA biosynthesis and cell before counting.

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VOL. 108, 1971 ANTIBACTERIAL EFFECTS OF NITROAKRIDIN 3582

Control

so / t-I

2-

70 2.5nI0-IS 457 10 3 NA . ProteinGo 20

30

TlblEINWIINUTES O lo~~~~4100

2i50E'c 50 cel

contng prlieato inEceihauonC2a untosoh

40c 70 NA

~~~~~~~~~~~~~~~~~~~~~90

95

20 - 96 w iIXq-M 99

10-~~~~~~~~~~~~~~~1TIME IN MINUTES o-50cr

FIG. 7. Effect of NA on incorporation of ofC-uracilM NA

into Escherichia coli C-2 growing as in Fig. 4. Cold tri- FIG. 9. Percentages of inhibition of DNA biosyn-_chioroacetic acid-soluble material was removed before thesis, RNA biosynthesis, protein biosynthesis, and cell

counting. proliferation in Escherichia coli C-2 as functions of the

concentration of NA.

too- proliferation were inhibited by approximately

40%. Clearly, decreases in rates of bacterial pro-

liferation by subinhibitory concentrations of NA

Contr were correlated with proportional decreases in

2.5xlOIM rates of DNA biosynthesis, and partial inhibitionsof RNA and protein biosyntheses were closely

a70 // 5i-M correlated.

DISCUSSION

The present investigation showed that the anti-bacterial action of NA can increase from a slight

a / // reduction of bacterial growth rates to completebacteriostasis and, with increases in drug concen-

40- // /tration by a factor of not more than 10, to a

marked bactericidal effect. Reversible and irre-2 30 // i versible inhibition of bacterial growth for con-

centrations of quinacrine differing by a factor of20 IXlsq4, only 4 was observed previously in one of our

laboratories.The extent of the reversible effects of NA on

2XI0-4M the growth of E. coli C-2 correlated with propor-

0 15 45 tional inhibitions of DNA biosynthesis. This cor-

TIME IN MINUTESrelation is plausible, as it is widely held that bac-terial cell division is causally related to and must

FIG. 8. Effect of NA on incorporation of 14C-iso- occur after the completion of one discrete roundleucine into Escherichia coli C-2 growing as in Fig. 4. of chromosomal DNA replication. When DNAHot trichloroacetic acid-soluble material was removed synthesis was inhibited by 90%, bacteriostasisbefore counting. occurred, and prolonged incubation of the test

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WOLFE, COOK, AND HAHN

bacterium in the presence of the static concentra-tion of NA (I x 10-' M) resulted in the forma-tion of long bacterial filaments. This indicationof continued biosynthesis of cell-wall polymer issupported by the continued biosyntheses of RNAand protein initially at rates of 25% of those innoninhibited control cultures.A bactericidal concentration of NA (2 x 10-4

M) inhibited DNA biosynthesis entirely. How-ever, increasing this concentration by a factor ofonly two produced a further increase in the rateof killing of E. coli C-2 (Fig. 4). Similar observa-tions were made with quinacrine. DNA biosynthe-sis cannot be inhibited by more than 100%. Thus,increases in the rates of killing of bacterial cul-tures at NA concentrations greater than thatwhich inhibits DNA biosynthesis completelysuggest that the accelerated bactericidal effectdoes not result solely from the cessation of DNAbiosynthesis.

In certain instances, inhibition or nonoccur-rence of DNA biosynthesis is lethal to bacteria.Logically, irreversible inhibition of DNA syn-thiesis through covalent cross-linking of thedouble helix by reduced mitomycin C (20) isbactericidal, as such DNA is permanently inca-pacitated to undergo the strand separation pre-requisite to chromosomal DNA replication. Thenonoccurrence of DNA synthesis in thymine-deprived E. coli 1ST- results in "thyminelessdeath" (3) unless all individual acts of DNAreplication go to completion before thymine iswithdrawn (22); the underlying mechanism re-mains hypothetical.

However, the present findings, together withthe analogous observations with quinacrine of an"overkill" by antibacterial acridines at concen-trations greater than those which block DNAbiosynthesis and the additional observation thatthe nonoccurrence of RNA and protein bio-syntheses did not protect bacteria from this le-thal effect, suggest that such acridines do not actmerely as metabolic poisons which induce unbal-anced growth and bacterial death. A differentexplanation of the lethal effect of acridines mustbe sought.Our present observations do not offer this ex-

planation. As acridines bind to double-helicalDNA by intercalation and also bind to the "cellenvelope" (31), they may alter either the sec-ondary structure of DNA or the association ofDNA with a membrane site of DNA replicationin a manner that renders these structural changesirreversible and, therefore, lethal. Hence, theobserved inhibition of bacterial DNA biosyn-thesis in the presence of NA may be regardednot as the cause of the lethality of the drug, butrather as a biochemical indicator of some direct

effect, with bactericidal consequences, on thestructure of DNA or on the state of cellular inte-gration.

ACKNOWLEDGMENT

We thank the Medical Audio-Visual Services Department,Walter Reed Army Institute of Research, for producing thefigures.

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