APPLIED MICROBIOLOGY, May, 1965Copyright @ 1965 American Society for Microbiology

Vol. 13, No. 3Printed in U.S.A.

Effect of Naturally Occurring Xanthines on Bacteria

I. Antimicrobial Action and Potentiating Effect on Antibiotic Spectra

C. V. SUNDAR RAJ AND SALIM DHALA

Department of Microbiology, Bhavan's College, Andheri, Bombay, India

Received for publication 4 January 1965

ABSTRACT

SUNDAR RAJ, C. V. (Bhavan's College, Bombay, India), AND SALIM DHALA. Effectof naturally occurring xanthines on bacteria. I. Antimicrobial action and potentiatingeffect on antibiotic spectra. Appl. Microbiol. 13:432-436. 1965.-The effect of xan-

thines on various microorganisms was studied. The antibacterial effect was not high;most of the test organisms could easily withstand a concentration of 2,500 Ag/ml.Caffeine was more antibacterial than theophylline, and the latter more than theo-bromine. Caffeine citrate exhibited greater inhibitory effect than did pure caffeine.The effect was both bacteriostatic and bactericidal against susceptible organisms.The susceptibility of organisms to xanthines differed greatly even in related species.The morphology of Aerobacter aerogenes and A. cloacae was affected under the influenceof caffeine; filamentation of cells followed sublethal doses. Potentiation was seen withantibiotics and caffeine; resistant strains were killed with a lower dose of drug in thepresence of caffeine. This potentiating effect was pronounced with the tetracyclines;with streptomycin, the effect was the contrary.

Caffeine and natural alkaloids like pyridine,guanidine, and chinoline have been reported tocause morphological changes in certain symbi-otic rhizobia (Waksman, 1952). Henis, Tagari,and Volcani (1964) recently reported the anti-bacterial effect of tannins, and found extractscontaining tannic acid to induce morphologicalchanges in gram-negative bacteria. Considerableattention has been focussed on the effect of plantpolyphenols on cellular functions (Pridham,1960). Caffeine has also been used in mutationalstudies. Novick and Szilard (1951) found it to bemutagenic to bacterial cells. According to Gyorffy(1960), caffeine treatment increases the numberof streptomycin-resistant mutants occurring incultures of Xanthomonas phaseoli. Witkin (1959),Lieb (1961), and Shankel (1961) also reportedthe effect of caffeine on ultraviolet-induced mu-tants. Doneson and Shankel (1964) observedmutational synergism between radiation andmethylated purines in Escherichia coli.The question therefore arises as to their effect

on human flora, since the xanthines are consumedby man in sizeable doses and their mutageniceffect has already been indicated. The presentpaper describes the antimicrobial action of thexanthines and the potentiating effect of caffeineon antibiotic substances.

MATERIALS AND METHODS

The xanthines used were caffeine, caffeinecitrate, theophylline, and theobromine (B.P.

Qualities, Rhodia, France). Dilutions tested forantimicrobial activity were prepared in 0.1 Mphosphate buffer (pH 7.6), and were sterilized bySeitz filtration.The test organisms were: Bacillus subtilis

NCTC 3690, Staphylococcus aureus NCTC 7447,Sarcina lutea, Streptococcus pyogenes NCTC 8370,Diplococcus pneumoniae NCTC 7465, Corynebac-terium diphtheriae type mitis, Mycobacterium phleiNCTC 8151, M. smegmatis ATCC 607, Candidaalbicans LSHTM 3119 (London School of Hygieneand Tropical Medicine), Escherichia coli NCTC9002, Aerobacter aerogenes, A. cloacae, Klebsiellapneumoniae, Proteus vulgaris, Salmonella typhosaNCTC 8222, S. schottmuelleri NCTC 5704, Shigelladysenteriae NCTC 4837, and Pseudomonas aeru-ginosa.

All of the test organisms were grown in nutrientbroth (1% peptone, 0.3% meat extract, 0.5%NaCl; pH 7.6) and tested in Penassay Broth,Penassay Base Agar, and Penassay Seed Agar(Difco). For C. diphtheriae, S. pyogenes, and D.pneumoniae, the media were enriched with 5%rabbit serum; for C. albicans, nutrient broth wasenriched with 1% dextrose, and the pH was ad-justed to 6.0.

Antimicrobial potency was expressed as theminimal inhibitory concentration (MIC). Doubledilutions of the xanthines were made in the assaymedium; sets were run in duplicate in test tubes,each containing 2 ml of the assay medium. Thetubes were inoculated to give 2 X 104 cells permilliliter of the test organism. To obtain a uniformnumber of living organisms, the test cultures were

passed in the assay medium for three consecutive

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ANTIMICROBIAL EFFECT OF XANTHINES

TABLE 1. Antimicrobial activity of xanthines

Minimal inhibitory concn (jsg/ml)

Organism Caf- Caf- Theo- Theo-Caine feine phyl- bro-feine citrate Plhine mine

Staphylococcus aureus 2, 500 2, 500 2, 500 -

Sarcina lutea......... 2,500 1,250 5,000Streptococcus pyogenes 5,000 5,000 -Diplococcus pneu-moniae ........... 2,500

Corynebacterium diph-theriae type mitis. . 2,500 5,000

Bacillus subtilis....... 2,500 312 5,000Mycobacterium phlei . 2,500 1,250 - 5,000M. smegmatis......... 5,000 2,500 5,000Candida albicans ....5,000 5,000 -Escherichia coli ....... 5,000 5,000 -

Aerobacter aerogenes .. 5,000 1,250 5,000A. cloacae ............ 5,000 2,500 5,000Klebsiella pneumoniae. 5,000 1,250 2,500Salmonella typhosa.... 5,000 2,500 5,000 -

S. schottmuelleri . .... 2,500 1,250 5,000Proteus vulgaris....... 2,500 2,500 5,000 2,500Shigella dysenteriae 1,250 1,250 5,000 -Pseudomonas aerugi-

nosa .............. 5,000 2,000 -

* Not inhibited by 5,000 jug/ml.

days before use, and were not used after 13 pas-

sages. The cultures were incubated at 37 C for24 hr. Results were determined in terms of opticaldensity in a Klett-Summerson colorimeter at450 mju.

Caffeine citrate was selected for testing theeffect of xanthines on the antibacterial action ofvarious antimicrobial agents. The antibacterialsused were: penicillin G, sulfisoxazole, chlor-amphenicol, tetracycline hydrochloride, oxytetra-cycline hydrochloride, streptomycin, chlortetra-cycline hydrochloride, and a 1:1 mixture ofoleandomycin and oxytetracycline.

Caffeine citrate was added to the assay mediumin a final concentration of 1,000 jug/ml. The stand-ard disc-agar diffusion method preceded turbidi-metric assay, with S. aureus and E. coli as the testorganisms. In the disc-agar method, the concen-

trations per disc were: penicillin, 0.6 jAg;sulfisoxazole, 125 ,g; other agents, 25 Ag. In thetube dilution method, the concentrations variedaccording to the nature and action of the drugs;the selected ranges are shown in Table 2.

RESULTS AND DIscuSSION

The organisms tested were not inhibited athigh xanthine dilutions; most of the organismscould well tolerate a concentration of 2,500jig/ml (Table 1). B. subtilis, S. aureus, S. lutea,M. smegmatis, M. phlei, A. aerogenes, K. pneu-moniae, S. dysenteriae, P. vulgaris, and S. schott-muelleri were found to be sensitive; C. diphtheriae,

S. pyogenes, C. albicans, E. coli, and P. aeruginosawere not inhibited at concentrations lower than5,000 Ag/ml. B. subtili was most susceptible.S. aureus and S. lutea were quite sensitive, butthe resistance of S. pyogenes and C. diphtheriaeindicates that, among the gram-positive group,some are susceptible and others are not. Similarlythe gram-negative bacilli differed in their sus-ceptibility to caffeine. The inhibitory effect wasfound to be bactericidal in high concentrationsand bacteriostatic in sublethal doses, as observedby streaking loopfuls from dilution tubes ontoagar surfaces.

Caffeine was more effective than theophyllineand theobromine; caffeine citrate exhibited agreater action than pure caffeine, and, beingmore soluble, was used for further work.

It was noticed that pigmentation of S. aureus,S. lutea, and Serratia marcescens was not inhib-ited, but P. aeruginosa could not produce itspigment in the presence of 2,000 ,ug/ml of caf-feine, although there was no reduction in thenumber of cells and growth was otherwise un-affected. In separate experiments it was notedthat sublethal doses of caffeine affected indolesynthesis and lactose fermentation by E. coli;the organism produced indole much later thanin the absence of caffeine. Lactose was fermentedonly after 24 hr; the effect was temporary andmay give some clue to the mode of action ofxanthines on bacteria.A few organisms showed elongation of cells

leading to filamentation (Fig. 1 and 2). A. aero-genes and A. cloacae developed long filaments inthe presence of 1,000 ,pg/ml of caffeine. Heniset al. (1964) found similar effects on P. fluorescensand E. coli when they were subjected to theaction of tannins from carob extracts.

Caffeine was found to exert an effect on theaction of various antimicrobial agents. Whenincorporated in a final concentration of 1,000,ug/ml, caffeine citrate alone had no inhibitoryeffect on any of the test organisms used in theexperiments. However, this concentration of caf-feine rendered the test organisms more sensitiveto the action of antibacterial agents (Table 2).With the agar diffusion method, chloramphenicolshowed only a slight increase in the zones ofinhibition when caffeine was present in the me-dium. With chlortetracycline and oxytetracy-cline, the inhibition zones were markedly in-creased against both S. aureus and E. coli. Tetra-cycline showed increased action against S. aureusonly. In the tube dilution method, action ofchloramphenicol and oxytetracycline was dou-bled against both the test organisms; the organ-isms showed a 10-fold increase in susceptibilityto tetracycline; and, with chlortetracycline, thefavorable effect of caffeine was evident only

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FIG. IA (top, left). Aerobacter aerogenes, 48-hr-old culture, stained with methylene blue, X 1,600.Thick, short bacilli are seen with capsule.

FIG. 1B (top, right). Aerobacter aerogenes, 48-hr-old culture, grown in the presence of caffeine citrate(1,000 ,ug/ml). The cells appear elongated and filamentous, with uneven staining. Methylene blue, X 1,600.

FIG. 2A (bottom, left). Aerobacter cloacae, 48-hr-old culture. Well-capsulated rods appear singly, inpairs, and in very short chains. Methylene blue, X 1,500.

FIG. 2B (bottom, right). Aerobacter cloacae, 48-hr-old culture grown in the presence of caffeine citrate(1,000 Ag/rml). Capsule formation is suppressed;filamentation and branching are pronounced.

against S. aureus. Thus, a synergism was observed mycin, there was no such effect; on the contrary,between caffeine and some antibacterial agents. some organisms could resist the action of strepto-This effect was more pronounced with the tetra- mycin in the presence of caffeine. Doneson andeyclines than with other agents. With strepto- Shankel (1964) found a greater percentage of

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ANTIMICROBIAL EFFECT OF XANTHINES 435

TABLE 2. Potentiation of Antimicrobial action by caffeine*

Disc agar method (zones in mm) Tube dilution methodt

Antibacterial agent Amt/disc S. aureus E. coli S. aureus E. coli

A A+ C A A+ C A A+ C A A+ C

Penicillin G ................ 0.6 22 27 - 0.125 0.031Sulfisoxazole ............... 125 14 18 125 125Streptomycin .............. 25 11 13 13 13 25 30 15 25Chloramphenicol ........... 25 22 24 18 20 10 5 5 2.5Chlortetracycline hydro-

chloride .................. 25 13 29 10 18 25 5 25 10Oxytetracycline hydrochlo-

ride .................... 25 14 25 12 17 20 10 20 10Tetracycline hydrochloride. 25 12 25 10 10 10 1 10 1Synermycint ............... 25 21 23 20 15

* A = antibiotic alone; A + C = antibiotic + 1,000 ,Ag/ml of caffeine citrate in assay medium.t Results expressed in terms of minimal inhibitory concentration (Ag/ml).I Mixture (1:1) of oleandomycin and oxytetracycline (Pfizer, India).

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FiG. 3. Turbidity changes of Staphylococcusaureus and Pseudomonas aeruginosa in media con-taining tetracycline (Achromycin) and tetracyclineplus caffeine.

streptomycin-resistant mutants of E. coli whenthe culture was irradiated with ultraviolet lightin the presence of 500 ,ug/ml of caffeine. An an-

tagonism may exist between streptomycin andcaffeine; this possibility needs further attention.

Experiments with resistant strains of S. aureus

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FIG. 4. Influence of caffeine on inhibition ofStaphylococcus aureus and Pseudomonas aeruginosaby tetracycline (Achromycin).

and P. aeruginosa clearly point out the favorableeffect of caffeine in restricting the growth of or-

ganisms in an antibiotic-containing medium (Fig.3). The lag phase in control tubes of S. aureus

and P. aeruginosa was 2 and 4 hr, respectively.In the presence of antibiotic alone, the lag phasewas protracted to 6 hr in S. aureus and remainedthe same for P. aeruginosa. Combination of anti-biotic and caffeine delayed growth of the formerfor 10 hr and that of the latter for 8 hr.Whereas 40 mg/ml of tetracycline (Achromycin,

Lederle Laboratories, India) reduced growth ofS. aureus by 63.9% in the first 12 hr (Fig. 4), in-

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corporation of 1,000 ,ug/ml of caffeine in theassay medium raised the inhibition to 88.9%. Thispotentiating effect showed an upward trend withlapse of time; in 48 hr, 94.1% reduction was seenas against 80.2% when the antibiotic acted alone.Similarly, a resistant strain of P. aeruginosa wassubdued further by tetracycline in the presenceof caffeine; in the first 12 hr, the number of sur-vivors was reduced from 54.1 to 16.6% whencaffeine was incorporated. Even at 24 hr, whenthe antibiotic was most effective, 22% of the cellssurvived, but caffeine reduced the growth toonly 6%. After 48 hr, the antibiotic alone hadpermitted 55.2% growth, but caffeine restricted itto 13.9%.

It is thus evident that caffeine affects the re-sistance of an organism to some drugs. In vivostudies and clinical trials are in progress to un-fold further the relationships observed during thepresent studies.

LITERATURE CITED

DONESON, I. N., AND D. M. SHANKEL. 1964. Muta-tional synergism between radiations andmethylated purines in Escherichia coli. J. Bac-teriol. 87:61-67.

GYORFFY, B. 1960. The effect of some chemicalmutagens upon Xanthomonas phaseoli var.fuscans. Abhandl. Deut. Akad. Wiss. BerlinKl. Med., p. 110-115.

HENIS, Y., H. TAGARI, AND R. VOLCANI. 1964.Effect of water extracts of carob pods, tannicacid, and their derivatives on the morphologyand growth of microorganisms. Appl. Microbiol.12:204-209.

LIEB, M. 1961. Enhancement of ultraviolet-in-duced mutation in bacteria by caffeine. Z.Vererbungslehre 92:416-429.

NOVICK, A., AND L. SZILARD. 1951. Experiments onspontaneous and chemically induced mutationsof bacteria growing in the chemostat. ColdSpring Harbor Symp. Quant. Biol. 16:337-343.

PRIDHAM, J. B. [ed.] 1961. Phenolics in plants inhealth and disease. Pergamon Press, Inc., NewYork.

SHANKEL, D. M. 1961. Effects of metaboliteanalogues on development of mutations inducedby "Noncidal" amounts of ultraviolet light.Bacteriol. Proc., p. 99.

WAKSMAN, S. A. 1952. Soil microbiology, p. 214.John Wiley & Sons, Inc., New York.

WITKIN, E. M. 1959. Post-irradiation metabolismand the timing of ultraviolet-induced mutationsin bacteria. Proc. Intern. Congr. Genet., 10th,Montreal, 1958 1:280-299.

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