Effect of Naturally Occurring Xanthines on Bacteria

Transcription

1 APPLED MCROBOLOGY, May, American Society for Microbiology Vol. 13, No. 3 Printed in U.S.A. Effect of Naturally Occurring Xanthines on Bacteria. Antimicrobial Action and Potentiating Effect on Antibiotic Spectra C. V. SUNDAR RAJ AND SALM DHALA Department of Microbiology, Bhavan's College, Andheri, Bombay, ndia Received for publication 4 January 1965 ABSTRACT SUNDAR RAJ, C. V. (Bhavan's College, Bombay, ndia), AND SALM DHALA. Effect of naturally occurring xanthines on bacteria.. Antimicrobial action and potentiating effect on antibiotic spectra. Appl. Microbiol. 13: The effect of xanthines 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 theobromine. 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 influence of caffeine; filamentation of cells followed sublethal doses. Potentiation was seen with antibiotics and caffeine; resistant strains were killed with a lower dose of drug in the presence 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 to cause morphological changes in certain symbiotic rhizobia (Waksman, 1952). Henis, Tagari, and Volcani (1964) recently reported the antibacterial effect of tannins, and found extracts containing tannic acid to induce morphological changes in gram-negative bacteria. Considerable attention has been focussed on the effect of plant polyphenols on cellular functions (Pridham, 1960). Caffeine has also been used in mutational studies. Novick and Szilard (1951) found it to be mutagenic to bacterial cells. According to Gyorffy (1960), caffeine treatment increases the number of streptomycin-resistant mutants occurring in cultures of Xanthomonas phaseoli. Witkin (1959), Lieb (1961), and Shankel (1961) also reported the effect of caffeine on ultraviolet-induced mutants. Doneson and Shankel (1964) observed mutational synergism between radiation and methylated purines in Escherichia coli. The question therefore arises as to their effect on human flora, since the xanthines are consumed by man in sizeable doses and their mutagenic effect has already been indicated. The present paper describes the antimicrobial action of the xanthines and the potentiating effect of caffeine on antibiotic substances. MATERALS AND METHODS The xanthines used were caffeine, caffeine citrate, theophylline, and theobromine (B.P. Qualities, Rhodia, France). Dilutions tested for antimicrobial activity were prepared in 0.1 M phosphate buffer (ph 7.6), and were sterilized by Seitz filtration. The test organisms were: Bacillus subtilis NCTC 3690, Staphylococcus aureus NCTC 7447, Sarcina lutea, Streptococcus pyogenes NCTC 8370, Diplococcus pneumoniae NCTC 7465, Corynebacterium diphtheriae type mitis, Mycobacterium phlei NCTC 8151, M. smegmatis ATCC 607, Candida albicans LSHTM 3119 (London School of Hygiene and Tropical Medicine), Escherichia coli NCTC 9002, Aerobacter aerogenes, A. cloacae, Klebsiella pneumoniae, Proteus vulgaris, Salmonella typhosa NCTC 8222, S. schottmuelleri NCTC 5704, Shigella dysenteriae NCTC 4837, and Pseudomonas aeruginosa. All of the test organisms were grown in nutrient broth (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 was enriched with 1% dextrose, and the ph was adjusted to 6.0. Antimicrobial potency was expressed as the minimal inhibitory concentration (MC). Double dilutions of the xanthines were made in the assay medium; sets were run in duplicate in test tubes, each containing 2 ml of the assay medium. The tubes were inoculated to give 2 X 104 cells per milliliter of the test organism. To obtain a uniform number of living organisms, the test cultures were passed in the assay medium for three consecutive 432

2 VOL. 13, 1965 TABLE 1. Antimicrobial activity of xanthines ANTMCROBAL EFFECT OF XANTHNES Minimal inhibitory concn (jsg/ml) Organism Caf- Caf- Theo- Theo- Caine feine phyl- brofeine citrate Plhine mine Staphylococcus aureus 2, 500 2, 500 2, Sarcina lutea... 2,500 1,250 5,000 Streptococcus pyogenes 5,000 5,000 - Diplococcus pneumoniae... 2,500 Corynebacterium diphtheriae type mitis.. 2,500 5,000 Bacillus subtilis... 2, ,000 Mycobacterium phlei. 2,500 1,250-5,000 M. smegmatis... 5,000 2,500 5,000 Candida albicans...5,000. 5,000 - Escherichia coli... 5,000 5,000 - Aerobacter aerogenes.. 5,000 1,250 5,000 A. cloacae... 5,000 2,500 5,000 Klebsiella pneumoniae. 5,000 1,250 2,500 Salmonella typhosa... 5,000 2,500 5,000 - S. schottmuelleri.... 2,500 1,250 5,000 Proteus vulgaris... 2,500 2,500 5,000 2,500 Shigella dysenteriae 1,250 1,250 5,000 - Pseudomonas aeruginosa... 5,000 2,000 - * Not inhibited by 5,000 jug/ml. days before use, and were not used after 13 passages. The cultures were incubated at 37 C for 24 hr. Results were determined in terms of optical density in a Klett-Summerson colorimeter at 450 mju. Caffeine citrate was selected for testing the effect of xanthines on the antibacterial action of various antimicrobial agents. The antibacterials used were: penicillin G, sulfisoxazole, chloramphenicol, tetracycline hydrochloride, oxytetracycline hydrochloride, streptomycin, chlortetracycline hydrochloride, and a 1:1 mixture of oleandomycin and oxytetracycline. Caffeine citrate was added to the assay medium in a final concentration of 1,000 jug/ml. The standard disc-agar diffusion method preceded turbidimetric assay, with S. aureus and E. coli as the test organisms. n the disc-agar method, the concentrations per disc were: penicillin, 0.6 jag; sulfisoxazole, 125,g; other agents, 25 Ag. n the tube dilution method, the concentrations varied according to the nature and action of the drugs; the selected ranges are shown in Table 2. RESULTS AND DscuSSON The organisms tested were not inhibited at high xanthine dilutions; most of the organisms could well tolerate a concentration of 2,500 jig/ml (Table 1). B. subtilis, S. aureus, S. lutea, M. smegmatis, M. phlei, A. aerogenes, K. pneumoniae, S. dysenteriae, P. vulgaris, and S. schottmuelleri were found to be sensitive; C. diphtheriae, 433- S. pyogenes, C. albicans, E. coli, and P. aeruginosa were not inhibited at concentrations lower than 5,000 Ag/ml. B. subtili was most susceptible. S. aureus and S. lutea were quite sensitive, but the resistance of S. pyogenes and C. diphtheriae indicates that, among the gram-positive group, some are susceptible and others are not. Similarly the gram-negative bacilli differed in their susceptibility to caffeine. The inhibitory effect was found to be bactericidal in high concentrations and bacteriostatic in sublethal doses, as observed by streaking loopfuls from dilution tubes onto agar surfaces. Caffeine was more effective than theophylline and theobromine; caffeine citrate exhibited a greater action than pure caffeine, and, being more soluble, was used for further work. t was noticed that pigmentation of S. aureus, S. lutea, and Serratia marcescens was not inhibited, but P. aeruginosa could not produce its pigment in the presence of 2,000,ug/ml of caffeine, although there was no reduction in the number of cells and growth was otherwise unaffected. n separate experiments it was noted that sublethal doses of caffeine affected indole synthesis and lactose fermentation by E. coli; the organism produced indole much later than in the absence of caffeine. Lactose was fermented only after 24 hr; the effect was temporary and may give some clue to the mode of action of xanthines on bacteria. A few organisms showed elongation of cells leading to filamentation (Fig. 1 and 2). A. aerogenes and A. cloacae developed long filaments in the presence of 1,000,pg/ml of caffeine. Henis et al. (1964) found similar effects on P. fluorescens and E. coli when they were subjected to the action of tannins from carob extracts. Caffeine was found to exert an effect on the action of various antimicrobial agents. When incorporated in a final concentration of 1,000,ug/ml, caffeine citrate alone had no inhibitory effect on any of the test organisms used in the experiments. However, this concentration of caffeine rendered the test organisms more sensitive to the action of antibacterial agents (Table 2). With the agar diffusion method, chloramphenicol showed only a slight increase in the zones of inhibition when caffeine was present in the medium. With chlortetracycline and oxytetracycline, the inhibition zones were markedly increased against both S. aureus and E. coli. Tetracycline showed increased action against S. aureus only. n the tube dilution method, action of chloramphenicol and oxytetracycline was doubled against both the test organisms; the organisms showed a 10-fold increase in susceptibility to tetracycline; and, with chlortetracycline, the favorable effect of caffeine was evident only

3 434 SUNDAR RAJ AND DHALA APPL. MCROBOL.. '_ ¼k 'WV./k oo"rj.n i e.-f t..w 4~~~~~~~~~~~~~~~~~~~~~~. i.- / ' *w Downloaded from -~~~~~~N p t -S oi.u on July 4, 2018 by guest FG. A (top, left). Aerobacter aerogenes, 48-hr-old culture, stained with methylene blue, X 1,600. Thick, short bacilli are seen with capsule. FG. 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. FG. 2A (bottom, left). Aerobacter cloacae, 48-hr-old culture. Well-capsulated rods appear singly, in pairs, and in very short chains. Methylene blue, X 1,500. FG. 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 and eyclines than with other agents. With strepto- Shankel (1964) found a greater percentage of

4 VOL. 13, 1965 ANTMCROBAL EFFECT OF XANTHNES 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 Sulfisoxazole Streptomycin Chloramphenicol Chlortetracycline hydrochloride Oxytetracycline hydrochloride Tetracycline hydrochloride Synermycint * 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). Mixture (1:1) of oleandomycin and oxytetracycline (Pfizer, ndia). C. C) )CONTROL c o 50 *Q 40 Achromycin 40 ALg/ml. - ( z 30 *CONTROL 20 / Achromycin 40 A1g /ml. / + CaffeinoeOoOA4g/ml. 0 -'j / ;;-= *oachromycin 40 Alg/mi. 'chromycin 40 kag/ml. O OO O 4_l o + Caffeine1ooo jig/m l. HOURS FiG. 3. Turbidity changes of Staphylococcus aureus and Pseudomonas aeruginosa in media containing tetracycline (Achromycin) and tetracycline plus caffeine. streptomycin-resistant mutants of E. coli when the culture was irradiated with ultraviolet light in the presence of 500,ug/ml of caffeine. An antagonism may exist between streptomycin and caffeine; this possibility needs further attention. Experiments with resistant strains of S. aureus 10 o o Ps. aeruginoso *-- - _ S.aureus Achromycin % Achromycin Achromycin+Caffeine A1crom HOURS FG. 4. nfluence of caffeine on inhibition of Staphylococcus aureus and Pseudomonas aeruginosa by tetracycline (Achromycin). and P. aeruginosa clearly point out the favorable effect of caffeine in restricting the growth of organisms 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. n the presence of antibiotic alone, the lag phase was protracted to 6 hr in S. aureus and remained the same for P. aeruginosa. Combination of antibiotic and caffeine delayed growth of the former for 10 hr and that of the latter for 8 hr. Whereas 40 mg/ml of tetracycline (Achromycin, Lederle Laboratories, ndia) reduced growth of S. aureus by 63.9% in the first 12 hr (Fig. 4), in o 60 M z,= e

5 436 SUNDAR RAJ AND DHALA APPL. MCROBOL. corporation of 1,000,ug/ml of caffeine in the assay medium raised the inhibition to 88.9%. This potentiating effect showed an upward trend with lapse of time; in 48 hr, 94.1% reduction was seen as against 80.2% when the antibiotic acted alone. Similarly, a resistant strain of P. aeruginosa was subdued further by tetracycline in the presence of caffeine; in the first 12 hr, the number of survivors was reduced from 54.1 to 16.6% when caffeine was incorporated. Even at 24 hr, when the antibiotic was most effective, 22% of the cells survived, but caffeine reduced the growth to only 6%. After 48 hr, the antibiotic alone had permitted 55.2% growth, but caffeine restricted it to 13.9%. t is thus evident that caffeine affects the resistance of an organism to some drugs. n vivo studies and clinical trials are in progress to unfold further the relationships observed during the present studies. LTERATURE CTED DONESON,. N., AND D. M. SHANKEL Mutational synergism between radiations and methylated purines in Escherichia coli. J. Bacteriol. 87: GYORFFY, B The effect of some chemical mutagens upon Xanthomonas phaseoli var. fuscans. Abhandl. Deut. Akad. Wiss. Berlin Kl. Med., p HENS, Y., H. TAGAR, AND R. VOLCAN Effect of water extracts of carob pods, tannic acid, and their derivatives on the morphology and growth of microorganisms. Appl. Microbiol. 12: LEB, M Enhancement of ultraviolet-induced mutation in bacteria by caffeine. Z. Vererbungslehre 92: NOVCK, A., AND L. SZLARD Experiments on spontaneous and chemically induced mutations of bacteria growing in the chemostat. Cold Spring Harbor Symp. Quant. Biol. 16: PRDHAM, J. B. [ed.] Phenolics in plants in health and disease. Pergamon Press, nc., New York. SHANKEL, D. M Effects of metabolite analogues on development of mutations induced by "Noncidal" amounts of ultraviolet light. Bacteriol. Proc., p. 99. WAKSMAN, S. A Soil microbiology, p John Wiley & Sons, nc., New York. WTKN, E. M Post-irradiation metabolism and the timing of ultraviolet-induced mutations in bacteria. Proc. ntern. Congr. Genet., 10th, Montreal, : Downloaded from on July 4, 2018 by guest