189775a0Nature1894766196103047757760028-0836196110.1038/189775a0ukNatureNatureNATUREnatureNature is a weekly international journal publishing the finest peer-reviewed research in all fields of science and technology on the basis of its originality, importance, interdisciplinary interest, timeliness, accessibility, elegance and surprising conclusions. Nature also provides rapid, authoritative, insightful and arresting news and interpretation of topical and coming trends affecting science, scientists and the wider public./nature/journal/v189/n4766issueJournal homeArchiveCurrent issueAdvance online publicationPrivacy policySubscribeNature Publishing GroupCurrent issue189775a0Chloramphenicol, a Simultaneous Carbon and Nitrogen Source for a Streptomyes sp. from Egyptian Soil
AU  - ABD-EL-MALEK, Y.
AU  - MONIB, M.
AU  - HAZEM, A.Department of Agricultural Bacteriology, Faculty of Agriculture, University of Cairo.IN work concerning the decomposition of antibiotics in soil and micro-organisms capable of this process, a chloramphenicol solution (250 [micro]gm. chloramphenicol/gm. soil) was percolated through 25-gm. sieved fresh garden soil, PH 7.5, in an apparatus similar to that described by Lees and Quastel1. The chloramphenicol was applied in three doses; the first dissolved in 50 ml. sterile distilled water and the others in 5 ml. added to the percolate in the reservoir. The antibiotic was bioassayed daily against Bacillus subtilis. The first dose disappeared after 11 days and the second and third after 3 days.Isolation of the micro-organisms involved in the decomposition of chloramphenicol was then carried out by plating the enriched soil in a medium containing the antibiotic as the sole organic carbon source. The medium (ammonium nitrate chloramphenicol medium) contained, in gm. per 100 ml., crystalline magnesium sulphate, 0 -5; crystalline ferrous sulphate, 0-003; calcium chloride, 0-005; potassium dihydro-gen phosphate, 0-1; dipotassium hydrogen phosphate, 0-25; ammonium nitrate, 0-05; agar, 1-3 adjusted to pH. 7-0-7-2, dispensed in 100 ml. quantitles and autoclaved at 15 Ib. for 15 min. Before use, the medium was melted, cooled to 50 C. and chloram-phenicol (100 (jigm./ml.) added. Plates were incubated at 30 C. for 15 days. Only one type of organism appeared on the plates of the highest dilution (10~5), a Streptomyces producing greyish-white thin mycelium and chains of chalky white rod-shaped conidia (1-6 X 0-8(1 in stained preparations) borne on long open-spiral conidiophores (section Spira in the classification of Pridham, Hesseltine and Benedict2). Its cultural characteristics on the media proposed by Waksman3 were: mycelium greyish-white on Czapek's and starch agar and nutrient gelatin, orange on glucose asparagine, glycerol asparagine and yeast glucose agar media, and lemon-yellow on nutrient agar. Chalky white conidia were produced on all the media except on nutrient gelatin where the growth was markedly poor. Pigmentation of the mycelium appeared to be connected with the heaviness of growth; on media showing non-pig-mented mycelia the growth was poorer than that produced on media that revealed pigmentation. Growth in liquid media was restricted to a thin unpigmented pellicle consisting of more or less separate colonies which acquired a chalky white surface when conidia were formed. None of the media tested induced the formation of diffusible pigments. Olucose, saccharose, lactose and mannitol were not utilized. There was no hydrolysis of starch, liquefaction of gelatin, digestion of casein or cellulose, production of ammonia from peptone or reduction of nitrate. Litmus milk turned alkaline after 15 days. Growth took place at 18 and 30 but not at 55 C. The organism resisted pasteurization in glucose broth at 60 C. for 30 min. It had no antagonistic properties when tested against Staphylococcus aureus, JBacillus subtilis, Escherichia coli and Mycobacteriwn phlei.
Repeated sub-culture (6 times) in the ammonium nitrate chloramphenicol liquid medium, using straight wire inoculation, always resulted in growth accompanied by the inactivation of the antibiotic, that is, the organism is capable of utilizing chloramphenicol.as the sole source of carbon. On sub-culturing in the.same manner (6 times) in the liquid medium without ammonium nitrate (chloramphenicol medium) growth.and inactivation of the antibiotic again occurred, that is, the organism is capable of utilizing chloramphenicol also as the sole source of nitrogen. This property was confirmed when the growth in the chloramphenicol medium was centrifuged, twice washed with sterile distilled water and used for inoculating fresh media (Table 1). Repeated sub-culturing decreased the inactivation period and improved growth. Inoculation of the liquid medium without chloramphenicol failed to produce visible growth after periods of incubation up to one month.
Table 1. DISAPPEARANCE OF CHLORAMPBCENICOL ON REPEATED SUB-CULTURING WITH WASHED Streptomyces, INCUBATION AT 30 C.
		nl.*		ml.*
	Inactivation period (days)	Visible growthf	Inactivation period (days)	Visible growthf
First sub-culture Second Third Fourth 	7 3 3	20 7 5 2	+ + +
In non-inoculated controls the variation in diameter of inhibition es did not exceed 0-5 mm. in 20 days incubation, t Seven days growth on glucose broth + + + +.
The ability of the organism to grow in increasing concentrations of chloramphenicol as sole carbon and nitrogen source was tested by inoculating 100 ml. quantities of medium containing 100, 250, 350, 400, 500, 550, 600 and 1,000 ptgm. chloramphenicol/ml. with a loopful of 7-day culture in chloramphenicol liquid medium (100 (jigm./ml.). Growth of the organism and inactivation of the antibiotic took place in all concentrations up to 600 jAgm./ml., where the antibiotic completely disappeared after 13 days. No growth or reduction in activity could be detected within one month in the medium containing 1,000 fjtgm./ml.
Finally, the purity of the Z-chloramphenicol used (Kemicetine, Carlo Erba) was verified by the determination of its melting point, carbon, nitrogen, hydrogen and chloride contents and by checking the weight in the capsules supplied.
Work is in progress to elucidate the mechanism of the decomposition of chloramphenicol by the isolate.
We wish to express our thanks to Dr. T. Gibson of the School of Agriculture, Edinburgh, for constructive criticism.Lees, , H., and Quastel, , J. H., Biochem. J., 40, 803 (1946).ISIPridham, , T. G., Hesseltine, , C. W., and Benedict, , R. G., App. Microbiol., 6, 52 (1958).ISIChemPortWaksman, , S. A., Bact. Rev., 21, 1 (1957).PubMedISIChemPort