1841743b0Nature184470019591128174317440028-0836195910.1038/1841743b0ukNatureNatureNATUREnatureNature 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/v184/n4700issueJournal homeArchiveCurrent issueAdvance online publicationPrivacy policySubscribeNature Publishing GroupCurrent issue1841743b0Nicotinamidase in Mycobacteria: A Method for Distinguishing Bovine Type Tubercle Bacilli from other Mycobacteria AU - KONNO, KIYOSHI AU - NAGAYAMA, HIDEO AU - OKA, SUTEMIResearch Institute for Tuberculosis and Leprosy, Tohoku University, Kitayobancho, Sendai.BOVINE type tubercle bacilli have been differentiated from other Mycobacteria by means of growth characteristics and pathogenecity for animals. They usually grow dysgonic in its original culture and are pathogenic for guinea pigs and rabbits. However, many laboratory strains of bovine tubercle bacilli now grow eugonic and can not be differentiated from growth characteristics, and furthermore, avirulent strains of bovine type tubercle bacilli like BCG or isoniazid resistant bovine tubercle bacilli have been developed, and they can not be differentiated from animal pathogenecity. Konno et al.1,2 suggested simple chemical method to distinguish human tubercle bacilli from bovine tubercle bacilli or atypical Mycobacteria applying high nicotinic acid production of human tubercle bacilli. Halpern and Grossowicz3 studied nicotinamidase activity of the extracts of M. phlei and BCG and reported M. phlei revealed high nicotinamidase activity than BCG. Bonicke and Lisboa4 compared nicotinamidase of emulsion of human tubercle bacilli and bovine tubercle bacilli and reported high nicotinamidase of human tubercle bacilli.The present investigation aims at distinguishing bovine type tubercle bacilli from other Mycobacteria (human tubercle bacilli, bovine tubercle bacilli, atypical acid-fast bacilli and non-pathogenic acid-fast bacilli) by nicotinamidase activity. Fifteen strains of various types of Mycobacteria grown on the surface of Sauton's synthetic liquid medium for 3 weeks (one week for non-pathogenic acid-fast bacilli) were filtered off, washed three times with water. Cell-free extracts were prepared by grinding mechanically the washed bacteria in a cold mechanical mortar with half the amount of sea-sand for 30 min. Cell debris was removed by centrifuging in the Hitachi preparative ultra-centrifuge in the cold at 10,000 g for 40 min. Then the cell-free extracts were dialyzed overnight against distilled water at 6 . The protein content of the extracts was determined by Mehl's biuret method5. The bacterial extracts which contain 2 mgm. of protein served for the determination of nicotinamidase. Ten [ moles of nicotinamide in 0-05 M of phosphate buffer (pH. 7-0) were added to each bacterial extract to reach a total volume of 2 ml. As a control, phosphate buffer with bacterial extracts without nicotinamide was used. They were placed in incubator at 37 for 3 hr. Ammonia formed in the system were distillated by Folin's method into JV/40 sulphuric acid and then determined colorimetrically by Russel's phenol-hypochlorite method6. As shown in Table 1, when 10 [ moles of nicotinamide were used as a substrate, human tubercle bacilli produced 3-2-3-7 [xmoles of ammonia, bovine tubercle bacilli were only 0-1 (moles, avian tubercle bacilli were 3-8 (moles, atypical acid-fast bacilli were 3-2-3-3 (moles, and non-pathogenic acid-fast bacilli were 6-8-7-4 (moles. Ammonia formation by bovine tubercle bacilli, either virulent or attenuated, was less than one thirtieth that of human, avian and atypical a/cid-fast bacilli, and about one sixtieth of non-pathogenic acid-fast bacilli. For simplifying this procedure, qualitative method using bacterial emulsion was tried. About 100 mgm. of bacteria grown in Sauton's liquid medium was emulsified and centrifuged at 3,000 r .p.m. for 20 min. in physiological saline solution three times to remove ammonia from the surface of bacteria. Then, a bacterial emulsion of about 20 mgm. per ml. in physiological saline solution was made. Two test tubes containing each 1 ml. of emulsion served for the test. Three hundred (gm. of nicotinamide in 1 ml. of 0-02 M phosphate buffer (pIL 7-0) was added to a test tube. Another control test tube contained bacterial emulsion in phosphate buffer without nicotinamide. Table 1. NICOTINAMIDASE ACTIVITY OF EXTRACTS FROM VARIOUS MYCOBACTERIA Types Human tubercle bacilli Bovine tubercle bacilli Avian tubercle bacilli Atypical acid-fast bacilli Non-pathogenic acid-fast bacilli Strains H37Rv INH-R H37Ra RI Rv Ravenel Bavenel INH-R BCG (Japan) BCG (Copenhagen) 4110 4121 P8 P 22 M.phlei M. 607 M. smegmatis Substrate Nicotin- Ammonia amide formed (moles) (moles) 10 3-3 10 3-2 10 3-7 10 3'6 10 0-1 10 0-1 10 0-1 10 0-1 10 3-8 10 3-8 10 3-3 10 3-2 10 6-8 10 6-8 io - : 7-4 Table 2. QUALITATIVE NICOTINAMIDASE ACTIVITY OF EMULSION FROM VARIOUS MYCOBACTERIA Types Human tubercle bacilli Bovine tubercle bacilli Avian tubercle bacilli Atypical acid-fast bacilli Non-pathogenic acid-fast bacilli Total Strains Virulent 10 INH resistant 2 attenuated 3 Virulent 7 attenuated 3 40 Nicotin-amidase test positive 10 K [plusmn]) 0 5 31 Nicotin-amidase negative 9 i>H 7-0 : incubation at 37 for 3 hr. >H 7-0, incubation at 37 for 12 hr. (substrate nicotinamide 300). Positive nicotinamidase : green to blue colour Negative nicotinamidase : amber colour These test tubes were incubated at 37 for 12 hr. and ammonia produced in the test tube was determined qualitatively by Russel 's phenol-hypochlorite method. Positive test showed green blue to deep blue colour development; on the other hand, the control was amber. Forty strains of various kinds of mycobacteria were tested for nicotinamidase qualitatively. As shown in Table 2, only bovine tubercle bacilli gave a negative test except one questionable positive strain. All other Mycobacteria gave a positive test irrespective of virulence. On performing this test, it is recommended to wash out the ammonia on the surface of bacteria thoroughly before adding nicotinamide. If ammonia remains, it will disturb the test.Konno, , K., Science, 124, 985 (1956).PubMedISIChemPortKonno, , K., Kurzmann, , R., Bird, , K. T., and Sbarra, , A., Amer. Rev. Tuberc., 77, 669, 675 (1958).ISIChemPortHalpern, , Y. S., and Grossowicz, , N., Biochem. J., 65, 716 (1957).PubMedISIChemPortBonicke, , R., and Lisboa, , B. P., Tbk. arzt., 13, 377 (1959).Mehl, , J. W., J. Biol. Chem., 157, 173 (1945).ISIChemPortRussel, , J. A., J. Biol. Chem., 156, 457 (1944).ChemPort