Radiometric studies on the oxidation of (1-14c) fatty acids by drug-susceptible and drug-resistant mycobacteria

Camargo, M.d., Edwaldo E.; Kopajtic, Theresa M.; Hopkins, Glinda K.; Cannon, Nancy P.; Wagner Jr., M.d., Henry N.

doi:10.1590/S0036-46651987000100002

Abstracts

A radiometric assay system has been used to study oxidation patterns of (1-14C) fatty acids by drug-susceptible and drug-resistant organisms of the genus Mycobacterium. Two strains of M. tuberculosis susceptible to all drugs, H37Rv and Erdman, were used. Drug-resistant organisms included in this investigation were M. tuberculosis H37Rv resistant to 5 ug/ml isoniazid, M. bovis, M. avium, M. intracellular, M. kansasii and M. chelonei. The organisms were inoculated in sterile reaction vials containing liquid 7H9 medium, 10% ADC enrichment and 1.0 uCi of one of the (1-14C) fatty acids (butyric, hexánoic, octanoic, decanoic, lauric, myristic, palmitic, stearic, oleic, linoleic, linolenic). Vials were incubated at 37°C and the 14CO2 envolved was measured daily for 3 days with a Bactec R-301 instrument. Although each individual organism displayed a different pattern of fatty oxidation, these patterns were not distinctive enough for identification of the organism. No combination of fatty acids nor preferential oxidation of long chain or of short chain fatty acids were able to separate susceptible from resistant organisms. Further investigation with a larger number of drug susceptible mycobacteria including assimilation studies and oxidation of other substrates may be required to achieve a distinction between drug-susceptible and drug-resistant mycobacteria.

Mycobacteria; Oxidation patterns; Radiometric system; Drug-resistance

Um sistema radiométrico foi utilizado para estudar os padrões de oxidação dos (1-14C) ácidos graxos por microorganismos do gênero Mycobacterium sensíveis e resistentes a drogas. Foram usadas duas cepas do M. tuberculosis sensíveis a todas as drogas, H37Rv e Erdman. As micobactérias resistentes foram M. tuberculosis H37Rv resistente a 5 ug/ml de hidrazida, M. bovis, M. avium, M. intracellulare, M. kansasii e M. chelonei. As micobactérias foram inoculadas em frascos estéreis contendo o meio líquido 7H9 com 10% do complexo albumina-dextrose-catalase e 1,0 uCi de um dos (1-14C) ácidos graxos (butírico, hexanoico, octanoico, decanóico, láurico, mirístico, plamítico, esteárico, olêico, linolêico, linolênico). Os frascos foram incubados a 37°C e o 14C0(2) produzido pelas micobactérias foi medido durante 3 dias, com uma máquina Bactec-R-301. Embora cada micobactéria apresentasse um padrão distinto de oxidação de ácido graxo, estes padrões não foram suficientemente diferentes para identificá-la. Nenhuma combinação de ácidos graxos nem a oxidação preferencial de ácidos graxos de cadeias longas ou curtas foi capaz de separar as micobactérias resistentes das sensíveis. Outras experiências com um maior número de micobactérias sensíveis, incluindo estudo da assimilação de substâncias marcadas, são necessárias para se tentar a diferenciação entre as micobactérias sensíveis e as resistentes a drogas.

14C-Fatty Acids

ARTIGOS ORIGINAIS

Radiometric studies on the oxidation of (1-¹⁴c) fatty acids by drug-susceptible and drug-resistant mycobacteria

Esudos radiométricos sobre a oxidação de (1-¹⁴C) ácidos graxos por micobactérias sensíveis e resistentes a drogas

Edwaldo E. Camargo, M.d.; Theresa M. Kopajtic; Glinda K. Hopkins; Nancy P. Cannon; Henry N. Wagner Jr., M.d.

From the Division of Nuclear Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205, U.S.A. Supported in part by USPHS Grant GM 10548

^{Address for correspondence} Address for correspondence: Edwaldo E. Camargo M.D., Centro de Medicina Nuclear da Universidade de São Paulo. Caixa Postal 22022. CEP 01499 São Paulo, SP., Brasil.

ABSTRACT

A radiometric assay system has been used to study oxidation patterns of (1-¹⁴C) fatty acids by drug-susceptible and drug-resistant organisms of the genus Mycobacterium.

Two strains of M. tuberculosis susceptible to all drugs, H₃₇Rv and Erdman, were used. Drug-resistant organisms included in this investigation were M. tuberculosis H₃₇Rv resistant to 5 ug/ml isoniazid, M. bovis, M. avium, M. intracellular, M. kansasii and M. chelonei. The organisms were inoculated in sterile reaction vials containing liquid 7H9 medium, 10% ADC enrichment and 1.0 uCi of one of the (1-¹⁴C) fatty acids (butyric, hexánoic, octanoic, decanoic, lauric, myristic, palmitic, stearic, oleic, linoleic, linolenic). Vials were incubated at 37°C and the ¹⁴CO₂ envolved was measured daily for 3 days with a Bactec R-301 instrument.

Although each individual organism displayed a different pattern of fatty oxidation, these patterns were not distinctive enough for identification of the organism. No combination of fatty acids nor preferential oxidation of long chain or of short chain fatty acids were able to separate susceptible from resistant organisms. Further investigation with a larger number of drug susceptible mycobacteria including assimilation studies and oxidation of other substrates may be required to achieve a distinction between drug-susceptible and drug-resistant mycobacteria.

Key words: Mycobacteria; ¹⁴C-Fatty Acids; Oxidation patterns; Radiometric system; Drug-resistance.

RESUMO

Um sistema radiométrico foi utilizado para estudar os padrões de oxidação dos (1-¹⁴C) ácidos graxos por microorganismos do gênero Mycobacterium sensíveis e resistentes a drogas. Foram usadas duas cepas do M. tuberculosis sensíveis a todas as drogas, H₃₇Rv e Erdman. As micobactérias resistentes foram M. tuberculosis H₃₇Rv resistente a 5 ug/ml de hidrazida, M. bovis, M. avium, M. intracellulare, M. kansasii e M. chelonei. As micobactérias foram inoculadas em frascos estéreis contendo o meio líquido 7H9 com 10% do complexo albumina-dextrose-catalase e 1,0 uCi de um dos (1-¹⁴C) ácidos graxos (butírico, hexanoico, octanoico, decanóico, láurico, mirístico, plamítico, esteárico, olêico, linolêico, linolênico). Os frascos foram incubados a 37°C e o ¹⁴C0₂ produzido pelas micobactérias foi medido durante 3 dias, com uma máquina Bactec-R-301.

Embora cada micobactéria apresentasse um padrão distinto de oxidação de ácido graxo, estes padrões não foram suficientemente diferentes para identificá-la. Nenhuma combinação de ácidos graxos nem a oxidação preferencial de ácidos graxos de cadeias longas ou curtas foi capaz de separar as micobactérias resistentes das sensíveis. Outras experiências com um maior número de micobactérias sensíveis, incluindo estudo da assimilação de substâncias marcadas, são necessárias para se tentar a diferenciação entre as micobactérias sensíveis e as resistentes a drogas.

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Recebido para publicação, em 6/2/1986.

1. ANTONINE, A. D. & TEPPER, B. S. - Enviromental control of glycogen and lipid content of Mycobacterium tuberculosis J. Bact., 100: 538-539, 1969.
2. BAILEY, W. R. & SCOTT, E. G. - Diagnostic microbiology 2nd ed. St. Louis, CV. Mosby, 1970c. p. 368.
3. BENNET, P. & ASSELINEAU, J. - Influence de l'age sur la teneur en acides gras a chaine ramifie' du bacile tuberculeux. Ann. Inst. Pasteur, 118: 324-329, 1970.
4. CAMARGO, E. E.; LARSON, S. M.; TEPPER, B. S.; CHARACHE, P. & WAGNER JR., H. N. - Automated radiometric detection of M. tuberculosis and M. lepraemurium J. nucl. Med., 15: 48, 1974.
5. CAMARGO, E. E.; LARSON, S. M.; TEPPER, B. S. & WAGNER JR., H. N. - Radiometric studies of Mycobacterium lepraemurium Int. J. Leprosy, 44: 294-300, 1976.
6. CAMARGO, E. E.; KERTCHER, J. A.; LARSON, S. M.; TEPPER, B. S.; WAGNER JR., H. N. - Radiometric measurement of differential metabolism of fatty acids by Mycobacterium lepraemurium Int. J. Leprosy, 47: 126-132, 1979.
7. CAMARGO, E. E.; KERTCHER, J. A. & LARSON, S. M. - Radiorespirometry in identification of mycobacteria. In: HORST, W.; WAGNER JR., H. N. & BU-CHANAN, J. W., ed. - Frontiers in Nuclear Medicine. Berlin, Springer-Verlag, 1980. p. 286-298.
8. CAMARGO, E. E.; MILKOWSKI, T. A. & WAGNER JR., H. N. - Relationship between amino acid metabolism and drug resistance of M. tuberculosis J. nucl. Med., 21: 59, 1980.
9. CAMARGO, E. E.; KERTCHER, J. A.; aARSON, S. M.; TEPPER, B. S. & WAGNER JR., H. N. - Radiometric measurement of differential metabolism of fatty acid by mycobacteria. Int. J. Leprosy, 50: 200-204, 1982.
10. DeBLANC JR., H. J.; DeLAND, F. H. & WAGNER JR., H. N. - Automated radiometric detection of bacteria in 2,967 blood cultures. Appl. Microbiol., 22: 846-849, 1971.
11. DeLAND, F. H. & WAGNER JR., H. N. - Automated radiometric detection of bacterial growth in cultures. J. Lab. clin. Med., 75: 529-534, 1970.
12. DUBOS, R. J. - The effect of organic acids on mammalian tubercle bacilli. J. exp. Med., 92: 319-332, 1950.
13. EBINA, T.; MUNAKATA, K. & MUTOMIYA, M. - Effect de l'isoniazide sur l'incorporation de l'acetate de sodium 1-¹⁴C dans les acides gras de la mycobacterie. C. R. Soc Biol. (Paris), 155: 1190-1192. 1961.
14. FERDINANDUS, J. & CLARKE, B. - Selective inhibition of bacterial enzymes by free fatty acids. J. Bact., 98: 1109-1113, 1969.
15. GOREN, M. B.; BROKL, O. & SCHAEFER, W. B. - Lipids of putative relevance to virulance in Mycobacterium tuberculosis: phthiocerol dimycocerosate and the attenuation indicator lipid. Infect. Immun., 9: 150-158, 1974.
16. KERTCHER, J A.: CHEN, M. D.; CHARACHE, P.; HWANGBO, C. C; CAMARGO, E. E.; McINTYRE, P. A. & WAGNER JR., H. N. - Rapid radiometric susceptibility testing of Mycobacterium tuberculosis Amer. Rev. resp. Dis., 117: 631-637, 1978.
17. KONDO, E. & KANAI, K. - The relationship between the chemical structure of fatty acids and their mycobactericidal activity. Jap. J. med. Sci BioL, 30: 171-178, 1977.
18. KUSAKA, T. - Fatty acid synthesizing enzyme activity of cultured Mycobacterium lepraemurium Int. J. Peprosy, 45: 132-138. 1977.
19. MCCARTHY, C. - Utilization of palmitic acid by Mycobacterium avium Infect. Immun., 4: 199-204, 1971.
20. MCCARTHY, C. - Effect of palmitic acid utilization on cell division in Mycobacterium avium Infect. Immun., 9: 363-372, 1974
21. MEDHI. I.; MURTHY, P. S. & VENKITASUBRAMANIAN, T. A. - Heat-stable stimulating factor for fatty acid synthetase from Mycobacterium tuberculosis H.₃₇ Rev. Amer. Rev. resp. Dis., 110: 201-202, 1974.
22. NANDEKKAR, A, K. N. - Fatty acid composition ot mycobacteria: An index of pathogenecity. Fed. Proc., 33: 1405, 1974.
23. OKUYAMA, H.; KAMEYAMA, Y.; FUJIKAWA, M.; YAMADA, K. & IKEZAWA, H. - Mechanism of diacyi-glycerophosphate synthesis in mycobacteria. J. biol Chem., 252: 6682-6686, 1977.
24. PIÉRARD, A. & GOLDMAN, D. S. - Enzyme system in mycobacteria. 14. Fatty acid synthesis in cell-free extracts of Mycobacterium tuberculosis Arch. Biochem. Biophys., 100: 56-65, 1963.
25. WANG, L.; KUSAKA, T. & GOLDMAN, D. S. - Elon gation of fatty acids in Mycobacterium tuberculosis. J. Bact., 101: 781-785, 1970.
26. WIMPENNY, J. W. T. - The uptake and fate of isoniazid in Mycobacterium tuberculosis var bovis BOG J. gen. Microbiol., 47: 389-403, 1967.
27 WINDER, F. G.; BRENNAN, P. & RATLEDGE, C. - Synthesis of fatty acids by extracts of mycobacteria and the absence of inhibition by isoniazid. Biochem. J., 93: 635-640, 1964.

Address for correspondence:

Edwaldo E. Camargo M.D.,

Centro de Medicina Nuclear da Universidade de São Paulo.

Caixa Postal 22022. CEP 01499 São Paulo, SP., Brasil.

Publication Dates

Publication in this collection
17 Feb 2011
Date of issue
Feb 1987

History

Received
06 Feb 1986

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. ANTONINE, A. D. & TEPPER, B. S. - Enviromental control of glycogen and lipid content of Mycobacterium tuberculosis J. Bact., 100: 538-539, 1969.

[2] 2. BAILEY, W. R. & SCOTT, E. G. - Diagnostic microbiology 2nd ed. St. Louis, CV. Mosby, 1970c. p. 368.

[3] 3. BENNET, P. & ASSELINEAU, J. - Influence de l'age sur la teneur en acides gras a chaine ramifie' du bacile tuberculeux. Ann. Inst. Pasteur, 118: 324-329, 1970.

[4] 4. CAMARGO, E. E.; LARSON, S. M.; TEPPER, B. S.; CHARACHE, P. & WAGNER JR., H. N. - Automated radiometric detection of M. tuberculosis and M. lepraemurium J. nucl. Med., 15: 48, 1974.

[5] 5. CAMARGO, E. E.; LARSON, S. M.; TEPPER, B. S. & WAGNER JR., H. N. - Radiometric studies of Mycobacterium lepraemurium Int. J. Leprosy, 44: 294-300, 1976.

[6] 6. CAMARGO, E. E.; KERTCHER, J. A.; LARSON, S. M.; TEPPER, B. S.; WAGNER JR., H. N. - Radiometric measurement of differential metabolism of fatty acids by Mycobacterium lepraemurium Int. J. Leprosy, 47: 126-132, 1979.

[7] 7. CAMARGO, E. E.; KERTCHER, J. A. & LARSON, S. M. - Radiorespirometry in identification of mycobacteria. In: HORST, W.; WAGNER JR., H. N. & BU-CHANAN, J. W., ed. - Frontiers in Nuclear Medicine. Berlin, Springer-Verlag, 1980. p. 286-298.

[8] 8. CAMARGO, E. E.; MILKOWSKI, T. A. & WAGNER JR., H. N. - Relationship between amino acid metabolism and drug resistance of M. tuberculosis J. nucl. Med., 21: 59, 1980.

[9] 9. CAMARGO, E. E.; KERTCHER, J. A.; aARSON, S. M.; TEPPER, B. S. & WAGNER JR., H. N. - Radiometric measurement of differential metabolism of fatty acid by mycobacteria. Int. J. Leprosy, 50: 200-204, 1982.

[10] 10. DeBLANC JR., H. J.; DeLAND, F. H. & WAGNER JR., H. N. - Automated radiometric detection of bacteria in 2,967 blood cultures. Appl. Microbiol., 22: 846-849, 1971.

[11] 11. DeLAND, F. H. & WAGNER JR., H. N. - Automated radiometric detection of bacterial growth in cultures. J. Lab. clin. Med., 75: 529-534, 1970.

[12] 12. DUBOS, R. J. - The effect of organic acids on mammalian tubercle bacilli. J. exp. Med., 92: 319-332, 1950.

[13] 13. EBINA, T.; MUNAKATA, K. & MUTOMIYA, M. - Effect de l'isoniazide sur l'incorporation de l'acetate de sodium 1-¹⁴C dans les acides gras de la mycobacterie. C. R. Soc Biol. (Paris), 155: 1190-1192. 1961.

[14] 14. FERDINANDUS, J. & CLARKE, B. - Selective inhibition of bacterial enzymes by free fatty acids. J. Bact., 98: 1109-1113, 1969.

[15] 15. GOREN, M. B.; BROKL, O. & SCHAEFER, W. B. - Lipids of putative relevance to virulance in Mycobacterium tuberculosis: phthiocerol dimycocerosate and the attenuation indicator lipid. Infect. Immun., 9: 150-158, 1974.

[16] 16. KERTCHER, J A.: CHEN, M. D.; CHARACHE, P.; HWANGBO, C. C; CAMARGO, E. E.; McINTYRE, P. A. & WAGNER JR., H. N. - Rapid radiometric susceptibility testing of Mycobacterium tuberculosis Amer. Rev. resp. Dis., 117: 631-637, 1978.

[17] 17. KONDO, E. & KANAI, K. - The relationship between the chemical structure of fatty acids and their mycobactericidal activity. Jap. J. med. Sci BioL, 30: 171-178, 1977.

[18] 18. KUSAKA, T. - Fatty acid synthesizing enzyme activity of cultured Mycobacterium lepraemurium Int. J. Peprosy, 45: 132-138. 1977.

[19] 19. MCCARTHY, C. - Utilization of palmitic acid by Mycobacterium avium Infect. Immun., 4: 199-204, 1971.

[20] 20. MCCARTHY, C. - Effect of palmitic acid utilization on cell division in Mycobacterium avium Infect. Immun., 9: 363-372, 1974

[21] 21. MEDHI. I.; MURTHY, P. S. & VENKITASUBRAMANIAN, T. A. - Heat-stable stimulating factor for fatty acid synthetase from Mycobacterium tuberculosis H.₃₇ Rev. Amer. Rev. resp. Dis., 110: 201-202, 1974.

[22] 22. NANDEKKAR, A, K. N. - Fatty acid composition ot mycobacteria: An index of pathogenecity. Fed. Proc., 33: 1405, 1974.

[23] 23. OKUYAMA, H.; KAMEYAMA, Y.; FUJIKAWA, M.; YAMADA, K. & IKEZAWA, H. - Mechanism of diacyi-glycerophosphate synthesis in mycobacteria. J. biol Chem., 252: 6682-6686, 1977.

[24] 24. PIÉRARD, A. & GOLDMAN, D. S. - Enzyme system in mycobacteria. 14. Fatty acid synthesis in cell-free extracts of Mycobacterium tuberculosis Arch. Biochem. Biophys., 100: 56-65, 1963.

[25] 25. WANG, L.; KUSAKA, T. & GOLDMAN, D. S. - Elon gation of fatty acids in Mycobacterium tuberculosis. J. Bact., 101: 781-785, 1970.

[26] 26. WIMPENNY, J. W. T. - The uptake and fate of isoniazid in Mycobacterium tuberculosis var bovis BOG J. gen. Microbiol., 47: 389-403, 1967.

[27] 27 WINDER, F. G.; BRENNAN, P. & RATLEDGE, C. - Synthesis of fatty acids by extracts of mycobacteria and the absence of inhibition by isoniazid. Biochem. J., 93: 635-640, 1964.

Brasil

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Radiometric studies on the oxidation of (1-14c) fatty acids by drug-susceptible and drug-resistant mycobacteria

Esudos radiométricos sobre a oxidação de (1-14C) ácidos graxos por micobactérias sensíveis e resistentes a drogas

Abstracts

Publication Dates

History