Radiometric studies of mycobacterium tuberculosis

Camargo, Edwaldo E.; Kertcher, Judith A.; Chen, Marianne F.; Charache, Patricia; Wagner Jr, Henry N.

doi:10.1590/S0036-46651987000100003

Abstracts

An in vitro assay system that included automated radiometric quantification of 14CO2 released as a result of oxidation of 14C- substrates was applied for studying the metabolic activity of M. tuberculosis under various experimental conditions. These experiments included the study of a) mtabolic pathways, b) detection times for various inoculum sizes, c) effect of filtration on reproducibility of results, d) influence of stress environment e) minimal inhibitory concentrations for isoniazid, streptomycin, ethambutol and rifampin, and f) generation times of M. tuberculosis and M. bovis. These organisms were found to metabolize 14C-for-mate, (U-14C) acetate, (U-14C) glycerol, (1-14C) palmitic acid, 1-14C) lauric acid, (U-14C) L-malic acid, (U-14C) D-glucose, and (U-14C) D-glucose, but not (1-14C) L-glucose, (U-14C) glycine, or (U-14C) pyruvate to 14CO2. By using either 14C-for-mate, (1-14C) palmitic acid, or (1-14C) lauric acid, 10(7) organisms/vial could be detected within 24 48 hours and as few as 10 organisms/vial within 16-20 days. Reproducible results could be obtained without filtering the bacterial suspension, provided that the organisms were grown in liquid 7H9 medium with 0.05% polysorbate 80 and homogenized prior to the study. Drugs that block protein synthesis were found to have lower minimal inhibitory concentrations with the radiometric method when compared to the conventional agar dilution method. The mean generation time obtained for M. bovis and different strains of M. tuberculosis with various substrates was 9 ± 1 hours.

Mycobacterium tuberculosis; 14C-substrates; Radiometric system; Drug-action; Generation time

A atividade metabólica do M. tuberculosis sob diversas condições experimentais foi estudada utilizando um sistema radiométrico automático, capaz de quantificar o 14CO2 produzido pela oxidação de substâncias marcadas com carbono-14. As experiências realizadas incluíram: a) vias metabólicas; b) determinação dos tempos de detecção para inoculações de diversas magnitudes; c) efeito da filtração sobre a reprodutibilidade dos resultados; d) influência de meio hostil; e) determinação das concentrações inibitórias mínimas para hidrazida, estreptomicina, etambutol e rifampicina f) tempo de duplicação para o M. tuberculosis e M. bovis. Estes microorganismos metabolizaram até 14CO2 o 14c-formato, (U-14C) acetato, (U-14C)glicerol, ácido palmítico, (1-14C) ácido láurico, ((U-14C) L-ácido málico, (U-14C) D glicose e (1-14C) D-glicose, mas não (1-14C) L-glicose, (U-14C) glicina ou (U-14C) piruvato. Usando 14C-formato, (1-14C) ácido palmítico ou (1-14C) ácido láurico foi possível detectar 10 bacilos/frasco em 24-48 horas e até 10 bacilos/ frasco em 16-20 dias. Resultados reprodutíveis foram obtidos sem filtrar a suspensão de bactérias, desde que cultivadas em meio 7H9 líquido com 0,05% de polissorbato 80 e homogenizadas antes da experiência. Drogas que bloqueiam a síntese protêica apresentaram concentração inibitória mínima menor com o método radiométrico do que com o convencional. O tempo médio de duplicação para o M. bovis e várias cepas de M. tuberculosis com diversas substâncias marcadas foi 9 ± 1 horas.

ARTIGOS ORIGINAIS

Radiometric studies of mycobacterium tuberculosis

Estudos radiométricos com o Mycobacterium tuberculosis

Edwaldo E. Camargo; Judith A. Kertcher; Marianne F. Chen; Patricia Charache; Henry N. Wagner Jr

The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205, USA

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

ABSTRACT

An in vitro assay system that included automated radiometric quantification of ¹⁴CO₂ released as a result of oxidation of ¹⁴C- substrates was applied for studying the metabolic activity of M. tuberculosis under various experimental conditions. These experiments included the study of a) mtabolic pathways, b) detection times for various inoculum sizes, c) effect of filtration on reproducibility of results, d) influence of stress environment e) minimal inhibitory concentrations for isoniazid, streptomycin, ethambutol and rifampin, and f) generation times of M. tuberculosis and M. bovis. These organisms were found to metabolize ¹⁴C-for-mate, (U-¹⁴C) acetate, (U-¹⁴C) glycerol, (1-¹⁴C) palmitic acid, 1-¹⁴C) lauric acid, (U-¹⁴C) L-malic acid, (U-¹⁴C) D-glucose, and (U-¹⁴C) D-glucose, but not (1-14C) L-glucose, (U-¹⁴C) glycine, or (U-¹⁴C) pyruvate to ¹⁴CO₂. By using either ¹⁴C-for-mate, (1-¹⁴C) palmitic acid, or (1-¹⁴C) lauric acid, 10⁷ organisms/vial could be detected within 24 48 hours and as few as 10 organisms/vial within 16-20 days. Reproducible results could be obtained without filtering the bacterial suspension, provided that the organisms were grown in liquid 7H9 medium with 0.05% polysorbate 80 and homogenized prior to the study. Drugs that block protein synthesis were found to have lower minimal inhibitory concentrations with the radiometric method when compared to the conventional agar dilution method. The mean generation time obtained for M. bovis and different strains of M. tuberculosis with various substrates was 9 ± 1 hours.

Key words: Mycobacterium tuberculosis; ¹⁴C-substrates; Radiometric system; Drug-action; Generation time.

RESUMO

A atividade metabólica do M. tuberculosis sob diversas condições experimentais foi estudada utilizando um sistema radiométrico automático, capaz de quantificar o ¹⁴CO₂ produzido pela oxidação de substâncias marcadas com carbono-14. As experiências realizadas incluíram: a) vias metabólicas; b) determinação dos tempos de detecção para inoculações de diversas magnitudes; c) efeito da filtração sobre a reprodutibilidade dos resultados; d) influência de meio hostil; e) determinação das concentrações inibitórias mínimas para hidrazida, estreptomicina, etambutol e rifampicina f) tempo de duplicação para o M. tuberculosis e M. bovis. Estes microorganismos metabolizaram até ¹⁴CO₂ o ¹⁴c-formato, (U-¹⁴C) acetato, (U-¹⁴C)glicerol, ácido palmítico, (1-¹⁴C) ácido láurico, ((U-¹⁴C) L-ácido málico, (U-¹⁴C) D glicose e (1-¹⁴C) D-glicose, mas não (1-14C) L-glicose, (U-14C) glicina ou (U-¹⁴C) piruvato. Usando ¹⁴C-formato, (1-¹⁴C) ácido palmítico ou (1-¹⁴C) ácido láurico foi possível detectar 10 bacilos/frasco em 24-48 horas e até 10 bacilos/ frasco em 16-20 dias. Resultados reprodutíveis foram obtidos sem filtrar a suspensão de bactérias, desde que cultivadas em meio 7H9 líquido com 0,05% de polissorbato 80 e homogenizadas antes da experiência. Drogas que bloqueiam a síntese protêica apresentaram concentração inibitória mínima menor com o método radiométrico do que com o convencional. O tempo médio de duplicação para o M. bovis e várias cepas de M. tuberculosis com diversas substâncias marcadas foi 9 ± 1 horas.

Texto completo disponível apenas em PDF.

Full text available only in PDF format.

ACKNOWLEDGEMENT

The authors are indebted to Dr. Gardner Middlebrook for supplying strains of M. tuberculosis Erdman and M. bovis BCG.

Recebido para publicação em 14/2/1986.

This work was supported by National Institutes of Health, Grant GM10548, U.S. Public Health Service

1. BAILEY, W. R. & SCOTT, E. G. - Diagnostic microbiology. 2nd. ed. St. Louis, CV. Mosby, 1970c. p. 368.
2. BUCHANAN, R. E. & GIBBONS, N. E., ed. - Bergey's manual of determinative bacteriology. 8th. ed. Baltimore, Williams and Wilkins, 1974. p. 683.
3. BUDDEMEYER, E. U.; HUTCHINSON, R. & COOPER, M. - Automatic quantitative radiometric assay of bacterial metabolism. Clin. Chem., 22: 1459-1464, 1976.
4. CAMARGO, E. E.; LARSON, S. M.; TEPPER, B. S. & WAGNER JR., H. N. - Radiometric measurement of metabolic activity of Mycobacterium lepraemurium Appl. Microbiol., 28: 452-455, 1974.
5. CAMARGO, E. E.; LARSON, S. M.; TEPPER, B. S. & WAGNER JR., H. N. - A radiometric method for predicting effectiveness of chemotherapeutic agents in murine leprosy. Int. J. Leprosy, 43: 234-238, 1975.
6. CAMARGO, E. E.; CHARACHE, P.; LARSON, S. M.; KERTCHER, J.; HWANGBO, C. C. & WAGNER JR., H. N. - Radiometric testing of susceptibility of M. tuberculosis to drugs. In: Interscience Conference on Amtimicrobial Agents and Chemotherapy, 15., Washington, D.C., September 1975.
7. CAMARGO, E. E.; LARSON, S. M.; TEPPER, B. S. Sc WAGNER JR., H. N. - Radiometric studies of Mycobacterium lepraemurium Int. J. Leprosy, 44: 294-300, 1976.
8. CAMARGO, E. E. & LARSON, S. M. - Radiomicro-biology. In: ROCHA, A. F. G. & HARBERT, J. C., ed. - Textbook of nuclear medicine: basic science. Philadelphia, Lea & Febiger, 1984. p. 339.
9. CUMMINGS, D. M.;; RISTROPH, D.; CAMARGO, E. E.; LARSON, S. M. & WAGNER JR., H. N. - Radiometric detection of the metabolic activity of Mycobacterium tuberculosis J. nucl. Med., 16: 1189-1191, 1975.
10. DeBLANC JR., H. J.; CHARACHE, P. & WAGNER JR., H. N. - Automated radiometric measurement ot antibiotic effect on bacterial growth. Antimicrob. Agents Chemother., 2: 360-336, 1972.
11. DEHYLE, R. L. & BARTON, L. L. - Nicotinamide adenine dinucleotide-independent formate dehydrogenase in Mycobacterium phlei Can ad. J. Microbiol., 23: 125-130, 1977.
12. DeLAND, F. H. & F.H WAGNER JR., H. N. - Automated radiometric detection of bacterial growth in blood cultures. J. Lab. clin. Med., 75: 529-534, 1970.
13. DeLAND, F. H. - Bacteriologic cultures and sensitivities. In: ROTHFELD, B., ed. - Nuclear medicine in vitro Philadelphia, J.B. Lippincot, 1974c. p. 384.
14. KERTCHER, J. A.; CHEN, M. F.; CHARACHE, P.; HWANGBO, C. C; CAMARGO, E. E.; McINTYRE, P. & WAGNER JR., H. N. - Rapid radiometric susceptibility testing of Mycobacterium tuberculosis. Amer. Rev. resp. Dis., 117: 631-637, 1978.
15. MIDDLEBROOK, G. & COHN, M. L. - Bacteriology of tuberculosis: laboratory methods. Amer. J. publ. Hlth., 48: 844, 1958.
16. MIDDLEBROOK, G. - Personal communication.
17. MIDDLEBROOK, G.; REGGIARDO, Z. & TIGERTT, W. - Automatable radiometric detection of growth of Mycobacterium tuberculosis in selective media. Amer. Rev. resp. Dis., 115: 1066-1069, 1977.
18. MORGAN, M. A.; HORSTMEIER, C. D.; DeYOUNG, D. R. & ROBERTS, G. D. - Comparison of a radiometrie method (BACTEC) and conventional culture media for recovery of mycobacteria from smear-negative specimens. J. clin. Miicrobiol., 18: 384-388, 1983.
19. SIDDIQI, S. H.; HWANGBO, C. C; SILCOX, V.; GOOD, R. C; SNIDER, D. E. & MIDDLEBROOK, G. - Rapid radiometric methods to detected and differentiate Mycobacterium tuberculosis/M. bovis from other mycobacterial species. Amer. Rev. resp. Dis., 130: 634-640, 1984.
20. TEPPER, B. S. & VARMA, K. G. - Metabolic activity of purified suspensions of Mycobacterium lepraemurium. J. gen. Microbiol., 73: 143-152, 1972.
21. WEISS, E. - Adenosine triphosphate and other requirements for the utilization of glucose by agents of the psittacosis-tracoma group. J. Bact., 90: 243-253, 1965.
22. YANGCO, B. G.; EIKMAN, E. A.; SOLOMON, D. A.; DERESINSKI, S. C. & MADDEN, J. A. - Rapid radiometric method for determining drug susceptibility of Mycobacterium avium-intracellulare Antimicrob. Agents Chemother., 19: 534-539, 1981.

Address for correspondence:

Edwaldo E. Camargo

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
14 Feb 1986

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

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

[2] 2. BUCHANAN, R. E. & GIBBONS, N. E., ed. - Bergey's manual of determinative bacteriology. 8th. ed. Baltimore, Williams and Wilkins, 1974. p. 683.

[3] 3. BUDDEMEYER, E. U.; HUTCHINSON, R. & COOPER, M. - Automatic quantitative radiometric assay of bacterial metabolism. Clin. Chem., 22: 1459-1464, 1976.

[4] 4. CAMARGO, E. E.; LARSON, S. M.; TEPPER, B. S. & WAGNER JR., H. N. - Radiometric measurement of metabolic activity of Mycobacterium lepraemurium Appl. Microbiol., 28: 452-455, 1974.

[5] 5. CAMARGO, E. E.; LARSON, S. M.; TEPPER, B. S. & WAGNER JR., H. N. - A radiometric method for predicting effectiveness of chemotherapeutic agents in murine leprosy. Int. J. Leprosy, 43: 234-238, 1975.

[6] 6. CAMARGO, E. E.; CHARACHE, P.; LARSON, S. M.; KERTCHER, J.; HWANGBO, C. C. & WAGNER JR., H. N. - Radiometric testing of susceptibility of M. tuberculosis to drugs. In: Interscience Conference on Amtimicrobial Agents and Chemotherapy, 15., Washington, D.C., September 1975.

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

[8] 8. CAMARGO, E. E. & LARSON, S. M. - Radiomicro-biology. In: ROCHA, A. F. G. & HARBERT, J. C., ed. - Textbook of nuclear medicine: basic science. Philadelphia, Lea & Febiger, 1984. p. 339.

[9] 9. CUMMINGS, D. M.;; RISTROPH, D.; CAMARGO, E. E.; LARSON, S. M. & WAGNER JR., H. N. - Radiometric detection of the metabolic activity of Mycobacterium tuberculosis J. nucl. Med., 16: 1189-1191, 1975.

[10] 10. DeBLANC JR., H. J.; CHARACHE, P. & WAGNER JR., H. N. - Automated radiometric measurement ot antibiotic effect on bacterial growth. Antimicrob. Agents Chemother., 2: 360-336, 1972.

[11] 11. DEHYLE, R. L. & BARTON, L. L. - Nicotinamide adenine dinucleotide-independent formate dehydrogenase in Mycobacterium phlei Can ad. J. Microbiol., 23: 125-130, 1977.

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

[13] 13. DeLAND, F. H. - Bacteriologic cultures and sensitivities. In: ROTHFELD, B., ed. - Nuclear medicine in vitro Philadelphia, J.B. Lippincot, 1974c. p. 384.

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

[15] 15. MIDDLEBROOK, G. & COHN, M. L. - Bacteriology of tuberculosis: laboratory methods. Amer. J. publ. Hlth., 48: 844, 1958.

[16] 16. MIDDLEBROOK, G. - Personal communication.

[17] 17. MIDDLEBROOK, G.; REGGIARDO, Z. & TIGERTT, W. - Automatable radiometric detection of growth of Mycobacterium tuberculosis in selective media. Amer. Rev. resp. Dis., 115: 1066-1069, 1977.

[18] 18. MORGAN, M. A.; HORSTMEIER, C. D.; DeYOUNG, D. R. & ROBERTS, G. D. - Comparison of a radiometrie method (BACTEC) and conventional culture media for recovery of mycobacteria from smear-negative specimens. J. clin. Miicrobiol., 18: 384-388, 1983.

[19] 19. SIDDIQI, S. H.; HWANGBO, C. C; SILCOX, V.; GOOD, R. C; SNIDER, D. E. & MIDDLEBROOK, G. - Rapid radiometric methods to detected and differentiate Mycobacterium tuberculosis/M. bovis from other mycobacterial species. Amer. Rev. resp. Dis., 130: 634-640, 1984.

[20] 20. TEPPER, B. S. & VARMA, K. G. - Metabolic activity of purified suspensions of Mycobacterium lepraemurium. J. gen. Microbiol., 73: 143-152, 1972.

[21] 21. WEISS, E. - Adenosine triphosphate and other requirements for the utilization of glucose by agents of the psittacosis-tracoma group. J. Bact., 90: 243-253, 1965.

[22] 22. YANGCO, B. G.; EIKMAN, E. A.; SOLOMON, D. A.; DERESINSKI, S. C. & MADDEN, J. A. - Rapid radiometric method for determining drug susceptibility of Mycobacterium avium-intracellulare Antimicrob. Agents Chemother., 19: 534-539, 1981.