750
ISOLATION OF LEGIONELLA PNEUMOPHILA FROM BLOOD PAUL H. EDELSTEIN
RICHARD D. MEYER SYDNEY M. FINEGOLD
Infectious Disease Section, Research and Medical Services, Veterans Administration Wadsworth Medical Center, Los Angeles, and Department of Medicine, University of California School of Medicine, Los Angeles, California
posed of Mueller-Hinton broth base (Difco, Detroit, Michigan), 20% fetal-calf serum (Irvine Biological Supply, Irvine, California), 0.03% sodium polyanethol sulphonate (Sigma Chemical, St. Louis, Missouri), 0.01% ferric pyrophosphate (Mallinckrodt, New York), and 0-04% L-cysteine monohvdrochloride (ICN Pharmaceuticals, Cleveland, Ohio). The final pH of the slant was 6.5and that of the broth was 7.0.5%CO, in air was added aseptically to the rubber-stoppered bottles.6 -
We had determined that
mixed with normal human at 37°C for 2 used to bottles. Inoculated botwas detect contaminated days tles were incubated at 35-36°C in 3.5% CO2, They were tilted once every 4-6 days to coat the agar surface with broth. Bottles were inspected daily for evidence of growth. Routine subcultures were not carried out. Methods for confirmation of identity of isolates published by the Center for Disease Control (C.D.C.) were followed. Dr Robert E. Weaver of C.D.C. performed confirmatory bacteriological tests,’ and Dr C. Wayne Moss of C.D.C. performed cellu-
blood,
Summary
Legionella pneumophila has been isolated, with an in-vitro method, from the
blood of a patient with fatal legionnaires’ disease. Introduction LEGIONNAIRES’ disease often causes systemic manifestations resembling those of gram-negative bacterasmia.’ A brief report without complete clinical information cited the recovery of a "rickettsia-like agent" from the blood of a febrile patient inoculated into guinea pigs.2 The investigators thought that the recovered organism was a contaminant, but it has been demonstrated to be a strain of Legionella pneumophila.3 We wish to report isolation of L. pneumophila from the blood of a patient with fatal legionnaires’ disease with in-vitro techniques. Patient and Methods A 50-year-old man was admitted to the V. A. Wadsworth Medical Center with metastatic lung carcinoma in October, 1978. He was given high-dosage corticosteroid and radiation
therapy.
grew well in the broth. Preincubation
lar-fatty-acid analysis by gas-liquid chromatography (G.L.c.).8 Knoxville antibody conjugate (serogroup 1) for D.F.A. examination was provided by the Biological Products Division, C.D.C.;9 and Los Angeles-1 (serogroup 4) and Togus (serogroup 2) antibody conjugates were provided by Dr Roger McKinney of C.D.C.IO Gelatinase production was tested with a heavy suspension of organisms in trypticase soy broth, incubated at 37° with undeveloped ’Plus-X-Pan’ film strips (Eastman Kodak, Rochester, N.Y.). Oxidase production was tested with Kovac’s reagent. Serum-antibody titres to L. pneumophila, Philadelphia-1 strain, were measured with an indirect fluorescent-antibody (LF.A.) technique" and confirmed by C.D.C.
He
developed pneumonia, diagnosed clinically as legionnaires’ disease, on Nov. 5, 1978. On day 1 of illness, before antibiotic therapy, 5 ml of aseptically collected blood was placed in the blood-culture medium. Sputum was collected through an endotracheal tube for direct fluorescent-antibody (D.F.A.) examination for legionnaires’ disease. He was treated with erythromycin but died on Nov. 9 of respiratory failure. Necropsy revealed a consolidating bronchopneumonia. A sample of involved lung was obtained aseptically for culture. Blood-culture bottles were prepared with an agar slant anc broth, similar to Casteneda medium. The slant and broth werf modifications of media formulated by Feeley and others.4,5 The slant (30 ml) was composed of charcoal/yeast-extract agar witl
2.0%, rather than 1.7%, agar. The broth (50 ml)
was com
8. Schteingart, D. E., Gregerman, R. I., Conn, J. W. Metabolism, 1963, 12, 484. 9. Schteingart, D. E., Conn, J. W. Ann N.Y. Acad. Sci. 1965, 131, 388. 10. Krieger, D. T., Howamtz, P. J., Frantz, A. G. J. clin. Endoc. Metab. 1976, 42, 260. 11. Krieger, D. T., Glick, S. Am. J. Med. 1972, 52, 25. 12. Krieger, D. T. Med. Clins N. Am. 1978, 62, 261 13. Macleod, R. M. Front. Neuroendocr. 1976, 4, 169. 14. MacIndoe, J. H., Turkington, R. W. J. clin. Invest. 1973, 52, 1972. 15. Smythe, G. A. Clin. Endocr. 1977, 7, 325. 16. Phillips, F., Crisp, A. H., McGuinness, B., Kalucy, E. G., Chen, C. N.,
Koral, J., Kalucy, R. S., Lacy, J. H. Lancet, 1975, ii, 723. 17. Parker, D. C., Rossman, L. G., Vanderhaan, E. F. J. clin. Endocr. Metab.
1974, 38, 646. 18. Hill, P., Wynder, E. Lancet, 1976, ii, 806. 19. Merimee, T. J., Fineberg, S. E. Metabolism, 1973, 22, 1491. 20. Copinschi, G., De Laet, M. H., Brion, J. P., Le Clercq, R., L’Hermite, M., Robyn, C., Virasoro, E., Van Cauter, E. Clin. Endocr. 1978, 9, 15. 21. Meier, A. H., Dusseau, T. W. Biol. Reprod. 1973, 8, 400. 22. Larson, B. A., Smha, Y. N., Vanderlaan, W. P. Endocrinology, 1976, 98, 139. 23. Smha, Y. N., Salocks, C. B., Vanderlaan, W. P. ibid. 1976, 99, 881. 24. Ball, M. F., El-Khodary, A. Z., Canary, J. J. J. clin. Endocr. 1972, 34, 498. 25. Harsoulis, P., Marshall, J. C., Ku Ku, S. F., Burke, C. W., London, D. R., Fraser, T. R. Br. med. J. 1973, iv, 326.
5 colony-forming units of L. pneu-
mophila, Philadelphia-1 strain,
Results Two white colonies were detected on the slant after 14 inoculation. The culture had not been inspected between the 8th and 14th day. The blood had hxmolysed and determination of turbidity was impossible. Gram smear of the colonies showed filamentous gramnegative bacilli 5-20 um long. Both colonies, as well as
days’
the broth, were subcultured onto charcoal/yeast-extract agar. All three subcultures produced heavy growth of gram-negative bacilli which were D.F.A.-positive with the Knoxville conjugate. These bacilli did not grow on subculture onto 5% sheep’s-blood agar incubated in 3.5% CO2 at 3S°C or on Mueller-Hinton agar with x and v factors after 3 days’ incubation. They were gelatinasepositive after 3 days’ incubation, catalase-positive, and weakly oxidase-positive. Neither the Los Angeles-1 nor the Togus antibody conjugates stained the organisms in a D.F.A. test. Fluorescence and browning were produced on Feeley-Gorman agar after 4 days’ growth.5 G,L.c. demonstrated a pattern of cellular fatty acids unique to L. pneumophila. Nine other bottles of this special medium made in the same batch were used to culture the blood of other pa-
tients with suspected legionnaires’ disease. One grew Listeria monocytogenes, and the other eight yielded no growth when held for 2 months and subcultured every 2 weeks onto charcoal/yeast-extract agar. D.F.A. examination of the sputum was positive with the Knoxville conjugate, with 7 fluorescent bacilli smear. Lung tissue was also Knoxville-D.F.A. positiBe. with 1-2 fluorescent bacilli/1200 x field, but negativee with Los Angeles-1 and Togus antibody conjugates. Culture of lung yielded Citrobacter diversus and L.
pneumophila.
751 I.F.A.
titres
were
ISOLATION OF LEGIONELLA PNEUMOPHILA FROM BLOOD PAUL H. EDELSTEIN
RICHARD D. MEYER SYDNEY M. FINEGOLD
Infectious Disease Section, Research and Medical Services, Veterans Administration Wadsworth Medical Center, Los Angeles, and Department of Medicine, University of California School of Medicine, Los Angeles, California
posed of Mueller-Hinton broth base (Difco, Detroit, Michigan), 20% fetal-calf serum (Irvine Biological Supply, Irvine, California), 0.03% sodium polyanethol sulphonate (Sigma Chemical, St. Louis, Missouri), 0.01% ferric pyrophosphate (Mallinckrodt, New York), and 0-04% L-cysteine monohvdrochloride (ICN Pharmaceuticals, Cleveland, Ohio). The final pH of the slant was 6.5and that of the broth was 7.0.5%CO, in air was added aseptically to the rubber-stoppered bottles.6 -
We had determined that
mixed with normal human at 37°C for 2 used to bottles. Inoculated botwas detect contaminated days tles were incubated at 35-36°C in 3.5% CO2, They were tilted once every 4-6 days to coat the agar surface with broth. Bottles were inspected daily for evidence of growth. Routine subcultures were not carried out. Methods for confirmation of identity of isolates published by the Center for Disease Control (C.D.C.) were followed. Dr Robert E. Weaver of C.D.C. performed confirmatory bacteriological tests,’ and Dr C. Wayne Moss of C.D.C. performed cellu-
blood,
Summary
Legionella pneumophila has been isolated, with an in-vitro method, from the
blood of a patient with fatal legionnaires’ disease. Introduction LEGIONNAIRES’ disease often causes systemic manifestations resembling those of gram-negative bacterasmia.’ A brief report without complete clinical information cited the recovery of a "rickettsia-like agent" from the blood of a febrile patient inoculated into guinea pigs.2 The investigators thought that the recovered organism was a contaminant, but it has been demonstrated to be a strain of Legionella pneumophila.3 We wish to report isolation of L. pneumophila from the blood of a patient with fatal legionnaires’ disease with in-vitro techniques. Patient and Methods A 50-year-old man was admitted to the V. A. Wadsworth Medical Center with metastatic lung carcinoma in October, 1978. He was given high-dosage corticosteroid and radiation
therapy.
grew well in the broth. Preincubation
lar-fatty-acid analysis by gas-liquid chromatography (G.L.c.).8 Knoxville antibody conjugate (serogroup 1) for D.F.A. examination was provided by the Biological Products Division, C.D.C.;9 and Los Angeles-1 (serogroup 4) and Togus (serogroup 2) antibody conjugates were provided by Dr Roger McKinney of C.D.C.IO Gelatinase production was tested with a heavy suspension of organisms in trypticase soy broth, incubated at 37° with undeveloped ’Plus-X-Pan’ film strips (Eastman Kodak, Rochester, N.Y.). Oxidase production was tested with Kovac’s reagent. Serum-antibody titres to L. pneumophila, Philadelphia-1 strain, were measured with an indirect fluorescent-antibody (LF.A.) technique" and confirmed by C.D.C.
He
developed pneumonia, diagnosed clinically as legionnaires’ disease, on Nov. 5, 1978. On day 1 of illness, before antibiotic therapy, 5 ml of aseptically collected blood was placed in the blood-culture medium. Sputum was collected through an endotracheal tube for direct fluorescent-antibody (D.F.A.) examination for legionnaires’ disease. He was treated with erythromycin but died on Nov. 9 of respiratory failure. Necropsy revealed a consolidating bronchopneumonia. A sample of involved lung was obtained aseptically for culture. Blood-culture bottles were prepared with an agar slant anc broth, similar to Casteneda medium. The slant and broth werf modifications of media formulated by Feeley and others.4,5 The slant (30 ml) was composed of charcoal/yeast-extract agar witl
2.0%, rather than 1.7%, agar. The broth (50 ml)
was com
8. Schteingart, D. E., Gregerman, R. I., Conn, J. W. Metabolism, 1963, 12, 484. 9. Schteingart, D. E., Conn, J. W. Ann N.Y. Acad. Sci. 1965, 131, 388. 10. Krieger, D. T., Howamtz, P. J., Frantz, A. G. J. clin. Endoc. Metab. 1976, 42, 260. 11. Krieger, D. T., Glick, S. Am. J. Med. 1972, 52, 25. 12. Krieger, D. T. Med. Clins N. Am. 1978, 62, 261 13. Macleod, R. M. Front. Neuroendocr. 1976, 4, 169. 14. MacIndoe, J. H., Turkington, R. W. J. clin. Invest. 1973, 52, 1972. 15. Smythe, G. A. Clin. Endocr. 1977, 7, 325. 16. Phillips, F., Crisp, A. H., McGuinness, B., Kalucy, E. G., Chen, C. N.,
Koral, J., Kalucy, R. S., Lacy, J. H. Lancet, 1975, ii, 723. 17. Parker, D. C., Rossman, L. G., Vanderhaan, E. F. J. clin. Endocr. Metab.
1974, 38, 646. 18. Hill, P., Wynder, E. Lancet, 1976, ii, 806. 19. Merimee, T. J., Fineberg, S. E. Metabolism, 1973, 22, 1491. 20. Copinschi, G., De Laet, M. H., Brion, J. P., Le Clercq, R., L’Hermite, M., Robyn, C., Virasoro, E., Van Cauter, E. Clin. Endocr. 1978, 9, 15. 21. Meier, A. H., Dusseau, T. W. Biol. Reprod. 1973, 8, 400. 22. Larson, B. A., Smha, Y. N., Vanderlaan, W. P. Endocrinology, 1976, 98, 139. 23. Smha, Y. N., Salocks, C. B., Vanderlaan, W. P. ibid. 1976, 99, 881. 24. Ball, M. F., El-Khodary, A. Z., Canary, J. J. J. clin. Endocr. 1972, 34, 498. 25. Harsoulis, P., Marshall, J. C., Ku Ku, S. F., Burke, C. W., London, D. R., Fraser, T. R. Br. med. J. 1973, iv, 326.
5 colony-forming units of L. pneu-
mophila, Philadelphia-1 strain,
Results Two white colonies were detected on the slant after 14 inoculation. The culture had not been inspected between the 8th and 14th day. The blood had hxmolysed and determination of turbidity was impossible. Gram smear of the colonies showed filamentous gramnegative bacilli 5-20 um long. Both colonies, as well as
days’
the broth, were subcultured onto charcoal/yeast-extract agar. All three subcultures produced heavy growth of gram-negative bacilli which were D.F.A.-positive with the Knoxville conjugate. These bacilli did not grow on subculture onto 5% sheep’s-blood agar incubated in 3.5% CO2 at 3S°C or on Mueller-Hinton agar with x and v factors after 3 days’ incubation. They were gelatinasepositive after 3 days’ incubation, catalase-positive, and weakly oxidase-positive. Neither the Los Angeles-1 nor the Togus antibody conjugates stained the organisms in a D.F.A. test. Fluorescence and browning were produced on Feeley-Gorman agar after 4 days’ growth.5 G,L.c. demonstrated a pattern of cellular fatty acids unique to L. pneumophila. Nine other bottles of this special medium made in the same batch were used to culture the blood of other pa-
tients with suspected legionnaires’ disease. One grew Listeria monocytogenes, and the other eight yielded no growth when held for 2 months and subcultured every 2 weeks onto charcoal/yeast-extract agar. D.F.A. examination of the sputum was positive with the Knoxville conjugate, with 7 fluorescent bacilli smear. Lung tissue was also Knoxville-D.F.A. positiBe. with 1-2 fluorescent bacilli/1200 x field, but negativee with Los Angeles-1 and Togus antibody conjugates. Culture of lung yielded Citrobacter diversus and L.
pneumophila.
751 I.F.A.
titres
were
