Laboratory of Bacteriology and Medical Mycology1 and Laboratory of Ultrastructures2, Istituto Superiore di Sanitá, Viale Regina Elena 299, 00161 Rome, Italy
Author for correspondence: Graziella Orefici. Tel: +39 06 49902333. Fax: +39 06 49387112. e-mail: marella{at}iss.it
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ABSTRACT |
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Keywords: Mycobacterium celatum, macrophage infection, mice infection model, drug susceptibility
Abbreviations: AMI, amikacin; AZI, azithromycin; CIP, ciprofloxacin; CLA, clarithromycin; CLO, clofazimine; EMB, ethambutol; INH, isoniazid; i.p., intraperitoneally; MAC, Mycobacterium aviumintracellulare complex; RFB, rifabutin; RMP, rifampicin; SO, smooth opaque; SPA, sparfloxacin; ST, smooth transparent.
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INTRODUCTION |
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Some authors (Butler et al., 1993 ) reported that M. celatum colonies were predominantly small, smooth and dome-shaped, and that flat transparent colonies were rarely observed. In other studies (Tortoli et al., 1995
), colonies were found to be polymorphic and similar to those of MAC and M. xenopi. Colonies of strains isolated from AIDS patients were reported to be transparent in the first period of growth but creamy white and pigmented after 812 weeks incubation (Bull et al., 1995
). The factors influencing the change in colony morphology in mycobacteria are not known, but studies on antimicrobial susceptibility and pathobiological significance of the smooth transparent (ST) and smooth opaque (SO) variants of MAC have been reported (Reddy et al., 1996
); in contrast, no information on the virulence and drug susceptibility of M. celatum colonial variants has been published.
The purpose of this investigation was to present data on the growth in vitro, ex vivo and in vivo of two colonial variants of a M. celatum strain isolated from an AIDS patient; in addition, the in vitro and in vivo susceptibility of the two morphotypes to antimicrobial agents is shown.
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METHODS |
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Infection studies in human macrophages.
Leukocyte buffy coats obtained from healthy donors were separated over FicollHypaque (Histopaque 1077; Sigma), as previously described (Fattorini et al., 1995 ). Briefly, suspensions of isolated peripheral blood mononuclear cells prepared in complete RPMI 1640 medium (Life Technologies) containing 10% heat-inactivated (56 °C, 30 min) fetal calf serum, 25 mM HEPES and 2 mM L-glutamine were adjusted to 1x107 cells ml-1 and distributed in 0·5 ml volumes in 24-well tissue culture plates. After 2 h incubation at 37 °C under a humidified 5% CO2 atmosphere, non-adherent cells were aspirated and the monocytes (24x105 per well) were washed extensively with warmed medium without antibiotics and counted by the method of Nakagawara & Nathan (1983
). After 4 d incubation, cells showed macrophage morphology and >98% of them were able to ingest neutral red. Monolayers were exposed to either ST or SO bacteria at a ratio of 500 or 50 c.f.u. per macrophage, respectively, incubated for 2 h, and washed extensively to remove extracellular bacteria. Cell viability was determined by the trypan blue exclusion method. At various times, infected macrophages were lysed to determine the number of c.f.u., as previously described (Fattorini et al., 1995
).
Transmission electron microscopy.
Samples for electron microscopy observations were fixed with 0·1 M sodium-cacodylate-buffered 4% paraformaldehyde/1·25% glutaraldehyde/10 mM CaCl2 for 24 h at 4 °C. Post-fixation with 1% OsO4 overnight at 4 °C was followed by dehydration through a graded series of ethanol solutions, 1:1 (v/v) ethanol and acetone, and pure acetone, and embedding in Spurr resin (Taab). In some experiments, macrophages were pulsed with cationized ferritin (Sigma) in RPMI 1640 medium at a final concentration of 0·2 mg ml-1 for 3 h at 37 °C before infection (Hart & Young, 1975 ). Sections obtained with an ultramicrotome MT-2B (LKB Pharmacia) were stained with uranyl acetate and lead citrate and examined with a Philips 208S transmission electron microscope.
Infection studies in mice.
Male BALB/c mice were obtained from Charles River. Mice were infected intraperitoneally (i.p.) with 0·5 ml of a bacterial suspension containing 1x107 ST or SO c.f.u. At different time points, mice were killed by cervical dislocation and 4 ml sterile PBS was injected into the peritoneum. The fluid was withdrawn and added to an equal volume of Middlebrook 7H9 broth. Suspensions were serially 10-fold diluted and plated onto 7H10 agar medium. The c.f.u. were counted after incubation of the plates for 1014 d at 37 °C in humidified air with 5% CO2. The organs, collected under aseptic conditions, were suspended in 7H9 medium, ground in homogenizers, briefly sonicated, and the number of c.f.u. was determined.
MIC determination by the agar dilution method.
MICs were determined by the twofold agar dilution technique using Middlebrook 7H10 agar. Inocula were prepared by suspending SO or ST colonies in Middlebrook 7H9 broth. Antibiotic-containing plates with drug concentrations ranging from 64 to 0·06 µg ml-1 were inoculated with 3x102 or 3x103 c.f.u. and incubated at 37 °C in plastic bags for 14 d. The MIC was defined as the lowest drug concentration at which no visible growth of the organism was observed.
Antimicrobial susceptibility in beige mice.
Male beige mice (C57BL/6/bgj/bgj) were obtained from Jackson Laboratories. Mice were infected i.p. with 0·2 ml of a bacterial suspension containing 1x107 ST c.f.u. (Gangadharam, 1995 ; Fattorini et al., 1998
). One day after infection, five mice were killed, and the organs were aseptically removed, homogenized in 1.5 ml Middlebrook 7H9 broth and sonicated for 10 s. To enumerate c.f.u., appropriate dilutions of the homogenates were plated onto Middlebrook 7H10 agar and, after 2 weeks incubation at 37 °C under a humidified 5% CO2 atmosphere, colonies were counted. The remaining mice were randomly allocated to an untreated control group and eight drug-treated groups. Starting from 1 d after infection, RMP, rifabutin (RFB), clarithromycin (CLA), azithromycin (AZI), ethambutol (EMB), ciprofloxacin (CIP) and INH were administered orally by gavage five times weekly at the following concentrations: RMP, 10 mg kg-1 (Ji et al., 1994
); RFB, 10 mg kg-1 (Ji et al., 1994
); CLA, 100 mg kg-1 (Ji et al., 1994
); AZI, 100 mg kg-1 (Cynamon & Klemens, 1992
); EMB, 100 mg kg-1 (Klemens et al., 1993
); CIP, 40 mg kg-1 (Inderlied et al., 1989
); INH, 50 mg kg-1 (Fattorini et al., 1998
). Amikacin (AMI) was injected subcutaneously five times weekly at 100 mg kg-1 (Fattorini et al., 1998
). c.f.u. in treated and untreated mice were determined on days 28 and 56 post-infection. Control untreated mice were injected with saline. To reduce the carry-over effects of the drugs in organs, treated mice were killed 72 h after administration of the last dose of the treatment (Ji et al., 1994
). Statistical evaluation of the differences in c.f.u. between treated and untreated mice was done by the Students t test. P<0·05 was considered significant.
Enumeration of resistant mutants.
Drug-resistant mutants were enumerated in the spleen suspensions from both drug-treated and untreated control mice on day 56. Besides the determination of the total number of c.f.u., 0·25 ml of the undiluted suspensions was plated onto Middlebrook 7H10 agar plates containing drug concentrations corresponding to eight times the MICs. The frequency of drug-resistant mutants in the bacterial population was defined as the ratio between the c.f.u. numbers of the resistant mutants and the total c.f.u. number.
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RESULTS |
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Growth of ST and SO variants in human macrophages
The growth of the two colonial variants of M. celatum in human macrophages is shown in Fig. 2.
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SO cells also multiplied efficiently within the macrophage, but the pattern of growth was different from that of ST cells. On day 0, the number of intracellular c.f.u. was 2±0·4x105 c.f.u. (ml macrophage lysate)-1, with about 50 % of macrophages infected and one to three mycobacteria per macrophage; about 1% dead macrophages was seen and the c.f.u. number in the supernatant was 0.2±0.04x105 ml-1. After 3 d, a rapid growth of SO mycobacteria in macrophages in the form of long filaments invading the cytoplasm (Fig. 2b) and completely destroying the monolayers after 7 d was observed.
By electron microscopy it was shown that the bacteria were located exclusively inside phagosomes (Fig. 3). Usually these vacuoles contained some ST cells (from one to four) on day 0 (Fig. 3a
) and a single bacterium on day 3 (Fig. 3b
). Ferritin labelling (Fig. 3d
) revealed that even though lysosomephagosome fusion had occurred, ST cells showed no visible signs of degradation. The labelling also showed that the bacteria were surrounded by a 4580-nm-thick electron-translucent zone resembling a capsule which separated the bacterial wall from the ferritin layer.
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Growth of ST and SO variants in BALB/c mice
The kinetics of M. celatum growth in BALB/c mice is shown in Fig. 4. After i.p. infection, about 4% of the ST inoculum and 2% of the SO inoculum was recovered in the spleen, liver and lung on day 1; furthermore, from 6 to 7% of ST cells and <1% of SO cells were found in the peritoneum. The ST variant multiplied in the spleen, liver and lung up to day 14, then a containment of the infection was observed. In infected mice, bacterial growth was associated with a 20% increase in the lung weight on day 7, and 40% and 24% increase, respectively, in the spleen and liver weights on day 14, in comparison with uninfected mice; after 2 weeks, organ enlargements slowly decreased but never returned to the uninfected mice levels. SO c.f.u. decreased by 80% in the liver and by 88 % in the spleen and lung until day 14, then slightly increased in the spleen and lung but not the liver; after this time, colonies with intermediate SOST morphology were observed in the viable count plates. SO variant infection was associated with a lower increase in organ weights up to day 14, in comparison with ST variant infection (14% increase in lung on day 7 and 29% and 10% increase in the spleen and liver weights, respectively, on day 14), followed by a rapid return to the uninfected mice levels. In the peritoneum, SO cells were cleared more rapidly than ST cells in the first day of infection, then a rapid decrease in ST and SO c.f.u. occurred.
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DISCUSSION |
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The strain of M. celatum used in the present investigation showed SO colonies similar to those of MAC (Butler et al., 1993 ; Tortoli et al., 1995
) and ST colonies with a moustached appearance. In MAC, ST>SO transition has been attributed to various reasons, including metabolic starvation (McCarthy, 1974
) and decreased temperature (Woodley & David, 1976
). The observation that in M. celatum ST>SO transition at 37 °C occurred in the third to fourth week supports the possibility that also in this organism starving conditions favour the growth of SO cells, which are nutritionally less demanding than cells of the ST type.
When human macrophages were infected with ST cells, mycobacteria multiplied efficiently inside the phagocytes. A different pattern was displayed by SO cells, as shown by the formation of long filaments inside the macrophage which rapidly invaded and destroyed them. Similar observations were reported in MAC-infected HeLa cells (Brosbe et al., 1962 ), but, to our knowledge, not in MAC-infected macrophages, in which mycobacteria were found to lie inside the limits of the phagocytes (Meylan et al., 1990
) and never exhibited a tendency toward branching (Crowle et al., 1986
). The M. celatum pattern is more similar to that observed in cells infected with Nocardia asteroides, in which aggregates of filaments were formed in glial cell cultures (Beaman & Beaman, 1993
) and human macrophages (unpublished observations of our group).
The ST variant was more virulent than the SO variant in the host, as reported for MAC (Reddy et al., 1996 ; Schaefer et al., 1970
). In contrast to what was observed in cultured macrophages, the growth of SO bacteria was rapidly contained in mice, thus indicating the presence of an intact immune system is an essential requirement for SO variant control. However, the formation of intermediate STSO colonies which occurred during the progress of the infection in organs infected with the SO variant indicates that the ST phenotype is selected for the maintenance of infection.
In comparison with what is known for MAC (Woodley & David, 1976 ), M. celatum shows an increased resistance to RMP and RFB. ST colonies are, in general, more resistant than SO colonies to many of the drugs tested; in contrast, it is interesting that in the SO variant the resistance to RFB and RMP was higher than in the ST variant.
Some discrepancies in the susceptibility of the clinical isolates of M. celatum have been reported (Piersimoni et al., 1997 ; Tortoli et al., 1995
) in comparison with initial studies (Butler et al., 1993
). This was believed to be due to either differences in the methods of testing or selection of strains representing different clones in the bacterial populations (Piersimoni et al., 1997
; Tortoli et al., 1995
). Our observations support the latter hypothesis and indicate that the presence of different proportions of ST and SO variants or intermediate forms in a bacterial population may affect drug susceptibility results; in vitro propagation or storage of clinical strains for an unspecified amount of time could be a cause of the differences in drug susceptibility results observed by various authors.
Therapy of M. celatum infections in AIDS patients is usually based upon administration of three to four anti-MAC agents, including CLA, AZI, RFB, CIP, AMI, EMB and CLO (Piersimoni et al., 1994 , 1997
; Gholizadeh et al., 1998
; Bonomo et al., 1998
; Bull et al., 1995
; Tortoli et al., 1995
; Zurawski et al., 1997
; Masur, 1993
). Our data showed that, in the beige mouse model, CLA, AZI and EMB are the most active antimicrobial agents among those tested, even if only a bacteriostatic activity was seen in the lung. However, while no drug-resistant mutants could be detected in the spleen of mice treated with CLA and AZI, many mutants were found in EMB-treated mice. Overall, our data indicate that CLA and AZI, which are drugs recommended as first-choice agents for therapy of MAC infections in AIDS patients (Masur, 1993
), could also be active against M. celatum infections in humans. The observation that CLA-containing regimens were usually beneficial for treatment of these infections in HIV-positive patients (Piersimoni et al., 1997
; Tortoli et al., 1995
; Zurawski et al., 1997
) is in keeping with our results. As expected, RMP, which showed high MIC values against both ST and SO variants, was not effective in vivo; unfortunately, RFB, which appeared to be more active than RMP in vitro, was also ineffective in mice. The latter observation is in keeping with a recent paper (Gholizadeh et al., 1998
) in which the occurrence of M. celatum infection in two HIV-positive patients treated prophylactically with RFB was reported. As for CIP, this drug is considered to be active in vitro against M. celatum strains (Butler et al., 1993
; Bonomo et al., 1998
; Bull et al., 1995
; Tortoli et al., 1995
) but, unexpectedly, showed low efficacy in mice. The discrepancy may be explained with the knowledge that, in vitro, the ST variant is 32 times more resistant than the SO variant to CIP. These observations clearly emphasize the need to use freshly isolated colonies for testing in vitro drug susceptibility of M. celatum.
Overall, our results indicate that the two colonial variants, ST and SO, of M. celatum can show differential cellular morphology, growth in macrophages, virulence in mice and drug susceptibility, and that CLA and AZI are, in the beige mouse model, the most effective drugs against the virulent ST variant.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bonomo, R. A., Briggs, J. M., Gross, W., Hassan, M., Graham, R. C., Butler, W. R. & Salata, R. A. (1998). Mycobacterium celatum infection in a patient with AIDS. Clin Infect Dis 26, 243-245.[Medline]
Brosbe, E. A., Sugihara, P. T. & Smith, C. R. (1962). Growth characteristics of Mycobacterium avium and group III nonphotochromogenic mycobacteria in HeLa cells. J Bacteriol 84, 1282-1286.[Medline]
Bull, T. J., Shanson, D. C., Archard, L. C., Yates, M. D., Hamid, M. E. & Minnikin, D. E. (1995). A new group (type 3) of Mycobacterium celatum isolated from AIDS patients in the London area. Int J Syst Bacteriol 45, 861-862.[Abstract]
Butler, W. R., OConnor, S. P., Yakrus, M. A. & 8 other authors (1993). Mycobacterium celatum sp. nov. Int J Syst Bacteriol 43, 539548.[Abstract]
Bux-Gewehr, I., Hagen, H. P., Rüsch-Gerdes, S. R. & Feurle, G. E. (1998). Fatal pulmonary infection with Mycobacterium celatum in an apparently immunocompetent patient. J Clin Microbiol 36, 587-588.
Crowle, A. J., Tsang, A. Y., Vatter, A. E. & May, M. H. (1986). Comparison of 15 laboratory and patient-derived strains of Mycobacterium avium for ability to infect and multiply in cultured human macrophages. Infect Immun 24, 812-821.
Cynamon, M. H. & Klemens, S. P. (1992). Activity of azithromycin against Mycobacterium avium complex infection in beige mice. Antimicrob Agents Chemother 36, 1611-1613.[Abstract]
Fattorini, L., Li, B., Piersimoni, C., Tortoli, E., Xiao, Y., Santoro, C., Ricci, M. L. & Orefici, G. (1995). In vitro and ex vivo activities of antimicrobial agents used in combination with clarithromycin, with or without amikacin, against Mycobacterium avium. Antimicrob Agents Chemother 39, 680-685.[Abstract]
Fattorini, L., Xiao, Y., Mattei, M., Li, Y., Iona, E., Ricci, M. L., Thoresen, O. F., Creti, R. & Orefici, G. (1998). Activity of isoniazid alone and in combination with other drugs against Mycobacterium avium infection in beige mice. Antimicrob Agents Chemother 42, 712-714.
Gangadharam, P. R. J. (1995). Beige mouse model of Mycobacterium avium complex disease. Antimicrob Agents Chemother 39, 1647-1654.
Gholizadeh, Y., Varnerot, A., Maslo, C., Salauze, B., Badaoui, H., Vincent, V. & Buré-Rossier, A. (1998). Mycobacterium celatum infection in two HIV-infected patients treated prophylactically with rifabutin. Eur J Clin Infect Dis 17, 278-281.
Hart, P. D. & Young, M. R. (1975). Interference with normal phagosome-lysosome fusion in macrophages, using ingested yeast cells and suramin. Nature 256, 47-49.[Medline]
Inderlied, C. B., Kolonowski, P. T., Wu, M. & Young, L. S. (1989). Amikacin, ciprofloxacin, and imipenem treatment for disseminated Mycobacterium avium complex infection of beige mice. Antimicrob Agents Chemother 33, 176-180.[Medline]
Ji, B., Lounis, N., Trouffot-Pernot, C. & Grosset, J. (1994). Effectiveness of various antimicrobial agents against Mycobacterium avium complex in the beige mouse model. Antimicrob Agents Chemother 38, 2521-2529.[Abstract]
Klemens, S. P., DeStefano, M. S. & Cynamon, M. H. (1993). Therapy of multidrug-resistant tuberculosis: lessons from studies with mice. Antimicrob Agents Chemother 37, 2344-2347.[Abstract]
McCarthy, C. (1974). Effect of palmitic acid utilization on cell division in Mycobacterium avium. Infect Immun 9, 363-372.[Medline]
Master, R. N. (1992). Mycobacteriology. In Clinical Microbiology Procedure Handbook, vol. 1, pp. 3.13.16. Edited by H. D. Isenberg. Washington, DC: American Society for Microbiology.
Masur, H. (1993). Recommendations on prophylaxis and therapy for disseminated Mycobacterium avium complex disease in patients infected with human immuno-deficiency virus. N Engl J Med 329, 898-904.
Meylan, P. R., Richman, D. D. & Kornbluth, R. S. (1990). Characterization and growth in human macrophages of Mycobacterium avium complex strains isolated from the blood of patients with acquired immunodeficiency syndrome. Infect Immun 58, 2564-2568.[Medline]
Nakagawara, A. C. & Nathan, C. F. (1983). A simple method for counting adherent cells: application to cultured human monocytes, macrophages and multinucleated giant cells. J Immunol Methods 56, 261-268.[Medline]
Piersimoni, C., Tortoli, E. & De Sio, G. (1994). Disseminated infection due to Mycobacterium celatum in patient with AIDS. Lancet 344, 332.[Medline]
Piersimoni, C., Tortoli, E., de Lalla, F., Nista, D., Donato, D., Bornigia, S. & De Sio, G. (1997). Isolation of Mycobacterium celatum from patients infected with human immunodeficiency virus. Clin Infect Dis 24, 144-147.[Medline]
Rastogi, N., Levy-Frebault, V., Blom-Potar, M. C. & David, H. (1989). Ability of smooth and rough variants of Mycobacterium avium and M. intracellulare to multiply and survive intracellularly: role of C-mycosides. Zentralbl Bakteriol Mikrobiol Hyg A 270, 345-360.[Medline]
Reddy, V. M., Luna-Heerera, J. & Gangadharam, P. R. J. (1996). Pathobiological significance of colony morphology in Mycobacterium avium complex. Microb Pathog 21, 97-109.[Medline]
Schaefer, W. B., Davis, C. L. & Cohn, M. L. (1970). Pathogenicity of transparent, opaque, and rough variants of Mycobacterium avium in chickens and mice. Am Rev Respir Dis 102, 499-506.[Medline]
Tortoli, E., Piersimoni, C., Bacosi, D. & 11 other authors (1995). Isolation of the newly described species Mycobacterium celatum from AIDS patients. J Clin Microbiol 33, 137140.[Abstract]
Woodley, C. L. & David, H. L. (1976). Effect of temperature on the rate of the transparent to opaque colony type transition in Mycobacterium avium. Antimicrob Agents Chemother 9, 113-119.[Medline]
Zurawski, C. A., Cage, G. D., Rimland, D. & Blumberg, H. M. (1997). Pneumonia and bacteremia due to Mycobacterium celatum masquerading as Mycobacterium xenopi in patients with AIDS: an underdiagnosed problem? Clin Infect Dis 24, 140-143.[Medline]
Received 10 April 2000;
revised 10 July 2000;
accepted 26 July 2000.