INFECTION AND IMMUNITY, June 1991, p. 2207-2214
Vol. 59, No. 6
0019-9567/91/062207-08$02.00/0 Copyright © 1991, American Society for Microbiology
Intramacrophage Growth of Mycobacterium avium during Infection of Mice CLAUDE FREHEL,* CHANTAL
CHASTELLIER, CATHERINE OFFREDO,
PATRICK BERCHE Laboratoire de Microbiologie, Faculte de Medecine Necker-Enfants Malades, 156, rue de Vaugirard, 75730 Paris Cedex 15, France DE
AND
Received 19 November 1990/Accepted 4 April 1991
Growth of the virulent Mycobacterium avium strain TMC 724 in host tissues during persistent infection of mice was studied. Following intravenous infection of C57BL/6 mice, the kinetics of bacterial growth was biphasic in the spleen and liver, with a significant reduction of the multiplication rate after day 21 to 28 of infection. An electron-microscopic study of the liver and spleen of infected mice showed that the bacteria were strictly intracellular. They were observed within inflammatory macrophages populating granulomas disseminated in host tissues. The bacteria were confined to the phagosome compartment, and they were encapsulated. Phagosome-lysosome fusions were encountered, but the bacteria showed no visible signs of degradation and continued to multiply. These results are the first in vivo evidence that virulent M. avium multiplies exclusively intracellularly and that encapsulated bacteria resist the microbicidal mechanisms of macrophages inside the phagosomal compartment.
Nontuberculous mycobacteria are opportunistic human pathogens capable of inducing disseminated infections in patients with underlying diseases (28). Among these mycobacteria, Mycobacterium avium is a major human pathogen mainly encountered in patients with AIDS (16, 31), although it can sporadically cause severe diseases in previously healthy patients (21). As M. avium is widespread in nature (11, 29), healthy populations frequently encounter this microorganism, which indicates why the source of infection is generally considered environmental (11). Once established, M. avium can persist in host tissues for long periods (28, 31). M. avium also multiplies and persists in tissues of experimentally infected immunocompetent mice (2). In the past years, mouse or human cultured macrophages have been used as experimental models to study the survival and multiplication of M. avium within macrophages (3, 7, 10, 25). Survival has been related to the resistance of this organism to lysosomal enzymes. First, bacteria can inhibit the transfer of hydrolytic enzymes to phagosomes by reducing phagosome-lysosome fusions (7). Second, the electron-translucent capsule surrounding bacteria (3, 9) can prevent or delay the diffusion of lysosomal enzymes toward bacteria if fusions have occurred (7). The protective role of the capsule had already been suggested by Draper and Rees (4, 5). These two strategies also account for the resistance of other mycobacteria, such as M. tuberculosis and M. leprae, to killing by cultured macrophages (5, 8, 13, 23). However, the relevance of these findings to the in vivo setting is not established. Knowledge of the exact replication site and the survival strategies of this organism within infected tissues could have important implications for the understanding of the process of bacterial virulence of M. avium. So far, it has only been suggested that M. avium could be associated with macrophages or circulating blood leukocytes, on the basis of light microscope examination of various biopsy specimens and blood films of patients with AIDS (6, 15, 22, 32). However, this approach does not allow determination of the replication site of M. avium. In this work, a bacteriological, histological, *
and ultrastructural study was performed to determine the replication site of M. avium in the organs of infected mice and the survival strategies used by this microorganism. Six- to eight-week-old specific-pathogen-free C57BL/6 female mice (Charles River, Saint-Aubin-les-Elbeuf, France) were inoculated intravenously (i.v.) with various doses (103, 104, or 105 bacteria per mouse) of M. avium TMC 724. This strain was obtained from the Trudeau Mycobacterial Culture Collection, Saranac Lake, N.Y., and was kindly provided by Frank Collins. This strain yielded smooth, transparent colonies on Middlebrook 7H11 agar (Difco Laboratories, Detroit, Mich.). The bacteria were grown (without any subculturing) in Middlebrook 7H9 broth (Difco) with Tween 80 added and stored in 1-ml vials at -80°C until required. The frozen suspension was quickly thawed, briefly sonicated, and adjusted to the desired titer in 0.15 M NaCl. Bacterial growth in the spleen, liver, and lungs was monitored for 75 days by sacrificing groups of five mice per time point. Organs were removed aseptically and homogenized. The number of bacteria per organ (liver, spleen, or lungs) was then estimated by plating 10-fold dilutions of the organ homogenates on Middlebrook 7H11 agar. Colonies were counted after incubation at 37°C for 21 days. The generation time was determined by calculating the average slope of the bacterial replication curve (log1o of the number of bacteria versus time) during the early phase (days 1 to 21) or the late phase (days 28 to 75) of infection. The doubling time was then assessed as the time required for an 0.3-log-unit increase in the bacterial population, as described by Crowle et al. (3). After i.v. infection, most bacteria were rapidly trapped in the spleen and liver (Fig. 1). The bacteria then multiplied in the spleen, liver, and lungs of mice during the 75-day experimentation period, with the growth curves showing a low level of variability in bacterial counts (Fig. 1). The spleen and liver appeared as the primary target organs of the i.v. infection, whereas lung infection was delayed, especially with the lowest inoculum. The kinetics of bacterial multiplication in the spleen and the liver was biphasic (Fig. 1; Table 1). In the early phase of infection, until day 21, the generation time varied between 1.9 and 3.1 days, depending upon the organ and inoculum. It is worth noting that the doubling
Corresponding author. 2207
2208
INFECT. IMMUN.
NOTES
z I0. 0
9.
b LVER
8I 7.'
6,
0 c-
9
5
10
20
30
60
s0
40
70
80
DAYS
FIG. 1. Growth of M. avium in the spleen (a), liver (b) (c) of C57BL/6 mice. The mice were inoculated i .v. with (A),
or
105 (I) bacteria of strain TMC 724.
an average of five determinations. standard error of the mean.
represents
the
E
a3nd lungs poi
Eror bars represent
time of strain TMC 724 was about 48 h in cultured bone marrow-derived mouse macrophages (unf)ublished results) and 40 h in cultured human blood monocyte s (3). This means that during the early phase of infection, t]he bacteria replicated almost without restriction in host nnacrophages at a rate similar to that found in nonactivated macrophages in culture. Starting on day 28, the rate of bacteria wad significantly reduced (four- to sevenfold) iInin the spleen and liver. This reduction in bacterial growth wa Ls probably due to
rtesplcation
TABLE 1. Generation time of M. avium TMIC 724 during the early and late phases of infection iin mice Generation time (days) in indlicated organ(s) indicated no. of days of infection
No. of bacteria used for challenge
105 104 103 a
Spleen >28
21 8 7.'7 20.(0
Lungs 28 2.8 4.2 3.1 4.6
21. 4
ND~ 4.7
Liver
Vol. 59, No. 6
0019-9567/91/062207-08$02.00/0 Copyright © 1991, American Society for Microbiology
Intramacrophage Growth of Mycobacterium avium during Infection of Mice CLAUDE FREHEL,* CHANTAL
CHASTELLIER, CATHERINE OFFREDO,
PATRICK BERCHE Laboratoire de Microbiologie, Faculte de Medecine Necker-Enfants Malades, 156, rue de Vaugirard, 75730 Paris Cedex 15, France DE
AND
Received 19 November 1990/Accepted 4 April 1991
Growth of the virulent Mycobacterium avium strain TMC 724 in host tissues during persistent infection of mice was studied. Following intravenous infection of C57BL/6 mice, the kinetics of bacterial growth was biphasic in the spleen and liver, with a significant reduction of the multiplication rate after day 21 to 28 of infection. An electron-microscopic study of the liver and spleen of infected mice showed that the bacteria were strictly intracellular. They were observed within inflammatory macrophages populating granulomas disseminated in host tissues. The bacteria were confined to the phagosome compartment, and they were encapsulated. Phagosome-lysosome fusions were encountered, but the bacteria showed no visible signs of degradation and continued to multiply. These results are the first in vivo evidence that virulent M. avium multiplies exclusively intracellularly and that encapsulated bacteria resist the microbicidal mechanisms of macrophages inside the phagosomal compartment.
Nontuberculous mycobacteria are opportunistic human pathogens capable of inducing disseminated infections in patients with underlying diseases (28). Among these mycobacteria, Mycobacterium avium is a major human pathogen mainly encountered in patients with AIDS (16, 31), although it can sporadically cause severe diseases in previously healthy patients (21). As M. avium is widespread in nature (11, 29), healthy populations frequently encounter this microorganism, which indicates why the source of infection is generally considered environmental (11). Once established, M. avium can persist in host tissues for long periods (28, 31). M. avium also multiplies and persists in tissues of experimentally infected immunocompetent mice (2). In the past years, mouse or human cultured macrophages have been used as experimental models to study the survival and multiplication of M. avium within macrophages (3, 7, 10, 25). Survival has been related to the resistance of this organism to lysosomal enzymes. First, bacteria can inhibit the transfer of hydrolytic enzymes to phagosomes by reducing phagosome-lysosome fusions (7). Second, the electron-translucent capsule surrounding bacteria (3, 9) can prevent or delay the diffusion of lysosomal enzymes toward bacteria if fusions have occurred (7). The protective role of the capsule had already been suggested by Draper and Rees (4, 5). These two strategies also account for the resistance of other mycobacteria, such as M. tuberculosis and M. leprae, to killing by cultured macrophages (5, 8, 13, 23). However, the relevance of these findings to the in vivo setting is not established. Knowledge of the exact replication site and the survival strategies of this organism within infected tissues could have important implications for the understanding of the process of bacterial virulence of M. avium. So far, it has only been suggested that M. avium could be associated with macrophages or circulating blood leukocytes, on the basis of light microscope examination of various biopsy specimens and blood films of patients with AIDS (6, 15, 22, 32). However, this approach does not allow determination of the replication site of M. avium. In this work, a bacteriological, histological, *
and ultrastructural study was performed to determine the replication site of M. avium in the organs of infected mice and the survival strategies used by this microorganism. Six- to eight-week-old specific-pathogen-free C57BL/6 female mice (Charles River, Saint-Aubin-les-Elbeuf, France) were inoculated intravenously (i.v.) with various doses (103, 104, or 105 bacteria per mouse) of M. avium TMC 724. This strain was obtained from the Trudeau Mycobacterial Culture Collection, Saranac Lake, N.Y., and was kindly provided by Frank Collins. This strain yielded smooth, transparent colonies on Middlebrook 7H11 agar (Difco Laboratories, Detroit, Mich.). The bacteria were grown (without any subculturing) in Middlebrook 7H9 broth (Difco) with Tween 80 added and stored in 1-ml vials at -80°C until required. The frozen suspension was quickly thawed, briefly sonicated, and adjusted to the desired titer in 0.15 M NaCl. Bacterial growth in the spleen, liver, and lungs was monitored for 75 days by sacrificing groups of five mice per time point. Organs were removed aseptically and homogenized. The number of bacteria per organ (liver, spleen, or lungs) was then estimated by plating 10-fold dilutions of the organ homogenates on Middlebrook 7H11 agar. Colonies were counted after incubation at 37°C for 21 days. The generation time was determined by calculating the average slope of the bacterial replication curve (log1o of the number of bacteria versus time) during the early phase (days 1 to 21) or the late phase (days 28 to 75) of infection. The doubling time was then assessed as the time required for an 0.3-log-unit increase in the bacterial population, as described by Crowle et al. (3). After i.v. infection, most bacteria were rapidly trapped in the spleen and liver (Fig. 1). The bacteria then multiplied in the spleen, liver, and lungs of mice during the 75-day experimentation period, with the growth curves showing a low level of variability in bacterial counts (Fig. 1). The spleen and liver appeared as the primary target organs of the i.v. infection, whereas lung infection was delayed, especially with the lowest inoculum. The kinetics of bacterial multiplication in the spleen and the liver was biphasic (Fig. 1; Table 1). In the early phase of infection, until day 21, the generation time varied between 1.9 and 3.1 days, depending upon the organ and inoculum. It is worth noting that the doubling
Corresponding author. 2207
2208
INFECT. IMMUN.
NOTES
z I0. 0
9.
b LVER
8I 7.'
6,
0 c-
9
5
10
20
30
60
s0
40
70
80
DAYS
FIG. 1. Growth of M. avium in the spleen (a), liver (b) (c) of C57BL/6 mice. The mice were inoculated i .v. with (A),
or
105 (I) bacteria of strain TMC 724.
an average of five determinations. standard error of the mean.
represents
the
E
a3nd lungs poi
Eror bars represent
time of strain TMC 724 was about 48 h in cultured bone marrow-derived mouse macrophages (unf)ublished results) and 40 h in cultured human blood monocyte s (3). This means that during the early phase of infection, t]he bacteria replicated almost without restriction in host nnacrophages at a rate similar to that found in nonactivated macrophages in culture. Starting on day 28, the rate of bacteria wad significantly reduced (four- to sevenfold) iInin the spleen and liver. This reduction in bacterial growth wa Ls probably due to
rtesplcation
TABLE 1. Generation time of M. avium TMIC 724 during the early and late phases of infection iin mice Generation time (days) in indlicated organ(s) indicated no. of days of infection
No. of bacteria used for challenge
105 104 103 a
Spleen >28
21 8 7.'7 20.(0
Lungs 28 2.8 4.2 3.1 4.6
21. 4
ND~ 4.7
Liver
