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Mycobacterium tuberculosis complex mycobacteria as amoeba-resistant organisms.

Mba Medie F, Ben Salah I, Henrissat B, Raoult D, Drancourt M - PLoS ONE (2011)

Bottom Line: M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts.Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde.Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle.

View Article: PubMed Central - PubMed

Affiliation: Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UMR 6236 CNRS-Université de la Méditerranée, IRD 189, IFR 48, Faculté de Médecine, Marseille, France.

ABSTRACT

Background: Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii.

Methodology/principal findings: Using optic and electron microscopy and co-culture methods, we observed that 89±0.6% of M. canettii, 12.4±0.3% of M. tuberculosis, 11.7±2% of M. bovis and 11.2±0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03), correlating with the significantly larger size of M. canetti organisms (P = 0.035). The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32±2% for M. canettii, 26±1% for M. tuberculosis, 28±2% for M. bovis and 36±2% for M. avium (P = 0.57). M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10(-6)) higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02) M. avium organisms (34×10(4) CFU/mL) than M. tuberculosis (42×10(1) CFU/mL) and M. bovis (35×10(1) CFU/mL) in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde.

Conclusions/significance: These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle.

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Survival of mycobacteria within A. polyphaga trophozoites at 33°C.Time zero represents 24 h after infection, immediately after treatment with streptomycin. Each bar represents the mean of duplicate wells, and error bars show the standard error of the mean. “*” denotes a statistically significant difference.
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pone-0020499-g003: Survival of mycobacteria within A. polyphaga trophozoites at 33°C.Time zero represents 24 h after infection, immediately after treatment with streptomycin. Each bar represents the mean of duplicate wells, and error bars show the standard error of the mean. “*” denotes a statistically significant difference.

Mentions: We observed that the length of mycobacterial cells used in this study was 2.56±0.5 µm for M. canettii organisms, 1.7±0.3 µm for M. tuberculosis organisms, 1.33±0.3 µm for M. bovis organisms, and 1.37±0.3 µm for M. avium organisms (Fig. S1); the length of M. canettii organisms was significantly larger than that of the three other mycobacteria (P = 0.035, one-way ANOVA). The four organisms were killed by 10 µg/mL streptomycin and M. avium and M. tuberculosis were killed also by 2.5% glutaraldehyde. Acid-fast stained A. polyphaga trophozoites exhibited intra-amoebal mycobacteria, regardless of the Mycobacterium species being studied. The proportion of inoculated mycobacteria phagocytozed by trophozoites was significantly higher (P = 0.03) for M. canettii organisms (89±0.6%) than for M. tuberculosis (12.4±0.3%), M. bovis (11.7±2%) and the M. avium control (11.2±0.5%) (Fig. 1). Phagocytosis yielded an insignificant difference in the percentage of infected amoeba, varying between 26±1% for M. tuberculosis, 28±2% for M. bovis, 32±2% for M. canettii and 36±2% for M. avium; the number of mycobacteria per trophozoite varied from 1 to 4 for M. tuberculosis, 1 to 6 for M. bovis and M. avium and from 1 to 10 for M. canettii (Fig. 2). Electron microscopy revealed that mycobacteria were residing inside one or several vacuoles without notable modification of the surrounding cytoplasm; vacuoles containing only one organism exhibited a close apposition of the vacuole membrane all over the mycobacterial cell surface (Fig. 2B). In some vacuoles, electron microscopy revealed morphological features that were compatible with mycobacterial division. Growth curves were not significantly different between the four species being studied, with the only observations being a small decrease in the number of CFUs at 24 h and minute variations in the number of CFUs from 24 h to 72 h (Fig. 3).


Mycobacterium tuberculosis complex mycobacteria as amoeba-resistant organisms.

Mba Medie F, Ben Salah I, Henrissat B, Raoult D, Drancourt M - PLoS ONE (2011)

Survival of mycobacteria within A. polyphaga trophozoites at 33°C.Time zero represents 24 h after infection, immediately after treatment with streptomycin. Each bar represents the mean of duplicate wells, and error bars show the standard error of the mean. “*” denotes a statistically significant difference.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3108610&req=5

pone-0020499-g003: Survival of mycobacteria within A. polyphaga trophozoites at 33°C.Time zero represents 24 h after infection, immediately after treatment with streptomycin. Each bar represents the mean of duplicate wells, and error bars show the standard error of the mean. “*” denotes a statistically significant difference.
Mentions: We observed that the length of mycobacterial cells used in this study was 2.56±0.5 µm for M. canettii organisms, 1.7±0.3 µm for M. tuberculosis organisms, 1.33±0.3 µm for M. bovis organisms, and 1.37±0.3 µm for M. avium organisms (Fig. S1); the length of M. canettii organisms was significantly larger than that of the three other mycobacteria (P = 0.035, one-way ANOVA). The four organisms were killed by 10 µg/mL streptomycin and M. avium and M. tuberculosis were killed also by 2.5% glutaraldehyde. Acid-fast stained A. polyphaga trophozoites exhibited intra-amoebal mycobacteria, regardless of the Mycobacterium species being studied. The proportion of inoculated mycobacteria phagocytozed by trophozoites was significantly higher (P = 0.03) for M. canettii organisms (89±0.6%) than for M. tuberculosis (12.4±0.3%), M. bovis (11.7±2%) and the M. avium control (11.2±0.5%) (Fig. 1). Phagocytosis yielded an insignificant difference in the percentage of infected amoeba, varying between 26±1% for M. tuberculosis, 28±2% for M. bovis, 32±2% for M. canettii and 36±2% for M. avium; the number of mycobacteria per trophozoite varied from 1 to 4 for M. tuberculosis, 1 to 6 for M. bovis and M. avium and from 1 to 10 for M. canettii (Fig. 2). Electron microscopy revealed that mycobacteria were residing inside one or several vacuoles without notable modification of the surrounding cytoplasm; vacuoles containing only one organism exhibited a close apposition of the vacuole membrane all over the mycobacterial cell surface (Fig. 2B). In some vacuoles, electron microscopy revealed morphological features that were compatible with mycobacterial division. Growth curves were not significantly different between the four species being studied, with the only observations being a small decrease in the number of CFUs at 24 h and minute variations in the number of CFUs from 24 h to 72 h (Fig. 3).

Bottom Line: M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts.Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde.Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle.

View Article: PubMed Central - PubMed

Affiliation: Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UMR 6236 CNRS-Université de la Méditerranée, IRD 189, IFR 48, Faculté de Médecine, Marseille, France.

ABSTRACT

Background: Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii.

Methodology/principal findings: Using optic and electron microscopy and co-culture methods, we observed that 89±0.6% of M. canettii, 12.4±0.3% of M. tuberculosis, 11.7±2% of M. bovis and 11.2±0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03), correlating with the significantly larger size of M. canetti organisms (P = 0.035). The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32±2% for M. canettii, 26±1% for M. tuberculosis, 28±2% for M. bovis and 36±2% for M. avium (P = 0.57). M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10(-6)) higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02) M. avium organisms (34×10(4) CFU/mL) than M. tuberculosis (42×10(1) CFU/mL) and M. bovis (35×10(1) CFU/mL) in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde.

Conclusions/significance: These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle.

Show MeSH
Related in: MedlinePlus