Low Concentrations of Nitric Oxide Exert a Hormetic Effect on Mycobacterium tuberculosis In Vitro
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微生物临床杂志 2005年第9期
Louisiana State University Health Sciences Center, School of Allied Health Professions, Department of Clinical Laboratory Sciences, New Orleans, Louisiana 70112-2223
ABSTRACT
Susceptibilities of 12 clinical Mycobacterium tuberculosis isolates to acidified sodium nitrite (ASN) were compared. The results demonstrate that 8 of the 12 isolates exhibited enhanced survival levels in 1.5 mM ASN compared to levels in medium alone, suggesting that low concentrations of reactive nitrogen intermediates have a hormetic effect on M. tuberculosis in vitro.
TEXT
Hormesis is a term given to the stimulatory effects caused by low levels of a potentially toxic agent. Specifically, hormesis is a dose-response relationship phenomenon characterized by low-dose stimulation and high-dose inhibition (2) and has been a common occurrence studied most extensively in the toxicology field. Many agents that elicit hormetic responses are environmental components, such as metals (e.g., mercury) and ionizing radiation, that are harmful or even fatal at high concentrations (9). Measures used to assess hormesis are mainly components of fitness, such as growth, longevity, fecundity, and survival (1, 13). Evidence from numerous studies demonstrates that growth hormesis has been observed to occur in a variety of organisms after exposure to a range of toxic agents (9, 12).
Considering that Mycobacterium tuberculosis is one of the few pathogens able to reside and replicate within the toxic environment of the macrophage, it is possible that this pathogen has adapted to its surroundings in a hormetic-like fashion. We previously demonstrated that low concentrations of reactive nitrogen intermediates (RNI) may inhibit replication (bacteriostatic) but do not eradicate M. tuberculosis (bactericidal) (5). The current study demonstrates that clinical M. tuberculosis isolates survive and reproduce in the presence of lower concentrations of RNI as well as or better than they survive and reproduce in medium alone. This new finding suggests that RNI exert a hormetic effect on M. tuberculosis in vitro. Consequently, a hormetic response to low concentrations of RNI may play a role in the ability of M. tuberculosis to persist for extended periods of time within the host.
The M. tuberculosis isolates used in this study were obtained from tuberculosis (TB) patients throughout Louisiana. Two isolates (designated R and S) were previously characterized and were used as controls for the assays. One of the controls (isolate R), CDC1551, was involved in a unique outbreak (17) and was previously shown to be highly resistant to acidified sodium nitrite (ASN) in vitro (4). The other control (isolate S), C.C. 22, a nonclustered strain isolated from a TB patient in northern California, was previously shown to be highly susceptible to ASN (4). The other clinical isolates used in this study were obtained from TB-positive patients living in various cities throughout Louisiana (isolates C, CC, and GG from New Orleans; isolates KK, Q, and HH from Shreveport; and isolates UU, P, and WW from unknown cities). All of the isolates used in this study demonstrated similar growth kinetics in 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with 0.5% glycerol, 0.02% Tween 80, and ADC enrichment (Becton Dickinson) (data not shown). Before the assays were performed, each isolate was given an alphabetical letter so that the technician performing the assays was blinded as to which strain or patient number corresponded to each culture. Single-cell suspensions of each isolate were prepared according to a previously published method (10) and quantified by enumeration of their CFU on Middlebrook 7H11 agar plates. A 0.1-ml aliquot of a freshly prepared single-cell suspension was added to 0.9 ml of Middlebrook 7H9 broth (pH 5.4) containing 1.5 mM or 6 mM sodium nitrite (Sigma, St. Louis, MO). Sodium nitrite dissolved in acidic pH generates nitric oxide, and in the presence of oxygen, it is converted into other reactive intermediates, such as nitrogen dioxide and peroxynitrite (3, 16). For the unexposed control group, each culture was added to 7H9 broth (pH 5.4) without the addition of sodium nitrite. Each suspension was incubated at 37°C for 16 h, plated onto Middlebrook 7H11 agar, and incubated at 37°C for 4 weeks. The number of CFU recovered from isolates exposed to ASN was compared to the number of CFU recovered from those not exposed, and the data were then calculated as percent survival [(no. of CFU at day 28 exposed/no. of CFU at day 28 unexposed) x 100]. The data represent results from triplicate experiments from a single-cell suspension per culture from at least three separate cultures for each isolate. The data are presented as mean percent survival (± standard error) for each isolate. Comparison of the percent survival of each isolate exposed to ASN with that of the same isolate unexposed was made by the Student t test, and a P value of less than 0.05 was considered to show a significant difference.
Our results demonstrate that each M. tuberculosis isolate varies regarding survival in ASN (Fig. 1 and Table 1). However, some clinical isolates were better able to survive in higher levels of ASN than others, which supports our previous studies using similar assays (4). For example, the percent survival of the isolates exposed to 6 mM ASN ranged from 74% to 0.07% (Fig. 1 and Table 2). The mean percent survival of each isolate exposed to 6 mM ASN was significantly less (P < 0.05) than the mean percent survival of the same isolate unexposed (Table 2). However, the survival differences observed were not due to differences in the amounts of bacteria used in the assays, since the bacterial CFU for each isolate were similar (Table 1) and an identical inoculum was utilized for each.
In contrast to the observations made at 6 mM ASN, most M. tuberculosis isolates were relatively resistant to 1.5 mM ASN, except for isolates S and Q (Fig. 1). Hence, 1.5 mM ASN is capable of killing susceptible M. tuberculosis strains. Although many isolates demonstrate an enhanced ability to survive in low RNI concentrations, the interesting finding from this study is that isolates appear to survive better in the presence of 1.5 mM ASN than when unexposed. For example, the mean percent survival of eight isolates exposed to 1.5 mM ASN exceeded 100%, with seven isolates demonstrating significantly (P < 0.05) enhanced survival compared to the same isolates unexposed (Table 2). These data, in combination with previous studies (5), suggest that RNI products may exert a hormetic effect at low concentrations (1.5 mM), a bacteriostatic effect at intermediate concentrations (3 mM), and a bactericidal effect at high concentrations (6 mM) in vitro.
The hormesis model states that substances which are toxic or even lethal at one dose can be beneficial at another, usually smaller, dose (7). For example, the clinical M. tuberculosis isolate UU had a mean percent survival of 9% when exposed to 6 mM ASN compared to the same isolate unexposed to ASN (Fig. 1 and Table 2). However, isolate UU demonstrated the highest mean percent survival when exposed to 1.5 mM ASN, with a mean percent survival of 128% compared to the same strain unexposed to ASN (Fig. 1 and Table 2). Hence, isolate UU demonstrated a hormetic response at 1.5 mM ASN and a bactericidal response at 6 mM ASN. These results are consistent with studies by Calabrese and Baldwin (2), who demonstrated that the hormetic response is inherently modest and almost never exceeds a factor of twofold greater than the control and usually no more than 130% to 160% (2).
In addition to the observed hormetic effect of RNI, our results demonstrate that some clinical M. tuberculosis isolates are able to survive in the presence of ASN (Fig. 1). These observations are consistent with numerous studies that have demonstrated strain-related differences regarding susceptibility to RNI (4, 6, 8, 11, 14, 15, 18) and suggest that certain M. tuberculosis strains are better able to resist the antibacterial mechanisms elicited by the host macrophage.
Collectively, our data indicate that under the in vitro conditions used in this study, exposure to a lower concentration of ASN resulted in hormesis of most clinical M. tuberculosis isolates. The amount of RNI added in the cell-free system used for the present study was higher than that expected to be generated in vivo. Therefore, the fitness of some clinical M. tuberculosis isolates may actually be enhanced when exposed to low concentrations of nitrosative intermediates in vivo. Consequently, a hormetic response to RNI may enable some M. tuberculosis strains to cause active disease more readily or persist for longer periods of time in the host.
ACKNOWLEDGMENTS
This study was supported by LSUHSC-SAHP grant 490114 and the Wetmore Foundation.
We thank Ronald Schiro, Rob Dellsperger, and Ann Ryan of the Louisiana Department of Health and Hospital (LA-DHH) Tuberculosis Laboratory for providing the Louisiana clinical M. tuberculosis isolates and Lisa Morici for careful review of the manuscript.
REFERENCES
Calabrese, E. J., M. E. McCarthy, and E. Kenyon. 1987. The occurrence of chemically-induced hormesis. Health Phys. 52:531-541.
Calabrese, E. J., and L. A. Baldwin. 2003. Hormesis: the dose-response revolution. Annu. Rev. Pharmacol. 43:175-197.
Doi, T., M. Ando, T. Akaike, M. Suga, K. Sato, and H. Maeda. 1993. Resistance to nitric oxide in Mycobacterium avium complex and its implication in pathogenesis. Infect. Immun. 61:1980-1989.
Firmani, M. A., and L. W. Riley. 2002. Mycobacterium tuberculosis CDC1551 is resistant to reactive nitrogen and oxygen intermediates in vitro. Infect. Immun. 70:3965-3968.
Firmani, M. A., and L. W. Riley. 2002. Reactive nitrogen intermediates have a bacteriostatic effect on Mycobacterium tuberculosis in vitro. J. Clin. Microbiol. 40:3162-3166.
Flesch, I., and S. H. E. Kaufmann. 1987. Mycobacterial growth inhibition by interferon--activated bone marrow macrophages and differential susceptibility among strains of Mycobacterium tuberculosis. J. Immunol. 138:4408-4413.
Flynn, J., and D. MacGregor. 2003. Commentary on hormesis and public risk communication: is there a basis for public discussions Hum. Exp. Toxicol. 22:31-34.
Friedman, C. R., G. C. Quinn, B. N. Kreiswirth, D. C. Perlman, N. Salomon, N. Schluger, M. Lutfey, J. Berger, N. Poltoratskaia, and L. W. Riley. 1997. Widespread dissemination of a drug-susceptible strain of Mycobacterium tuberculosis. J. Infect. Dis. 176:478-484.
Gerber, L. M., G. C. Williams, and S. H. Gray. 1999. The nutrient-toxin dosage continuum in human evolution and modern health. Q. Rev. Biol. 74:273-289.
Grover, A. A., H. K. Kim, E. H. Wiegeshaus, and D. W. Smith. 1967. Host-parasite relationships in experimental airborne tuberculosis. II. Reproducible infection by means of an inoculum preserved at –70°C. J. Bacteriol. 94:832-835.
Jagannath, C., J. K. Actor, and R. L. Hunter, Jr. 1998. Induction of nitric oxide in human monocytes and monocyte cell lines by Mycobacterium tuberculosis. Nitric Oxide 2:174-176.
Joiner, M. C. 1994. Induced radioresistance: an overview and historical perspective. Int. J. Radiat. Biol. 65:79-84.
Luckey, T. D. 1982. Physiological benefits from low-levels of ionizing radiation. Health Phys. 43:771-789.
O'Brien, L., J. Carmichael, D. B. Lowrie, and P. W. Andrew. 1994. Strains of Mycobacterium tuberculosis differ in susceptibility to reactive nitrogen intermediates in vitro. Infect. Immun. 62:5187-5190.
Rhoades, E. E., and I. M. Orme. 1997. Susceptibility of a panel of virulent strains of Mycobacterium tuberculosis to reactive nitrogen intermediates. Infect. Immun. 65:1189-1195.
Stuehr, D., and C. F. Nathan. 1989. Nitric oxide. A macrophage product responsible for the cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med. 169:1543-1555.
Valway, S. E., M. P. C. Sanchez, T. F. Shinnick, I. Orme, T. Agerton, D. Hoy, J. S. Jones, H. Westmoreland, and I. M. Onorato. 1998. An outbreak involving extensive transmission of a virulent strain of Mycobacterium tuberculosis. N. Engl. J. Med. 338:633-639.
Yu, K., C. Mitchell, Y. Xing, R. S. Magliozzo, B. R. Bloom, and J. Chan. 1999. Toxicity of nitrogen oxides and related oxidants on mycobacteria: M. tuberculosis is resistant to peroxynitrite anion. Tuber. Lung Dis. 79:191-198.(William Benjamin Brugmann)
ABSTRACT
Susceptibilities of 12 clinical Mycobacterium tuberculosis isolates to acidified sodium nitrite (ASN) were compared. The results demonstrate that 8 of the 12 isolates exhibited enhanced survival levels in 1.5 mM ASN compared to levels in medium alone, suggesting that low concentrations of reactive nitrogen intermediates have a hormetic effect on M. tuberculosis in vitro.
TEXT
Hormesis is a term given to the stimulatory effects caused by low levels of a potentially toxic agent. Specifically, hormesis is a dose-response relationship phenomenon characterized by low-dose stimulation and high-dose inhibition (2) and has been a common occurrence studied most extensively in the toxicology field. Many agents that elicit hormetic responses are environmental components, such as metals (e.g., mercury) and ionizing radiation, that are harmful or even fatal at high concentrations (9). Measures used to assess hormesis are mainly components of fitness, such as growth, longevity, fecundity, and survival (1, 13). Evidence from numerous studies demonstrates that growth hormesis has been observed to occur in a variety of organisms after exposure to a range of toxic agents (9, 12).
Considering that Mycobacterium tuberculosis is one of the few pathogens able to reside and replicate within the toxic environment of the macrophage, it is possible that this pathogen has adapted to its surroundings in a hormetic-like fashion. We previously demonstrated that low concentrations of reactive nitrogen intermediates (RNI) may inhibit replication (bacteriostatic) but do not eradicate M. tuberculosis (bactericidal) (5). The current study demonstrates that clinical M. tuberculosis isolates survive and reproduce in the presence of lower concentrations of RNI as well as or better than they survive and reproduce in medium alone. This new finding suggests that RNI exert a hormetic effect on M. tuberculosis in vitro. Consequently, a hormetic response to low concentrations of RNI may play a role in the ability of M. tuberculosis to persist for extended periods of time within the host.
The M. tuberculosis isolates used in this study were obtained from tuberculosis (TB) patients throughout Louisiana. Two isolates (designated R and S) were previously characterized and were used as controls for the assays. One of the controls (isolate R), CDC1551, was involved in a unique outbreak (17) and was previously shown to be highly resistant to acidified sodium nitrite (ASN) in vitro (4). The other control (isolate S), C.C. 22, a nonclustered strain isolated from a TB patient in northern California, was previously shown to be highly susceptible to ASN (4). The other clinical isolates used in this study were obtained from TB-positive patients living in various cities throughout Louisiana (isolates C, CC, and GG from New Orleans; isolates KK, Q, and HH from Shreveport; and isolates UU, P, and WW from unknown cities). All of the isolates used in this study demonstrated similar growth kinetics in 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with 0.5% glycerol, 0.02% Tween 80, and ADC enrichment (Becton Dickinson) (data not shown). Before the assays were performed, each isolate was given an alphabetical letter so that the technician performing the assays was blinded as to which strain or patient number corresponded to each culture. Single-cell suspensions of each isolate were prepared according to a previously published method (10) and quantified by enumeration of their CFU on Middlebrook 7H11 agar plates. A 0.1-ml aliquot of a freshly prepared single-cell suspension was added to 0.9 ml of Middlebrook 7H9 broth (pH 5.4) containing 1.5 mM or 6 mM sodium nitrite (Sigma, St. Louis, MO). Sodium nitrite dissolved in acidic pH generates nitric oxide, and in the presence of oxygen, it is converted into other reactive intermediates, such as nitrogen dioxide and peroxynitrite (3, 16). For the unexposed control group, each culture was added to 7H9 broth (pH 5.4) without the addition of sodium nitrite. Each suspension was incubated at 37°C for 16 h, plated onto Middlebrook 7H11 agar, and incubated at 37°C for 4 weeks. The number of CFU recovered from isolates exposed to ASN was compared to the number of CFU recovered from those not exposed, and the data were then calculated as percent survival [(no. of CFU at day 28 exposed/no. of CFU at day 28 unexposed) x 100]. The data represent results from triplicate experiments from a single-cell suspension per culture from at least three separate cultures for each isolate. The data are presented as mean percent survival (± standard error) for each isolate. Comparison of the percent survival of each isolate exposed to ASN with that of the same isolate unexposed was made by the Student t test, and a P value of less than 0.05 was considered to show a significant difference.
Our results demonstrate that each M. tuberculosis isolate varies regarding survival in ASN (Fig. 1 and Table 1). However, some clinical isolates were better able to survive in higher levels of ASN than others, which supports our previous studies using similar assays (4). For example, the percent survival of the isolates exposed to 6 mM ASN ranged from 74% to 0.07% (Fig. 1 and Table 2). The mean percent survival of each isolate exposed to 6 mM ASN was significantly less (P < 0.05) than the mean percent survival of the same isolate unexposed (Table 2). However, the survival differences observed were not due to differences in the amounts of bacteria used in the assays, since the bacterial CFU for each isolate were similar (Table 1) and an identical inoculum was utilized for each.
In contrast to the observations made at 6 mM ASN, most M. tuberculosis isolates were relatively resistant to 1.5 mM ASN, except for isolates S and Q (Fig. 1). Hence, 1.5 mM ASN is capable of killing susceptible M. tuberculosis strains. Although many isolates demonstrate an enhanced ability to survive in low RNI concentrations, the interesting finding from this study is that isolates appear to survive better in the presence of 1.5 mM ASN than when unexposed. For example, the mean percent survival of eight isolates exposed to 1.5 mM ASN exceeded 100%, with seven isolates demonstrating significantly (P < 0.05) enhanced survival compared to the same isolates unexposed (Table 2). These data, in combination with previous studies (5), suggest that RNI products may exert a hormetic effect at low concentrations (1.5 mM), a bacteriostatic effect at intermediate concentrations (3 mM), and a bactericidal effect at high concentrations (6 mM) in vitro.
The hormesis model states that substances which are toxic or even lethal at one dose can be beneficial at another, usually smaller, dose (7). For example, the clinical M. tuberculosis isolate UU had a mean percent survival of 9% when exposed to 6 mM ASN compared to the same isolate unexposed to ASN (Fig. 1 and Table 2). However, isolate UU demonstrated the highest mean percent survival when exposed to 1.5 mM ASN, with a mean percent survival of 128% compared to the same strain unexposed to ASN (Fig. 1 and Table 2). Hence, isolate UU demonstrated a hormetic response at 1.5 mM ASN and a bactericidal response at 6 mM ASN. These results are consistent with studies by Calabrese and Baldwin (2), who demonstrated that the hormetic response is inherently modest and almost never exceeds a factor of twofold greater than the control and usually no more than 130% to 160% (2).
In addition to the observed hormetic effect of RNI, our results demonstrate that some clinical M. tuberculosis isolates are able to survive in the presence of ASN (Fig. 1). These observations are consistent with numerous studies that have demonstrated strain-related differences regarding susceptibility to RNI (4, 6, 8, 11, 14, 15, 18) and suggest that certain M. tuberculosis strains are better able to resist the antibacterial mechanisms elicited by the host macrophage.
Collectively, our data indicate that under the in vitro conditions used in this study, exposure to a lower concentration of ASN resulted in hormesis of most clinical M. tuberculosis isolates. The amount of RNI added in the cell-free system used for the present study was higher than that expected to be generated in vivo. Therefore, the fitness of some clinical M. tuberculosis isolates may actually be enhanced when exposed to low concentrations of nitrosative intermediates in vivo. Consequently, a hormetic response to RNI may enable some M. tuberculosis strains to cause active disease more readily or persist for longer periods of time in the host.
ACKNOWLEDGMENTS
This study was supported by LSUHSC-SAHP grant 490114 and the Wetmore Foundation.
We thank Ronald Schiro, Rob Dellsperger, and Ann Ryan of the Louisiana Department of Health and Hospital (LA-DHH) Tuberculosis Laboratory for providing the Louisiana clinical M. tuberculosis isolates and Lisa Morici for careful review of the manuscript.
REFERENCES
Calabrese, E. J., M. E. McCarthy, and E. Kenyon. 1987. The occurrence of chemically-induced hormesis. Health Phys. 52:531-541.
Calabrese, E. J., and L. A. Baldwin. 2003. Hormesis: the dose-response revolution. Annu. Rev. Pharmacol. 43:175-197.
Doi, T., M. Ando, T. Akaike, M. Suga, K. Sato, and H. Maeda. 1993. Resistance to nitric oxide in Mycobacterium avium complex and its implication in pathogenesis. Infect. Immun. 61:1980-1989.
Firmani, M. A., and L. W. Riley. 2002. Mycobacterium tuberculosis CDC1551 is resistant to reactive nitrogen and oxygen intermediates in vitro. Infect. Immun. 70:3965-3968.
Firmani, M. A., and L. W. Riley. 2002. Reactive nitrogen intermediates have a bacteriostatic effect on Mycobacterium tuberculosis in vitro. J. Clin. Microbiol. 40:3162-3166.
Flesch, I., and S. H. E. Kaufmann. 1987. Mycobacterial growth inhibition by interferon--activated bone marrow macrophages and differential susceptibility among strains of Mycobacterium tuberculosis. J. Immunol. 138:4408-4413.
Flynn, J., and D. MacGregor. 2003. Commentary on hormesis and public risk communication: is there a basis for public discussions Hum. Exp. Toxicol. 22:31-34.
Friedman, C. R., G. C. Quinn, B. N. Kreiswirth, D. C. Perlman, N. Salomon, N. Schluger, M. Lutfey, J. Berger, N. Poltoratskaia, and L. W. Riley. 1997. Widespread dissemination of a drug-susceptible strain of Mycobacterium tuberculosis. J. Infect. Dis. 176:478-484.
Gerber, L. M., G. C. Williams, and S. H. Gray. 1999. The nutrient-toxin dosage continuum in human evolution and modern health. Q. Rev. Biol. 74:273-289.
Grover, A. A., H. K. Kim, E. H. Wiegeshaus, and D. W. Smith. 1967. Host-parasite relationships in experimental airborne tuberculosis. II. Reproducible infection by means of an inoculum preserved at –70°C. J. Bacteriol. 94:832-835.
Jagannath, C., J. K. Actor, and R. L. Hunter, Jr. 1998. Induction of nitric oxide in human monocytes and monocyte cell lines by Mycobacterium tuberculosis. Nitric Oxide 2:174-176.
Joiner, M. C. 1994. Induced radioresistance: an overview and historical perspective. Int. J. Radiat. Biol. 65:79-84.
Luckey, T. D. 1982. Physiological benefits from low-levels of ionizing radiation. Health Phys. 43:771-789.
O'Brien, L., J. Carmichael, D. B. Lowrie, and P. W. Andrew. 1994. Strains of Mycobacterium tuberculosis differ in susceptibility to reactive nitrogen intermediates in vitro. Infect. Immun. 62:5187-5190.
Rhoades, E. E., and I. M. Orme. 1997. Susceptibility of a panel of virulent strains of Mycobacterium tuberculosis to reactive nitrogen intermediates. Infect. Immun. 65:1189-1195.
Stuehr, D., and C. F. Nathan. 1989. Nitric oxide. A macrophage product responsible for the cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med. 169:1543-1555.
Valway, S. E., M. P. C. Sanchez, T. F. Shinnick, I. Orme, T. Agerton, D. Hoy, J. S. Jones, H. Westmoreland, and I. M. Onorato. 1998. An outbreak involving extensive transmission of a virulent strain of Mycobacterium tuberculosis. N. Engl. J. Med. 338:633-639.
Yu, K., C. Mitchell, Y. Xing, R. S. Magliozzo, B. R. Bloom, and J. Chan. 1999. Toxicity of nitrogen oxides and related oxidants on mycobacteria: M. tuberculosis is resistant to peroxynitrite anion. Tuber. Lung Dis. 79:191-198.(William Benjamin Brugmann)