Reactive Nitrogen Intermediates Have a Bacteriostatic Effect on Mycobacterium tuberculosis In Vitro

Susceptibility of six isolates of Mycobacterium tuberculosis(CB3.3, CDC1551, RJ2E, C.C.13, H37Rv, and H37Ra) and two isolatesof Mycobacterium bovis (Ravenel and BCG) to reactive oxygenintermediates (ROI) and reactive nitrogen intermediates (RNI)was determined by standard in vitro survival assays. After 21days of incubation, the survival of most strains exposed toeither acidified sodium nitrite (ASN) or hydrogen peroxide (H₂O₂)was significantly lower than the same strains unexposed to theseRNI or ROI products. However, after 50 days of incubation, thesedifferences in susceptibility became less apparent for strainsexposed to ASN but not for strains exposed to H₂O₂. The recoveryof these strains after exposure to RNI suggests that the effectof RNI on M. tuberculosis is bacteriostatic. The in vitro concentrationsof ROI and RNI used in these assays were higher than those expectedin vivo. These observations suggest that, in vivo, RNI expressionat physiologically achievable concentrations may keep M. tuberculosisfrom proliferating but that removal of RNI may allow the organismsto proliferate. Furthermore, the ability of some M. tuberculosisstrains to cause rapidly progressive disease may relate to theirintrinsic levels of RNI and ROI resistance.

INTRODUCTION

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Introduction

Although current estimates suggest that one third of the world'spopulation is infected with Mycobacterium tuberculosis, themajority of such infections remain clinically latent. Factorsthat contribute to the success of M. tuberculosis as a pathogeninclude its ability to resist the harsh environment of the hostmacrophage and to persist within immunocompetent hosts evenafter a clinical cure of the disease is achieved.

The ability of M. tuberculosis to cause disease in the humanor animal host may vary according to host susceptibility andto intrinsic biologic differences among clinical strains. Thelatter may reflect a particular strain's specific adaptationto the antimicrobial defenses of the host macrophage. For example,numerous studies have demonstrated that strains of Mycobacteriumdiffer in susceptibility to reactive oxygen intermediates (ROI)and reactive nitrogen intermediates (RNI) (7, 12, 16, 17, 18,20, 23, 26).

The role of ROI and RNI in controlling acute infections withM. tuberculosis has been fairly well studied in the murine system(1, 3, 4, 8, 9, 10, 19). However, little is known about therole of these intermediates during latent infections. Studieshave demonstrated that the inducible form of nitric oxide synthase(iNOS) expression is required to control mycobacterial infectionin mice (1, 9, 13, 19). However, the induction of iNOS doesnot lead to complete elimination of M. tuberculosis in vivo(22). In addition, Flynn et al. (11) demonstrated that M. tuberculosisreactivation occurs if the production of RNI is inhibited ina murine model of latency (11). This suggests that reactivationtuberculosis may result from the removal of host defense mechanisms,such as RNI, that keep M. tuberculosis from replicating in vivo.Consequently, it is possible that ROI and RNI may inhibit replication(bacteriostatic) but do not eradicate the bacteria (bactericidal).We decided to test this concept in vitro by using RNI and ROIsusceptibility assays applied to both clinical and laboratorystrains of mycobacteria.

MATERIALS AND METHODS

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Materials and Methods

The mycobacterial strains used in the present study are listedin Table 1. All strains of Mycobacterium were grown in Middlebrook7H9 broth with 0.5% glycerol, 0.02% Tween 80, and 10% ADC enrichmentto mid-log phase. Single-cell suspensions of each isolate wereprepared according to a previously published method (15) andquantified by enumeration of their CFU on Middlebrook 7H11 agarplates. A 0.1-ml aliquot of this suspension was added to 0.9ml of 7H9 broth (pH 5.3 for ASN assays or pH 7.0 for H₂O₂ assays)containing various concentrations of either sodium nitrite (NaNO₂)or hydrogen peroxide (H₂O₂). As a control, the culture was addedto 7H9 broth at the appropriate pH without NaNO₂ or H₂O₂ (unexposed).Each suspension was incubated at 37°C for 24 h, plated onto7H11 agar, and incubated at 37°C for 3 weeks. At day 21(or day 26 for H37Ra), the CFU recovered from isolates exposedto either ASN or H₂O₂ were marked, quantified, and comparedto the CFU for recovered isolates not exposed to ASN or H₂O₂;the data were then calculated as the percent survival ([numberof CFU exposed at day 21/number of CFU unexposed at day 21]x 100). Incubation at 37°C was allowed to continue to day50, at which time each plate was reexamined to enumerate additionalCFU recovered during the interval. The total number of CFU wasagain compared to the total number of CFU from recovered isolatesnot exposed to ASN or H₂O₂ at 50 days of incubation, and thedata were converted to percent survival ([number of CFU exposedat day 50/number of CFU unexposed at day 50] x 100). Each strainwas tested similarly in triplicate, and each assay was repeatedat least twice. Comparison of the mean CFU recovery of eachstrain was made by Student t test, and a P value of <0.05was considered to show a significant difference.

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TABLE 1. Mycobacterial strains used in this study

RESULTS

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Results

Each strain of mycobacteria demonstrated similar levels of growthbetween days 21 and 50 when not exposed to ASN or H₂O₂ (Fig.1A and 2A). At day 21, at a lower concentration of ASN (3 mM),the percent survival of all strains of mycobacteria was $<=$ 80%compared to the same strains not exposed to ASN. At day 50,however, the percent survival of all of the clinical strainsexceeded 80% (Fig. 1A). Compared to the laboratory strain H37Ra,at 3 mM ASN all of the clinical strains of M. tuberculosis andM. bovis (Ravenel and BCG) were significantly more resistantat day 21 or 50 of incubation (P < 0.05, Student t test)(Fig. 1B and Table 2). In addition, at 3 mM ASN the percentsurvival at day 21 versus that at day 50 was significantly differentfor each strain. On the other hand, at a higher concentrationof ASN (6 mM), the percent survival of the strains at days 21and 50 showed no significant difference, except for H37Ra andM. bovis Ravenel (Fig. 1B). At 6 mM ASN all of the strains,except M. bovis BCG, remained relatively resistant comparedto H37Ra (Table 2).

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FIG. 1. Mycobacterial ASN susceptibility assays. Mycobacterial isolates were incubated for 16 h in 7H9 medium (pH 5.3) and then plated onto 7H11 agar. CFU were determined at 21 and 50 days of incubation. (A) Isolates were incubated at 37°C in 7H9 broth at pH 5.3 without NaNO₂, and CFU were quantified at day 21 (shaded bars) and day 50 (hatched bars). The results are expressed as the CFU ± the standard error of the mean (SEM). (B) Isolates were exposed to 3 mM (day 21 [black diamonds] and day 50 [solid squares]) and 6 mM NaNO₂ (day 21 [gray triangles] and day 50 [gray circles]). The results are expressed as the percent survival based on the CFU recovered from ASN-exposed strains relative to the CFU of unexposed strains ± the SEM. Both graphs are representative of triplicate experiments and show mean values of triplicate cultures for each strain.

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FIG. 2. Mycobacterial H₂O₂ susceptibility assays. Mycobacterial isolates were incubated for 16 h in 7H9 medium (pH 7.0) and then plated onto 7H11 agar. CFU were determined at 21 and 50 days of incubation. (A) Isolates were incubated at 37°C in 7H9 broth at pH 7.0 without H₂O₂, and CFU were quantified at day 21 (shaded bars) and day 50 (hatched bars). The results are expressed as CFU ± the SEM. (B) Isolates were exposed to 2 mM (day 21 [black diamonds] and day 50 [black squares]) and 5 mM H₂O₂ (day 21 [gray triangles] and day 50 [gray circles]). The results are expressed as the percent survival based on CFU recovered from strains exposed to H₂O₂ relative to the CFU of unexposed strains ± the SEM. Both graphs are representative of triplicate experiments and show mean values of triplicate cultures for each strain.

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TABLE 2. Comparison to laboratory strain H37Ra of resistance levels of each strain to ASN and H₂O₂

At 2 mM H₂O₂, H37Ra and one clinical isolate (C.C.13) were mostsusceptible compared to the other strains tested, but no significantdifference was detected between the two time points of incubationfor each strain, except with H37Ra (Fig. 2B). At 5 mM H₂O₂ allof the strains except H37Rv and a clinical strain (C.C.13) remainedrelatively resistant compared to the laboratory strain H37Ra(Table 2). At 5 mM H₂O₂, no significant differences in the percentsurvival were observed for each individual strain at day 21versus day 50 except with H37Ra (Fig. 2B). Two of the clinicalisolates (CB3.3 and CDC1551) consistently exhibited the highestresistance to killing by 5 mM H₂O₂ or 6 mM ASN compared to allof the other strains tested in the present study (Fig. 1B and2B).

DISCUSSION

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Discussion

Our results demonstrate that, at a low concentration of ASN,the tested Mycobacterium strains display a greater level ofvariation in percent survival at 21 days of incubation comparedto that observed at 50 days of incubation. However, at highconcentrations of ASN, these differences in the percent survivalat both day 21 and day 50 disappeared for each strain. Significantdifferences in the percent survival of the clinical isolatesat the two time points were not observed when they were exposedto either concentration of H₂O₂. These data suggest that theRNI products, but not H₂O₂, exert a bacteriostatic effect atlow concentrations and a bactericidal effect at high concentrations.

Considering that most mycobacterial strains were able to recoverfrom exposure to lower concentrations of ASN when allowed toincubate beyond 3 weeks, it appears that the time of incubationafter which CFU data are analyzed is important when similarassays are used to determine mycobacterial susceptibility toany type of stress. Although all of the strains in the presentstudy exhibited some level of recovery to low concentrationsof H₂O₂, the differences in the percent survival between day21 and day 50 within each strain were not statistically significant,with the exception of one strain (H37Ra). One explanation forthis observation is that the concentrations of H₂O₂ we usedare bactericidal to Mycobacterium. Another possibility may bethat most strains of mycobacteria, except H37Ra, are more resistantto H₂O₂ at 2 mM, and thus they are able to grow within 3 weeks.Hence, the concentrations of H₂O₂ used in the present studymay have been either too low (2 mM) or too high (5 mM) to seesignificant differences among all of the strains when we comparedthe percent survival values at days 21 and 50. These hypothesesare in accord with other studies that have demonstrated eitherthat H₂O₂ is bactericidal in vitro or that M. tuberculosis isrelatively resistant to the effects of H₂O₂ in a cell-free system(2, 16, 21). Since H37Ra has a much slower growth rate thanthe other strains used, the significant level of recovery seenat all concentrations of ASN and H₂O₂ may be a consequence ofnot being able to visualize the colonies until much later. Wetried to minimize this effect by determining the initial CFUof H37Ra at day 26 instead of at day 21, thus allowing the organismsufficient time to grow.

Collectively, our data indicate that under the in vitro conditionsused here exposure to low concentrations of ASN resulted inthe stasis of most clinical isolates of M. tuberculosis. Incontrast to these data, a study by Chan et al. (2) found thatchemically generated RNI at concentrations of between 1.0 and10 mM are bactericidal to M. tuberculosis in vitro (2), althoughit should be noted that only one strain (Erdman) was analyzedand that the incubation period after which CFU were enumeratedwas not clearly defined. Our results are consistent with thoseof Rhoades and Orme (24), who demonstrated in vitro that theantimycobacterial activity of interferon-primed macrophageswas bacteriostatic rather than bactericidal (24). In addition,Rhoades and Orme found that a high concentration (10 mM) ofNaNO₂ was mycobactericidal in a cell-free system and that lowerconcentrations (0.1 to 5.0 mM) showed a range of tolerance byclinical mycobacterial isolates (24). It should be noted thatthe cell-free assay used in the Rhoads and Orme study, althoughsimilar, was not identical to ours. Our study exposed mycobacterialstrains for 16 h as opposed to a period of 10 days, and theCFU in the Rhoades and Orme study were determined at between3 and 4 weeks.

Despite the observed bacteriostatic effect of RNI, our resultsalso demonstrate that some clinical M. tuberculosis isolates(CDC1551 and CB3.3) are able to resist even high concentrationsof ASN or H₂O_2. These clinical isolates were previously shownto be associated with large outbreaks of tuberculosis and werefound to exhibit high levels of resistance to RNI and H₂O₂ comparedto other clinical isolates or laboratory strains of M. tuberculosis(6, 12, 25). These observations are consistent with numerousstudies that have demonstrated strain-related differences regardingsusceptibility to RNI and ROI (7, 12, 16, 17, 18, 20, 23, 24,26), and suggest that certain strains of M. tuberculosis haveevolved an enhanced level of resistance to the antibacterialmechanisms elicited by the host macrophage.

The physiologic concentrations of ROI and RNI within human macrophagesin vivo are not well established, although in vitro studieshave determined that ca. 3.2 nmol of NO_2^-/10⁵ cells and 287nmol of H₂O₂/mg of protein/h are generated from resident murinemacrophages stimulated with IFN- ${gamma}$ (5). In addition, other studieshave demonstrated that between 34 and 241 nmol of nitrite/10⁶cells in culture supernatants of human peripheral blood monocytesinfected with M. tuberculosis (17) and between 10 and 80 pmolof H₂O₂/µg of DNA from cells stimulated with polystyreneparticles (14) are generated. Hence, the amount of ROI and RNIadded in the cell-free system used in the present study is higherthan that expected to be generated in vivo. Therefore, relativelyresistant M. tuberculosis strains, such as CB3.3 and CDC1551,are less likely to be kept in check within the host. Consequently,one could postulate that such strains are more capable of causingactive disease more readily after an initial infection thanstrains that are relatively susceptible to RNI and ROI. In contrast,the proliferation of RNI- and ROI-susceptible strains may becontrolled as long as the host is able to maintain RNI and/orROI expression. In the absence of these stresses, such M. tuberculosisstrains may resume proliferation and cause the reactivationof disease.

While there may be multiple other host factors that contributeto bacteriostasis of M. tuberculosis in vivo, the observationof the bacteriostatic effect of RNI made in vitro, as well asthe wide range of RNI and/or ROI susceptibilities of the clinicalisolates, may provide new clues about the different clinicaloutcomes after M. tuberculosis infection.

ACKNOWLEDGMENTS

This study was supported by NIH grants HL51967 and F31 DA05874.

We thank Ed Desmond, Jennifer Flood, and Lisa Pascopella atthe California State Health Department and Sally Cantrell forproviding C.C.13 and Thomas Shinnick for kindly providing CDC1551.We also thank Lisa Morici for technical assistance and for criticalreview of the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: School of Public Health, Division of Infectious Diseases, 140 Warren Hall, University of California, Berkeley, Berkeley, CA 94720. Phone: (510) 642-9200. Fax: (510) 642-6350. E-mail: lwriley{at}uclink4.berkeley.edu.

REFERENCES

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