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. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: Pathog Dis. 2014 Feb 24;70(3):370–378. doi: 10.1111/2049-632X.12143

Reversal of Mycobacterium tuberculosis Phenotypic Drug Resistance by 2-Aminoimidazole Based Small Molecules

David F Ackart 1, Erick A Lindsey 2, Brendan K Podell 1, Roberta J Melander 2, Randall J Basaraba 1, Christian Melander 2,*
PMCID: PMC4367863  NIHMSID: NIHMS564252  PMID: 24478046

Abstract

The expression of phenotypic drug resistance or drug tolerance serves as a strategy for Mycobacterium tuberculosis to survive in vivo antimicrobial drug treatment; however the mechanisms are poorly understood. Progress toward a more in depth understanding of in vivo drug tolerance and the discovery of new therapeutic strategies designed specifically to treat drug-tolerant M. tuberculosis are hampered by the lack of appropriate in vitro assays. A library of 2-aminoimidazole based small molecules combined with the anti-tuberculosis drug isoniazid were screened against M. tuberculosis expressing in vitro drug-tolerance as microbial communities attached to an extracellular matrix derived from lysed leukocytes. Based on the ability of nine of ten 2-aminoimidazole compounds to inhibit M. smegmatis biofilm formation and three of ten molecules capable of dispersing established biofilms, two active candidates and one inactive control were tested against drug tolerant M. tuberculosis. The two active compounds restored isoniazid susceptibility as well as reduced the in vitro minimum inhibitory concentrations of isoniazid in a dose-dependent manner. The dispersion of drug tolerant M. tuberculosis with 2-aminoimidazole based small molecules as an adjunct to antimicrobial treatment has the potential to be an effective anti-tuberculosis treatment strategy designed specifically to eradicate drug-tolerant M. tuberculosis.

Keywords: tuberculosis, drug-tolerant, biofilm, 2-aminoimidazole, drug resistant, isoniazid

Introduction

One of the greatest challenges to controlling the spread of tuberculosis is the length of time required to effectively treat patients with active or latent Mycobacterium tuberculosis (M. tuberculosis) infections. Six to nine months of combination antimicrobial therapy is required to effectively treat patients infected with drug-susceptible M. tuberculosis (Mitchison & Davies, 2012). The treatment interval is further extended when more potent and toxic drug combinations are used to treat patients infected with drug-resistant strains (Kliiman & Altraja, 2009, Ahmad & Mokaddas, 2013). Even asymptomatic patients with latent tuberculosis that opt for treatment, require months of monotherapy with the first-line anti-tuberculosis drug isoniazid (INH) (Cruz, et al., 2013). These lengthy treatment regimes are thought necessary to ensure complete killing of persistent, drug-tolerant bacilli (Ahmad, et al., 2009). A major hurdle to developing more effective tuberculosis treatments is our poor understanding of how host and pathogen factors contribute to the expression of in vivo drug tolerance during M. tuberculosis infections.

A hallmark of tuberculosis in humans and some animals is the development of well-structured granulomatous inflammatory lung lesions in response to primary M. tuberculosis infections. In the early stages of infection, bacilli effectively evade host defenses by surviving intracellularly within macrophages and other host cells (Ehlers & Schaible, 2012, Lee, et al., 2013). Besides being an effective immune evasion strategy, intracellular bacilli are also less susceptible to antimicrobial drug therapy (Andreu, et al., 2012). Another potential explanation for poor in vivo drug treatment responses have been linked to the inability to achieve therapeutic concentrations of some drugs at the site of primary and secondary infections. Studies in humans, guinea pigs and rabbits have shown that effective drug delivery is attenuated in vivo especially in lesions with extensive cellular necrosis (Barclay, et al., 1953, Manthei, et al., 1954, Prideaux, et al., 2011). Another feature unique to necrotic granulomata that likely contributes to the expression of in vivo drug tolerance is the persistence of both intracellular and extracellular populations of bacilli (Lenaerts, et al., 2007, Basaraba, 2008, Ryan, et al., 2010). Multiple studies in guinea pigs, a model species that consistently develops necrotic lesions, support the assertion that the expression of in vivo drug tolerance is collectively due to the combined effects of multiple, heterogenous populations of bacilli that differ in drug susceptibility (Ahmad, et al., 2009, Ryan, et al., 2010, Hoff, et al., 2011, Shang, et al., 2012). Animal studies demonstrate that extracellular bacilli enmeshed in a complex extracellular matrix derived from necrotic or lysed host leukocytes represent at least one population of M. tuberculosis that survive aggressive in vivo drug treatment (Lenaerts, et al., 2007, Basaraba, 2008, Driver, et al., 2012).

There is mounting evidence that like other pathogenic bacteria, M. tuberculosis uses biofilm formation as a strategy to survive host immune pressure and antimicrobial drug therapy (Basaraba, 2008, Orme, 2014). Surface attachment, the production of a pathogen derived extracellular polymeric substance (EPS) and the formation of complex microbial communities, are all features of bacterial biofilms (Stewart & Franklin, 2008). Like known biofilm formatting bacteria, the growth of M. tuberculosis as a pellicle at the air-liquid interface of static, liquid cultures has been equated to a bacterial biofilm in which free mycolic acids contribute to the EPS (Ojha, et al., 2008, Pang, et al., 2012, Ramsugit, et al., 2013, Sambandan, et al., 2013). This model system however, fails to take into account the host contribution to the extracellular matrix, which may contribute to the establishment drug-tolerant microbial communities in vivo. To this end, our laboratory has developed an alternative model system that selects for the in vitro persistence of drug-tolerant microbial communities of virulent M. tuberculosis associated with lysed human leukocytes to more closely mimic the in vivo microenvironment (Ackart, et al., 2013). This in vitro model not only takes into account the importance of early surface attachment by bacilli, but also the host contribution to the establishment of microbial communities.

In these studies we have used this in vitro assay to determine whether 2-aminoimidazole (2-AI) based small molecules known to inhibit and disperse bacterial biofilms (Worthington, et al., 2012) are effective against drug-tolerant M. tuberculosis. We have established that the 2-AI class of anti-biofilm agents are capable of inhibiting and dispersing a wide variety of Gram-positive, Gram-negative bacterial and fungal biofilms as well as biofilms composed of a mixture of microbial pathogens (Ballard, et al., 2008, Richards, et al., 2008, Rogers & Melander, 2008, Rogers, et al., 2010). Our approach to the development of anti-biofilm compounds is underpinned by the identification of small molecules that inhibit biofilm formation as well as disperse established or mature biofilms through non-microbicidal mechanisms. The rationale is that these small molecules could be employed as “adjuvants” to potentiate the activity of existing and future antimicrobial drugs. This strategy has the potential to not only render current drugs more effective against drug-tolerant bacilli, but also fill the unmet need to discover and bring to market new classes of antimicrobial compounds.

Based upon the biological response that drives the broad-spectrum anti-biofilm activity exhibited by 2-AIs, we posited that a related organism, M. smegmatis, could serve as a surrogate in preliminary screens to identify small molecules that could then be advanced to assays using virulent M. tuberculosis. Herein, we report the first studies that utilize specific 2-AI based small molecules capable of inhibiting and dispersing M. smegmatis biofilms that also effectively restore drug susceptibility to attached microbial communities of M. tuberculosis expressing INH tolerance.

Methods

Compounds 1-10 were prepared synthetically and have been previously reported (1 and 2) (Ballard, et al., 2008), 3 (Worthington, et al., 2012), 4 (Rogers, et al., 2010), 5 and 6 (Harris, et al., 2012), 7 (Huigens, et al., 2010), 8 (Lindsey, et al., 2012), 9 and 10 (Lindsey, et al.). Compounds were dissolved in biological grade DMSO as 100 mM or 10 mM stock solutions.

Inhibition of M. smegmatis biofilm formation by 2-AI based small molecules

From an in-house library, we chose representative 2-AIs, as well as 2-aminobenzimidazoles (2-ABIs), and 2-aminopyrimidines (2-AP) (which have been designed as structural 2-AI mimics, Figure 1) to screen for activity against M. smegmatis. M. smegmatis (ATCC # 700084) was cultured in Middlebrook 7H9 media supplemented with ADC and allowed to grow at 37 °C for 48 h. Subcultures of M. smegmatis (OD600 of 0.01) were resuspended in Difco M9 minimal salts media and added to each well of a 96 well plate (100 μL) in the absence or presence of test compounds. Plates were sealed and incubated under stationary conditions for 48 h at 37 °C. At the end of the incubation period, media and planktonic bacteria were removed, and 110 μL of a 0.1% aqueous solution of crystal violet (CV) was added and then incubated an additional 30 min at room temperature. Plates were again washed and the remaining stain was solubilized with 95% ethanol (200 μL). A 125 μL sample of solubilized CV from each well was transferred to a second 96 well plate and the CV concentration quantified spectrophotometrically (A540). Dose response curves were then constructed and an IC50 value calculated for each compound. The IC50 is defined here as the concentration of compound required to inhibit M. smegmatis biofilm formation by 50% compared to an untreated control.

Figure 1.

Figure 1

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Representative examples of known biofilm inhibitors: 2-aminoimidazoles (2-AIs) 1-6, 2-aminobenzimidazoles (2-ABIs) 7 and 8, and 2-aminopyrimidines (2-APs) 9 and 10.

Dispersion of established M. smegmatis biofilms by 2-AI based small molecules

M. smegmatis was cultured in Middlebrook 7H9 media as described above and allowed to grow at 37°C for 48 hours. M. smegmatis was again seeded into 96 well plates in M9 minimal salts media (OD600 of 0.01) and biofilms were allowed to form for 48 h. The planktonic bacilli were then removed and the wells were washed leaving only surfaced attached bacilli. Each well was then treated either with media alone or media containing serial dilutions of compounds 1-10. After an additional 24 h, all wells were then washed, stained with CV, solubilized with 95% ethanol and the CV quantified spectrophotometrically (A540). Dose response curves were then constructed as above, and an EC50 value caluclated for each compound. The EC50 was definded as the concentration of compound required to disperse a an established biofilm by 50% as compared to a media only treated control.

Establishment of microbial communities of drug-tolerant M. tuberculosis

The H37Rv strain of M. tuberculosis was propagated in Proskauer and Beck liquid broth supplemented with 0.05% Tween-80 (Sigma, St. Louis, MO) at 37°C with shaking. Cultures were grown to an OD600 of 0.6–0.9, aliquoted and frozen at -80 °C. Bacilli from stock cultures were centrifuged and resuspended to a concentration of 1.5 × 107 CFU/mL in RPMI 1640 with L-glutamine, phenol red (Life Technologies, Carlsbad, CA) and 2% heat inactivated bovine platelet poor plasma (PPP, Bioreclamation, Liverpool, NY) referred from here on as complete RPMI-1640. Bacilli (7.5 × 106 bacilli/mL) were maintained in 96-well flat bottom plates (Becton Dickinson, Franklin Lakes, NJ) or Lab-Tek II Chamber Slides (Nunc, Rochester, NY) planktonically in complete RPMI-1640 supplemented with Tween-80 (final concentration of 0.05%) or under abiotic conditions in the absence of Tween-80. M. tuberculosis was also maintained under biotic conditions cultured in complete RPMI-1640 in the presence of lysed human leukocytes. Peripheral blood leukocytes enriched for neutrophils, were isolated from freshly collected whole blood from healthy volunteers by the plasma Percoll method. Isolated human peripheral blood leukocytes enriched for neutrophils were washed and resuspended in RPMI-1640 with 2% PPP (complete RPMI-1640) and seeded in each well of a 96-well plate at a concentration of 7.5 × 106 cells/mL. Cells were lysed by freezing and stored at -80 °C. Plates containing M. tuberculosis maintained planktonically, or as attached microbial communities in the absence of Tween-80 and leukocytes (abiotic) or attached to the extracellular matrix derived from lysed leukocytes (biotic) were incubated for seven days at 37 °C in a humidified incubator without supplemental carbon dioxide.

Combination 2-AI and antimicrobial drug treatment of drug-tolerant M. tuberculosis

Each of the three separate culture conditions were established for seven days then treated for an additional seven days with isoniazid (INH, 10μg/mL, Sigma, St. Louis, MO) dissolved in water, in the presence or absence of serial dilutions of a 250 μM stock solution of 2-AI compounds 1, 2 and 3. To determine the bactericidal capacity of combination drug therapy, at 24 h intervals, attached microbial M. tuberculosis communities were scraped from plate surfaces, dispersed into a single cell suspension mechanically through a 26 gauge hypodermic needle and plated on 7H11 agar and colony forming units (CFUs) counted. Data is expressed as log10 percent survival of the original inoculum on day seven of culture when drug treatment was initiated.

Minimum inhibitory concentration (MIC) of INH as a function of compound concentration

The MIC of INH was determined in the different culture conditions in the presence or absence of 2-AI based compounds using alamar blue (Life Technologies) as previously described (Franzblau, et al., 1998). On day five, 50 μL of a solution of a 5× alamar blue and 5% Tween–80 solution was added to wells and incubated at 37 °C. After 48 hours, the MIC was calculated based on the lowest drug concentration that does not result in alamar blue reduction as indicated by color change.

Laser Scanning Confocal Microscopy

In separate cultures prepared for fluorescence confocal microscopy, M. tuberculosis cultures were grown on Lab-Tek II chamber slides for seven days, treated an additional seven days with each of the 2-AI based small molecules (250 μM), then media was removed and attached microbial communities allowed to dry prior to fixation with 4% paraformaldehyde for 30 min. Slides were blocked with a serum free blocking solution (Dako) and then stained with Rhodamine B (Sigma) solution and decolorized with acid-alcohol. Slides were counterstained for nucleic acids with TO-PRO-3 (Life Technologies, Carlsbad, CA) and mounted with Prolong Gold (Life Technologies, Carlsbad, CA). Confocal stacks were captured with a Zeiss Laser Scanning Axiovert Confocal Microscope and analyzed with Volocity 6.3.0 software.

Results

Inhibition of M. smegmatis biofilm formation

The ability of 2-AI, 2-ABI and 2-AP based small molecules (Figure 1) to inhibit in vitro biofilm formation by M. smegmatis is shown in Table 1. With the exception of 2-AP 10, all compounds inhibited M. smegmatis biofilm formation, with 2-AP 9 being the most active, returning an IC50 value of 4.1 μM. Despite the structural differences, the majority of the compounds displayed potent biofilm inhibitory activity with seven of the ten having IC50 values in the low micromolar range.

Table 1.

IC50 and EC50 values for inhibition and dispersal of pre-formed of M. smegmatis biofilms respectively by compounds 1-10 (values in μM). Note: Highest concentration tested = 200 μM.

Compound IC50 EC50 Compound IC50 EC50
1 20.0 >200 6 5.3 >200
2 5.3 52.8 7 8.1 >200
3 8.0 >200* 8 7.1 >200
4 22.6 74.9 9 4.1 >200
5 4.5 180.5 10 >200 >200
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*

Compound 3 dispersed ca. 40% of a pre-formed biofilm at 50 μM; however this effect was not dose-responsive.

Dispersion of pre-formed biofilms by M. smegmatis

The ability of our compounds to disperse established M. smegmatis biofilm is shown in Table 1. In contrast to the M. smegmatis biofilm inhibitory activity, only three compounds were able to disperse pre-formed M. smegmatis biofilms in a dose-dependent fashion. Compounds 2, 4, and 5, were active with EC50 values of 53, 75, and 180 μM respectively. Compound 3 dispersed M. smegmatis biofilms; however activity plateaued at 40% dispersion at a concentration of 50 μM. Based upon the ability of selected compounds to both inhibit and disperse M. smegmatis biofilms, we chose 2-AIs 1, 2, and 3 to advance to studies with M. tuberculosis and did not pursue any 2-ABIs or 2-AP derivatives further.

Combination treatment of drug-tolerant M. tuberculosis

The ability of 2-AI based compounds to potentiate the antimicrobial activity of INH against drug-tolerant M. tuberculosis is shown in Figure 2. Based on the ability of 2-AI compounds to inhibit and disperse M. smegmatis biofilms, two compounds were chosen to test as adjunct therapy with INH in the in vitro model of M. tuberculosis drug tolerance. Based on the M smegmatis data, compounds 2 and 3 showed potent biofilm inhibitory activity (IC50 = 5.3 μM and 8.0 μM respectively) as well as the ability to disperse pre-formed biofilms. Compound 1 showed potent inhibitory activity, but did not have the ability to disperse pre-formed biofilms and served as our inactive control.

Figure 2. Combined treatment with 2-AI based small molecules potentiates the bactericidal activity of isoniazid (INH) against drug-tolerant M. tuberculosis.

Figure 2

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The H37Rv strain of M. tuberculosis was maintained (A) planktonically in the presence of Tween-80, (B) under abiotic conditions with microbial communities of bacilli attached to untreated wells or (C) under biotic conditions with microbial communities attached to human leukocyte lysate. (A) Bacilli maintained planktonically remained susceptible to INH (10 μg/mL) alone (▲), which was not significantly different when combined with compound 2 (▼), or compound 3 (♦). Compound 2 (●), compound 3 (□) or the INH carrier water (■) alone had no bactericidal activity. (B) Bacilli maintained under abiotic conditions expressed increased drug tolerance to INH (10 μg/mL) alone (filled triangles), but no significant increase in bactericidal activity of INH was seen when combined with compound 2 (solid inverted triangles), or compound 3 (solid diamonds). Similar to bacilli maintained planktonically, compound 2 (solid circles), 3 (open squares) or the INH carrier water (solid squares) alone had no bactericidal activity. (C) Bacilli maintained under biotic conditions with microbial communities attached to lysed human leukocytes also expressed increased drug tolerance to INH (10 μg/mL) alone (filled triangles), but combined treatment with compound 2 (solid inverted triangles), or compound 3 (solid diamonds) further potentiated the bactericidal activity of INH with an additional one log reduction in CFUs. Similar to bacilli maintained planktonically and abiotically, compound 2 (solid circles), compound 3 (open squares) or the INH carrier water (solid squares) alone had no bactericidal activity. Data is expressed as the mean CFU ± standard error of the mean from three separate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, p < 0.0001 when INH is compared to INH/Compound 2. †† p < 0.01, ††† p < 0.001, ††††p < 0.0001 when INH is compared to INH/Compound 3.

M. tuberculosis maintained planktonically in media supplemented with Tween-80 was highly susceptible to INH even though bacilli failed to reach log phase growth. The 2-AI compounds had no direct bactericidal activity (Figure 2), as evidenced by the fact that there was no significant difference in CFUs between 2-AI treated cultures and those treated with equal volumes of water, which served as the INH carrier control. However, INH treatment either alone or in combination with compounds 2 and 3, resulted in over a three log10 reduction in CFUs with no significant differences between treatment groups. In contrast, M. tuberculosis cultured under abiotic conditions in the absence of Tween and leukocyte lysate, expressed tolerance to INH with only a one log10 reduction in CFUs. Similar to planktonic cultures, there was no significant difference in the rate of killing by INH when combined with 2-AI compounds when M. tuberculosis was maintained under abiotic culture conditions. Also similar to the planktonic cultures, the 2-AI compounds had no direct bactericidal activity. Parallel to the abiotic cultures, M. tuberculosis maintained under biotic conditions in the presence of lysed human leukocytes also expressed drug tolerance, with a 1 log10 reduction in CFUs in response to INH alone. However, the combination of 2-AI compounds 2 or 3 and INH resulted in an additional 1 log10 reduction in CFUs, while compound 1 had no effect. There were statistically difference between the rate and degree of potentiation of INH bactericidal activity of compounds 2 and 3. As described previously, none of the compounds exhibited bactericidal activity when dosed alone. When M. tuberculosis was maintained as microbial communities in the presence of lysed leukocytes, dispersion of bacilli with 2-AI compounds alone was reflected by an increase in CFUs from approximately 30% for compound 2 and 17% for compound 3 (Figure 2).

MIC of INH as a function of compound concentration

The minimum inhibitory concentration (MIC) of INH against M. tuberculosis maintained under different culture conditions as a function of compound concentration is illustrated in Figure 3. In the absence of compound, 0.625 – 2.5 μg/mL INH was required to inhibit M. tuberculosis growth in the presence of lysed leukocytes, a ≥16-fold increase in concentration in comparison to bacilli cultured planktonically. At all concentrations of compound 1 (15.6 μM – 250 μM), the amount of INH required to reduce bacilli viability remained constant at 0.625 – 2.5 μg/mL. In the presence of either compound 2 or compound 3, there was a dose-dependent effect on INH bactericidal activity, such that at 250 μM and 125 μM, the MIC of INH was 0.039 μg/mL. Neither compound had any effect at 15.6 μM, while at 31.2 μM and 62.5 μM, both compounds had an intermediate effect.

Figure 3. Combined treatment with 2-AI based small molecules decreases the minimum inhibitory concentration (MIC) of isoniazid (INH) against drug-tolerant M. tuberculosis.

Figure 3

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The mean and range of the MIC of INH against the H37Rv strain of M. tuberculosis was determined in vitro on bacilli maintained (A-C) planktonically in the presence of Tween-80, (D-F) under abiotic conditions with microbial communities of bacilli attached to untreated wells, or (G-I) under biotic conditions with microbial communities attached to human leukocyte lysate. The MIC of INH against drug susceptible M. tuberculosis maintained planktonically was not influenced by the addition of compound 2 (A), compound 3 (B) or the negative control compound 1 (C) or when maintained under abiotic conditions in the presence of compound 2 (D), compound 3 (E) or the negative control compound 1 (F). However, the standardized MIC of INH decreased significantly against drug-tolerant bacilli maintained under biotic conditions in a dose-dependent manner when combined with compound 2 (G), or compound 3 (H), but was unchanged in the presence of the negative control compound (I). The mean and range of the standardized MIC was calculated from three separate experiments.

Dispersion of attached microbial communities of M. tuberculosis by 2-AI compounds

The phenotypic changes of attached communities of M. tuberculosis in response to 2-AI treatment are illustrated in Figure 4. Compounds 1, 2 and 3 had no effect on bacterial colony morphology when M. tuberculosis was maintained planktonically in the presence of Tween (Figure 4 A-C) or in abiotic cultures in the absence of Tween (Figure 4 D-F). Compounds 2 and 3 however, showed almost complete dispersion of M. tuberculosis maintained in biotic cultures in the presence of leukocyte lysate. Treatment of the attached communities of M. tuberculosis with compound 3, but not compound 2, also led to nearly complete digestion of the extracellular DNA component of the lysate. Treatment of lysate with compound 3 alone had no effect on the extracellular matrix, suggesting that the digestion of the host-derived matrix in the presence of compound 3 is occurring through a bacilli mediated pathway. The differential effects that 3 and 2 induce upon the cellular lysate in the presence of M. tuberculosis (but not the absence) indicate that these compounds are potentially affecting differential pathways to release M. tuberculosis from the biofilm-like state.

Figure 4. Treatment with 2-AI compounds alters colony morphology and significantly reduced persistent, drug tolerant bacilli maintained as attached microbial communities.

Figure 4

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The H37Rv strain of M. tuberculosis maintained (A-C) planktonically in the presence of Tween-80, (D-F) under abiotic conditions with microbial communities of bacilli attached to untreated wells or (G-I) under biotic conditions with microbial communities attached to human leukocyte lysate were treated for seven days with 2-AI compounds, paraformaldehyde fixed and stained with rhodamine (red) and the DNA stain TO-PRO-3 and viewed by fluorescence confocal microscopy. M. tuberculosis maintained planktonically with INH combined with (A) 2-AI compound 2, (B) compound 3 or the negative control compound 1 (C) had no significant effect on the dispersed aggregates of rhodamine positive and negative (green) bacilli. Predominately rhodamine positive bacilli maintained under abiotic conditions treated with (D) 2-AI compound 2, (E) compound 3 or the negative control (F) compound 1, persisted as attached microbial communities that were largely unaffected. Treatment of drug-tolerant bacilli attached to lysed human leukocytes with INH combined with (G) 2-AI compound 2 and (H) compound 3, significantly reduced the bacterial load compared to the negative control compound 1 (I). Extracellular DNA derived from lysed leukocytes in cultures treated with compound2 persisted but was significantly depleted when treated with compound 2 (H). Images are representative of two separate experiments.

Discussion

The overall goals of this study were to determine whether 2-AI based small molecules are effective at restoring antimicrobial drug susceptibility to M. tuberculosis expressing in vitro drug tolerance. The key finding is that compounds identified by the ability to disperse in vitro M. smegmatis biofilms, were also effective at restoring in vitro susceptibility of M. tuberculosis expressing phenotypic resistance to the first-line anti-tuberculosis drug INH. Moreover, we show that M. smegmatis can be used in initial screening assays to identify compounds that are active against virulent M. tuberculosis, even among compounds that appear to have different biological activities. Lastly, these data demonstrate the value of an in vitro assay of M. tuberculosis drug tolerance with modifications, has potential as a high throughput platform to screen new drug candidates or novel therapeutic strategies designed specifically to treat drug-tolerant M. tuberculosis.

From an in-house library of approximately 700 2-AI derivatives, ten representative compounds were tested for the ability to inhibit or disperse M. smegmatis biofilms in vitro. All but one of the compounds tested initially demonstrated the ability to inhibit in vitro biofilm formation albeit with a range of activity. Three of these compounds demonstrated relevant biological activity by dispersing established M. smegmatis biofilms. It is not surprising that fewer compounds were active under the more stringent assay conditions, given that mature biofilms are orders of magnitude more complex and present a more significant therapeutic challenge. Compounds 2 and 3 demonstrated both biofilm inhibition and dispersing activity against M. smegmatis, whereas compound 1 only exhibited inhibitory activity. The different biological activities of the various compounds against M. smegmatis were then tested against drug-tolerant M. tuberculosis. As was anticipated, compounds 2 and 3 were effective at potentiating the bactericidal activity and reducing the MIC of INH against drug-tolerant M. tuberculosis. Despite the differences in biological activity, compound 3 failed to completely disperse M. smegmatis, however both compounds 2 and 3 were equally effective against drug tolerant M. tuberculosis. Despite having M. smegmatis biofilm inhibitory activity, compound 1 had no dispersing activity which was also reflected in the M. tuberculosis bactericidal and MIC assay. Therein, compound 1 was viewed as a structurally related negative control. These data illustrate that while the M. smegmatis assay has predictive value in the early stages of 2-AI screening, it is necessary to conduct secondary screening against M. tuberculosis maintained under more complex culture conditions as is represented here by the extracellular matrix derived from lysed human leukocytes.

Previous in vitro and in vivo studies from our laboratory have demonstrated that 2-AI based compounds alone have no direct bactericidal activity at the concentrations at which they exhibit biofilm inhibitory and dispersing activities, which was further confirmed in these studies (Ballard, et al., 2008, Rogers, et al., 2010). One of the most significant findings of this study is that compounds 2 and 3 showed significantly more biological activity against M. tuberculosis maintained as attached microbial communities in the presence of human leukocyte lysate. Compared to abiotic cultures, compounds 2 and 3 significantly decreased the MIC of INH in biotic cultures in a dose-dependent fashion. Moreover, only in the biotic cultures did both compounds potentiate the bactericidal activity of INH against M. tuberculosis. It is presumed that the attachment and formation of complex microbial communities of bacilli in the absence of leukocyte lysate more closely equates to growth and maintenance of M. tuberculosis as a pellicle in liquid media as described in previous studies (Ojha, et al., 2008, Pang, et al., 2012). The significance of these data is that biological activity of the 2-AI compounds were demonstrated only in the more complex and stringent assay system that takes into account the host contribution to the expression of drug-tolerance by M. tuberculosis. The possibility exists that screening compounds under these in vitro conditions will more accurately predict in vivo biological activity, which is currently being tested in our laboratory. Recently, M. tuberculosis was shown to express in vitro drug tolerance when cultured in the presence of viable human peripheral blood mononuclear cells, which also supports the importance of the host contribution to the in vitro expression of drug tolerance (Kapoor, et al., 2013).

Confocal microscopic images of treated bacilli also suggest that compounds 2 and 3 have different biological activity. Compound 2 was effective at dispersing established communities of M. tuberculosis associated with leukocyte lysate but appeared to have minimal effect on the host-derived extracellular matrix. The ability of 2-AI compounds to disperse M. tuberculosis was also reflected by an increase in CFUs when used alone compared to the INH carrier controls. In contrast, besides dispersing bacilli, compound 3 induced the dissolution of the extracellular matrix as determined by the loss of host-derived extracellular DNA in the presence of the bacilli, but not the absence. Currently, studies are being conducted to determine the molecular and biochemical pathways of drug-tolerant M. tuberculosis that are targeted by the compounds 2 and 3. Preliminary mechanisms of action studies on other bacterial pathogens have indicated that effective 2-AI compounds bind and interact with bacterial response regulators, (Thompson, et al., 2012) which along with membrane-bound histidine kinase sensors comprise bacterial two-component regulatory systems that allow bacteria to sense and respond to environmental cues (Worthington, 2013). The possibility exists that combining 2-AI small molecules that are effective against drug-tolerant M. tuberculosis through different molecular pathways, will further improve the biological activity as an adjunct to conventional antimicrobial therapy in vitro and in vivo.

In conclusion, these studies demonstrate that like other pathogenic bacteria, M. tuberculosis expresses in vitro drug-tolerance by forming biofilm-like microbial communities and are susceptible to the activity of 2-AI based anti-biofilm agents. Similar to our previous studies, the potential therapeutic value of these compounds is not based on direct bactericidal or bacteriostatic activity, but on the ability to disperse attached communities, thus rendering bacilli again susceptible to antimicrobial drugs. This approach is attractive in that these small molecules, which do not target bacterial growth or metabolic pathways directly, are less likely to result in the emergence of antimicrobial drug resistance. The combined use of the in vitro assays described in these studies have the potential to identify additional compounds that can be used alone or in combination with existing or future antimicrobial drugs to improve tuberculosis treatment by shortening the treatment interval and possibly drug concentrations required to effectively treat patients with both active and latent tuberculosis.

Acknowledgments

The authors would like to thank Dr. Mercedes Gonzalez-Juarrero for technical assistance in acquiring confocal microscopic images and other members of the Mycobacterium Research Laboratories for valuable discussions and manuscript critiques. This research is supported by NIH grant 1R01AI106733 awarded to RJB and CM.

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