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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):359–369. doi: 10.1111/2049-632X.12144

Expression of Antimicrobial Drug Tolerance by Attached Communities of Mycobacterium tuberculosis

David F Ackart 1, Laurel Hascall-Dove 1, Silvia M Caceres 2, Natalie M Kirk 1, Brendan K Podell 1, Christian Melander 3, Ian M Orme 1, Jeff G Leid 4, Jerry A Nick 2, Randall J Basaraba 1,*
PMCID: PMC4361083  NIHMSID: NIHMS564253  PMID: 24478060

Abstract

There is an urgent need to improve methods used to screen anti-tuberculosis drugs. An in vitro assay was developed to test drug treatment strategies that specifically target drug-tolerant Mycobacterium tuberculosis. The H37Rv strain of M. tuberculosis survived antimicrobial treatment as attached microbial communities when maintained in tissue culture media (RPMI-1640) with or without lysed human peripheral blood leukocytes. When cultured planktonically in the presence of Tween-80, bacilli failed to form microbial communities or reach logarithmic phase growth yet remained highly susceptible to antimicrobial drugs. In the absence of Tween, bacilli tolerated drug therapy by forming complex microbial communities attached to untreated well surfaces or to the extracellular matrix derived from lysed human leukocytes. Treatment of microbial communities with DNase I or Tween effectively dispersed bacilli and restored drug susceptibility. These data demonstrate that in vitro expression of drug tolerance by M. tuberculosis is linked to the establishment of attached microbial communities and that dispersion of bacilli targeting the extracellular matrix including DNA restores drug susceptibility. Modifications of this in vitro assay may prove beneficial in a high throughput platform to screen new anti-tuberculosis drugs especially those that target drug tolerant bacilli.

Keywords: Mycobacterium, tuberculosis, drug resistant, drug tolerant, biofilm, microbial communities

Introduction

Current first-line tuberculosis treatment consist of a minimum of 6-9 months of combination antimicrobial drug therapy, which is presumed necessary to completely eradicate persistent populations of drug-tolerant bacilli (Mitchison & Davies, 2012). In vivo drug tolerance is not only expressed during active disease in humans but can be modeled in a variety of laboratory animal species and in vitro. The in vitro susceptibility of Mycobacterium tuberculosis (M. tuberculosis) to antimicrobial drugs is influenced by specific nutrient depletion, decreasing environmental oxygen concentrations or a combination of nutrient depletion, low oxygen and the introduction of other stress conditions that exist during M. tuberculosis infection (Deb, et al., 2009, Patel, et al., 2011). The persistence of extracellular bacilli sequestered within necrotic lung granulomata represents at least one population of bacilli that may contribute significantly to the overall expression of phenotypic drug resistance or drug tolerance in vivo but the mechanisms are poorly understood (Ulrichs & Kaufmann, 2006, Basaraba, 2008).

M. tuberculosis is considered an obligate aerobe and the inherent slow rate of growth is further reduced when bacilli are maintained under low oxygen conditions. Using an in vitro model, Wayne et al. demonstrated that the gradual consumption of oxygen by M. tuberculosis forces bacilli into multiple transition phases of non- or slowly replicating persistence, which coincides with decreased antimicrobial drug susceptibility (Wayne & Hayes, 1996, Wayne & Sohaskey, 2001). It is the slow rate of bacterial replication that is thought to limit the effectiveness of some antimicrobial drugs especially those that target cell wall synthetic pathways (Erdemli, et al., 2012). The original Wayne model with and without modifications are still commonly used to better understand the factors that influence in vitro bacterial growth and M. tuberculosis drug susceptibility (Leistikow, et al., 2010). However, the design and complexity of these assays are not well suited for high throughput screening of anti-tuberculosis drugs or novel treatment strategies. Moreover, these assays fail to take into account host factors that likely contribute to the expression of in vivo drug tolerance in humans and animals.

The Wayne model is designed to specifically mimic the low oxygen concentration measured in lung lesions of M. tuberculosis humans and some animal models (Tsai, et al., 2006, Via, et al., 2008, Heng, et al., 2011, Harper, et al., 2012). Prolonged hypoxia of inflammatory cells that respond to lung infection likely contributes to individual cell death and lesion necrosis, which releases bacilli from infected cells into the extracellular matrix (Basaraba, 2008). A feature of necrotic lesions therefore is the coexistence of both intracellular and extracellular populations of bacilli (Lenaerts, et al., 2007, Hoff, et al., 2011). In vivo, extracellular bacilli survive entrapped in an extracellular matrix composed of macromolecules derived primarily from necrotic and lysed leukocytes, which we hypothesize contributes to the expression of antimicrobial drug tolerance (Canetti, 1955, Lenaerts, et al., 2007, Basaraba, 2008). The other consequence of necrotic lesions is blood flow is limited which not only reduces oxygen availability, but further limits the delivery of therapeutic drug concentrations directly to the site of infection (Barclay, et al., 1953, Manthei, et al., 1954, Prideaux, et al., 2011). Therefore the in vivo expression of phenotypic resistance by M. tuberculosis is multifactorial in which lesion necrosis is central to the pathogenesis (Lenaerts, et al., 2007, Ahmad, et al., 2009).

To more closely mimic the host contribution to the expression of in vivo drug tolerance, we developed a simple assay that enriches for extracellular M. tuberculosis that expresses in vitro drug tolerance in the presence of macromolecules derived from lysed human cells. This model system takes into account the possible contribution host-derived macromolecules play in the establishment of in vitro and in vivo drug tolerance. Through the use of this assay we determined that the ability of M. tuberculosis to attach to abiotic or biotic surfaces is an important determinant of the expression of drug tolerance, which we show here, can be reversed by dispersion of attached microbial communities.

Materials and Methods

Bacterial strains

The H37Rv and HN878 strains of M. tuberculosis were propagated in Proskauer and Beck liquid broth supplemented with 0.05% Tween-80 (Sigma, St. Louis, MO) at 37°C with shaking. Cultures were aliquoted and frozen at -80°C from cultures grown to an optical density 600 (OD600) of 0.6–0.9. For in vitro assays, bacilli were thawed and resuspended at 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) (complete RPMI-1640).

Human leukocyte isolation

Peripheral blood leukocytes enriched for neutrophils, were isolated from freshly collected whole blood from healthy volunteers by the plasma Percoll method as previously described (Walker, et al., 2005). These studies were conducted in accordance with the Declaration of Helsinki, approved by the Colorado State University Biosafety Committee and the National Jewish Health Institutional Review Board. Written informed consent was obtained from all clinical healthy leukocyte donors. 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. Lysis and the loss of viability were confirmed microscopically using the trypan blue dye exclusion technique.

M. tuberculosis cultures and the establishment of drug tolerance

M. tuberculosis H37Rv or HN878 (7.5 × 106 bacilli/ml) was cultured in 96-well flat bottom plates (Becton Dickinson, Franklin Lakes, NJ) or Lab-Tek II Chamber Slides (Nunc, Rochester, NY) in complete RPMI-1640 with and without Tween-80 at a final concentration of 0.05%. M. tuberculosis was also cultured in complete RPMI-1640 in the presence of lysed human leukocytes. Plates were incubated for seven days at 37°C in a humidified incubator without supplemental carbon dioxide. To determine the growth rates from seven to fourteen days in the presence or absence of drug treatment or drug carriers, attached communities of bacilli were scraped from well surfaces, dispersed mechanically into a single cell suspension and serial dilutions plated on 7H11 agar. Data is expressed as CFU/ml or log10 percent survival.

Antimicrobial drug treatment

Each of the three separate culture conditions, complete RPMI-1640 with Tween-80, complete RPMI-1640 without Tween-80 and complete RPMI-1640 with lysed leukocytes, were established for seven days then treated for an additional seven days with isoniazid (INH, 10μg/ml, Sigma, St. Louis, MO) or rifampin (RIF, 25μg/ml, Sigma, St. Louis, MO) alone or in combination with pyrazinamide (PZA, 3mg/ml Sigma, St. Louis, MO), dissolved in water (INH and PZA) or DMSO (RIF). In separate experiments, molecular grade, protease free DNase I (0.5mg/ml, Sigma, St. Louis, MO) was added on days seven and ten of culture to established drug tolerant cultures with or without INH and incubated to day fourteen. Also in a separate experiment, Tween-80 (0.05% final concentration) was added to established drug tolerant cultures of M. tuberculosis with and without INH and incubated for seven days. At twenty-four hour intervals bacilli were scraped, mechanically dispersed and plated on 7H11 agar and CFUs counted. The CFU data was expressed as percent survival of the original inoculum or on day seven of culture when drug treatment was initiated. The minimum inhibitory concentrations (MIC) of INH and RIF were determined in the different culture conditions using the alamar blue (Life Technologies, Carlsbad, CA) as previously described (Franzblau, et al., 1998). Starting on day seven, cultures were treated for five days with two-fold serial dilutions of INH or RIF. On day five, 50μl 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 defined as 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 microscopy, media was removed from M. tuberculosis cultures grown on Lab-Tek II chamber slides and allowed to dry prior to fixation with 4% paraformaldehyde for 30 minutes. Slides were blocked with a serum free blocking solution (Dako, Carpinteria, CA) and then stained with Rhodamine B (Sigma, St Louis MO) solution. After decolorization with acid-alcohol, slides were stained with biotinylated wheat germ agglutinin (Vector Laboratories, Burlingame, CA) and visualized with an avidin conjugated AMCA (Vector Laboratories, Burlingame, CA). Slides were counterstained for nucleic acids with TO-PRO-3 (Life Technologies, Carlsbad, CA) and mounted with Prolong Gold (Life Technologies, Carlsbad, CA). To assess the spatial distribution of viable and non-viable bacilli within attached communities, the LIVE/DEAD BacLight Bacterial Viability assay (Life Technologies, Carlsbad, CA) was utilized per manufactures instructions prior to paraformaldehyde fixation. Confocal stacks were captured with a Zeiss Laser Scanning Axiovert Confocal Microscope and analyzed with Nikon NIS Elements AR imaging software version 3.20.00 or Volocity 6.3.0.

Statistical Analysis

SAS 9.3 software was used for statistical analysis with an alpha equal to 0.05. CFUs were normalized to day 7 starting values and log transformed to correct for unequal variance. A three-way factor analysis of variance was used to determine differences between growth conditions. A Tukey adjusted least squares means method was employed to determine statistical differences between growth conditions for each treatment-time point combination. Differences within growth conditions were determined using a two-way ANOVA and Tukey adjusted for multiple comparisons between treatments for each time point.

Results

M. tuberculosis cultured in nutrient-rich mammalian cell culture media fails to reach log phase growth in vitro

The relative rate of growth and survival of the H37Rv strain of M. tuberculosis cultured in complete RPMI-1640 with and without Tween-80 and lysed human leukocytes is shown in FIGURE 1. Despite being maintained in nutrient rich media, bacilli not only failed to reach a logarithmic stage of growth but there was an approximate one log10 reduction in viable bacilli by day seven as determined by counting CFUs. Data were expressed as CFUs per ml (FIGURE 1A), which was used to calculate percent survival of the original inoculum (FIGURE 1B) and responses to drug treatment. There were no significant differences in the viability of bacilli between the three different culture conditions across all time points even out to fourteen days in culture. Even in media further enriched by a complex mixture of macromolecules derived from lysed human leukocytes, there was no net growth increase. However, in the presence of lysed leukocytes bacilli numbers returned to that of the original inoculum by day seven compared to bacilli grown in the presence or absence of Tween-80, but differences between treatment groups were not statistically significant. In contrast, the HN878 strain of M. tuberculosis showed evidence of slow growth by day fourteen under all three different culture conditions, yet also failed to reach log phase growth (SUPPLEMENTAL FIGURE 1).

Figure 1. M. tuberculosis cultured in nutrient rich mammalian cell culture media fails to proliferate in vitro.

Figure 1

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The H37Rv strain of M. tuberculosis was cultured in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) with and without Tween-80 and in the presence of lysed human leukocytes show no net increase in viable bacilli numbers out to seven days. Data is expressed as (A) mean log10 colony forming units (CFUs) ± standard error of the mean (SEM) or (B) mean percent survival ± SEM of CFUs cultured on 7H11 agar. n=3.

M. tuberculosis expresses differences in tolerance to INH and RIF monotherapy in vitro

The differences in susceptibility of M. tuberculosis exposed to high doses of INH (10μg/ml) and RIF (50μg/ ml) alone under different in vitro growth conditions are shown in FIGURE 2 and FIGURE 3 respectively. Despite the lack of log phase growth of bacilli out to fourteen days carrier controls treated with water for INH and DMSO for RIF, M. tuberculosis cultured in complete RPMI-1640 supplemented with Tween-80 (FIGURE 2A, 3A) were significantly more susceptible to individual drugs alone. The rapid two log10 reduction in CFUs, which occurred in the first four days of treatment, reached a plateau phase of survival by day eleven leaving approximately one percent of the original number of bacilli viable, which persisted throughout the remainder of the culture period. In contrast, the survival of M. tuberculosis cultured in the absence of Tween-80 and lysed leukocytes (FIGURE 2B, 3B) was significantly higher between days ten through fourteen (p ≤ 0.0001) compared to bacilli cultured in Tween-80 supplemented media. Tolerance to monotherapy was further enhanced when M. tuberculosis was cultured in the presence of lysed human leukocytes without Tween-80 (FIGURE 2C, 3C), which showed less than a 0.5 log10 reduction in viable bacilli out to day fourteen. The rapid ca. 2.5 log10 reduction in viable bacilli treated with RIF continued out to day thirteen but like INH treatment, there were approximately 0.3 percent of bacilli that survived drug treatment and persisted throughout the remainder of the culture period. The survival of M. tuberculosis in the presence of lysed leukocytes and treated with drugs alone were significantly higher between days ten and fourteen compared to bacilli cultured in media supplemented with Tween-80 (p ≤ 0.0001). In a separate set of experiments, Tween-80 and INH were added to drug-tolerant cultures of M. tuberculosis H37Rv that had been established for seven days. The dispersion of established communities of bacilli with Tween-80 restored drug susceptibility, which was significantly different from controls by days thirteen and fourteen both in the absence (SUPPLEMENTAL FIGURE 1A) or presence of lysed human leukocytes (SUPPLEMENTAL FIGURE 1B). Similar to H37Rv, the HN878 strain of M. tuberculosis also expressed tolerance to INH in the absence of Tween-80 and in the presence of lysed human leukocytes. Similar to the laboratory-adapted strain, HN878 was also susceptible to INH in media supplemented with Tween-80 but not in the absence of Tween-80 or the presence of leukocyte lysate (SUPPLEMENTAL FIGURE 2).

Figure 2. M. tuberculosis cultured in nutrient rich mammalian cell culture media express differences in tolerance to isoniazid (INH) in vitro.

Figure 2

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The H37Rv strain of M. tuberculosis was cultured in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%) and (C) in the presence of lysed human leukocytes was treated for seven days with INH (10μg/ml) or equal volumes of the carrier water. Data is expressed as the mean percent survival of viable colony forming units (CFUs) ±standard error of the mean (SEM) on 7H11 agar. n=6 separate experiments. ** = P≤ 0.01, *** = P≤ 0.001 compared to carrier controls.

Figure 3. M. tuberculosis cultured in nutrient rich mammalian cell culture media express differences in tolerance to rifampin (RIF) in vitro.

Figure 3

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The H37Rv strain of M. tuberculosis was cultured for seven days in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%) and (C) in the presence of lysed human leukocytes. Cultures were treated with RIF (50μg/ml) or equal volumes of the carrier dimethyl sulfoxide (DMSO, < 0.01%). Data is expressed as the mean percent survival of viable colony forming units (CFUs) ± standard error of the mean (SEM) on 7H11 agar. n=3 separate experiments. * = P≤ 0.05 ** = P≤ 0.01, *** = P≤ 0.001 compared to carrier controls.

M. tuberculosis expresses differences in tolerance to combination isoniazid, rifampin and pyrazinamide (RHZ) in vitro

The differences in susceptibility of M. tuberculosis to combination INH (10μg/ml), RIF (50μg/ ml) and PZA (50mg/ml) drug therapy under different in vitro growth conditions are shown in FIGURE 4. Similar to what was seen in response to INH and RIF monotherapy, treatment of M. tuberculosis cultured in complete RPMI-1640 supplemented with Tween-80 (FIGURE 4A) were highly susceptible to RHZ combination therapy compared to the DMSO carrier control. The rapid ca. 2.5 log10 reduction in viability continued out to day eleven but like INH and RIF monotherapy, approximately 0.1 percent of bacilli survived drug treatment and persisted throughout the culture period. Compared to bacilli cultured in media supplemented with Tween-80, the survival of M. tuberculosis cultured in the absence of Tween-80 (FIGURE 4B) and the presence of lysed leukocytes (FIGURE 4C) was significantly higher on days eight and nine (p ≤ 0.0001), ten (p ≤ 0.001), eleven and twelve (p ≤ 0.0001), thirteen (p ≤ 0.001) and fourteen (p ≤ 0.0001). Also like INH and RIF monotherapy, M. tuberculosis cultured in complete RPMI-1640 in the absence of Tween-80 was significantly higher with only one log10 net reduction in CFUs out to day fourteen. The expression of tolerance of M. tuberculosis to RHZ when cultured in the presence of lysed leukocytes was similar to that of bacilli cultured in the absence of Tween-80.

Figure 4. M. tuberculosis cultured in nutrient rich mammalian cell culture media express differences in tolerance to combination antimicrobial drug treatment in vitro.

Figure 4

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The H37Rv strain of M. tuberculosis was cultured for seven days in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%) and (C) in the presence of lysed human leukocytes. Cultures were treated for seven days with of isoniazid (INH, 10μg/ml), rifampin (50μg/ml) and pyrazinamide (PZA, 3mg/ml) combined (RHZ) or equal volumes of the carrier dimethyl sulfoxide (DMSO, < 0.01%). Data is expressed as the mean percent survival of viable colony forming units (CFUs) ± standard error of the mean (SEM) on 7H11 agar. n=2 separate experiments. * = P≤ 0.05 ** = P≤ 0.01, *** = P≤ 0.001 compared to carrier controls.

The MIC of INH and RIF against M. tuberculosis differs under different growth conditions

The differences in MIC of INH and RIF against M. tuberculosis under different in vitro growth conditions are shown in TABLE 1. In the absence of Tween-80, the MIC for INH increased up to seven-fold compared to media containing Tween-80. In the absence of Tween-80 and presence of lysed leukocytes, INH MIC increased up to sixty two-fold compared to M. tuberculosis cultured in the presence of Tween-80. For RIF, the increase in MIC was less marked and increased up to four-fold in media in the absence of Tween-80 and an eight- to fifteen-fold increase in media without Tween-80 but in the presence of lysed leukocytes.

Table 1. The minimum inhibitory concentration of INH and RIF in increased when M. tuberculosis is cultured as attached microbial communities.

In the absence of Tween-80, the MIC for INH increased one- to seven-fold compared to media containing Tween-80. In the absence of Tween-80 and presence of lysed leukocytes, the MIC of INH increased ranged from thirty one- to sixty two-fold compared to M. tuberculosis cultured in the presence of Tween-80. For RIF, the increase in MIC was less marked and ranged from a two- to four-fold increase in media in the absence of Tween-80 and an eight- to fifteen-fold increase in media without Tween-80 but in the presence of lysed leukocytes. Data is expressed as the mean range of MIC from three separate experiments.

Minimum Inhibitory Concentration (MIC)
INH (μg/mL) RIF (μg/mL)
RPMI w/ Tween-80 <0.04 0.06 – 0.12
RPMI w/o Tween-80 0.04 – 0.31 0.12 -0.23
Leukocyte Lysate 1.25 – 2.5 0.47 – 0.94
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M. tuberculosis forms attached microbial communities in the absence of Tween-80 and the presence of lysed leukocytes

The relative difference in colony morphology of attached communities of M. tuberculosis observed by fluorescence confocal microscopy is shown FIGURE 5. In the presence of Tween-80 (FIGURE 5A), bacilli were attached to tissue culture well surfaces as individual or small clusters of bacilli and failed to form complex microbial communities (maximal Z stack height 6.4μm). The majority of bacilli cultured in the presence of Tween-80 stained poorly by the fluorescent acid-fast technique (Rhodamine B) and stain only with the DNA stain TO-PRO3. Individual or small clusters of bacilli were either acid-fast positive and stain only with Rhodamine or were positive for both stains (yellow). In contrast, the vast majority of bacilli cultured in the absence of Tween-80 (FIGURE 5B) were acid-fast positive with Rhodamine and form raised complex microbial communities (maximal Z stack height 20.6μm). In addition, there were dual stained bacilli that are stratified near the attachment surface. Acid-fast bacilli cultured in the absence of Tween-80 and the presence of lysed leukocytes (FIGURE 5C) formed complex communities of M. tuberculosis (maximum Z stack height 29.8μm) attached to an extracellular matrix composed of leukocyte-derived macromolecules including extracelluar DNA (eDNA). In addition to host derived DNA and acid fast bacilli, microbial communities of M. tuberculosis in the presence of lysed leukocytes contained non-acid fast bacilli that stain with TO-PRO-3 only as well as complex carbohydrates (SUPPLEMENTAL FIGURE 3).

Figure 5. The expression of in vitro antimicrobial drug tolerance by M. tuberculosis is associated with attached communities of bacilli with different staining characteristics.

Figure 5

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The H37Rv strain of M. tuberculosis was cultured for fourteen days in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%) and (C) in the presence of lysed human leukocytes, paraformaldehyde fixed and stained with rhodamine (red) and the DNA stain TO-PRO-3 and viewed by confocal microscopy. (A) In the presence of Tween-80, bacilli stain poorly with rhodamine and are attached as individual or small clusters of bacilli (Z stack height, 6.4μm). (B) In the absence of Tween-80 and lysed leukocytes, the majority of bacilli stain with rhodamine and are firmly attached to well surfaces to form complex colonies of bacilli (Z stack height, 20.6μm). (C) In the presence of lysed human leukocytes, rhodamine positive bacilli form complex raised colonies (Z stack height, 29.8μm) attached to host-derived extracellular DNA. Grid = 45μm square.

Viable and non-viable bacilli are spatially stratified when cultured as attached communities of M. tuberculosis

The spatial distribution of viable and non-viable bacilli cultured as attached microbial communities for seven days in media supplemented with Tween-80 and in the absence of Tween-80 is shown in FIGURE 6. Out to seven days in culture, M. tuberculosis failed to form complex microbial communities when cultured in media supplemented with Tween-80 (FIGURE 6A) and viable and non-viable bacilli were admixed and evenly dispersed as individual bacilli or small clusters. In contrast, in the absence of Tween-80 (FIGURE 6B), viable bacilli aggregated to form raised, complex microbial communities, which were attached and spatially stratified from non-viable bacilli at the attachment surface of tissue culture wells. The orthogonal view demonstrates that the DNA of non-viable bacilli stained with propidium iodide were concentrated at the well surface and served as an attachment matrix for viable bacilli in the upper layers, which comprise the majority of the microbial community.

Figure 6. Viable and non-viable bacilli are spatially stratified when cultured as attached communities of M. tuberculosis.

Figure 6

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The H37Rv strain of M. tuberculosis was cultured in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%). The H37Rv strain of M. tuberculosis was cultured for seven days in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%) and stained with the live-dead (Syto 9 and propidium iodide, Bac-Light, Life Technologies) prior to fixation with paraformaldehyde and viewed by confocal microscopy.

Treatment of drug tolerant M. tuberculosis with DNase I partially restores in vitro susceptibility to INH

The influence that dispersion of attached communities of M. tuberculosis with DNase I had on the in vitro susceptibility to INH grown under different culture conditions is shown in FIGURE 7. The split treatment of M. tuberculosis cultures with DNase I on days seven and ten in combination with INH, increased the susceptibility of drug tolerant bacilli when cultured in media supplemented with Tween-80 (FIGURE 7A) resulting in an additional one log10 reduction of viability by day fourteen. The increased susceptibility to INH in the presence of DNase I was statistically significant on days thirteen and fourteen but no effect of DNase I treatment alone was seen in the presence of the water carrier control (FIGURE 7A). The killing of M. tuberculosis by INH was also potentiated by DNase I when cultured in media without Tween-80 (FIGURE 7B). Treatment with DNase I further enhanced the bactericidal capacity of INH with an additional 0.5 log10 reduction by day fourteen. Compared to INH alone treated cultures, the percent survival was significantly different from day 10 through fourteen when DNase I was combined with INH. In contrast to M. tuberculosis cultured in the presence of Tween-80 (FIGURE 7A), DNase I treatment alone also significantly reduced the survival from days twelve to fourteen compared to water treated controls. When cultured in the presence of lysed leukocytes, (FIGURE 7C) treatment with INH combined with DNase I had a transient effect, reducing survival from days eight to twelve, but there were no significant statistical differences on days thirteen and fourteen compared to cultures treated with INH alone. Similar to M. tuberculosis cultured in the presence of Tween-80, treatment of M. tuberculosis attached to lysed leukocytes with DNase I had no effect on viability compared to the water control cultures. DNase I digestion of extracellular DNA was effective as evidenced by the lack of extracellular DNA staining with the DNA stain TO-PRO3 in the different culture conditions as determined by fluorescence confocal microscopy (SUPPLEMENTAL FIGURE 4).

Figure 7. Treatment of drug tolerant M. tuberculosis with DNase I partially restores in vitro susceptibility to isoniazid.

Figure 7

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The H37Rv strain of M. tuberculosis was cultured in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%) and (C) in the presence of lysed human leukocytes. Cultures were treated for seven days with INH (10μg/ml) or equal volumes of the carrier water with or without DNase I (0.5 mg/ml) added on day seven and ten. Data is expressed as the mean percent survival of viable colony forming units (CFUs) ± standard error of the mean (SEM) on 7H11 agar. n=6 separate experiments. * = P≤ 0.05 ** = P≤ 0.01, *** = P≤ 0.001 compared to carrier controls.

Discussion

The goal of these studies was to develop a simple assay that better mimics the expression of phenotypic drug resistance or tolerance by M. tuberculosis in humans and animal models. The assay was adapted from that of studies by Walker and colleagues who demonstrated that the inclusion of human neutrophils promotes in vitro biofilm formation and the expression of antimicrobial drug tolerance by Pseudomonas aeruginosa (Walker, et al., 2005, Parks, et al., 2009). The key findings in the current study are that 1) even in nutrient-rich media, M. tuberculosis failed to reach log phase growth yet remained susceptible to mono- or combination therapy with first anti-tuberculosis drugs; 2) the formation of attached microbial communities and the expression of in vitro drug tolerance was prevented when M. tuberculosis was maintained planktonically in media containing Tween-80; 3) inclusion of a complex mixture of macromolecules derived from lysed leukocytes promotes the formation of attached communities and the expression of drug tolerance and 4) dispersion of attached communities of M. tuberculosis enzymatically with DNase or nonspecifically with Tween-80 restores in vitro drug susceptibility.

Among the most significant findings were that maintaining planktonic bacilli in media supplemented with Tween-80 prevented the attachment and establishment of drug-tolerant communities of M. tuberculosis. The use of mycobacterial media containing non-ionic detergents is widely practiced and has the desired effect of preventing the inherent tendency of mycobacteria to clump or attach to plastic or glass surfaces in vitro (Franzblau, et al., 2012). In the early development of artificial mycobacterial media bacilli it was determined that maintaining bacilli as a single cell suspension (planktonic) by either agitation or the inclusion of detergents were necessary to achieve the shortest time to logarithmic phase growth (Dubos, 1946, Dubos & Davis, 1946). Prior to the routine use of commercially available detergents, glycerinated bovine bile functioned as a surfactant to prevent clumping and to promote in vitro growth especially in the development of the Bacillus Calmette-Guérin (BCG) vaccine. However, numerous studies have demonstrated that the presence of Tween-80 alters in vitro drug susceptibility but the mechanisms are unknown (Dubos & Davis, 1946, Sattler & Youmans, 1948). The inclusion of Tween-80 has been shown to serve as an alternative carbon source to promote growth and to alter M. tuberculosis cell wall structure by stripping the outer cell wall envelope potentially influencing drug penetration (Sani, et al., 2010, Stoops, et al., 2010). Conflicting studies have demonstrated that the inclusion of Tween limits the accumulation of some drugs in M. tuberculosis (Piddock, et al., 2000, Sarathy, et al., 2013). The in vivo relevance of the effect of surfactants on M. tuberculosis drug tolerance is supported by the observations that poloxamer surfactants potentiate the in vivo antimicrobial activity against M. tuberculosis in vivo (Stoops, et al., 2010).

Results of the current studies suggest that Tween-80 prevents bacilli attachment and the establishment of mature microbial communities of drug-tolerant bacilli. Attachment to abiotic or biotic surfaces, establishment of complex microbial communities, production of an extracellular polymeric substance (EPS) and the expression of drug tolerance are all features of bacterial biofilms, which remains controversial in regards to M. tuberculosis (Ojha, et al., 2008, Pang, et al., 2012, Sambandan, et al., 2013). Attachment is an early and necessary step in the establishment of bacterial biofilms, which has been suggested recently to be due to expression of pili by pellicle grown M. tuberculosis (Ramsugit, et al., 2013). The growth of M. tuberculosis as a pellicle results in the expression of drug tolerance, which has been attributed to an EPS composed of free mycolic acids and phenolic glycolipids (Ojha, et al., 2008, Pang, et al., 2012, Sambandan, et al., 2013).

Similar to our data, previous studies have demonstrated that M. tuberculosis fails to replicate in nutrient rich RPMI-1640 even in the presence of macrophage lysate (Armitige, et al., 2000). In contrast, another study proposed the use the mammalian cell culture media (F12) to enhance M. tuberculosis growth as an improved method over traditional mycobacterial growth media to rapidly screen antimicrobial drugs in vitro (Nozawa & Yokota, 1983). The conflicting data between these studies is likely due to differences in the basic media formulations or related to the media supplements which included five percent heat inactivated fetal bovine serum and 0.2% Tween-80 in the former compared to two percent bovine plasma and 0.05% Tween-80 in the current study. Others have also shown that RPMI-1640 supplemented with ten percent fetal bovine serum also failed to support M. tuberculosis growth in vitro (Zhang, et al., 1998).

Our data show that similar to traditional mycobacterial growth media small populations of drug-tolerant bacilli survive in vitro antimicrobial drug treatment even despite the presence of Tween-80 (Gruppo, et al., 2006). By eleven days in culture, one percent of the original population express in vitro tolerance to INH, 0.3 percent for RIF monotherapy and 0.1 percent persisted throughout the remainder of the culture period when combined with PZA. The ability of M. tuberculosis to establish attached microbial communities even in Tween containing media may be related to the production and secretion of cutinase-like phospholipases that deplete Tween in late stage cultures (Parker, et al., 2007). This fact would also explain the ability of M. tuberculosis to eventually express drug-tolerance in the current studies and to ultimately form a biofilm-like pellicle in media containing Tween-80 (Ojha, et al., 2008). This possibility is further supported by our confocal data showing that attached but immature communities of M. tuberculosis are present by day fourteen in culture even in media supplemented with Tween-80.

Fluorescent confocal microscopic images of M. tuberculosis maintained as abiotic cultures showed that after seven days, viable and non-viable bacilli were spatially stratified within attached microbial communities. Non-viable bacilli or M. tuberculosis extracellular DNA (eDNA) stained with propidium iodide served as an attachment matrix for viable bacilli. The presence of eDNA derived from non-viable bacilli was further supported by the susceptibility to dispersion with DNase I, which partially restored drug susceptibility. The detection of M. tuberculosis DNA or DNA fragments from human tuberculosis patients especially in culture negative samples is presumed to originate from non-viable bacilli however the possibility of secreted DNA cannot be discounted (van Staden, et al., 1998, Fenhalls, et al., 2002, Green, et al., 2009). Pathogen derived eDNA secreted by some pathogenic and non-pathogenic bacteria is known to function as an attachment matrix in the early stages of in vitro biofilm formation (Whitchurch, et al., 2002). Host derived eDNA especially is abundant in vivo associated with necrotic tuberculosis granulomata (Lenaerts, et al., 2007, Basaraba, 2008, Ordway, et al., 2010, Hoff, et al., 2011).

The combined treatment of drug tolerant M. tuberculosis with DNase I partially restored susceptibility to INH under all culture conditions. Of interest and unexpected was that DNase I alone was as effective as INH alone in M. tuberculosis grown under abiotic conditions in the absence of Tween-80. This response is not likely due to a direct toxic effect of DNase, since DNase I alone had no effect on the viability of bacilli maintained under planktonic conditions. Because the attachment and formation of microbial communities provides a significant survival advantage, we interpreted the loss of M. tuberculosis in the presence of DNase and absence of drugs as a loss of in vitro fitness of bacilli detached by enzymatic activity (Ramasubbu, et al., 2005, Tetz, et al., 2008, Kaplan, 2009).

In the presence of lysed leukocytes, the increased susceptibility to INH conferred by DNase I was transient suggesting that host or pathogen derived macromolecules other than eDNA support drug tolerant communities of M. tuberculosis. Despite the short half-life, multiple of DNase treatments was effective at digesting host- and pathogen-derived eDNA as evidenced by the lack of DNA staining viewed by confocal microscopy. The potential involvement of host or bacilli derived macromolecules other than eDNA in the establishment of drug tolerant communities of M. tuberculosis is supported by the pellicle growth model system which demonstrates the presence of bacilli-derived free mycolic acids (Ojha, et al., 2008, Sambandan, et al., 2013) and by our data showing the presence of complex carbohydrates associated with drug-tolerant microbial communities of M. tuberculosis.

It is unclear whether M. tuberculosis like other pathogenic bacteria actively secretes eDNA (Yang, et al., 2007, Mann, et al., 2009) however these studies suggest that DNA derived from non-viable bacilli or from the host may function to promote the establishment of drug-tolerant microbial communities in vitro and in vivo (Wilson & Schwabacher, 1935). Our previous studies have shown that M. tuberculosis-derived eDNA is a major component of the extracellular matrix that supports the expression of drug tolerant bacilli grown under low oxygen conditions in vitro (Ryan, et al., 2010). Recently, is has been demonstrated that M. tuberculosis grown as a suspension secretes stable double stranded RNA fragments into the culture media (Obregon-Henao, et al., 2012). The in vivo relevance of bacteria interacting with nucleic acids specifically eDNA is a major contributor to the viscosity of inflammatory exudate in patients with cystic fibrosis (CF) and other chronic lung diseases (Fahy, et al., 1993, Puchelle, et al., 1996). Recombinant human DNase I (Pulmzyme®, Dornase alfa, Genentech USA, Inc.) is commonly used therapeutically as an adjunct to antimicrobial drug therapy to reduce airway secretion viscosity associated with eDNA in CF patients (Pressler, 2008, Kaplan, 2009, Bakker, et al., 2011).

The use of leukocytes enriched for neutrophils as the source of leukocyte lysate in these studies also has in vivo relevance. Neutrophils in vivo are short lived and are among the first immune cells to encounter bacilli following mycobacterial infections (Canetti, 1955, Abadie, et al., 2005). Moreover, M. tuberculosis has been shown to survive extracellular killing by neutrophil extracellular nets (NETs) in vitro (Ramos-Kichik, et al., 2009, Wong & Jacobs, 2013). The extrusion of eDNA from neutrophils and other cells is thought to be an alternative defense mechanism against extracellular bacteria but can also be damaging by contributing to the pathogenesis of infectious and non-infectious inflammatory diseases (Brinkmann, et al., 2004, Wartha, et al., 2007, Ma & Kubes, 2008). It is also of interest that M. tuberculosis and other mycobacteria express secreted or surface exposed DNA-binding proteins that may facilitate the interaction with host or pathogen derived eDNA (Prabhakar, et al., 2004, Yeruva, et al., 2006). Antibodies directed against DNABII family members of bacterial DNA-binding proteins have been shown to be effective at disrupting bacterial biofilms and to render drug tolerant pathogens again susceptible to antimicrobial drugs (Goodman, et al., 2011). The targeting of DNA binding proteins of M. tuberculosis may be beneficial in preventing the establishment of drug tolerant communities of bacilli and an attractive target in the development of new tuberculosis vaccines (Chen, et al., 2008, Kaplan, 2009, Gordon, et al., 2010, Liu & Gordon, 2012).

We describe here a simple in vitro assay that favors the establishment of drug tolerant M. tuberculosis, which does not require specific nutrient depletion, the need to maintain cultures under controlled low oxygen conditions or to introduce other stress factors to induce in vitro drug tolerance. Recent studies have demonstrated that M. tuberculosis cultured in the presence of viable human peripheral blood mononuclear cells also express in vivo drug tolerance (Kapoor, et al., 2013). The poorly defined extracellular matrix derived from viable or lysed leukocytes is a limitation to the use of current assay design as a high throughput drug-screening platform. Our data suggest that eDNA is an important extracellular matrix in vitro and in vivo and we are currently exploring whether purified DNA alone is sufficient to promote the in vitro expression of M. tuberculosis drug tolerance. Adapting this assay using a well-defined attachment matrix in a high throughput platform has the potential to fill a critical unmet need to identify urgently needed new drugs or novel therapeutic strategies specifically targeting drug tolerant M. tuberculosis. Modifications of this assay system have been recently used to identify novel anti-tuberculosis drugs that specifically target copper metabolism (Speer, et al., 2013). Currently, we are using this assay system to discover small molecules that disperse bacilli from lysed leukocytes as a strategy to restore drug susceptibility of drug tolerant mycobacterium in vivo.

Supplementary Material

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Acknowledgments

This work was supported by NIH NIAID Grants AI083856 (RJB), 1RO1AiI106733 (RJB and CM) AI070456 (IMO), 1R01HL090991 (JAN), Science Foundation Arizona (JGL) and Rebecca Runyon Bryan Chair for CF, Ira and Libbie Pink Charitable Fund (JAN).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Fig S1-S4
NIHMS564253-supplement-Supp_Fig_S1-S4.pdf (1MB, pdf)
Supp Figure S1 Legend
NIHMS564253-supplement-Supp_Figure_S1_Legend.docx (13.3KB, docx)
Supp Figure S2 Legend
NIHMS564253-supplement-Supp_Figure_S2_Legend.docx (13.2KB, docx)
Supp Figure S3 Legend
NIHMS564253-supplement-Supp_Figure_S3_Legend.docx (13.5KB, docx)
Supp Figure S4 Legend
NIHMS564253-supplement-Supp_Figure_S4_Legend.docx (13.1KB, docx)

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