Bactericidal activity of nitrofurans against growing and dormant Mycobacterium bovis BCG

Bernadette Murugasu-Oei and Thomas Dick*

Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Singapore


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Depletion of oxygen triggers the shift-down of Mycobacterium bovis BCG to a state of dormancy. Bacilli in their dormant state are resistant to standard anti-mycobacterials. The nitroimidazole metronidazole was the first compound identified to show bactericidal activity against dormant tubercle bacilli. In contrast to metronidazole's selective toxicity for dormant bacilli, we report here that the nitrofurans nitrofurantoin, furaltadone and nitrofurazone showed bactericidal activity against dormant and growing bacteria. Importantly, the bactericidal effect of nitrofurans on dormant bacilli was 35- to 250-fold higher compared with metronidazole.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A key problem in tuberculosis control is the persistence of Mycobacterium tuberculosis, despite prolonged chemotherapy. The development of physiological (as opposed to genetic) drug resistance is thought to be a contributing factor.1 Mycobacteria are obligate aerobes. However, tubercle bacilli encounter hypoxic environments in vivo.1,2 Upon depletion of oxygen in culture the bacillus terminates growth and develops into a dormant form.3,4 Importantly, the dormant form of the bacterium was found to be resistant to the standard anti-mycobacterials.

Metronidazole is the first compound that shows activity against dormant tubercle bacilli, although growing bacteria are not affected by the drug.35 The compound acts as a pro-drug that requires reduction of its nitro-group to a reactive intermediate that then causes damage to DNA and subsequent cell death.6 However, the bactericidal activity of metronidazole against dormant bacilli is poor.3,4,7 The finding that metronidazole, a nitroimidazole, showed some bactericidal activity against dormant mycobacteria prompted us to question whether nitrofurans, the other class of medically important nitroheterocyclic antimicrobials, might possess higher anti-dormancy activity. The nitrofurans, like the nitroimidazoles, require reduction of the nitro group to exert their antimicrobial activity.8 Previous reports had demonstrated that nitrofurans show activity against growing M. tuberculosis with an MIC of 10–25 mg/L.9,10

Here, we report the comparative analysis of the anti-microbial activity of nitrofurantoin (a widely used representative of nitrofurans),11 two other nitrofurans, and metronidazole, on the growing and dormant form of the BCG strain of the bovine tubercle bacillus Mycobacterium bovis. To grow dormant BCG we employed the dormancy culture system that was developed by Wayne for M. tuberculosis.3,4 Wayne's system is based on growth of the bacilli in sealed stirred tubes. Sealing the cultures limits the amount of total oxygen and stirring prevents the formation of spatial oxygen gradients. Initially the cultures grow exponentially and consume oxygen rapidly. A temporal oxygen gradient is generated and the culture enters stationary phase when the oxygen concentration reaches a microaerobic threshold level. The hypoxic stationary-phase mycobacteria do not synthesize DNA, i.e. they are in a non-replicating or dormant state.3,4


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Nitrofurantoin, furaltadone, nitrofurazone and metronidazole were obtained from Sigma (St Louis, MO, USA). Stock solutions were prepared in dimethylformamide (BDH Laboratory Supplies, Poole, UK). All experiments were conducted with M. bovis BCG Pasteur ATCC 35734.

Liquid culture experiments were conducted at 37 °C in Dubos Tween-albumin broth (BD Biosience, Sparks, MD, USA). The medium was dispensed in 17 mL aliquots to screw-cap glass tubes, 20 x 125 mm. Bacilli were subcultured once in liquid medium until they reached an early exponential growth phase (OD600 = 0.2–0.3) before inoculation to an experimental culture. To grow experimental exponential phase bacilli, pre-cultures were diluted to OD600 = 0.05 (5 x 106 cfu/mL). Drugs (170 µL) were added and caps were loosely screwed down allowing exchange of air. Cultures were aerated by incubation on a shaker-incubator at 250 rpm. For MIC determination, cultures were exposed to various drug concentrations. The effect of the drugs was monitored by measuring the optical density of the cultures in a Bacharach spectrophotometer (Thomas Scientific, Swedesboro, NJ, USA) after 24 and 48 h of drug exposure. The lowest drug concentration that caused inhibition of growth was recorded as the MIC. To produce dormant bacilli, pre-cultures were diluted to OD600 = 0.005 (5x105 cfu/mL). Magnetic stirrers were added, the caps (with rubber septa) were tightly screwed down to seal the tubes and the cultures were incubated on stirring platforms at 170 rpm. After 20 days under these oxygen-limited conditions the bacilli were in their dormant state4 (OD600 = 0.3, 108 cfu/mL) and drugs (170 µL) were injected by needle. Oxygen depletion in the sealed cultures was monitored using methylene blue (Sigma). Reduction and fading of this dye served as a visual indication of hypoxia.4

In preparation for the counting of colonies on agar, dilutions of liquid culture were made in Dubos Tween–albumin broth. Dubos oleic-albumin agar (BD Biosience) was dispensed in 3 mL amounts to the wells of tissue culture plates. The agar surfaces were inoculated in triplicate with 20 µL of selected dilutions, and the plates were incubated at 37°C under normal atmosphere. Colonies were counted with a dissecting microscope 14 and 21 days after inoculation. Viable count determinations of control cultures that were mock-injected with 170 µL dimethylformamide were indistinguishable from the ‘Drug-free control’ values. Significant clumping or pH change were not observed in any culture. Typically, cultures were diluted >103-fold before plating. Thus, considering the MIC of nitrofurantoin (50 µM) and the maximum concentration used (500 µM), the culture dilution for plating contained <1% of the MIC. However, to examine the possibility that drug carry-over might affect cfu determinations, the following control experiment was carried out. An exponential growth phase BCG culture was diluted in Dubos Tween–albumin broth containing either no, or 50 or 500 µM nitrofurantoin, and 20 µL samples were plated on 3 mL agar wells. The numbers of cfu/well were 40 ± 16 (no drug), 41 ± 9 (50 µM nitrofurantoin) and 36 ± 7 (500 µM nitrofurantoin). This result shows that effects on cfu based on drug carry-over were negligible.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Nitrofurantoin had an MIC of c. 50 µM (12 mg/L). To determine the effect of the drug on the viability of exponentially growing bacilli, actual survival was established by plating the cultures. The FigureGo (a) shows that nitrofurantoin at its MIC appears to be bacteriostatic. In contrast, incubation of growing cultures with 10 x MIC nitrofurantoin (500 µM) resulted in 103-fold and 104-fold decrease in viability after 24 and 48 h, respectively. Two other nitrofurans, furaltadone and nitrofurazone, showed similar bactericidal activities on growing bacilli. Metronidazole at 500 µM did not have any inhibitory effect on growing cultures. Growth was not inhibited by the nitroimidazole up to the highest concentration tested (MIC for metronidazole >1000 µM).



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Figure. Effect of nitrofurans and metronidazole on the viability of (a) aerobic growing and (b) hypoxic dormant BCG cultures. NIT, nitrofurantoin; FTD, furaltadone; NFS, nitrofurazone; MTZ, metronidazole. Experiments were repeated three times and each experiment was carried out with duplicate cultures. Mean values and standard deviations are shown. {blacksquare}, 0 h; {square}, 24 h; {blacksquare}, 48 h.

 
The FigureGo (b) shows the effect of the nitroheterocycles on dormant bacilli. The bacilli in the hypoxic stationary-phase culture showed a 4-fold and a 70-fold reduction of viable counts after 24 and 96 h of exposure to 500 µM nitrofurantoin. Exposure to 500 µM furaltadone and nitrofurazone for 96 h resulted in a 300- and 500-fold reduction of viable counts, respectively. Consistent with previous reports,3,4 metronidazole showed only a moderate bactericidal activity for hypoxic dormant bacilli. Five hundred micromolar metronidazole reduced viability of the hypoxic dormant culture <2-fold after exposure for 96 h. These results suggest that the bactericidal activity of nitrofurans on dormant bacilli was 35- to 250-fold higher than that of metronidazole.

In summary, the comparison of the bactericidal effect of the nitrofurans nitrofurantoin, furaltadone and nitrofurazone and the nitroimidazole metronidazole on dormant bacilli revealed that nitrofurans had a higher antidormancy activity than the nitroimidazole. Furthermore, in contrast to metronidazole, nitrofurans showed substantial bactericidal activity against growing bacilli. The similarity of the bactericidal activity of the three tested nitrofurans suggests that this effect is characteristic of this class of compounds. The reason for the higher bactericidal activity of nitrofurans against growing and dormant BCG remains to be elucidated. It is possible that the difference in the redox potential of the two nitroheterocyclic compound classes plays a role. The redox potential of nitrofurans is higher (e.g. –257 mV for nitrofurazone) compared with the redox potential of the nitroimidazoles (e.g. –486 mV for metronidazole).6 Therefore, the nitrofurans might be more easily reduced and activated. Nitrofurantoin is well absorbed by mouth, but rapidly broken down in the tissues and eliminated. High concentrations are only achieved in the urine, hence, the clinical use of this drug against urinary-tract infections.11 Considering the comparably high concentrations of the nitrofurans required to show significant bactericidal activity against growing or dormant tubercle bacilli in vitro, it is evident that the application of these drugs against tuberculosis is limited. However, the drastically increased anti-dormancy activity of nitrofurans compared with the activity of the first anti-dormancy lead metronidazole suggests the potential of nitroheterocyclic compounds in the development of new anti-dormancy leads.12


    Acknowledgments
 
We thank Hock Leng Peh for help with the experiments. This study was supported by the Institute of Molecular and Cell Biology (IMCB).


    Notes
 
* Corresponding author. Tel: +65-874-8606; Fax: +65-779-1117; E-mail: mcbtd{at}imcb.nus.edu.sg Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Wayne, L. G. (1994). Dormancy of Mycobacterium tuberculosis and latency of disease. European Journal of Clinical Microbiology and Infectious Diseases 13, 908–14.[ISI][Medline]

2 . Weber, I., Fritz, C., Ruttkowski, S., Kreft, A. & Bange, F. C. (2000). Anaerobic nitrate reductase (narGHJI) activity of Mycobacterium bovis BCG in vitro and its contribution to virulence in immunodeficient mice. Molecular Microbiology 35, 1017–25.[ISI][Medline]

3 . Wayne, L. G. & Hayes, L. G. (1996). An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of non replicating persistence. Infection and Immunity 64, 2062–9.[Abstract]

4 . Lim, A., Eleuterio M., Hutter, B., Murugasu-Oei, B. & Dick, T. (1999). Oxygen depletion-induced dormancy in Mycobacterium bovis BCG. Journal of Bacteriology 181, 2252–6.[Abstract/Free Full Text]

5 . Wayne, L. G. & Sramek, H. A. (1994). Metronidazole is bactericidal to dormant cells of Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 38, 2054–8.[Abstract]

6 . Smith, M. A. & Edwards, D. I. (1995). Redox potential and oxygen concentration as factors in the susceptibility of Helicobacter pylori to nitroheterocyclic drugs. Journal of Antimicrobial Chemotherapy 35, 751–64.[Abstract]

7 . Brooks, J. V., Furney, S. K. & Orme, I. M. (1999). Metronidazole therapy in mice infected with tuberculosis. Antimicrobial Agents and Chemotherapy 43, 1285–8.[Abstract/Free Full Text]

8 . McOsker, C. C. & Fitzpatrick, P. M. (1994). Nitrofurantoin: mechanism of action and implications for resistance development in common uropathogens. Journal of Antimicrobial Chemotherapy 33, Suppl. A, 23–30.[ISI][Medline]

9 . Wayne, L. G. & Russell, R. L. (1966). Selective effects of nitrofuraldoxime-anti on isoniazid resistant M. tuberculosis. Scandinavian Journal of Respiratory Disease 47, 18–26.

10 . Sun, Z. & Zhang, Y. (1999). Antituberculosis activity of certain antifungal and antihelmintic drugs. Tubercle and Lung Disease 79, 319–20.[Medline]

11 . Reynolds, J. E. F (1996). Martindale, the extra pharmacopoeia, 31st edn. Royal Pharmaceutical Society, London.

12 . Stover, C. K., Warrener, P., Vandevanter, D. R., Sherman, D. R., Arain, T. M., Langhorne, M. H. et al. (2000). A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature 405, 962–6.[ISI][Medline]

Received 5 June 2000; returned 3 August 2000; revised 16 August 2000; accepted 30 August 2000