Plate-based dormancy culture system for Mycobacterium smegmatisand isolation of metronidazole-resistant mutants

Amanda Lim, Thomas Dick *Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Singapore

Received 26 March 2001; received in revised form 9 May 2001; accepted 11 May 2001

First published online 5 June 2001

Abstract

Mycobacterium smegmatis is an obligate aerobe. However, growth analyses in oxygen-limited liquid cultures have shown that the bacillusis able to survive anoxia with a half-life of 4 days by shifting down to a drug-resistant, dormant state. Metronidazole is the first lead againstdormant bacilli and shows selective toxicity for this physiological state. Here, we report a plate-based dormancy culture system employinganoxic jars for M. smegmatis. Its usefulness for the genetic analysis of dormancy was demonstrated by isolating the first metronidazole-resistant mutants. Highly resistant mutants formed slightly yellow (as opposed to creamy) colonies. Furthermore, high-level metronidazoleresistance correlated with an increased half-life of 12 days under anoxic conditions. This suggests a link between metronidazole susceptibilityand anaerobic survival. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Dormancy; Metronidazole; Drug resistance

1. Introduction

Mycobacteria require oxygen for growth. However, theslow-growing parasitic Mycobacterium tuberculosis [1], itsattenuated relative Mycobacterium bovis BCG [2] and thefast-growing saprophyte Mycobacterium smegmatis [3] allhave the capability to adapt to anoxia by developing ade¢ned non-replicating or dormant form. For the tuberclebacilli, the dormant state could play a role in the survivalof hypoxic environments encountered in the human host.For M. smegmatis the ability to shift down to dormancymay be involved in the survival of changing oxygen avail-ability in the soil. Importantly, the dormant state is resis-tant against conventional anti-mycobacterials. Therefore,dormant tubercle bacilli could, at least in part, be respon-sible for the observed persistence of infection during che-motherapy [4].

To study the dormancy response we apply the dor-mancy culture system that was developed by Wayne andHayes for M. tuberculosis [1] on M. smegmatis [3]. Wayneand Hayes' dormancy culture system is based on growth

of the bacilli in liquid medium under oxygen-limited con-ditions in sealed, stirred tubes. Initially the cultures growexponentially and consume the available oxygen. A tem-poral oxygen gradient is generated and the cultures termi-nate growth when the oxygen concentration reaches a hy-poxic threshold level. The bacilli in the anoxic stationaryphase are in a reversible, non-replicating, apparently dip-loid, synchronised state of low metabolic activity, in whichthe cultures maintain viability for extended periods [1^3].In contrast to slow, gradual depletion of oxygen that al-lows adaptation to anoxia, rapid oxygen depletion resultsin death of the bacilli [1,3]. This observation implies thatthe bacteria possess a yet unknown `dormancy pro-gramme' that requires time to be executed and ensuresanoxic survival of the bacilli.

The nitroimidazole metronidazole (MTZ) is the ¢rstlead against dormant mycobacteria [2,3,5,6]. MTZ hasbeen used for a long time for the treatment of anaerobicbacteria, luminal parasites and Helicobacter pylori. Inthese organisms the compound acts as a pro-drug thatrequires reduction of its nitro-group to exert cidal activity[7]. The mechanism of action of MTZ in mycobacteria isnot known. This is, in part, due to the di¤culty associatedwith the isolation of MTZ-resistant (Mtzr) mutants. Thecidal activity of MTZ in Wayne and Hayes' dormancy

0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 2 3 1 - 2

* Corresponding author. Tel. : +65 874 8606; Fax: +65 779 1117;E-mail : mcbtd@imcb.nus.edu.sg

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culture system is relatively poor. Exposure of M. smegma-tis to 100 Wg ml31 MTZ for 10 days results in only 100-fold reduction in the number of viable bacilli ([3], see Fig.3). Increasing the concentration of the drug does not resultin a reduction of the number of wild-type bacilli to a levelthat makes testing of survivors for MTZ resistance feasible(see Section 3.2). Thus, the liquid medium-based dor-mancy culture system is not suitable for the isolation ofMTZr mycobacteria. The isolation of Mtzr mutants bysimply spreading culture on drug-containing agar andpicking colonies is not possible because MTZ sensitivityis expressed speci¢cally in the dormant stage. Growingbacilli are not a¡ected by the drug [2,3,5].

To facilitate genetic analyses of dormant mycobacteria,we report here a solid medium-based dormancy culturesystem for M. smegmatis and demonstrate the usefulnessof the system by isolating the ¢rst Mtzr mutants in myco-bacteria.

2. Materials and methods

All experiments were conducted with M. smegmatismc2155 [8] at 37³C. Dubos Tween^albumin broth (BectonDickinson) was dispensed in 17-ml aliquots to screw-capglass tubes, 20U125 mm. To grow experimental exponen-tial-phase bacilli, pre-cultures were diluted to OD600 = 0.05and caps were loosely screwed down allowing exchange ofair. Cultures were aerated by incubation on a shaker^in-cubator at 250 rpm. To grow dormant bacilli, pre-cultureswere diluted to OD600 = 0.005. Magnetic stirrers (andMTZ if appropriate) were added, the caps were tightlyscrewed down to seal the tubes and the cultures werestirred at 170 rpm [3]. For cultivation of bacilli on solidmedium plates (85 mm diameter) with 30 ml Dubos oleic^albumin agar were used. Plates containing a drug gradientfrom 0 to 500 Wg ml31 MTZ were prepared by ¢rstly

pouring 15 ml of drug-free agar into the slightly tiltedPetri dish. After the agar had solidi¢ed the dish was puton a £at surface and 15 ml of agar containing 500 Wg ml31

MTZ was added. Anoxic atmosphere for solid mediumexperiments was generated using GasPak 100 anaerobicjars according to the instructions supplied by the manu-facturer (Becton Dickinson). This system is based on ahydrogen generator envelope containing sodium borohy-dride. The addition of water starts a H2 generating reac-tion. The reduction of O2 by H2 is catalysed by palladium.Anoxia was monitored using the oxygen indicator meth-ylene blue (Sigma). Methylene blue was diluted in themedium (broth or agar) to yield a concentration of 1.5Wg ml31 [3]. Anoxia in the atmosphere of the GasPakjars was monitored by methylene blue strips supplied bythe manufacturer (Becton Dickinson). MTZ was obtainedfrom Sigma. 10 mg ml31 stock solutions were prepared inwater.

3. Results and discussion

3.1. Solid medium dormancy culture system

To establish a dormancy culture system based on solidmedium we tested growth and survival of M. smegmatison agar plates under hypoxic conditions. Anoxic atmos-phere was generated in jars using the GasPak system inwhich O2 is reduced to water in a palladium-catalysedreaction with H2 as described in Section 2. Diluted culturesamples containing 300 cfu were spread on plates andtransferred into jars. The hydrogen generating reactionwas started and the jars were sealed. The generation ofanoxic conditions in the atmosphere of the jars was moni-tored using an oxygen indicator strip containing methyleneblue. Within 30 min the strips turned white, indicatinganoxia in the gas phase. Methylene blue-containing agarplates decoloured after 5^6 days. After a total of 10 days,the jars were opened. Inspection of the agar surfaces undera dissection microscope did not show any growth. Todetermine whether any bacilli on the agar surface hadsurvived the oxygen-starvation treatment the plates wereincubated under normal air. Colony count after 3 daysshowed that 90% of the initially plated cfu had survivedthe 10 days of hypoxia (Fig. 1). Next we tested a keyfeature of hypoxic dormant bacilli, the development ofsensitivity to MTZ. The anoxic jar experiment was carriedout in the same way, but using agar plates containing 100Wg ml31 MTZ. In contrast to the anoxic survival of thebacilli on drug-free plates, incubation for 10 days in theanoxic jars on plates containing MTZ resulted in a 100%killing of the bacilli (Fig. 1). This result showed that thebacilli in the jar developed sensitivity to MTZ. In contrastto the cidal e¡ect of MTZ on the non-growing bacteria inthe anoxic jar the drug did not show any e¡ect on thecolony formation of bacilli incubated under normal at-

Fig. 1. Growth and survival of M. smegmatis on agar plates as a func-tion of oxygen and MTZ. 300 cfu ( = 100%) from exponentially growingcultures were spread on agar plates with and without MTZ and incu-bated in normal atmosphere (+O2) for 3 days or under anoxic atmos-phere in jars (3O2). After 10 days incubation under anoxic conditionsplates were exposed for 3 days to normal atmosphere. Numbers on theplates shows percentage of plated cfu that was observed to form colo-nies after each incubation step. The experiment was repeated once withthe same results.

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mosphere (Fig. 1). Taken together, our experiments sug-gest that bacilli sitting on the agar surface exposed to theanoxic atmosphere generated in the jars shifted down to astate of MTZ-sensitive dormancy that is similar to thestate of bacilli in the anoxic stationary phase generatedin the liquid medium-based Wayne and Hayes dormancyculture model.

3.2. Isolation and characterisation of spontaneousMTZ-resistant mutants

To determine whether the plate-based dormancy culturesystem is suitable for the selection of spontaneous Mtzr

mutants an increased number (107 cfu) of bacilli werespread on agar plates containing 100 and 250 Wg ml31

MTZ. Growth was examined after incubation in anoxicjars for 10 days and exposure to normal atmosphere for

3 days. The selection at 100 Wg ml31 MTZ appeared to besomewhat leaky upon plating large numbers of bacilli.However, selection on agar containing 250 Wg ml31

MTZ eliminated the background of wild-type bacilli(from 107 cfu plated, only 100 cfu formed colonies; seebelow). In parallel experiments employing the liquid dor-mancy culture system, an increase of the MTZ concentra-tion from 100 to up to 500 Wg ml31 did not result in theelimination of wild-type bacilli. Both drug concentrationsreduced viability only about 100-fold after 10 days of in-cubation (from about 107 cfu ml31 to 105 cfu ml31). Thus,MTZ appears to be more e¡ective in the anoxic jars com-pared with the liquid dormancy culture system. A possiblereason for this di¡erence could be that MTZ exerts itscidal activity only in a strictly anoxic environment, asde¢ned by the complete decolourisation of methyleneblue (H.L. Peh and T. Dick, unpublished data). In theWayne and Hayes dormancy culture system, oxygen isdepleted slowly over a period of 7^8 days (see Fig. 4).In contrast, oxygen is removed rapidly from the gas phasein the solid medium-based culture system and the gener-ation of anoxia in the agar takes only 5^6 days. Therefore,bacilli on the agar surface are exposed to anoxia earlierand (because the total cultivation time (10 days) in bothsystems is kept constant) longer than bacilli in the sealedliquid culture system. Taking these observations together,it is conceivable that di¡erent oxygen depletion kinetics inthe two culture systems are responsible for the di¡erencein the cidal e¡ect of MTZ.

To isolate spontaneous Mtzr mutants, 5U107 cfu werespread on agar plates containing 250 Wg MTZ ml31, in-cubated for 10 days in anoxic jars, and then exposed tonormal atmosphere to detect surviving, presumably Mtzr

bacilli. One of 105-plated cfu formed a colony. 3% of theapparent Mtzr colonies were found to be slightly yellow

Fig. 2. Hypoxic MTZ gradient plate streak assay. Streaks of M. smeg-matis wild-type and Mtzr cultures along a gradient from 0 to 500 Wgml31 MTZ are shown. After streaking the strains, the plate was incu-bated for 10 days in anoxic atmosphere in a jar followed by 3 days ex-posure to normal atmosphere. 1: representative of the Mtzr mutantsthat retained wild-type colour; 2: wild-type; 3: mtz-1 representative ofthe highly resistant Mtzr mutants that showed slightly yellow (Yeo) col-ony colour. A typical example of a streak test is shown.

Fig. 3. MTZ susceptibility of M. smegmatis wild-type (clear bars) andmtz-1 mutant (¢lled bars) in the liquid dormancy culture system. Wild-type and mtz-1 were grown in sealed, stirred tubes in the presence ofvarious MTZ concentrations. After 10 days, viable counts were deter-mined by plating and colony count. Mean values and standard devia-tions are shown from two experiments with duplicate cultures.

Fig. 4. Growth and survival of M. smegmatis wild-type (squares) andmtz-1 (triangles) in the liquid dormancy culture system. Wild-type andmtz-1 were grown in sealed, stirred tubes. Viable counts at various timepoints were determined by plating and colony count (clear symbols).Filled symbols represent turbidity measurements of the cultures. Notethe similar generation time (3 h) of the two strains in the aerobic expo-nential growth phase that is observed at the beginning of the experi-ment. f, d: fading and decolouration of the oxygen indicator methyleneblue. Mean values and standard deviations are shown from two experi-ments with duplicate cultures. All liquid cultures in this study werechecked microscopically for clumping. Signi¢cant clumping was not ob-served.

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(Yeo) as opposed to the creamy colour of the wild-type.Colony puri¢cation of mutants on drug-free plates showedthat the yellow colour was independent of MTZ. To de-termine relative resistance levels of the apparent Mtzr mu-tants, hypoxic survival of 50 mutants (15 Mtzr-Yeo, 35Mtzr) was tested on agar plates containing a gradient ofthe drug. Mutants were streaked out along the gradientand incubated for 10 days in anoxic jars. Then the plates,which did not show any growth after removal from thejars, were exposed to normal atmosphere for 3 days. Therationale behind this experiment was that extent and den-sity of growth of the streak along the MTZ gradient underoxic conditions should re£ect the survival of the non-growing bacilli exposed to increasing MTZ concentrationsunder hypoxic conditions. Fig. 2 shows that Mtzs wild-type bacilli survived hypoxia only along the section ofthe streak that was exposed to low concentrations of theMTZ gradient. All 15 Mtzr-Yeo mutants formed densestreaks up to the highest MTZ concentration of the gra-dient. Extent and density of growth of the streaks of the35 Mtzr mutants that had retained their wild-type colourvaried (i.e. was strain-speci¢c) and covered the whole spec-trum from the weak growth, shown by the wild-type, tothe strong growth shown by the Mtzr-Yeo mutants. TheMtzr mutant isolation and characterisation experimentwas repeated once in the same way yielding similar results.The frequency of Mtzr cfu was found to be about 1035.6% of the Mtzr colonies showed a Yeo phenotype andwere highly MTZ-resistant in the gradient plate assay.

One mutant, mtz-1, that displayed the Mtzr-Yeo pheno-type was selected for further analysis in the liquid me-dium-based dormancy culture system employing sealedtubes. To con¢rm the high MTZ resistance level of themutant that was indicated by the MTZ gradient plateassay, sealed cultures were grown in the presence of var-ious MTZ concentrations. After 10 days, survival was de-termined by plating and colony count. Fig. 3 shows thatthe wild-type was killed 100-fold by 100 Wg ml31 MTZ. Incontrast mtz-1 was hardly a¡ected by the drug, thus val-idating the results from the drug gradient plate assay. Todetermine whether mtz-1 was a¡ected in the survival underanoxic conditions, long-term drug-free sealed cultureswere grown and viability was determined at various timepoints by colony count. Fig. 4 shows that the half-life ofanoxic culture of mtz-1 was increased three-fold (t0:5 = 12days) compared to that of the wild-type (t0:5 = 4 days).Thus, the high level of MTZ resistance of mtz-1 appearsto be associated with increased viability of dormant cul-ture under drug-free conditions.

Upon plating of anoxic cultures of mtz-1 for cfu deter-mination, a number of apparent revertants were observedthat had lost their Yeo phenotype. This high reversionfrequency (1032) was observed in both MTZ-containingand MTZ-free cultures and was thus independent of thedrug. To determine whether the high frequency of rever-sion of the Yeo phenotype was speci¢c to anoxic cultures

of mtz-1, the frequencies of Yeo reversion in growing andanoxic culture of mtz-1 were compared. A pre-culture wassplit and used to generate aerated exponentially growingcultures (OD600 = 0.2) and sealed 10-day-old anoxic cul-tures. Culture samples from both growth phases werespread on plates and the frequencies of appearance ofcreamy colonies were observed. No Yeo revertant colonieswere detected in 104 cfu plated from growing culture.Thus, the reversion frequency was less than 1034 and theYeo phenotype appeared to be stable in the exponentialgrowth phase. As before, about one in 100 colonies fromanoxic culture were observed to have reverted to creamycolony colour. This experiment was repeated once, yield-ing the same results. Thus, the frequency of Yeo reversionappears to be at least 100-fold higher in non-growing an-oxic culture compared to growing culture. It is importantto note that yellow colonies were not observed upon plat-ing of dormant wild-type or dormant Yeo revertant cul-tures. Thus, dormancy-dependent instability appears to bespeci¢c to mtz-1. Recently, it was shown that brief expo-sure of M. smegmatis to hypoxic conditions stimulates thetransposition of the M. tuberculosis insertion sequenceIS6110 [9]. Furthermore, it was demonstrated that the sta-tionary growth phase of M. smegmatis is associated withhypermutability [10]. Whether dormancy is associated withgenetic instability has not been determined and the mech-anism responsible for the apparent dormancy-speci¢c in-stability of mtz-1 remains to be elucidated. Furthermore, itremains to be established whether the reversion frequencyof mtz-1 is increased only in anoxic dormant culture orgenerally in the stationary phase.

To con¢rm that the three observed phenotypes of mtz-1(Mtzr, Yeo and increased anoxic survival) were geneticallylinked, we determined MTZ susceptibility and half-life ofanoxic culture of one Yeo revertant, mtz-1rev, in sealedliquid culture experiments. mtz-1rev was found to displaywild-type sensitivity to MTZ and wild-type survival ofanoxic culture. The concurrent reversion of all three phe-notypes suggests that the traits were indeed due to thesame mutation.

In conclusion, we report a culture system for dormancyin M. smegmatis that is based on solid medium and thusfacilitates genetic analysis of dormancy. We demonstratedits usefulness by isolating the ¢rst mutants that are resis-tant against MTZ. High-level MTZ resistance was associ-ated with yellow colony colour and with an increase inviability of anoxic culture. This suggests a link betweenMTZ susceptibility, metabolism (accumulation of a yellowmetabolite?) and dormancy survival. It is interesting tonote that in a parallel e¡ort to isolate Mtzr mutants byTn5-based [11] transposon mutagenesis 35 000 insertionmutants were generated and subjected to selection forMTZ resistance. However, no Mtzr colonies were ob-tained. This could suggest that the genes conferringMTZ susceptibility are essential for viability. Experimentsare now under way to identify the gene(s) involved in

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spontaneous MTZ resistance. This work will reveal themolecular nature of MTZ susceptibility, shed light on itslinkage with dormancy viability, and should lead to anunderstanding of the mechanism underlying the dor-mancy-dependent high reversion frequency of the Mtzr-Yeo mutant mtz-1.

Acknowledgements

We would like to thank Pamela Thayalan for help withthe transposon mutagenesis and Bernadette Murugasu-Oeifor comments on the manuscript. This study was sup-ported by the Institute of Molecular and Cell Biology.

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[9] Ghanekar, K., McBride, A., Dellagostin, O., Thorne, S., Mooney, R.and McFadden, J. (1999) Stimulation of transposition of the insertionsequence IS6110 by exposure to a microaerobic environment. Mol.Microbiol. 33, 982^993.

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