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This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyright Author's personal copy A novel screening method based on menadione mediated rapid reduction of tetrazolium salt for testing of anti-mycobacterial agents Upasana Singh 1 , Shamim Akhtar 1 , Abhishek Mishra 1 , Dhiman Sarkar ⁎ Combi. Chem. Bio Resource Center, Organic Chemistry Division, Dr. Homi Bhabha Road, National Chemical Laboratory (NCL), Pune-411008, Maharastra, India a b s t r a c ta r t i c l e i n f o Article history: Received 4 June 2010Received in revised form 8 November 2010Accepted 19 November 2010Available online 1 December 2010 Keywords:Mycobacterium tuberculosis Tetrazolium saltMenadioneXRMA A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2, 3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determinetheviabilityof Mycobacteriumtuberculosis , Mycobacteriumbovis BCGand Mycobacteriumsmegmatis bacilliinmicroplate format. In general, XTT reduction is an extremely slow process which takes almost 24 h toproduce a detectable signal. Menadione could drastically induce this reduction to an almost equal extentwithin a few minutes in a dose dependent manner. The reduction of XTT is directly proportional to the cellconcentration in the presence of menadione. The standardized protocol used 200 μ M of XTT and 60 μ M of menadione in 250 μ l of cell suspension grown either in aerobic or anaerobic conditions. The cell suspensionof M. bovis BCG and M. tuberculosis were incubated for 40 min before reading the optical density at 470 nmwhereas M. smegmatis was incubated for 20 min. Calculated Signal/Noise (S/N) ratios obtained by applyingthis protocol were 5.4, 6.4 and 9.4 using M. bovis BCG, M. tuberculosis and M. smegmatis respectively. Thecalculated Z ′ factors were N 0.8 for all mycobacterium bacilli indicating the robustness of the XTT ReductionMenadione Assay (XRMA) for rapid screening of inhibitors. The assay protocol was validated by applying10 standard anti-tubercular agents on M. tuberculosis , M. bovis BCG and M. smegmatis . The MinimumInhibitoryConcentration(MIC)valueswerefoundtobesimilartoreportedvaluesfromColonyFormingUnit(CFU)andREMA(resazurinmicroplateassay)assays.Altogether,XRMAisprovidinganovelanti-tubercularscreening protocol which could be useful in high throughput screening programs against different physio-logical stages of the bacilli.© 2010 Elsevier B.V. All rights reserved. 1. Introduction Morethan2 billionpeoplearecarrying Mycobacteriumtuberculosis that causestuberculosis. Onein 10 amongthemcan develop active TBwhich kills 4400 people per day i.e. 1.6 million per year (WHO report,2009; Caccamo et al., 2009). Incomplete treatment due to long termtherapiesiscausingtheemergenceofmulti-drugresistant(MDR)andextremely drug resistant (XDR) TB (Dye, 2006; Alexander and De,2007). Tuberculosis (TB) is the largest cause of death in individualscarrying immunode ciency virus type 1 (HIV-1) infection. This co-infectionhasclaimeddeathofanestimatedonethirdtoonehalfofthe30 million AIDS patients worldwide (Ranjbar et al., 2009). No newdrug has been developed for tuberculosis in the last 40 years (Warnerand Mizrahi, 2004). The current ‘ short-term ’ treatment for tubercu-losisalsolastsfor6 months(WHOreport,2009).Thecurrentsituationrequires the speeding up of the anti-tubercular identi cation anddevelopment program using novel approaches.Inordertodevelopantitubercularscreeningassaysmanyattemptswere made using redox dyes like alamar blue/resazurin, tetrazolium,etc. on aerobic cultures of mycobacterium (Taneja and Tyagi, 2007;Collins and Franzblau, 1997; Palomino et al., 2007). The search fordrugs targeting mycobacterium is delayed because of either continu-ing CFU or following unexpectedly longer incubation with the dye asthe main assessment technique, both of which are primarily designedfor very low throughput screening. Apart from these disadvantages,incubation of the dye for a longer period could changes in metabolicand physiological status of the bacilli under investigation. Currentlyavailable assays are not capable enough to identify inhibitors of dormant stage mycobacteria. In one of our earlier attempts, Mycobacterium bovis BCG was used to develop a screening protocolwhich is convenient and applicable on dormant culture (Khan andSarkar,2008).Duetothehigherlevelofbasalnitratereduction,itsusein anti-tubercular screening remained limited to only M. bovis BCG.The present study described the application of tetrazolium salt(XTT) along with menadione to estimate viable bacilli during thescreening of chemical libraries. Due to the addition of menadione inthis assay, superoxide was generated in the bacilli which rapidlyreduced XTT to produce the color. Hence, XTT reduction assay wascarried out on bacilli obtained from Wayne's dormancy model to Journal of Microbiological Methods 84 (2011) 202 – 207 ⁎ Corresponding author. Tel.: +91 020 2590 2400; fax: +91 020 2590 2664. E-mail addresses:
[email protected] (U. Singh),
[email protected](S. Akhtar),
[email protected] (A. Mishra),
[email protected] (D. Sarkar). 1 Tel.: +91 020 2590 2447; fax: +91 020 2590 2664.0167-7012/$ – see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.mimet.2010.11.013 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth Author's personal copy develop a robust whole cell assay to screen anti-tubercular com-pounds against all possible physiological stages of bacilli (Wayne andHayes, 1996). However, the use of tetrazolium salts is a well knownmethod for analyzing M. tuberculosis strains while the addition of menadione is a new development in this context (Martin et al., 2007;Logu et al., 2001). 2. Materials and methods 2.1. Bacterial strains and growth conditions Inordertopreparethestockcultures, M. tuberculosis H37Ra(ATCC25177), M. bovis BCG (ATCC 35755) and Mycobacterium smegmatis mc 2 155 were grown to logarithmic phase (O.D. 595 ~1.0) in a de nedmedium ( M. pheli medium) following a described method earlier(Khan and Sarkar, 2008). The stock culture was maintained at − 70 °Cand sub-cultured once in M. pheli medium before inoculation intoexperimental culture. The bacterial culture was grown in M. pheli medium under aerobic conditions in a shaker incubator (ThermoElectronCorporationModel481)maintainedat150 rpmand37 °Ctilllogarithmic phase (O.D. 595 ~1.0) was reached. After growth, theculture was sonicated for 2 min using a water bath sonicator(BANDELYN electronic Model RK 1028c).For the cultivation of anaerobic dormant bacilli, Wayne 0.5 HSR model was followed wherein 20×125 mm tubes with 17 ml of themediumwhichwasinoculatedwith1%v/vofO.D. 595 ~1.0(WayneandHayes, 1996). In these tubes, 8 mm magnetic spin bars were alreadyplaced and gently stirred at 100 rpm on a magnetic stirrer. Aftermaking the tubes air tight with rubber septa, they were incubated at37 °Cfor12 daysincaseof M. tuberculosis and M. bovis BCGand5 daysfor M. smegmatis . Viable cells were counted from CFU methoddescribed earlier (Khan and Sarkar, 2008). For aerobic cells, 50 ml of M. pheli medium was inoculated with 1% v/v log phase culture of thebacilli. For initial optimization the cells were grown in identicalconditions as mentioned above for both aerobic and anaerobic bacilli.Generally, O.D. 470 ~0.25 cells were taken for initial optimizationunlessotherwisementioned.Dilutionsofthecellsweredonebyusingsterile M. pheli medium. 2.2. Drug and reagent preparation Rifampicin, Streptomycin, Ethambutol, Isoniazid, Pyrazinamide,O oxacin, p-Amino salicylic acid, Vancomycin, Clotrimazole andFluconazole were procured from Sigma. Drugs were solubilizedaccording to the manufacturer's recommendations and were storedin aliquots at − 20 °C. XTT sodium salt powder (Sigma) was preparedas a 1.25 mM stock solution in sterile 1× PBS which was stored at − 20 °C, for not more than 10 days. Menadione (Sigma) was preparedas a 6 mM solution in Dimethyl Sulfoxide (DMSO) and was stored atroom temperature for 2 weeks. 2.3. Reduction of 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide by M. smegmatis For optimization of XTT and menadione concentrations, cells weresonicated for 2 min and 250 μ l of cells was taken in sterile 96 wellplates. Cells in M. pheli medium with quadruplicate wells were usedfor each of the experimental conditions. Unless otherwise mentioned,XTT was added at 200 μ M nal concentration and incubated for20 min. At the end of 20 min incubation, different concentrations(30 μ M,60 μ Mand80 μ M)ofmenadionewereaddedandtheincreasein optical density at 470 nm was observed with continuous mixing atinterval of 30 seconds in Spectramax pro5 plate reader (MolecularDevices Inc). In blank wells, XTT and menadione were added in M. pheli medium without bacilli. 2.4. In vitro culture of aerobic and anaerobic cells in micro plate In order to avail of aerobic as well as anaerobic bacilli in microplate format, a modi ed method reported earlier was followed (Khanand Sarkar, 2008). Mycobacterium cells were mostly remainedaggregated under aerobic condition in the M. pheli medium. Beforeinoculation, the log phase cells were sonicated at 50 kHz for 2 min inwater bath sonicator. These sonicated cells were used for inoculationin microplatewells. 250 μ lofthe culturecontaining~10 5 cells/mlwasadded to each well of 96 well plates which maintained at 0.5 HSR. Airsupply of the culture in micro plate was blocked by applying microplate sealer (Nunc Inc.). Then, the plate was incubated in a CO 2 incubator at 37 °C. 2.5. Final XTT Reduction Menadione Assay protocol 2.5.1. M. tuberculosis and M. bovis BCG ForXRMAagainstaerobicculture,theplatesweretakenoutonthe8thdayofincubationtoremovethesealandtomeasuretheviablecellcounts. The optical density of the culture was measured beforeaddition of XTT at 470 nm which was served as a blank for the MICcalculationsusingEq.(1)(mentionedinSection2.4).200 μ MXTTwasadded and incubated for 20 min at 37 °C after shaking for 1 min. After20 min of incubation, 60 μ M menadione was added and incubated at37 °C for 40 min after mixing of 1 min. Finally, the optical density of the suspension was measured at 470 nm by using microplate reader.For XRMA against hypoxia induced dormant culture, the plateswere taken out on the 12th day of incubation to measure the viablecellcounts.Asmentionedearlier,opticaldensitywasmeasuredbeforeaddition of XTT at 470 nm and a similar method was repeated asmentioned above. 2.5.2. M. smegmatis For XRMA on the 3rd day of incubation, the microplate was takenout to remove the seal and measure the viable cells. Optical Densitywas measured before the addition of XTT at 470 nm. 200 μ M XTT wasadded and incubated for 20 min at 37°C after shaking for 1 min. After20 min incubation, 60 μ M menadione was added and mixed for 1 minand then incubated at 37 °C for another 20 min. The optical densitywas measured at 470 nm by using a micro plate reader. For Hypoxiainduced XTT reduction microplate assay (HXRMA) on the 7th day of incubation, the plates were taken out and the seal was removed. Asimilar protocol was repeated as mentioned for aerobically grown M. smegmatis . 2.6. Calculation for determination of Minimum Inhibitory Concentration The MIC was de ned as the lowest drug concentration effectinggrowth inhibition of ≥ 90% relative to the growth for the drug-freecontrols. The MICs were numerically extrapolated from transformedinhibition – concentration plots derived from the equation as follows% Inhibition = 100 − A1 − Blank ð Þ A2 − Blank ð Þ × 100 ð 1 Þ where,A1 Culture absorbance at 470 nm in the presence of the com-pound after addition of menadioneA2 Culture absorbance at 470 nm (DMSO solvent control) afteraddition of menadioneBlank Culture absorbance at 470 nm of the respective data pointsbefore addition of XTT/menadione.Brie y, the numeric approach uses Origin 6.1 software to calculatethe average difference in growth indicators (percent inhibition in the 203 U. Singh et al. / Journal of Microbiological Methods 84 (2011) 202 – 207 Author's personal copy concentration intervals between the test culture and the DMSOsolvent control culture and linear approximation of the concentra-tions effecting a 90% reduction). 3. Results The major obstacles in developing a simple protocol for drugscreening were the varying aggregation, generation time andmetabolic activity between replicate and dormant stage of differentmycobacterium bacilli. The screening protocol earlier developed byour group using M. bovis BCG clearly demonstrated the establishmentof culture conditions for both dormant and replicate stage of thebacilli. This microplate format provided a platform which could becoupledwithdifferentdetectiontechniquestoobtainbetterresults inthe screening of anti-mycobacterial compounds. In the present study,the assay was modi ed by using XTT to monitor the viability of thebacilli irrespective of its replicate or non-replicate stage. The watersolubilityandcolorimetricdetectionofXTTat470 nmhavealsomadethe method more convenient to rapidly carry out the screeningprogram (Logu et al., 2001; Kisaburo et al., 1989). The earlier reportsas well as our present study indicated that mycobacterium cultureswere very poor reducers of tetrazolium dyes (Fig. 1) (Martin et al., 2007; Logu et al., 2001). For this reason, we included menadione intothis assay to enhance XTT reduction, so that its detection could befaster and easier. Therefore, the effect of menadione was checked totest this hypothesis. 3.1. Menadione concentration Initially, a dose response experiment was carried out to nd thesensitivityofmenadionetowardsXTTreduction(datanotshown)and nally three different concentrations were selected for kinetic studies(Fig. 1). The kinetic data observed from XTT reduction using M.smegmatis cells indicated that maximum reduction was attainedwithin 30 min of incubation in the presence of 60 μ M menadione.Molar extinction coef cient ( ∆ ɛ ) of XTT reduction was around8.3×10 4 M − 1 S − 1 (Sutherland and Learmonth, 1997). In this exper-iment, it was observed that XTT reduction was directly proportionalto the concentration of menadione applied. The linearity of XTTreduction at lower concentrations of menadione was maintainedbeyond 30 min of incubation. The linearity of the same wasterminated within 10 min when 80 μ M of menadione was added. As60 μ M was the maximum concentration where the linearity wasmaintained up to a substantial period of time along with the ac-ceptable rate of XTT reduction, this concentration was accepted asconvenient for carrying out subsequent experiments. 3.2. Incubation time Even though the XTT reduction continued for 30 min, the resultindicated that it maintained the linearity till 20 min after the additionof menadione when M. smegmatis cell suspension was used (Fig. 2).Hence,incubationtimefor M.smegmatis was xedat20 min.Identicalprotocol was used to compare the kinetics of XTT reduction by M.tuberculosis and M. bovis BCG. The results also indicated that theextent of XTT reduction by adding menadione to the non-pathogenic M. smegmatis was~3.5×greaterthanboth M. tuberculosis and M. bovis BCG (Fig. 2). Therefore, reduction of XTT was signi cantly faster in M. smegmatis than M. tuberculosis and M. bovis BCG. Linearity wasgrossly maintained for M. bovis BCG and strictly for M. tuberculosis upto 45 min of incubation with menadione. Hence, the incubation timefor both M. tuberculosis and M. bovis BCG cells were xed at 40 minbefore read out at 470 nm. 3.3. Cell density for aerobic cultures Initially,aerobicculturesfrom M. smegmatis , M. tuberculosis and M.bovis BCGweretakenatdifferentconcentrationstocheckthelinearityof XTT reduction (Fig. 3). For M. smegmatis , the cell suspension wasincubated for 20 min whereas for M. tuberculosis and M. bovis BCG, itwas incubated for 40 min. The results indicated that all threemycobacterium cultures were showing linearity in XTT reductionwiththe increasein O.D 470 ofthe culture taken. The XTT reductions inall these cultures were directly proportional to the cell density. 3.4. Cell density for anaerobic cultures In order to understand the pattern of XTT reduction, anaerobiccultures of different mycobacterium bacilli grown in Wayne 0.5 HSR 0 5 10 15 20 25 300.00.51.01.52.02.53.03.5 X T T r e d u c t i o n ( 4 7 0 n m ) Time (min.) Fig. 1. Kinetics of XTT reduction in the presence of varying menadione concentrationson M. smegmatis . The cell suspensions with ~0.2 O.D 620 of density were separatelytreated with 30 μ M ( ● ), 60 μ M ( ■ ), 80 μ M ( ▲ ) and none ( ▼ ) of menadione along with200 μ M XTT. Kinetic studies of samples were done at 4 min of interval using microplatereader at O.D 470 . Further details of the experiment were mentioned in Section 2.Experiments were carried out 4 times and results are mean±standard deviation using8 identical wells of a microplate from each experiment. 0 10 20 30 40 50 600.00.51.01.52.02.53.0 X T T r e d u c t i o n ( 4 7 0 n m ) Time (min.) Fig. 2. The time kinetics of XTT reduction by different mycobacterial cells. M. smegmatis ( ■ ), M. tuberculosis ( ● ) and M. bovis BCG ( ▲ ) cell suspensions at ~0.25 O.D 620 wereincubated with 60 μ M menadione and 200 μ M XTT. Kinetic studies of samples weredone at interval of 4 min immediately after addition of menadione. Further details of theexperimentwerementionedinSection2.Experimentswerecarriedout4timesandresults are mean±standard deviation using 8 identical wells of a microplate from eachexperiment.204 U. Singh et al. / Journal of Microbiological Methods 84 (2011) 202 – 207 Author's personal copy modelwereimmediatelyusedinthesameassayprotocol(Fig.4). Ourunpublished observation suggested that these hypoxia induceddormant bacilli when exposed to air do not revert to metabolicallyactive stage at least within 2 h. Interestingly, though the linearity inXTT reduction was maintained in all the cultures used, the extent of reduction was signi cantly similar with their respective aerobiccultures. The extent of XTT reduction in all these cultureswas directlyproportional to the culture density. 3.5. Z ′ factor and statistical analysis for quality assessment S/N ratio and Z ′ factor were the two essential components con-sidered during the development of a robust assay protocol for rapidscreening of compounds (Zhang et al., 1999). The S/N ratio wascalculated for M. smegmatis cells using the optimized assay protocol,werefoundtovarybetween8.0and10.0.TheZ ′ valuesfor XRMA werefound to be ~0.9 for the whole range of cell density evaluated(Table1). The determined S/N ratioswere N 6.0 for M. tuberculosis and N 5.0 for M. bovis BCG respectively. The Z ′ factor values obtained fromsimilar experiments using M. tuberculosis and M. bovis BCG werevarying around 0.80 and 0.90 respectively. The S/N ratio and Z ′ factorvalues obtainedfrom the experiments using the optimizedconditionsindicated that the protocol was robust for carrying out highthroughput screening of the compound library. 3.6. Validation with standard inhibitors The MIC values were determined by using the calculationsmentionedinEq.(1).10antimycobacterialagentsincludingRifampin,Streptomycin, Isoniazid, Ethambutol and Pyrazinamide were used todetermie the MIC values using this protocol. All these inhibitors arespeci c to the aerobic stage of the bacilli. The major obstacle forvalidation with anaerobic cells is the unavailability of latent stagespeci c inhibitors in the literature. Brie y, 2.5 μ l of these inhibitorsolutions were added in a total volume of 250 μ l of M. pheli mediumconsisting of bacilli, XTT and menadione. The incubation wasterminated on the 4th and 8th day for M. smegmatis and M.tuberculosis / M. bovis BCG cultures respectively. The XRMA was thencarried out to estimate viable cells present in different wells of theassay plate. Growth inhibition of the bacilli was calculated byfollowing Eq. (1) (Section 2.6). The concentration of inhibitor at which N 90% inhibition of growth was seen was considered as the MICvalue of the respective anti-tubercular compound. The calculated MICvalueswerefoundtobefairlyconsistentwiththereportedMICvaluesusing CFU, REMA and NR assays (Taneja and Tyagi, 2007; Khan andSarkar, 2008). 4. Discussion In conclusion, we developed and validated a robust assay protocolbasedonthereductionofXTTthatallowsrapidscreeningofinhibitorsagainst mycobacterial cultures (Table 2). Dye-based protocols took atleast 12 h to produce a minimum signal which makes it little dif cultto apply on bacilli particularly from anaerobic cultures (Taneja andTyagi, 2007; Collins and Franzblau, 1997; Palomino et al., 2007). Itbecomes evenmore dif cult to apply these protocolson M. smegmatis because most of the cells multiply during incubation as well.Generally, reduction of XTT in mycobacterial culture is a very slowprocess and hence, the application of menadione has increased theXTT reduction to such an extent that the assay incubation takes only20 minfor M.smegmatis while40 minfor M. tuberculosis / M. bovis BCG 0.05 0.10 0.15 0.20 0.25 0.300.00.51.01.52.02.53.0 Cell density (470 nm) X T T r e d u c t i o n ( 4 7 0 n m ) Fig. 3. XTT reduction as a function of cell density for aerobically grown M. smegmatis , M.tuberculosis and M. bovis BCG. Aerobic cell suspensions of bacilli were incubated with200 μ M ofXTT and 60 μ M of menadione to measure the XTT reduction. M. smegmatis ( ■ )were incubated for 20min whereas M. tuberculosis ( ● ) and M. bovis BCG ( ▲ ) wereincubatedfor40min afteraddition of menadione.Furtherdetailsoftheexperimentwerementioned in Section 2. Experiments were carried out 4 times and results are mean±standard deviation using 8 identical wells of a microplate from each experiment. 0.00.51.01.52.02.53.0 X T T r e d u c t i o n ( 4 7 0 n m ) Cell density (470 nm) 0.05 0.10 0.15 0.20 0.25 0.30 Fig. 4. XTT reduction as a function of cell density for anaerobic M. smegmatis , M.tuberculosis and M. bovis BCG. Anaerobic cellsuspensions of bacilli wereincubated with200 μ M of XTT and 60 μ M of menadione to measure the XTT reduction at 470 nm. M.smegmatis ( ■ )wereincubated for20 minwhereas M. tuberculosis ( ● )and M. bovis BCG( ▲ ) were incubated for 40 min after addition of menadione as described in Fig. 3.Further details of the experiment were mentioned in Section 2. Experiments werecarried out 4 times and results are mean±standard deviation using 8 identical wells of a microplate from each experiment. Table 1 S/N ratio and Z factor for M. smegmatis — varying cell concentrations of M. smegmatis were incubated with 200 μ M of XTT followed by addition of 60 μ M menadione. The S/Nratio and Z ′ factors of M. smegmatis cells were determined using the data points of 20 min after addition of menadione. Experiments were carried out 4 times and resultsare mean±standard deviation using 8 identical wells of a microplate from eachexperiment.S no. Cell O.D S/N ratio ⁎ Z ′ factor ⁎⁎ 1. 0.07 9.4±0.049 0.95±0.0492. 0.11 9.7±0.072 0.96±0.1203. 0.15 9.3±0.054 0.85±0.0544. 0.20 9.5±0.096 0.86±0.0965. 0.25 9.3±0.026 0.76±0.0266. 0.31 9.0±0.035 0.67±0.035 ⁎ S/Nratioistheratiobetween control andblank(wheremenadione isnot added)O.D 470 at a particular time point. ⁎⁎ Z ′ factor=1 − (3×standard deviation of control+3×standard deviation of blank)/(mean value of control − mean value of blank).205 U. Singh et al. / Journal of Microbiological Methods 84 (2011) 202 – 207
