Dipartimento di Scienze Biomediche, sez. Microbiologia, Università degli Studi di Trieste, Trieste, Italy
Received 30 July 2002; returned 6 February 2003; revised 21 February 2003; accepted 9 August 2003
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Abstract |
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Keywords: M. tuberculosis, resazurin, MIC evaluation, microdilution assay, first-line antitubercular drugs
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Introduction |
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The aim of this study was to develop a new, simple and rapid microdilution plate method, employing resazurin to evaluate the MICs of the first-line antitubercular drugs isoniazid, rifampicin, ethambutol and streptomycin for M. tuberculosis reference and clinical strains. Resazurin is an oxidationreduction indicator used for the evaluation of cell proliferation and microbial growth.16,17 It is a blue non-fluorescent dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells. A resazurin reduction test has also been used for decades to demonstrate bacterial and yeast contamination of milk.18,19
We investigated the growth conditions of different M. tuberculosis strains, the growth incubation time of each culture, the concentration of mycobacterium inoculum, and the incubation time of the microplate before and after resazurin addition. The results obtained were compared with those obtained by means of the reference agar dilution method; the accuracy of the method was verified by viable counting from the test wells.
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Materials and methods |
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Thirteen M. tuberculosis strains were clinical isolates cultured from specimens submitted to the Modulo di Microbiologia Polmonare, Ospedale di Cattinara, Trieste, Italy, for investigation of mycobacterial disease, and consisted of respiratory isolates from different patients. Strains were identified by classical culture and biochemical characteristics and by DNA probe (GeneProbe, San Diego, CA, USA); M. tuberculosis reference strain H37Rv was also employed. All strains were maintained on LowensteinJensen medium and grown for 728 days in Middlebrook 7H9 broth, supplemented with 10% OADC, 0.2% glycerol and 0.1% Bacto Casitone (Difco). Stock cultures containing 1 x 107 cfu/mL of each mycobacterium strain were saved frozen at 80°C, thawed when required to perform the test and grown for 2 days in complete 7H9 medium. The cultures obtained were vortexed, large agglomerates allowed to sediment completely and the supernatant further diluted 1:5, 1:50 and 1:500 in complete 7H9 broth. Titres were determined by viable counting on 7H11 agar plates, giving 0.51 x 104, 0.51 x 103 and 0.51 x 102 cfu/mL, respectively. These strain dilutions were used as inoculum in the microdilution plate assay. Alternatively to frozen stock cultures, suspensions from LowensteinJensen slants in complete 7H9 broth were vortexed, adjusted to a turbidity equivalent to that of a 0.5 McFarland standard, diluted as described previously and used as the inoculum in the microdilution plate assay.
Chemicals
Isoniazid, rifampicin, streptomycin sulphate and ethambutol hydrochloride were obtained from SigmaAldrich. Solutions were prepared in sterile water, except for rifampicin, which was diluted in dimethyl sulphoxide, to obtain 10 mg/L stock solutions. Resazurin was obtained from SigmaAldrich and prepared as 10 g/L sterile water stock solution, saved frozen at 20°C, thawed and diluted 1:10 in sterile water when required.
Agar proportion test
The agar proportion susceptibility test was performed according to established procedures20 in Middlebrook 7H11 medium. Briefly, the test was performed in quadrant Petri plates, drugs were diluted in order to obtain 0.2 and 1 g/L isoniazid, 1 mg/L rifampicin, 7.5 mg/L ethambutol, and 2 and 10 mg/L streptomycin; drug-free controls were included. Each strain was classified as susceptible to a drug if the number of colonies that grew on the drug-containing medium was <1% of the number of colonies grown on the control plate and resistant if the number of colonies was >10%. In cases where two drug concentrations were tested in the agar proportion method, a strain was classified as intermediate if it showed resistance to the lower drug concentration but was susceptible to the higher drug concentration.
Microdilution Resazurin Assay (MRA)
The microdilution test was performed in 96-well plates. Two-fold dilutions of each drug were prepared in the test wells in complete 7H9 broth, the final drug concentrations being: isoniazid 1280.00625 mg/L, rifampicin 1280.0625 mg/L, streptomycin 1280.125 mg/L and ethambutol 1280.25 mg/L. Twenty microlitres of each bacterial suspension was added to 180 µL of drug-containing culture medium. Control wells were prepared with culture medium only and bacterial suspension only. The plates were sealed and incubated for 714 days at 37°C. After each incubation time, 5 µL of resazurin solution were added per well, colouring them blue. Plates were incubated at 37°C for additional 24, 48 or 72 h. After each incubation time intervals plates were read for colour change from blue to pink in live mycobacterium-containing wells. MIC was defined as the lowest drug concentration that prevented resazurin colour change from blue to pink. Each MIC was determined three times in duplicate experiments. Viable counting from control wells and from test wells was performed onto 7H11 agar plates.
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Results |
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Different growth conditions of M. tuberculosis H37Rv were evaluated in the MRA. In preliminary experiments, no difference in mycobacterial growth and killing was observed after 714 days of plate incubation; a 7 day incubation time was therefore selected for all further experiments. Further reading of resazurin colour change in mycobacterium-containing wells gave the same results after an additional 72 h incubation as after 48 h incubation; 48 h was therefore always selected as evaluation end time. Visual observation of colour change from blue to pink to assess MICs was adequate, as confirmed by viable counting; results were always assessed without the need for instrumentation for photometric or fluorimetric reading.19
The MICs of the drugs obtained by MRA are given in Table 1, employing bacterial suspensions prepared at different dilutions obtained either from LowensteinJensen slants or from complete 7H9 broth cultures. The results obtained using suspensions from LowensteinJensen medium as the inoculum at concentrations of 0.51 x 104 cfu/mL indicated that the range of MICs was no more than one two-fold dilution for isoniazid, rifampicin and ethambutol. For streptomycin, a wider MIC range was determined, ranging from 0.125 to 1 mg/L. Results obtained using suspensions from 7H9 broth as the inoculum at concentrations of 0.51 x 104 and 0.51 x 103 cfu/mL indicate a limited or absent variability in MICs of all drugs, except for streptomycin. A 10-fold dilution of bacterial suspension, corresponding to 0.51 x 102 cfu/mL, always determined resazurin colour change from blue to pink only after additional 5 days incubation of the micro-plate.
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Table 3 reports the MICs obtained for isoniazid, rifampicin, streptomycin and ethambutol for M. tuberculosis strain H37Rv by the agar proportion susceptibility test performed in 7H11 agar, and the MICs obtained by MRA. A 14-day-old (21-day-old to evaluate streptomycin) M. tuberculosis H37Rv culture, diluted 1:50 and containing 0.51 x 104 cfu/mL live mycobacterium, was employed in MRA. The microplate was incubated for 7 days and read for colour change after addition of resazurin and an additional 24 h incubation time. MICs obtained by MRA were one two-fold dilution lower than MICs obtained with the agar proportion test. Viable counting performed on blue-coloured wells showed 99100% inhibition.
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The MICs of isoniazid, rifampicin, streptomycin and ethambutol for 13 M. tuberculosis clinical strains as measured by MRA are reported in Table 4, together with the results obtained by agar proportion test. The assay was performed as indicated for M. tuberculosis H37Rv reference strain; each determination was obtained in duplicate experiments. Five clinical strains out of the 13 tested were multidrug-resistant according to clinical laboratory indications and the agar proportion test. Three of these strains, H160, H190 and H320, were resistant to all the drugs tested; one strain, H200, was resistant to isoniazid and rifampicin; and one strain, H231, was resistant to isoniazid, streptomycin and ethambutol according to the agar proportion test. The MICs of clinical strains determined by MRA were lower than the critical concentration employed in the agar proportion method, as indicated, ranging from 0.0125 to 0.05 mg/L for isoniazid, from 0.0625 to 0.125 mg/L for rifampicin, from 0.25 to 1 mg/L for streptomycin, and from 1 to 4 mg/L for ethambutol. The MICs obtained on the multidrug-resistant strains confirmed resistance to all drugs; strain H320 showed an intermediate susceptibility to streptomycin, as did strains H260 and H310.
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Discussion |
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The streptomycin MICs depended strictly on the growth phases of the mycobacteria, as shown in Table 2, owing to the fact that both the transport of streptomycin across the bacterial walls and its binding to the 30S ribosomal subunit are energy-dependent processes.21 The low metabolic activity of mycobacteria during their very early growth and the slowing metabolic activity of mycobacteria during the stationary phase of growth may limit the entrance of streptomycin into bacteria, resulting in higher MICs. Both the growth incubation time and the inoculum size of the culture have been standardized to allow correct determination of streptomycin MICs.
The resazurin microdilution plate method, MRA, proved to be a reliable and reproducible assay. As shown in Table 3, MRA gave good, reproducible results compared with those obtained by the reference agar proportion method; results were obtained after 89 days incubation, with convenient shortening of the evaluation time. MRA performed on clinical strains (Table 4) again gave good, reproducible results, including when employing inoculum suspensions prepared directly from LowensteinJensen slants. It is noteworthy that MIC determinations for isoniazid, rifampicin and ethambutol were always scarcely affected by culture conditions and inoculum size.
Different methods have been described to detect antibiotic resistance of M. tuberculosis complex clinical strains, some of which employed colour development like the Alamar Blue assay,11,12,22 which is a proprietary reagent that requires stabilizing agents, or tetrazolium salt assay,23 which requires lysing buffer for colour development. Both tests employed solutions containing either Tween 80, ethanol, formamide or SDS, which are foaming agents that do not ensure complete safety when testing mycobacterium, and could furthermore interfere with mycobacterium viability. The MRA method proved to be a safe, rapid and reliable assay; the resazurin solution we employed in the microplate test MRA did not need stabilizing agents, its stability being always ensured by diluting the frozen stock solution when needed for the test.
New candidate drugs are needed to treat multidrug-resistant tubercular bacilli, and it is therefore necessary to have a screening assay to evaluate the mycobacterium-inhibiting activity of new chemicals. In a preliminary series of experiments, we have evaluated using MRA the MICs of a number of new molecules24 that have already been described for their interesting in vitro antitubercular activity.25 Resazurin was employed as an oxidationreduction indicator at a final concentration of 25 mg/L19 and no interaction with drugs or any other chemical was ever detected. Intermediate-coloured wells have sometimes been observed after 48 h incubation in the presence of resazurin when evaluating multidrug-resistant strains; prolonged incubation up to 72 h resulted in intermediate-coloured wells changing to pink; therefore, intermediate colours obtained after 48 h can be consequently interpreted as positive growth, as was also determined by viable counting. Visual observation of colour change from blue to pink was adequate: results were always assessed without instrumentation for photometric or fluorimetric reading.
In conclusion, MRA is a rapid, inexpensive, low technology procedure, suitable for susceptibility testing of first- and second-line antitubercular drugs and for screening new antitubercular compounds against M. tuberculosis clinical strains. The interesting results obtained prompt us to evaluate by means of MRA the drug susceptibility of different Mycobacterium species, including Mycobacterium avium.
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Acknowledgements |
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Footnotes |
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References |
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