Evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assays
Background: Fluoroquinolones (FQs) are being used as second-line agents in the treatment of tuberculosis caused by multidrug-resistant strains. Ofloxacin (OFX) is being tried as a part of modified multidrug therapy regimens for leprosy. A preliminary study was carried out to evaluate the accumulatio...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wolters Kluwer Medknow Publications
2020-01-01
|
| Series: | International Journal of Mycobacteriology |
| Subjects: | |
| Online Access: | http://www.ijmyco.org/article.asp?issn=2212-5531;year=2020;volume=9;issue=1;spage=34;epage=38;aulast=Deo |
| _version_ | 1811222040658575360 |
|---|---|
| author | Nirmala Deo Divakar Sharma Deepa Bisht Krishnamurthy Venkatesan |
| author_facet | Nirmala Deo Divakar Sharma Deepa Bisht Krishnamurthy Venkatesan |
| author_sort | Nirmala Deo |
| collection | DOAJ |
| description | Background: Fluoroquinolones (FQs) are being used as second-line agents in the treatment of tuberculosis caused by multidrug-resistant strains. Ofloxacin (OFX) is being tried as a part of modified multidrug therapy regimens for leprosy. A preliminary study was carried out to evaluate the accumulation of FQs – OFX, levofloxacin (LFX), norfloxacin (NFX), and ciprofloxacin (CIF) in Mycobacterium smegmatis. Methods: M. smegmatis were grown in Sauton's medium till log phase, harvested and resuspended in phosphate buffer (0.1 M, pH 7.2, Optical Density (OD) of 0.4–0.5) The suspensions were incubated with OFX, LFX, NFX, and CIF (10 μg/ml) at 37°C. The drugs were estimated in the supernatants using spectrofluorimeteric methods. The experiments were also conducted with the addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a proton motive force inhibitor, at 100 μM, 10 min before and/or immediately after the addition of the drugs. Results: The time taken to achieve a Steady State Concentration (SSC) of OFX in M. smegmatis was 3 min and the level of accumulation was 102 ng/mg dry weight of the bacilli; with LFX the time for SSC was 5 min and the level of accumulation was 90 ng/mg; in case of NFX the accumulation to SSC was 87 ng/mg in 3 min. CIF accumulation attained a steady state (SSC level of 79 ng/mg) in 4 min. The accumulation kinetics for NFX in M. smegmatis using the spectrofluorimetric method is comparable with radioactive assays. Dose-related accumulation was observed with 10 μg/ml exposure concentrations. The addition of CCCP failed to influence the accumulation of each of these quinolones. Conclusion: The findings of dose-related accumulation of OFX, LFX NFX, and CIF suggest simple diffusion as the possible mechanism of transport of these drugs. |
| first_indexed | 2024-04-12T08:09:52Z |
| format | Article |
| id | doaj.art-2f2e16d4d7514a128c70521c91ae08b7 |
| institution | Directory Open Access Journal |
| issn | 2212-5531 2212-554X |
| language | English |
| last_indexed | 2024-04-12T08:09:52Z |
| publishDate | 2020-01-01 |
| publisher | Wolters Kluwer Medknow Publications |
| record_format | Article |
| series | International Journal of Mycobacteriology |
| spelling | doaj.art-2f2e16d4d7514a128c70521c91ae08b72022-12-22T03:41:01ZengWolters Kluwer Medknow PublicationsInternational Journal of Mycobacteriology2212-55312212-554X2020-01-0191343810.4103/ijmy.ijmy_194_19Evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assaysNirmala DeoDivakar SharmaDeepa BishtKrishnamurthy VenkatesanBackground: Fluoroquinolones (FQs) are being used as second-line agents in the treatment of tuberculosis caused by multidrug-resistant strains. Ofloxacin (OFX) is being tried as a part of modified multidrug therapy regimens for leprosy. A preliminary study was carried out to evaluate the accumulation of FQs – OFX, levofloxacin (LFX), norfloxacin (NFX), and ciprofloxacin (CIF) in Mycobacterium smegmatis. Methods: M. smegmatis were grown in Sauton's medium till log phase, harvested and resuspended in phosphate buffer (0.1 M, pH 7.2, Optical Density (OD) of 0.4–0.5) The suspensions were incubated with OFX, LFX, NFX, and CIF (10 μg/ml) at 37°C. The drugs were estimated in the supernatants using spectrofluorimeteric methods. The experiments were also conducted with the addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a proton motive force inhibitor, at 100 μM, 10 min before and/or immediately after the addition of the drugs. Results: The time taken to achieve a Steady State Concentration (SSC) of OFX in M. smegmatis was 3 min and the level of accumulation was 102 ng/mg dry weight of the bacilli; with LFX the time for SSC was 5 min and the level of accumulation was 90 ng/mg; in case of NFX the accumulation to SSC was 87 ng/mg in 3 min. CIF accumulation attained a steady state (SSC level of 79 ng/mg) in 4 min. The accumulation kinetics for NFX in M. smegmatis using the spectrofluorimetric method is comparable with radioactive assays. Dose-related accumulation was observed with 10 μg/ml exposure concentrations. The addition of CCCP failed to influence the accumulation of each of these quinolones. Conclusion: The findings of dose-related accumulation of OFX, LFX NFX, and CIF suggest simple diffusion as the possible mechanism of transport of these drugs.http://www.ijmyco.org/article.asp?issn=2212-5531;year=2020;volume=9;issue=1;spage=34;epage=38;aulast=Deociprofloxacin and carbonyl cyanide m-chloro phenyl-hydrazonelevofloxacinmycobacterianorfloxacinofloxacin |
| spellingShingle | Nirmala Deo Divakar Sharma Deepa Bisht Krishnamurthy Venkatesan Evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assays International Journal of Mycobacteriology ciprofloxacin and carbonyl cyanide m-chloro phenyl-hydrazone levofloxacin mycobacteria norfloxacin ofloxacin |
| title | Evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assays |
| title_full | Evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assays |
| title_fullStr | Evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assays |
| title_full_unstemmed | Evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assays |
| title_short | Evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assays |
| title_sort | evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assays |
| topic | ciprofloxacin and carbonyl cyanide m-chloro phenyl-hydrazone levofloxacin mycobacteria norfloxacin ofloxacin |
| url | http://www.ijmyco.org/article.asp?issn=2212-5531;year=2020;volume=9;issue=1;spage=34;epage=38;aulast=Deo |
| work_keys_str_mv | AT nirmaladeo evaluationoftheintracellularaccumulationoffluoroquinolonesinmycobacteriabyfluorometricassays AT divakarsharma evaluationoftheintracellularaccumulationoffluoroquinolonesinmycobacteriabyfluorometricassays AT deepabisht evaluationoftheintracellularaccumulationoffluoroquinolonesinmycobacteriabyfluorometricassays AT krishnamurthyvenkatesan evaluationoftheintracellularaccumulationoffluoroquinolonesinmycobacteriabyfluorometricassays |