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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 1  |  Page : 34-38

Evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assays


Department of Biochemistry, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, India

Date of Submission18-Dec-2019
Date of Acceptance31-Dec-2019
Date of Web Publication6-Mar-2020

Correspondence Address:
Nirmala Deo
Department of Biochemistry, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra - 282 004, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_194_19

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  Abstract 


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.

Keywords: Ciprofloxacin and carbonyl cyanide m-chloro phenyl-hydrazone, levofloxacin, mycobacteria, norfloxacin, ofloxacin


How to cite this article:
Deo N, Sharma D, Bisht D, Venkatesan K. Evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assays. Int J Mycobacteriol 2020;9:34-8

How to cite this URL:
Deo N, Sharma D, Bisht D, Venkatesan K. Evaluation of the intracellular accumulation of fluoroquinolones in mycobacteria by fluorometric assays. Int J Mycobacteriol [serial online] 2020 [cited 2020 Apr 10];9:34-8. Available from: http://www.ijmyco.org/text.asp?2020/9/1/34/280148




  Introduction Top


The mycobacterial cell wall has low permeability and that this reduced permeability plays a major role in the intrinsic resistance of mycobacteria to most antibiotics.[1],[2],[3] Very few antimicrobial agents are effective against Mycobacterium tuberculosis and even fewer are effective against atypical mycobacteria, such as Mycobacterium avium complex. Tuberculosis (TB) is one of the major causes of death worldwide. There are relatively few antimicrobial agents that are clinically active against M. tuberculosis.[4] Prolonged antibiotic treatment is also required due to the ability of the bacterium to enter a dormant, antibiotic-resistant phase. Fluoroquinolones (FQs) exhibit potentin vitro andin vivo antimycobacterial activity against several mycobacteria.[5],[6] They are being used as second-line agents in the treatment of TB caused by multi drug-resistant strains. Most strains of M. tuberculosis, Mycobacterium leprae, Mycobacterium bovis, Mycobacterium kansasii, Mycobacterium marinum, and Mycobacterium xenopi are inhibitedin vitro by concentrations of ciprofloxacin (CIP) or ofloxacin (OFX) ranging between 0.5 mg/L and 2.0 mg/L.[7],[8] The target of FQs is they inhibit the topoisomerase II enzyme DNA gyrase, which catalyzes the unwinding or negative supercoiling of double-stranded DNA during replication.[9] Topoisomerase IV is encoded by parC and parE is proposed as a secondary target of FQs in  Escherichia More Details coli when a susceptible DNA gyrase is lacking.[10],[11] To exert their antibacterial effect, the FQs must cross the bacterial cell wall as DNA gyrase is intracellular. The role of permeability exerts a major role in the accumulation of fluoroquinolones in mycobacteria.[12] Ofloxacin (OFX) is being tried as a part of modified multidrug therapy regimens for leprosy. A preliminary study was carried out to estimate the level of intracellular accumulation of FQs – OFX, levofloxacin (LFX), norfloxacin (NFX), and ciprofloxacin (CIF) in Mycobacterium smegmatis allowing its uptake to be evaluated by fluorometric assays. M. smegmatis was chosen because it is a fast-growing mycobacterium which produces homogeneous suspensions after culture in the solid medium[13] more easily than other species. The general chemical model of the cell envelope of mycobacteria proposed by Minnikin applies to M. smegmatis and to most mycobacteria studied.[14] Moreover, M. smegmatis has also been used recently as a model for studies of mycobacterial genetics.[15] The purpose of the present study was to measure the intracellular levels of the accumulation of OFX, LFX, NFX, and CIF in M. smegmatis to determine whether the methods commonly applied to other bacteria can be used to evaluate quinolone accumulation in mycobacteria.[16]


  Materials and Methods Top


Antibiotics and chemicals

  • FQs (OFX, LFX, NFX and CIF from Sigma)
  • CCCP (carbonyl cyanide m-chlorophenyl-hydrazone, Sigma)
  • 50 mM Sodium Phosphate Buffer (pH 7) (Merck)
  • M Glycine HCl (Sigma)
  • Distilled water Central Drug House (CDH).


Bacterial strain

Standard strain of M. smegmatis (ATCC-607) which is sensitive for OFX, LFX, NFX, and CIF was obtained from Mycobacterial Repository Centre, National JALMA Institute for Leprosy, and Other Mycobacterial Diseases, Agra, India.

Method of the accumulation of ofloxacin, levofloxacin, norfloxacin, and ciprofloxacin in Mycobacterium smegmatis

The method employed to determine the accumulation of either OFX, LFX, NFX, or CIF was the one established by Williams et al.[17] for NFX accumulation in M. smegmatis and Mycobacterium aurum. In brief, M. smegmatis were grown in Sauton's medium till the log phase, harvested and resuspended in phosphate buffer (0.1M, pH 7.2, OD of 0.4–0.5) and incubated with OFX, LFX, NFX and CIF (10 μg/ml) at 37°C. One milliliter aliquots were drawn at different time intervals (in min) for about 20 min, centrifuged and the drugs were estimated in the supernatants using spectrofluorimeteric methods. The experiments were also conducted with the addition of CCCP, a proton motif force inhibitor, at 100 μM, 10 min before and/or immediately after the addition of the drugs.

Cell dry weight

For each set of experiments aliquots of 1 ml/each of M. smegmatis suspension used for incubation studies were taken in 3 Eppendorf tubes. Cells were centrifuged at 3003 × g for 15 min at 4°C. Cell pellets were washed with 10 ml of sterile distilled water and again centrifuged at 3003 × g for 15 min at 4°C. Cell pellets were dried overnight at 60°C in a hot air oven and weighed. The mean dry weight was recorded.


  Results Top


Accumulation of ofloxacin, levofloxacin, norfloxacin, ciprofloxacin

For better fluorescence response, optimum concentration for accumulation studies was 10 μg/ml. The data on OFX accumulation kinetics are presented in [Table 1] and [Figure 1]. For LFX the accumulation kinetics are presented in [Table 2] and [Figure 2]. For NFX accumulation, kinetics are presented in [Table 3] and [Figure 3] and the data for CIF accumulation kinetics are presented in [Table 4] and [Figure 4].
Table 1: Accumulation of ofloxacin in Mycobacterium smegmatis

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Figure 1: Accumulation of ofloxacin in Mycobacterium smegmatis (ng/mg dry cell weight)

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Table 2: Accumulation of levofloxacin in Mycobacterium smegmatis

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Figure 2: Accumulation of levofloxacin Mycobacterium smegmatis(ng/mg dry cell weight)

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Table 3: Accumulation of norfloxacin in Mycobacterium smegmatis

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Figure 3: Accumulation of norfloxacin in Mycobacterium smegmatis (ng/mg dry cell weight)

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Table 4: Accumulation of ciprofloxacin in Mycobacterium smegmatis

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Figure 4: Accumulation of ciprofloxacin in Mycobacterium smegmatis(ng/mg dry cell weight)

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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 cells. LFX accumulation attained a steady state (SSC level of 90 ng/mg) in 5 min. NFX accumulation attained a steady state (SSC level of 87 ng/mg) in 3 min. CIF accumulation attained a steady state (SSC level of 79 ng/mg) in 4 min. The data are shown in [Table 5].
Table 5: Summary of accumulation of fluroquinolones in Mycobacterium smegmatis (conc 10 μg/ml). The addition of carbonyl cyanide m-chlorophenyl hydrazine failed to influence the accumulation of either of these quinolones

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The addition of CCCP (100 μM) 10 min before the addition of OFX, LFX, norfloxacin, CIF in respective experiments did not influence the accumulation kinetics to a large extent. Details of the data interpretation have been shown in the supplementary file.


  Discussion Top


FQs are broad-spectrum antimicrobial agents having high activity against Gram-negative aerobes.[18],[19],[20] Because of spectrum and physicochemical properties of fluoroquinolones, they are the most useful class of antimicrobials today in human and animal medicine, due to which there is an increase in the popularity in clinical situations.[21] There is a promising group of antimicrobial agents (FQs) by which they have become popular during the last decade as alternatives to treat bacterial infections which are resistant to aminoglycosides, third-generation cephalosporins, extended-spectrum penicillins, other β-lactam antibiotics, folic acid antagonists, and macrolides.[22],[23] They also show high tissue and intracellular distribution.[24],[25] They are highly effective at extremely low concentrations compared to other groups of antibacterial agents. The minimum inhibitory concentration (MIC) values of FQs range from 0.001 to 1.0 μg/ml against most of the susceptible microbes.[20] One of the unique advantages of these antimicrobialsis prolonged postantibiotic effect of FQs against some of the susceptible microorganisms. They act on bacterial DNA gyrase which is an intracellular enzyme. FQs enter the bacteria through pores[26] and rapidly accumulate within the bacteria with the SSCs reaching within a few minutes.[27] By action on DNA gyrase, they inhibit the replication of bacteria. The binding of FQs is of cooperative manner to a pocket of single-strand DNA created by DNA gyrase by electrostatic interaction. This suggests that the binding kinetics of these antimicrobials have four molecules that can stereochemically fit into the pocket to inhibit DNA gyrase.[28] Type II isomerase plays an important role in unwinding, cutting, and consecutive resealing of DNA. The inhibition of resealing leads to the liberation of fragments which subsequently are destroyed by bacterial exonucleases.[29],[30] The resistance genes targeting the FQs are found on the bacterial genome and plasmid-mediated resistance against them have not been documented.[31]

FQs exhibit potentin vitro andin vivo antimycobacterial activity.[5],[6] There is a significant effort to include FQs as new front-line agents, CIP, OFX, and moxiffloxacin as second-line agents.[32],[33],[34],[35] There is also a considerable effort in the industry to discover and develop newer FQs, and some of them might have value in the treatment of TB. However, the choice of ffluoroquinolone for the treatment of TB is dictated by its efficacy with murine models, and as a consequence, only a very few members of the class are tested due to the complexities of the animal models for TB. Mycobacterial cell wall consists nearly 40% of lipids by dry weight and has been used in the chemotaxonomy and pathogenesis.[36] The permeability varies greatly in hydrophilic agents. The permeability of Mycobacterium chelonae to β-lactams is about three orders of magnitude lower than that of the E. coli outer membrane and ten times lower than the permeability of Pseudomonas aeruginosa.[1] It is proposed that the uptake of quinolones by Gram-negative bacteria is by passive diffusion through porin channels or the lipid bilayer, depending on the hydrophobicity of the quinolone,[37] whereas in Gram-positive bacteria, it occurs through simple diffusion across the cytoplasmic membrane.[38] Norffloxacin is a very hydrophilic quinolone, with a partition coefficient in n-octanol–phosphate buffer (pH 7.2) of 0.022.[39] Norffloxacin accumulation by M. aurum was nonsaturable over the concentration range studied (0–100 mg/ml), and transport was apparently unaffected by the presence of CCCP. Therefore, it is proposed that norffloxacin enters M. aurum by an energy-independent, nonsaturable porin pathway. Mycobacterial porins have been characterized in M. chelonae and M. smegmatis and are thought to exist in all mycobacterial species, but they are proposed to be both less abundant and less efficient than other bacterial porins.[40],[41] OFX is a second-generation fluoroquinolone, being a broader-spectrum analog of norfloxacin. The uptake could be related to an amino acid transport system.[42] LFX has an extended spectrum of activity compared with older-generation FQs (CIF, OFX), with improved activity against Gram-positive bacteria and excellent activity against Gram-negative bacteria and atypical organisms. However, at least for OFX, LFX, norffloxacin, and CIF, the mycobacterial cell wall does not act as a significant barrier to drug accumulation.

This study shows that the level of the accumulation of OFX, LFX, NFX, and CIF in M. smegmatis. The SSCs of norffloxacin in M. smegmatis mc2155 reported by Liu et al.[43] were similar to those obtained in the current study. However, Corti et al.[16] reported an SSC of 35 ng of norffloxacin/mg of cells for a different strain of M. smegmatis (strain CNCM 7326; MIC,4 or 5 mg/ml). Kocagöz et al.[44] reported an SSC of 110 pmol of norffloxacin/mg of cells (approximately 35 ng of norffloxacin/mg of cells) for M. tuberculosis H37 Ra, suggesting that M. tuberculosis is less permeable to norffloxacin than M. aurum and M. smegmatis. The SSC of OFX in M. smegmatis was 3 min and the level of accumulation was 102 ng/mg dry cells. LFX accumulation attained a steady state (SSC level of 90 ng/mg) in 5 min. NFX accumulation attained a steady state (SSC level of 87 ng/mg) in 3 min. CIF accumulation attained a steady state (SSC level of 79 ng/mg) in 4 min. Clearly, the permeability of mycobacteria to OFX, LFX, norffloxacin, and CIF and presumably other agents, varies not only between mycobacterial species but also between different strains of the same species.


  Conclusion Top


OFX, LFX, NFX, and CIF appear to be transported across the cell envelopes of M. smegmatis strain used in the study by simple diffusion. The lack of any effect on the intracellular drug levels by the added energy inhibitor suggests noninvolvement of efflux pumps in the transport of these quinolones in these drug-sensitive strains. Further studies are required with quinolones-resistant strains.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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