Preliminary evaluation of anti-tuberculosis potential of siderophores against drug-resistant Mycobacterium tuberculosis by mycobacteria growth indicator tube-drug sensitivity test

Background Alternative treatment strategies have become essential in overcoming the problem of drug-resistant Mycobacterium tuberculosis (Mtb). In this preliminary in vitro study, the anti-tuberculosis (anti-TB) activity of exogenous iron chelators (xenosiderophores) such as Exochelin-MS (Exo-MS) and Deferoxamine-B (DFO-B) was evaluated against ten multi-drug-resistant (MDR) and seven pyrazinamide-resistant (PZA R) Mtb isolates. Methods Mycobacteria Growth Indicator Tube-Drug Susceptibility Test was used to assess the anti-TB effect of Exo-MS or DFO-B individually and their combinations with isoniazid (INH), rifampicin (RIF) and pyrazinamide (PZA). Results For the MDR-Mtb isolates, Exo-MS alone inhibited two out of the five isolates tested. Whereas, DFO-B alone inhibited nine out of the ten MDR isolates tested. For PZA-resistant Mtb isolates, both Exo-MS and DFO-B individually inhibited five out of the seven isolates. The MIC of Exo-MS in combination with INH, RIF and PZA remained the same. The MIC of DFO-B decreased when tested in combination with INH, RIF and PZA. Conclusions Exo-MS and DFO-B were shown to have activity against drug-resistant Mtb isolates. Therefore, these xenosiderophores may be useful adjuncts to antibiotics in overcoming the problem of drug-resistant Mtb in clinical setting.


Background
Tuberculosis (TB) is one of the leading infectious diseases in the world today. Mycobacterium tuberculosis (Mtb), the causative agent of TB, is mainly transmitted from person to person through aerosols. Infections caused by TB bacteria are usually treated with the firstline antituberculosis drugs, namely, isoniazid (INH), rifampicin (RIF), ethambutol (EMB), pyrazinamide (PZA), and streptomycin (STR).
However, Mtb bacteria have acquired resistance over the years to many of these drugs due to inappropriate use, mutations in Mtb, etc. These factors have resulted in the emergence of multi-drug-resistant TB (MDR-TB) and extensively-drug-resistant TB (XDR-TB), which have rendered current treatment strategies ineffective.
In MDR-TB, the organisms develop resistance to INH and RIF, the two most important primary drugs. In XDR-TB, the bacteria are resistant to the first-line as well as to the second-line anti-TB drugs. XDR-TB develops in about 9% of MDR-TB patients and is more challenging to treat [1]. In 2014, globally 4,80,000 cases of MDR-TB were reported with mortality of 1,90,000. XDR-TB patients have been identified in about 100 countries. As per this WHO report, India, along with other third world countries is a high-burden country for TB. With the aim of ending TB by 2035, WHO has initiated an "End TB Strategy" [2].
Iron is a vital nutrient for all living organisms necessary for life-sustaining cellular processes such as cell growth, DNA synthesis, electron transport, oxygen transport, etc. The mammalian host maintains low levels of iron using its iron-binding proteins; about 10 −18 M of circulating free ferric (Fe 3+ ) ions. Microorganisms synthesize small molecules called siderophores (Greek "iron-carrier") which scavenge iron from the host to promote their own growth [3]. Siderophore-Fe 3+ ion complexes that are formed are then transported intracellularly via specific outer membrane receptor proteins by bacteria.
If Fe 3+ ion is complexed with xenosiderophores (not "self") for a given microorganism, iron uptake may be affected due to the absence of specific receptors in most bacteria. A decrease in the availability of iron would probably be detrimental to the growth of pathogens including the drug-resistant ones. A phytosiderophore extracted from plant root washings has been reported to inhibit the in vitro growth of H 37 Ra strain of Mtb [4].
M. smegmatis produces three siderophores: mycobactin S (S for smegmatis), carboxymycobactin S and Exochelin MS (Exo-MS; MS for M. smegmatis). Mtb expresses mycobactin T (T for tuberculosis) and carboxymycobactin T. However, it does not produce exochelin. Therefore, Exo-MS is a xenosiderophore for Mtb. Similarly, the commercially available Deferoxamine mesylate (DFO-B), originally extracted from Streptomyces pilosus, is also a xenosiderophore for Mtb. Desferri forms of Exo-MS and DFO-B were tested for their anti-TB potential alone and in combination with drugs against MDR and PZA R -Mtb isolates by the Mycobacteria Growth Indicator Tube -Drug Susceptibility Test (MGIT TM -DST) [5].
MGIT-DST is a qualitative test used for antimycobacterial susceptibility testing of Mtb developed by Becton Dickinson. A fluorescent oxygen-quenched sensor (Tris 4, 7-diphenyl-1, 10 phenanthroline ruthenium chloride pentahydrate) embedded in silicone is present at the base of a tube. The principle underlying the test is that the initial concentration of dissolved oxygen in the Middlebrook 7H9 broth quenches the emission of fluorescence from this sensor and therefore is not visualized under UV light. However, when actively growing Mtb bacilli consume the available oxygen, the sensor fluoresces and is visualized under UV light. The intensity of this fluorescence is directly proportional to the amount of oxygen consumed by the Mtb bacilli for growth. The amount of oxygen utilized by Mtb for growth in the medium is monitored by the BACTEC MGIT system which automatically interprets results as 'S'(susceptible) or 'R' (resistant) based on the extent of fluorescence [5].

Methods
Extraction and purification of Exo-MS from M. smegmatis mc 2 155 M. smegmatis was grown in an iron-deficient medium [6,7] for 10 days at 37°C with aeration [For details of Exo-MS extraction, refer to Gokarn et al., 2017, BCAM DOI:10.1186/s12906-017-1657-8]. Briefly, Exo-MS was recovered from the culture supernatant using the benzyl alcohol extraction procedure [8]. Purification was carried out on an alumina column [9], which removed most of the hydrophobic impurities; Exo-MS was then eluted using a mixture of methanol and formic acid. HR-LCMS was carried out to determine the purity of Exo-MS.  Table 3. Similarly, its concentration alone and in combination with PZA against PZA-resistant isolates are shown in Table 4.
For each isolate, one set of tubes was supplemented with excess ferric ammonium citrate as control to ascertain whether the growth inhibition was solely due to iron deprivation. All the tubes were mixed by inverting several times and then placed in set carriers before transferring them into the MGIT instrument maintained at 37°C. The first tube in the set carrier is always the Growth Control (GC) tube. In MGIT-DST, the GC should have a Growth Unit (GU) value of 400 or more. If the GU value of the drug-containing tube is <100, the interpretation "S" is read by the MGIT instrument. If the GU value of the drug-containing tube is ≥ 100, the reading "R" is given by the instrument [5]. When the GU value of the GC was 400 or greater, the tubes were removed, scanned and a report was generated. iv Test for bactericidal or bacteriostatic effect of the siderophores: Determination of minimum bactericidal concentration was carried out to test whether the concentration of the siderophores used had a bacteriostatic (inhibiting), or bactericidal (killing) effect. For this test, 0.5 mL aliquots from the tubes showing GU value <100 after 14 days were re-inoculated into fresh Middlebrook 7H9 broth without any siderophores and antibiotics; the tubes were incubated in the MGIT instrument for 35 days.

Results
Extraction and purification of Exo-MS from M. smegmatis        The control tubes with ferric citrate added showed GU value of 400 for all the isolates.

Bacteriostatic or bactericidal effect of the siderophores in vitro
When the inhibited isolates showing GU value of <100 were re-inoculated into fresh siderophore-free and drugfree Middlebrook 7H9 medium, the distinction between bacteriostatic and bactericidal effect of the siderophores (and their combinations) could be inferred based on the GU values. If the GU value of re-inoculated tube was <100, the original siderophore-drug combination was considered bactericidal. If the GU value of re-inoculated tube was 400, the original siderophore-drug combination was considered bacteriostatic.
After 14 days of re-inoculation into fresh medium, the GU values were <100 for two isolates inhibited by Exo-MS + INH, three of the nine isolates inhibited by DFO-B + RIF and three of the five isolates inhibited by DFO-B + PZA. However, at the end of 35 days, all concentrations of siderophores and their combinations with drugs were found to be bacteriostatic for all isolates, with GU values of 400.

Discussion
Tubercle bacilli acquire iron from a mammalian host for their growth. Excess iron in vivo promotes Mtb infection and may impair macrophage function affecting innate immunity. A study using female Balb/C mouse infected with Mtb showed that iron overloading significantly reduced the bactericidal activity of INH and completely neutralized the bacteriostatic activity of ethambutol [10].   There are various strategies that the human body has adapted to safeguard the iron. Iron retention by the reticuloendothelial system [11] and mild anemia which is a common occurrence in TB patients are examples of host mechanisms to ensure iron-deficient conditions in the body [12,13]. Though these natural processes exist, restricting the spread of MDR-Mtb in the human population is still a major problem.
MDR-Mtb strains are defined as those that are resistant to INH and RIF, both of which are important anti-TB drugs. PZA is also a first-line anti-TB drug. All the three drugs are bactericidal to susceptible strains of Mtb and hence were chosen for this study. Ethambutol was not selected since it is bacteriostatic to susceptible Mtb strains.
For the first time, MIC of siderophores has been determined using the MGIT-DST method for drug-resistant Mtb isolates. It is important to note that the anti-TB activities of Exo-MS and DFO-B by themselves were significant.
Inhibitory effect of Exo-MS on MDR-Mtb isolates was determined in the Middlebrook 7H9 medium with a neutral pH. Two of the five MDR-Mtb isolates tested were susceptible to Exo-MS. The same numbers were susceptible to its combination with INH and RIF. Due to limited availability of Exo-MS, only five isolates of MDR-Mtb could be tested with it.
When the inhibitory effect of DFO-B on MDR-Mtb isolates was determined, nine out of ten isolates of MDR-Mtb tested were found to be susceptible to DFO-B. The same numbers were susceptible to its combination with INH and RIF, though at a lower DFO-B concentration for some.
Inhibitory effect of Exo-MS and DFO-B on PZA-resistant Mtb isolates was determined in a modified Middlebrook 7H9 medium with pH 5.9. The standard growth medium used for testing INH and RIF is not used for PZA, since PZA requires acidic pH for activity in vitro. Five of the seven PZA-resistant Mtb isolates were susceptible to Exo-  A significant observation was that the inhibitory activity of the siderophores was abolished when excess iron was added in the medium. This proves that the anti-TB effect of the siderophores is solely due to their ability to deprive pathogens of iron. This makes it imperative to use the desferri form of siderophores in such studies.
For all isolates, the siderophore concentrations used were found to be bacteriostatic. This could be beneficial in restricting multiplication of the pathogen in vivo, thus participating in controlling the infection.
The tubercle bacilli down-regulate iron-containing proteins during iron-deficiency [14]. Therefore, irondeprivation by Exo-MS and DFO-B may result in inhibition or inactivation of proteins and enzymes involved in vital functions required for drug resistance such as cell wall integrity. Perhaps Exo-MS and DFO-B may act as facilitators for antibiotics across the cell membrane due to increased cell permeability. Therefore, in the presence of exogenous iron chelators, drug-resistant Mtb could once again become susceptible to the same drugs.
Middlebrook 7H9 medium contains high levels of iron which may not represent true physiological concentrations of ferric ions. It could be one of the reasons why the MIC of the siderophores was high in vitro. Such susceptibility testing predicts the possible antimicrobial agent for the treatment of an infection. The clinical outcome may not be the same due to factors such as host physiology or other interventions, which cannot be simulated in laboratory tests. In vivo studies are required to validate these most promising in vitro results. While DFO-B is already approved for use to treat iron overload in thalassemic patients, safety of Exo-MS for therapeutic use needs to be determined.
We have shown in another study that Exo-MS and DFO-B do not have any cytotoxic effects in vitro on normal mammalian cell lines, such as human embryonic kidney cell line HEK-293 and mouse fibroblast cell line NIH/ 3 T3 [Gokarn etal., 2017, BCAM DOI:10.1186/s12906-017-1657-8]. From the results of the anti-TB activity as well as the effect on normal mammalian cells, it can be concluded that Exo-MS at 0.5 mg/mL and DFO-B at 1.0 mg/mL selectively inhibit Mtb without significantly harming normal mammalian cells.

Conclusions
Our study is the first ever investigation done to evaluate siderophores as potential agents against drug-resistant Mtb by MGIT-DST method. Since the work was exploratory in nature, few drug-resistant isolates of Mtb were used. Nonetheless, it has provided a "proof of concept" that exogenous siderophores such as Exo-MS and DFO-B could be valuable additions to fight drug-resistant M. tuberculosis. In view of the encouraging preliminary results, new in vitro study with large number of M. tuberculosis isolates can be carried out.