Keywords: Antibacterial; Mithramycin; MRSA, Streptomyces; VRE
Although majority of work related to mithramycin is concerned with its antitumor properties, it is also known to exhibit activity against Gram positive bacteria [13,14]. However, the organisms, Staphylococcus aureus or Bacillus subtilis, have merely been used as a tool in bioassay guided purification of mithramycin [2].
Globally, in last two decades, prevalence of the antibiotic resistance among the MRSA, penicillin-resistant Streptococcus pneumoniae (PRSP) and vancomycin resistant enterococci (VRE) has been observed against variety of antibiotics [15-19]. Moreover, incidences of resistance of Gram positive bacterial infections against the new drugs such as Linezolid and Daptomycin have also been published [20,21]. To combat such infections, discovery of novel drugs and/or novel scaffolds is of utmost importance. However new drug discovery is a long term and uncertain process with high expenditure. Another alternative to tackle such problem could be repositioning of the existing bioactive compounds in which known drugs are screened to establish their new bioactivity of desired interest [22]. For example, Rapamycin, a known immunosuppressant drug had been reported to exhibit anticancer activity against primary chronic lymphocytic leukemia cells [23]. Recently new anti-amyloidogenic activity was reported from previously known antibacterial antibiotic tetracycline [24]. Moreover mithramycin as a new candidate for developing new therapeutic drugs for neurodegenerative diseases has also been proposed [25]. Mithramycin has its beneficial effect for not only neurodegenerative diseases, but also a host of others, like cartilage degeneration in osteoarthritis, ER stress mediated apoptosis in hippocampus, erythroid differentiation and fetal haemoglobin production in thalassemia and sickle cell anaemia [26-28]
In the current study, we had isolated, purified and characterized mithramycin from a Streptomyces sp. PM1129877 isolated from a playa region of Thar Desert in Rajasthan, India. This playa region was hardly explored for isolation of actinomycetes producing bioactive compounds. During screening, we observed that mithramycin exhibited potent in-vitro antibacterial activity against variety of Gram positive pathogenic strains of MRSA and VRE. We confirmed these observations by In vitro testing of the compound against variety of Gram positive and Gram negative bacteria including resistant strains obtained from clinics and hospitals. This is the first report on the novel activity of mithramycin against the MRSA and VRE strains.
The normal phase flash chromatography was performed on CombiFlash Rf (Teledyne isco) using Redicep Rf 20 g silica column. HPLC analysis and UV spectra were monitored on Shimadzu LC-2010- PDA instrument (Shimadzu, Kyoto, Japan). The data were processed with LC-solution software (Shimadzu, Kyoto, Japan). Distilled LR grade solvents were used for column chromatography and TLC, HPLC grade solvents from Merck were used for preparative and analytical HPLC. TLC Silica gel 60 F254 plates were from Merck Specialties Pvt. Ltd, India. The NMR spectra were recorded on Bruker Avance at 500 MHz
Approximately 0.5 g of the collected sandy soil was suspended in 5 ml sterile saline (8.5 g/l NaCl in demineralised water) and vortexed for 1 min. This was diluted serially 1:10 up to 10-3. 200 μl of 10-3 dilution of the soil suspension was surface spread on modified Bennet's agar medium containing (g/l), Glucose 10, Casamino acids 2, Yeast Extract 2, Beef Extract 2, Agar powder 15, final pH 7.2-7.5, supplemented with 50 g/l NaCl. The plates were incubated at 25 ± 1°C for 2-3 weeks and observed regularly for appearance of actinomycetes colonies. The producer strain of the mithramycin was one such isolate. The isolate was picked up, purified and maintained on modified ISP2 medium slants containing (g/l), Glucose 40, Yeast extract 40, Malt extract 100, Agar powder 15, final pH 7.5, supplemented with 50 g/l NaCl. The isolate was designated with code PM1129877.
A loopful of growth from 15 days old slant of this strain PM1129877 was inoculated in 274 (1) seed medium containing (g/l), Glucose 15, Peptone 7.5, Yeast extract 7.5, Corn steep liquor 5, NaCl 5, CaCO3 2, pH was adjusted to 7.5. The culture was incubated on shaker at 200 rpm for 72 h at 30°C. 4% (v/v) of seed inoculum was added in 20 L production medium 1M containing (g/l), Glycerol 30, Glucose 3, Peptone 3, Yeast extract 2, CaCO3 3, NaNO3 1, NaCl 30, final pH 7.5. The medium was supplemented with 15 g/l additional NaCl. The 200 production medium flasks (each containing 100 ml medium in 1 L capacity flasks) were incubated on shaker at 200 rpm for 96 h at 30°C. After incubation, contents of the flasks were pooled together and representative sample (100 ml) of this whole broth was extracted with equal volume of methanol under shaking condition for 1.5 h at 30°C. The contents were centrifuged and supernatant (Methanolic extract of whole broth, as mentioned in Table 1) was screened against Gram positive and Gram negative bacterial test cultures and yeasts and fungal test cultures. Antimicrobial activity was determined by whole cell agar well diffusion bioassay by boring 6 mm diameter wells in agarified medium and adding 50 μl samples to be tested in it [30]. Vancomycin (20 μg/ml), Gentamicin (50 μg/ml) and Amphotericin B (20μg/ml) were used as standard antibiotics for Gram positive bacteria, Gram negative bacteria and fungi respectively. Absolute methanol was used as solvent control.
Antimicrobial activity of fermented broth of Streptomyces sp. PM1129877 (zone of inhibition around the well in mm, well diameter 6 mm) |
|||||||||
Details of samples used in whole cell agar well diffusion assay |
S. aureus 209P, MSSA |
S. aureus ATCC 33591, MRSA |
E. faecium R-2-323, VRE |
E. faecalis ATCC 51575, VRE |
E. coli ATCC20732 |
C. albicas HMR |
C. krusei GO3, FlucR |
C. glabrata HO5, FlucR |
A. fumigatus, HMR |
Methanolic whole broth of the organism |
29 |
28 |
25 |
22 |
- |
9vh |
9h |
- |
12h |
Vancomycin (20 µg/ml) |
15 |
14 |
14 |
9h |
NT |
NT |
NT |
NT |
NT |
Gentamicin (50 µg/ml ) |
NT |
NT |
NT |
NT |
15 |
NT |
NT |
NT |
NT |
Amphotericin B (20 µg/ml) |
NT |
NT |
NT |
NT |
NT |
18 |
16 |
17 |
19 |
Methanol |
- |
- |
- |
- |
- |
- |
- |
- |
- |
FlucR: Fluconazole resistant
vh: very hazy zone of inhibition
h: hazy zone of inhibition
-: No zone of inhibition
Gram negative strains (total 8) obtained from in house strain bank of Hoechst Marion Roussel Ltd. India, included Escherichia coli ATCC 20732, E. coli ATCC 25922, E. coli ATCC 35218, Pseudomonas aeruginosa M-35; standard strains procured from the American Type Culture Collection (ATCC), Manassas, USA included, P. aeruginosa ATCC 27853, P. aeruginosa ATCC BAA 47, Acinetobacter baumannii ATCC 19606 and A.baumannii ATCC BAA 747.
The fungal test strains used in agar well diffusion assay of fermented broth included Candida albicans HMR, Candida krusei GO3 FlucR, Candida glabrata HO5 FlucR and Aspergillus fumigatus HMR. These test strains were obtained from in house strain bank of Hoechst Marion Roussel Ltd, India.
The sample generated from methanolic whole broth extraction of PM1129877, had yellow color and exhibited exclusive and potent activity against Gram positive test cultures with very slight or no activity against yeasts/fungal and Gram negative test cultures (Table 1). For bioassay guided isolation of the compound, only Gram positive bacterial test cultures were selected.
Physical and chemical properties of mithramycin |
|
Appearance |
Yellow solid |
Molecular formula |
C52H76O24 |
Molecular weight |
1084.5 |
LC-MS (m/z) |
1083.5 [M+H]- |
UV λmax Neutral nm |
229,272,317 and 412 |
Solubility |
Methanol, DMSO |
Melting point |
180-184°C |
Apart from MIC estimation, mithramycin was also subjected to agar well diffusion assay against 8 Gram negative strains (Table 4) up to 256 μg/ml concentration. There was no zone of inhibition around any agar well for any of these test cultures, indicating mithramycin was ineffective against Gram negative test organisms. These results indicated that perhaps mithramycin was unable to penetrate the outer cell membrane barrier of the Gram negative test organisms and hence exhibited specific in vitro activity against different Gram positive test organisms only.
We started the present study with a hope to find a new antimicrobial compound, from microorganism, isolated from soil collected from untapped natural habitat, such as from playa region. In this journey, although we discovered a known molecule, mithramycin, it exhibited novel bioactivity against MRSA and VRE strains. Mithramycin with brand name Mithracin® was available in the market and was also FDA approved for the treatment of testicular cancer and for the treatment of hypercalcemia [35]. Despite hepatotoxicity and nephrotoxicity problems and other side effects, recently there
1H Data of mithramycin (DMSO - d6, 500 MHz) |
1H Data of mithramycin (DMSO - d6, 500 MHz) |
||||
Position |
δH |
Multiplicity |
Position |
δH |
Multiplicity |
1 |
- |
- |
1B |
4.63 |
dd |
2 |
4.64 |
d |
2Ba |
1.65 |
ddd |
3 |
2.75 |
dddd |
2Be |
2.46 |
ddd |
4 |
3.1 |
dddd |
3B |
4.9 |
ddd |
4' |
2.97 |
dddd |
4B |
4.71 |
dd |
4a |
- |
- |
5B |
3.46 |
dq |
5 |
6.87 |
s |
6B |
1.21 |
d |
6 |
- |
- |
1C |
4.99 |
dd |
7 |
- |
- |
2Ca |
1.73 |
ddd |
CH3-7 |
2.06 |
s |
2Ce |
2.63 |
ddd |
8 |
- |
- |
3C |
4.09 |
ddd |
8-OH |
9.81 |
s |
4C |
4.64 |
dd |
9 |
- |
- |
5C |
3.46 |
dq |
9-OH |
15.57 |
s |
6C |
1.21 |
d |
10 |
6.82 |
s |
1D |
4.57 |
dd |
10a |
- |
- |
2Da |
1.73 |
ddd |
1' |
4.59 |
d |
2De |
1.97 |
ddd |
OCH3-1' |
Overlapped with solvent |
3D |
4 |
dt |
|
2' |
- |
- |
4D |
4.99 |
dd |
3' |
5.17 |
d |
5D |
3.56 |
dq |
4' |
5.3 |
d |
6D |
1.16 |
d |
5' |
1.36 |
d |
1E |
4.89 |
dd |
1A |
4.96 |
dd |
2Ea |
1.5 |
dd |
2Aa |
1.97 |
ddd |
2Ee |
1.99 |
dd |
2Ae |
2.46 |
ddd |
3E |
- |
- |
3A |
4 |
ddd |
3E-CH3 |
1.12 |
s |
4A |
4.71 |
dd |
4E |
4.57 |
d |
5A |
3.63 |
dq |
5E |
3.78 |
dq |
6A |
1.18 |
d |
6E |
1 |
D |
In vitro antibacterial activity of mithramycin (MIC expressed in µg/ml) |
||||||
Test culture (n) |
Mithramycin |
Linezolid |
||||
|
MIC range |
MIC50 |
MIC90 |
MIC range |
MIC50 |
MIC90 |
S. aureus MRSA* (15) |
0.125-0.250 |
0.125 |
0.25 |
2.00-8.00 |
4.00 |
4.00 |
S. aureus 209P MSSA (1) |
0.125 |
- |
- |
4.00 |
- |
- |
E.faecalis VRE (3) |
2.00 |
- |
- |
4.00 |
- |
- |
E.faecalis VSE (1) |
2.00 |
- |
- |
4.00 |
- |
- |
E.faecium VRE (2) |
1.00-16 |
- |
- |
2.00-4.00 |
- |
- |
E.faecium VSE (1) |
4.00 |
- |
- |
2.00 |
- |
- |
Clinical Enterococci spp.VRE (10) |
1.00-2.00 |
1.00 |
2.00 |
2.00-4.00 |
2.00 |
4.00 |
Bacillus spp. (4) |
0.031-1.00 |
- |
- |
1.00-2.00 |
- |
- |
Gram Negatives [E.coli strains (3), Acinetobacter baumanii strains (2), Pseudomonas spp.(3)] (8) |
>16 |
- |
- |
NT |
NT |
NT |
MSSA: methicillin-sensitive Staphylococcus aureus
VRE: vancomycin-resistant enterococci
VSE: vancomycin susceptible enterococcus
n: number of test strains
*: including 13 clinical strains and two standard ATCC strains
NT: Not tested
Development of a drug as an antibiotic needs mode of action studies along with in vivo susceptibility data. At present outcome of such studies for mithramycin cannot be predicted. However, considering the ever increasing cost of new drug discovery program all over the world, reports of challenges faced by Linezolid [20] and Daptomycin [21] and over all the grave problem of resistance of staphylococci and enterococci to these established drugs [16], this study can make scientists revisit the use of mithramycin scaffold, despite its toxicity and side effects.
As confirmed by In vitro studies, mithramycin has potent antibacterial activity. This may be one of the important multiple factors, facilitating dose reduction, required for treating infected animals as compared to high dose regimen of the compound required for the treatment of cancer. Moreover, cancer treatment being a long term process, frequency of occurrence of unpleasant side effects of mithramycin is much more. In contrast, treatment for infectious diseases can be completed in limited period of time and toxic side effects of mithramycin can be minimized considerably.
Recently Nunez et al. [39] reported novel mithramycin analogue (demycarosyl-3D-β-d-digitoxosylmithramycin SK) with high antitumor activity and less toxicity. So alternatively, mithramycin can be used as a 'much needed potential scaffold' for synthesis of less toxic analogs which can also act as anti- MRSA drugs. As mithramycin is a known compound, the time and money required for new drug development in infectious disease area can be reduced substantially and in future mithramycin and its analogs would be new leads for the treatment of variety of Gram positive infectious diseases, associated with resistant strains of MRSA and VRE.
In conclusion, mithramycin obtained from Streptomyces sp. PM1129877, isolated from playa region of Thar Desert in Rajasthan, India, was purified and identified using spectroscopic methods. MIC estimations revealed that mithramycin exhibited anti-MRSA and anti-VRE activity, which was very potent compared to Linezolid. As regimen for infectious disease can be of limited period as compared to lengthy cancer treatment, authors propose and hope that in future mithramycin and its analogs could be potential candidates for developing a suitable lead for treatment of such diseases.
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