PHYTOTHERAPY RESEARCH Phytother. Res. (2013) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5090
Antitubercular and Antibacterial Activity of Quinonoid Natural Products Against Multi-Drug Resistant Clinical Isolates Diganta Dey,1,2 Ratnamala Ray2 and Banasri Hazra1* 1
Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India Department of Microbiology, Ashok Laboratory Clinical Testing Centre Private Limited, Kolkata 700068, India
2
Multi-drug resistant Mycobacterium tuberculosis and other bacterial pathogens represent a major threat to human health. In view of the critical need to augment the current drug regime, we have investigated therapeutic potential of five quinonoids, viz. emodin, diospyrin, plumbagin, menadione and thymoquinone, derived from natural products. The antimicrobial activity of quinonoids was evaluated against a broad panel of multi-drug and extensively drugresistant tuberculosis (M/XDR-TB) strains, rapid growing mycobacteria and other bacterial isolates, some of which were producers of β-lactamase, Extended-spectrum β-lactamase (ESBL), AmpC β-lactamase, metallo-betalactamase (MBL) enzymes, as well as their drug-sensitive ATCC counterparts. All the tested quinones exhibited antimycobacterial and broad spectrum antibacterial activity, particularly against M. tuberculosis (lowest MIC 0.25 μg/mL) and Gram-positive bacteria (lowest MIC emodin ~ menadione ~ thymoquinone > diospyrin, whereas their antibacterial efficacy was plumbagin > menadione ~ thymoquinone > diospyrin > emodin. Furthermore, this is the first evaluation performed on these quinonoids against a broad panel of drug-resistant and drug-sensitive clinical isolates, to the best of our knowledge. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: Mycobacterium tuberculosis; quinonoids; natural products; multi-drug resistance; clinical isolates.
INTRODUCTION Infectious diseases continue to be a leading cause of mortality around the world, particularly in developing countries. Bacterial diseases are rampant in India, and it is enlisted as one of the 22 countries with high tuberculosis (TB) burden (WHO, 2011, 2012). India, along with China, accounted for almost 40% of the total number of TB victims in the world, estimated as nine million new cases and 1.4 million deaths in 2011 (WHO, 2012). Moreover, clinical bacteria are becoming increasingly resistant due to antibiotic selection pressure. About a decade back, methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus spp. had raised a point of concern. Subsequently, multi-drug resistant (MDR) Gram-negative bacteria emerged, posing a greater threat to public health, since their increase in resistance would be faster than the Gram-positive ones, and there are fewer new antibiotics under development to provide sufficient therapeutic coverage in the near future (Kumarasamy et al., 2010). Again, the widespread appearance of MDR-TB further aggravated the scenario, particularly for those affected with the human immunodeficiency virus (HIV) infection (Zumla and Grange, 1998). In 2011, almost 60% of the notified pulmonary MDR-TB cases in the world were contributed by India, China and the Russian Federation (WHO, 2012).
* Correspondence to: Banasri Hazra, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India. E-mail: [email protected]
Copyright © 2013 John Wiley & Sons, Ltd.
Usually, the treatment of TB involves a combination regimen of four anti-TB drugs (ATDs), viz. isoniazid, rifampicin, ethambutol and pyrazinamide, administered over a period of six to eight months. Nevertheless, there is an urgent need to augment the current drug regime in view of the emerging drug resistance and co-infection with HIV (Mukanganyama et al., 2012; Guzman et al., 2012). In order to discover newer antibacterial therapeutics, it would be worthwhile to investigate selected plant products, as many antimicrobial agents, including ATDs like rifampicin, streptomycin, capreomycin and cycloserine, have been derived from natural products (Newton et al., 2000; Guzman et al., 2012). Worldwide exploration of certain medicinal plants generated many novel products, which, in turn, also validated the ethnomedical resources in terms of their key constituents possessing potent antimicrobial activity (Cowan, 1999). No wonder that the primary health care of 65–75% of the world’s population continue to be served by the age-old herbal formulations which are thriving even to this day (Newton et al., 2000). Plants have an amazing ability to synthesize secondary metabolites, mainly flavonoids, tannins and phenols or their oxygen-substituted quinonoid derivatives possessing unique structural features. In general, terpenoids endow the plants with distinctive odors, while quinones and tannins are responsible for the coloring pigments. Some such products also serve to defend their hosts from plant pathogens, signifying their capability to fight against infectious microbial diseases. In our laboratory, phytochemical screening and semi-synthetic derivatisation could unravel the pharmacological prospect of phenolics and quinones isolated from traditional medicinal Received 12 August 2013 Revised 28 October 2013 Accepted 06 November 2013
D. DEY ET AL.
plants (Hazra et al., 2002; Basu et al., 2005; Dey et al., 2012). Presently, we have evaluated five bioactive quinonoids (Fig. 1) against drug-resistant bacteria including Mycobacterium spp. characterized from clinical isolates. The plant-derived quinonoids, viz. emodin (1), diospyrin (2), plumbagin (3) and thymoquinone (5) have been identified as the active constituents in certain age-old Oriental medicinal formulations still popular in the Indian sub-continent (Hazra et al., 2004). However, menadione or vitamin K3 (4; a synthetic version of vitamin K2) was included due to its clinical implication and structural analogy with plumbagin (3). Preliminary findings on antimycobacterial (Newton et al., 2000; Guzman et al., 2012) and antibacterial (Cowan, 1999) activity of some of these compounds were reported by previous researchers. Here, we have performed a broad spectrum study on the inhibitory effect of the quinones against clinically significant drug-resistant strains sparsely investigated so far.
MATERIALS AND METHODS Test compounds. Emodin (1) was isolated from the stem bark of Ventilago madraspatana Gaertn. by the procedure described earlier (Basu et al., 2005). Plumbagin (3) was obtained from the root of Plumbago indica Linn. (syn. P. rosea Linn.), and diospyrin (2) from the stem-bark of Diospyros montana Roxb., following the respective methods described previously (Hazra et al., 2002). The products were characterized through spectroscopic analysis and direct comparison with previously authenticated samples. The plant materials were collected from Bolangir district, Orissa, and the voucher specimens were authenticated at the Botanical Survey of India, Kolkata. Menadione (4) and thymoquinone (5) were purchased from Sigma–Aldrich Co., MO, USA.
Isolation of mycobacteria. Detection of mycobacteria in clinical specimen was performed through enrichment in broth culture using BacT/ALERT 3D system (bioMérieux, Inc., Durham, NC, USA), after the sample was decontaminated by conventional N-acetyl-L-cysteine (NALC)-soduium hydroxide (NaOH) protocol (Collee et al., 1996). Decontaminated specimen (0.5 mL) was
Figure 1. Chemical structure of the studied quinones: emodin (1), diospyrin (2), plumbagin (3), menadione (4) and thymoquinone (5). Copyright © 2013 John Wiley & Sons, Ltd.
aseptically injected to the BacT/ALERT MP bottle containing 10 mL of aqueous media consisting of middlebrook 7H9 broth, pancreatic digest of casein, bovine serum albumin and catalase under vacuum. Lyophilized MB/BacT antibiotic supplement was reconstituted with 10 mL of MB/BacT reconstitution fluid and re-constituted solution (0.5 mL) was injected back into the preinoculated BacT/ALERT MP bottle for incubation within BacT/ALERT 3D Mycobacteria Detection System. The gas-permeable colorimetric sensor installed in the bottom of each culture bottle transmitted the increase of reflectance units proportional to the carbon dioxide produced by Mycobacterium growth, leading to a positive detection at approximately 106–107 colony forming units (CFUs) per mL (Thorpe et al., 1990).
Mycobacterial species identification. The culture broth from the BacT/ALERT MP bottle with positive growth of Mycobacterium was inoculated in Löwenstein–Jensen (LJ) slant (HiMedia, Mumbai), and identified by phenotypic tests and routine biochemical methods, viz., nitrate reduction, pyrazinamidase, catalase, urease and niacin accumulation tests using kits procured from Himedia, Mumbai (Collee et al., 1996). Additionally, genotypic confirmation of Mycobacterium tuberculosis complex (MTC) was accomplished by using artus® M. tuberculosis RG PCR kit (QIAGEN, Hilden). For this purpose, culture media (500 μL) collected from positive BacT/ALERT MP bottle was centrifuged at 13,000 rpm for 10 min. The resultant pellet was dissolved in 180 μL lysozyme (SRL, Mumbai; 20 mg/mL) and incubated at 37 °C for 45 min, followed by DNA extraction using QIAamp DNA Mini Kit (QIAGEN, Hilden), keeping appropriate internal control as per protocol. Real-time DNA detection was performed using the Rotor-Gene Q analyzer (QIAGEN, Hilden). The specific amplicons (a 159 base pair region of the mycobacterial genome) were detected directly using the 5′- nuclease (TaqMan) probe labelled with FAM dye (Hur et al., 2011).
Anti-tubercular drug (ATD) susceptibility. Susceptibility testing of isolated M. tuberculosis strains was carried out against first line (isoniazide, ethambutol, pyrazinamide, refampicin, streptomycin) and second line (kanamycin, amikacin, ethionamide, D-cycloserine, clarithromycin, p-amino salicylic acid, rifabutin) ATDs, following LJ proportion method on drug- incorporated LJ slants (HiMedia, Mumbai). The organism was considered to be resistant to a particular drug when the percentage ratio of the number of colonies on the drugcontaining slant and the control slant (without drug) was greater than 1%, sensitive in case of less than 1%, and intermediate when equal to 1% (Collee et al., 1996).
Antimycobacterial activity. Tetrazolium microplate assay with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, MO, USA) was done by following the method of Caviedes et al. (2002), with minor modification, for testing the susceptibility of five quinonoid test compounds against selected strains of Mycobacteria spp. Accordingly, mycobacterial suspension was taken from the tested BacT/ALERT MP Phytother. Res. (2013)
ANTIMICROBIAL ACTIVITY OF QUINONOID NATURAL PRODUCTS
bottle, turbidity was adjusted to 1 McFarland standard and subsequently diluted to 1:25 by adding Middlebrook 7H9 broth (bioMérieux BacT/ALERT MP), and used as inoculum in the microplate assay. In the 96-well plate, only sterile water (200 μL) was added to the upper and lower rows (A1-12, and H1-12, labelled as commercially stamped on the plates), and Middlebrook 7H9 broth (100 μL) was added to the rest of the wells. Serial 2× dilutions of each compound were prepared directly on the plate by adding 100 μL of the working solutions (1.024 mg/mL in dimethyl sulfoxide; DMSO) in five horizontal rows (B to F) of the microtitre plate. The residual row of 12 wells (G1-12) contained only DMSO as the ‘control’ serially diluted to the concentration corresponding to the respective wells used for MIC determination. Finally, the aforesaid inoculum (100 μL) was added to each well except those in A and H-rows, so that the final concentration range of the test compounds was 256 – 0.125 μg/mL. The microtitre plates were sealed with parafilm and incubated in plastic bags at 37 °C. The mycobacterial growth was detected by MTT solution (15 μL; 5 mg/mL MTT dissolved in ethanol: water =1:9), which was added to a single well in the ‘control’ row (G1-12). In case of positive growth, the colour turned purple after 24 h of incubation, when MTT was added to rest of the wells, and the colour change recorded after further 24 h of incubation. However, in case of incomplete growth, the ‘control’ well would remain yellow, and hence MTT was added to the next well for further incubation. In this way, the colour change was monitored until the tubercular growth appeared on days 8/9/10/14/16, depending on the nature of the particular strain. However, the rapid grower strains were monitored every 24 h starting from day 2, as these strains would grow faster (2 – 6 days) than M. tuberculosis (7 – 15 days; Collee et al., 1996). The lowest concentration, at which the mycobacterial growth was inhibited, as indicated by the colour change of MTT, was noted as the minimum inhibitory concentration (MIC) of the compound. To determine the minimum bactericidal concentration (MBC), a loop-full of culture from each well containing different dilutions of the tested compounds was streaked by a 4 mm loop (HiMedia, Mumbai; calibrated to 0.01 mL) on solid LJ medium base (HiMedia, Mumbai) containing glycerol. The lowest concentration of the tested compound that did not support the growth of visible mycobacterial colony on LJ medium was recorded as the MBC (Collee et al., 1996).
Isolation and characterization of the bacterial isolates. A total of 35 bacteria (13 Gram positive and 22 Gram negative), comprising of eight American Type Culture Collection (ATCC) strains and 27 pathogenic bacteria isolated from clinical specimen were included in this study. All the clinical isolates were identified by routine phenotypic and biochemical methods, as well as, agglutination with specific polyvalent and monovalent antisera (Collee et al., 1996). β-Lactamase production was identified by sharply demarcated zone edge around a penicillin G disc (10 U; HiMedia, Mumbai) (Collee et al., 1996; CLSI 2013) and inducible AmpC β-lactamase production was determined by blunting of cefotaxime (30 μg) zone adjacent Copyright © 2013 John Wiley & Sons, Ltd.
to the cefoxitin (30 μg) disc on a Mueller Hinton agar (MHA; HiMedia, Mumbai) plate as a result of their antagonism (Collee et al., 1996). Extended-spectrum β-lactamase (ESBL) production was identified by the enhancement of zone diameter of cephalosporin/ clavulanate disc in comparison to the cephalosporin disc alone. Metallo-β-lactamase (MBL) production was characterized by Imipenem-EDTA combined and double disc diffusion as well as by the modified Hodge test (Dey et al., 2012; CLSI 2013).
Antibacterial drug susceptibility. Antibacterial susceptibility studies were carried out by Kirby and Bauer disk diffusion technique using commercially available antibiotic discs (HiMedia, Mumbai) and interpretation of inhibition zone data were done by according CLSI guidelines (Dey et al., 2012; CLSI 2013).
Antibacterial activity. The broth micro-dilution assay was used for the determination of minimum inhibitory concentration (MIC) of five quinonoids against each of the selected bacteria (eight ATCC strains and 27 clinical isolates) to find the lowest concentration within the range of 512 – 4 μg/mL at which no growth was visible following the established procedure (Dey et al., 2012; CLSI 2013). To determine MBC, a loop-full of culture was taken from each well containing different dilutions of the tested compounds, and inoculated by a 4 mm loop (calibrated to 0.01 mL) on MHA plate. The lowest concentrations of the tested compounds that did not support the growth of visible bacterial colony were recorded as the MBC of the respective compounds (Collee et al., 1996).
RESULTS Antimycobacterial activity In the present study, five quinonoid compounds (1 – 5) were evaluated for antimycobacterial activity against one ATCC strain (H37Ra) and four clinical strains of M. tuberculosis, as well as two rapid-grower isolates of M. chelonei and M. fortuitum, in terms of MIC and MBC. The M. tuberculosis strains were isolated from pulmonary tuberculosis cases, while the two atypical rapid grower mycobacteria were taken from subcutaneous infections (Table 1). All these strains were selected from the assortment of patient samples routinely characterized in our clinic, and tested individually for resistance pattern against first- and second-line ATDs, using L-J proportional method (Collee et al., 1996). Thus, two out of the four M. tuberculosis strains selected for this study originated from MDR-TB cases, while the rest were from extensively drug resistant tuberculosis (XDR-TB; Table 1). According to WHO, MDR-TB is caused by strains of M. tuberculosis that are resistant to isoniazid and rifampicin, while additional resistance to a fluoroquinolone, such as, ciprofloxacin, and at least one second-line injectable agent, as for example, amikacin, kanamycin, signify XDR-TB (WHO, 2010). Phytother. Res. (2013)
D. DEY ET AL.
Table 1. Anti-tuberculous drug (ATD) resistance and minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) of emodin (1), diospyrin (2), plumbagin (3), menadione (4) and thymoquinone (5) against the tested mycobacterial strains MICa and MBC of tested quinonoids (μg/mL) Sl. no.
Species
Source
First line ATD resistance
Second line ATD resistance
1
2
3
5
None
4
16
EMB, INH, RMP
CS
8 4
32 4
M. tuberculosis (MDR) Pleural fluid
EMB, INH, PZA, RMP
CLR, RFB
8 16
16 64
1 8
8 16
8 8
4.
M. tuberculosis (XDR)
Sputum
INH, PZA, RMP, SM
AN, CIP, CS, KM
16 8
64 8
8 2
16 8
32 8
5.
M. tuberculosis (XDR)
Sputum
EMB, INH, RMP, SM
AN, CIP, ETA, KM
16 16
16 32
4 4
16 32
16 16
6.
M. chelonei (Rapid grower)
Wound discharge
EMB, INH, PZA, RMP
AN, CLR, CS, ETA, KM, PAS, RFB
32 16
64 128
8 16
32 32
32 16
7.
M. fortuitum (Rapid grower)
Pus from abscess
EMB, INH, PZA, RMP, SM CLR, CS, ETA, KM, PAS, RFB
64 32
128 256
64 32
64 64
64 32
128
>256
64
128
128
1.
M. tuberculosis (Reference)
H37Ra (ATCC 25177) None
2.
M. tuberculosis (MDR) Sputum
3.
8
4 8
8
16 16 0.25 4
16 4
a
Shaded row: MIC MDR: Multi-drug resistant; XDR: Extensively drug resistant; AN: Amikacin (700 μg/mL); CIP: Ciprofloxacin (12.5 μg/mL); CLR: Clarithromycin (8 μg/mL); CS: D-Cycloserine (30 μg/mL); ETA: Ethionamide (20 μg/mL); EMB: Ethambutol (2 μg/mL); INH: Isoniazide (0.2 μg/mL); KM: Kanamycin (30 μg/mL); PAS: p-Amino salicylic acid (2.5 μg/mL); PZA: Pyrazinamide (200 μg/mL); RFB: Rifabutin (0.5 μg/mL); RMP: Rifampicin (40 μg/mL); SM: Streptomycin (4 μg/mL)
The two rapid-grower strains were found to be resistant to most of the first-line and second-line ATDs (Table 1). All the tested quinones were found to show potential anti-TB activity with MIC values ranging between 0.25 and 64 μg/mL, although the MIC range was found to be comparatively higher (16 – 256 μg/mL) against the two rapid grower strains. Among all the compounds, plumbagin was found to be the most potent (MIC = 0.25 – 16 μg/mL), while menadione, emodin and thymoquinone showed more or less similar inhibitory activity against M. tuberculosis (MIC = 4 – 32 μg/mL). Diospyrin was found to be the least potent against M. tuberculosis as well as the rapid grower strains with the MIC range of 4 – 256 μg/mL. The other four quinonoids exhibited moderate activity against M. chelonei and M. fortuitum (MIC =16 – 32 μg/mL) (Table 1). Antibacterial activity The antibacterial efficacy of quinones (1 – 5) was evaluated against a broad spectrum of 35 bacteria, comprising 27 clinical isolates and eight ATCC strains (Table 2). The bacteria were isolated from different patients suffering from superficial wounds, urinary, enteric and pulmonary infections, and bacteremia (Table 2). All isolates were checked in vitro for antibiotic resistance property following CLSI guidelines (CLSI, 2013). Furthermore, the bacteria were selected in consideration of their clinical significance in the context of enzyme production related to their multi-drug resistant phenotypes. Hence, a vigorous screening of available clinical samples was undertaken to select one E. coli and one Pseudomonas aeruginosa as producers of AmpC β-lactamase and MBL enzymes, respectively. Similarly, a couple of Staphylococcus aureus and Morexella catarrhalis, in addition to one ATCC strain of Escherichia coli, were taken for their Copyright © 2013 John Wiley & Sons, Ltd.
ability to produce β-lactamase. Again, two clinical isolates and one ATCC strain of Klebsiella spp., and one Proteus mirabilis were chosen as ESBL producers (Table 2). In Table 2, the results of antibacterial evaluation, expressed as MIC and MBC, revealed the broadspectrum activity of the quinonoid compounds (1 – 5), albeit with variable efficacy. In general, the Grampositive bacteria exhibited greater susceptibility to the tested quinones in comparison to the Gram-negative bacteria. Among all the tested compounds, plumbagin was the most potent against both Gram-positive (MIC 512 μg/mL, and 8 – >512 μg/mL against the spectrum of tested bacteria.
DISCUSSION Antimycobacterial activity Naturally occurring quinones are ubiquitously distributed in living organisms. These compounds are characteristically capable of generating reactive oxygen species through redox cycling mechanism, easily undergoing either one-electron reduction to yield the semiquinone free radical, or two-electron reduction directly to the hydroquinone (Nohl et al., 1986). Thus, quinonoid natural Phytother. Res. (2013)
ANTIMICROBIAL ACTIVITY OF QUINONOID NATURAL PRODUCTS
Table 2. Antibiotic resistance pattern and minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) of emodin (1), diospyrin (2), plumbagin (3), menadione (4) and thymoquinone (5) against the bacterial strains tested in vitro MICa and MBC of tested quinonoids (μg/mL) Sl. no.
1.
Bacteria (source)
Enzyme produced
Gram positive bacteria Staphylococcus aureas (ATCC 25923)
–
Antibiotic resistance in vitro
1
2
3
4
5
None
>512
>512
16
32
32
>512 256
32 32
64 32
32 64
2.
Streptococcus pneumoniae (ATCC 27336)
–
None
>512 256
3.
S. pyogenes (ATCC 12384)
–
None
512 512
512 256
32 16
64 32
128 32
4.
Enterococcus faecalis (ATCC 29212)
–
None
>512 >512
512 256
32 32
32 256
64 16
5.
Staphylococcus aureus (Pus)
>512 256
>512 64
32 8
256 32
32 256
>512
128
16
64
256
6.
S. saprophyticus (Urine)
β-lactamase producer –
Cx, E, Cfx, Ca, Cd, Ox, As, Amc, Pt Ca, Cip, Nx
>512
64
64
128
128
128 128
64 32
128 64
256 128
7.
S. epidermidis (Wound swab)
–
None
>512 256
8.
Streptococcus pneumoniae (Sputum)
–
None
>512 128
256 256
32 64
64 64
256 64
9.
S. pyogenes (Throat swab)
–
None
128 256
512 512
64 32
64 64
128 128
10.
S. agalactiae (Pus)
–
None
>512 256
>512 64
64 32
128 64
128 64
11.
Bacillus subtilis (Faeces)
–
None
512 128
128 64
32 16
64 32
128 64
12.
B. cereus (Faeces)
–
P, Ca
256 8
128 64
16 512
128 128
8 64
32 512
32 128
>512
256
64
>512
256
14.
Gram negative bacteria Escherichia coli (ATCC 35218) β-lactamase producer
None
>512
256
16
64
256
Ao, As, Ca, Cfx, Do
>512 512
256 >512
16 128
64 128
512 128
None
>512 >512
>512 >512
128 128
256 128
256 128
None
>512 >512
>512 >512
256 32
256 32
512 64
>512 256
>512 >512
64 64
64 128
128 256
512 >512
>512 >512
128 64
256 128
256 256
>512 512
>512 >512
128 128
256 128
512 256
>512 128
>512 256
256 64
128 128
256 256
256 256
512 512
128 64
128 64
512 128
512 512
>512 256
64 128
128 128
256 256
>512 256
512 256
128 32
256 64
256 128
512 512
512 >512
64 64
128 128
256 128
>512
>512
64
128
256
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Klebsiella pneumoniae (ATCC 700603)
ESBL producer
Pseudomonas aeruginosa (ATCC 27853)
–
Proteus vulgaris (ATCC 6896)
–
Escherichia coli (Urine) Klebsiella pneumoniae (Sputum) K. oxytoca (Urine) Enterobacter aerogenes (Urine) Citrobacter freundii (Urine) Proteus mirabilis (Urine) P. vulgaris (Urine) Serratia marcescens (Blood)
AmpC β- lactamase producer
Amc, Cfx, Cl, Ao, Cip, Nx, Ca, Do, Nf, cfm
ESBL producer
As, Cfx, Cip, Cl, Lv, Mr, Nx, Of, Ca, Ao, Cfm
ESBL producer – – ESBL producer – –
As, Cfx, Cip, Cl, Do, Of, Nf, Ca, Ao, Cfm Cip, Nx, Ca, Gen Nx, Ca, Do Cfx, Cl, Ca, Ao, Cip, Gen, Pt, As Do Cfx
(Continues) Copyright © 2013 John Wiley & Sons, Ltd.
Phytother. Res. (2013)
D. DEY ET AL.
Table 2. (Continued) MICa and MBC of tested quinonoids (μg/mL) Sl. no. 26.
Bacteria (source) Pseudomonas aeruginosa (Urine)
Enzyme produced MBL producer –
Antibiotic resistance in vitro
1
2
3
4
5
Gen, Mr, Nx, Of, Ca
>512
256
128
128
128
Ca, Gen, Cip, Ak
>512 256
512 >512
128 128
256 128
256 256
Amp, Ca
512 256
>512 128
256 64
128 32
512 128
256 512
256 256
64 32
64 64
128 256
>512 >512
>512 >512
64 64
64 64
256 128
27.
Acinetobacter baumannii (Blood)
28.
Morexella catarrhalis (Throat swab)
29.
Vibrio cholerae (Faeces)
–
Ct
30.
Salmonella typhi (Blood)
–
As, Amc, Gen, Nx, Of, Ak, Ao, C
31.
S. paratyphi A (Blood)
–
None
>512 512
>512 >512
64 32
64 64
256 128
32.
Shigella flexneri (Faeces)
–
Amp, Nx, Ct
>512 256
>512 256
64 64
64 32
256 64
33.
Shigella sonnei (Faeces)
–
None
512 >512
512 512
64 32
32 64
64 256
34.
Burkholderia cepacia (Blood)
–
Ca, Ct
>512 256
>512 256
32 64
64 32
512 128
35.
Stenotrophomonas maltophilia (Pus)
–
Ca
512 256
512 >512
64 128
32 128
256 128
512
>512
128
256
128
β-lactamase producer
a
Shaded row: MIC Ak: Amikacin (30 μg), Amc: Amoxy-Clavulanic acid (20/10 μg), Amp: Ampicillin (10 μg), Ao: Aztreonam (30 μg), As: Ampicillin/Sulbactum (10/10 μg), C: Chloramphenicol (30 μg), Ca: Ceftazidime (30 μg), Cd: Clindamycin (2 μg), Cfm: Cefixime (5 μg), Cfx: Cefuroxime (30 μg), Cip: Ciprofloxacin (5 μg), Cl: Ceftriaxone (30 μg), Ct: Co-trimoxazole (Sulfamethoxazole/trimethoprim) (1.25/23.75 μg), Cx: Cefoxitin (100 μg), Do: Doxycycline (30 μg), E: Erythromycin (15 μg), Gen: Gentamicin (10 μg), Lv: Levofloxacin (5 μg), Mo: Moxifloxacin (5 μg), Mr: Meropenem (10 μg), Nf: Nitrofurantoin (300 μg), Nx: Norfloxacin (10 μg), Of: Ofloxacin (5 μg), Ox: Oxacillin (1 μg), P: Penicillin (100 μg), Pt: Piperacillin/Tazobactum (100/10 μg), Tp: Teicoplanin (30 μg)
products, particularly the anthracycline antibiotics, have been developed into anticancer agents and antimicrobials, as they could bind irreversibly with nucleophilic amino acids, often leading to inactivation of proteins and loss of function, with enormous toxicological and pharmacological implication. Again, ubiquinones (coenzyme Q) and plastoquinones are known to play vital roles in the biochemistry of energy production in mammalian and plant cells, respectively, by shuttling electrons between the membrane-bound proteins in the electron transport chain, while in bacteria, menaquinone (vitamin K2), too, is involved in the functioning of respiratory pathways. It was found that E. coli can utilize both ubiquinone and menaquinone, while only menaquinone is involved in mycobacteria. Therefore, menaquinone biosynthesis pathway is considered as an attractive target for developing novel inhibitors as antimycobacterial agents (Mathew et al., 2010). Menadione is a precursor of vitamin K2 as it undergoes alkylation to yield menaquinones. Thus, it would be worthwhile to study the selected quinonoids, representing benzo-, naphtho- and anthraquinones (Fig. 1), against a series of drug-resistant bacteria, both Gram-positive and negative, as well as Mycobacterium strains critically selected from clinical isolates. Emodin and its anthraquinonoid analogues are the ‘marker’ constituents in several herbal preparations popularly dispensed as Ayurveda and Unani medicines in India, Pen Ts’ao in China, Kampo in Japan and Copyright © 2013 John Wiley & Sons, Ltd.
numerous traditional systems in other Asian countries (Hazra et al., 2004). Diospyrin and plumbagin were identified to be the active principles found in medicinal plants traditionally used in ancient Ayurveda and Chinese medicines, and folklore systems in Africa and Latin America for treatment of miscellaneous ailments, such as skin lesions, febrile conditions, parasitic infestation, renal disease, dental problems, diarrhea, pneumonia, syphylis and tumors (Mallavadhani et al., 1998). Thymoquinone is an active ingredient isolated from Nigella sativa, or black cumin, used as a common spice in Mediterranean countries and Indian sub-continent. Historically, the use of its seed oil was recommended for the treatment of bronchitis, asthma, rheumatism, diarrhea and skin disorders, which had been mentioned in ancient Latin, Arabic and Sanskrit literature (Salem, 2005). Presently, multi-drug resistance has become a burning issue in the public health domain as the total number of XDR-TB in 84 countries comprises nearly 9.0% of the average proportion of MDR-TB cases (WHO, 2012). Therefore, a well-proven combination regimen for the treatment of MDR- and XDR-TB needs to be established. Previously, the antimycobacterial activity of some plant-derived quinonoids and derivatives have been demonstrated mostly against non-pathogenic reference and atypical strains of mycobacteria, but not in the clinical scenario (Pullen et al., 2002; Tran et al., 2004; Kanokmedhakul et al., 2005; Mathew et al., 2010; Mukanganyama et al., 2012). Therefore, it was Phytother. Res. (2013)
ANTIMICROBIAL ACTIVITY OF QUINONOID NATURAL PRODUCTS
important to extend our study to M. tuberculosis strains from MDR- and XDR-TB cases, as well as rapidly growing mycobacteria. The latter represented a wide variety of opportunistic pathogens responsible for pulmonary diseases, and primary skin and soft tissue infections usually found in patients with systemic impairment of immunity (Collee et al., 1996; van Ingen et al., 2010). Previously, the antimycobacterial activity of plumbagin against M. avium and M. smegmatis (MIC = 12.5 μg/mL) was reported by Tran et al. (2004). However, they found the response of plumbagin against M. tuberculosis H37Ra strain to be substantially lower (MIC = 400 μg/mL), which did not agree with our result (MIC = 8 μg/mL). Again, Mossa et al. (2004) and Mathew et al. (2010) found the MIC of plumbagin to be diospyrin > emodin. Furthermore, this is the first evaluation performed on these quinonoids against a broad panel of bacteria comprising drug-susceptible as well as resistant clinical bacteria in addition to their ATCC counterparts, to the best of our knowledge.
Acknowledgements D.D. acknowledges technical and management support provided by Ashok Laboratory Clinical Testing Centre Private Limited, Kolkata. Research Scientist Grant from the University Grants Commission, New Delhi, received by B.H.
Conflict of Interest The authors declare that there is no conflict of interest.
REFERENCES Adeniyi BA, Fong HH, Pezzuto JM, Luyengi L, Odelola HA. 2000. Antibacterial activity of diospyrin, isodiospyrin and bisisodiospyrin from the root of Diospyros piscatoria (Gurke) (Ebenaceae). Phytother Res 14: 112–117. Basu S, Ghosh A, Hazra B. 2005. Evaluation of the antibacterial activity of Ventilago madraspatana Gaertn., Rubia cordifolia Linn. and Lantana camara Linn.: isolation of emodin and physcion as active antibacterial agents. Phytother Res 19: 888–894. Caviedes L, Delgado J, Gilman RH. 2002. Tetrazolium microplate assay as a rapid and inexpensive colorimetric method for determination of antibiotic susceptibility of Mycobacterium tuberculosis. J Clin Microbiol 40: 1873–1874. Clinical and Laboratory Standards Institute. 2013. Performance standards for antimicrobial susceptibility testing; twentythird informational supplement (Document M100-S23). CLSI: Wayne, PA. Collee JG, Fraser AG, Marmion BP, Simmons A. 1996. Mackie and McCartney Practical Medical Microbiology, 14th edn. Churchill Livingstone: London. Cowan MM. Plant products as antimicrobial agents. 1999. Clin Microbiol Rev 12: 564–582. Dey D, Debnath S, Hazra S, Ghosh S, Ray R, Hazra B. 2012. Pomegranate pericarp extract enhances the antibacterial activity of ciprofloxacin against extended-spectrum β-lactamase [ESBL] and metallo-β-lactamase [MBL] producing Gramnegative bacilli. Food Chem Toxicol 50: 4302–4309. Guzman JD, Gupta A, Bucar F, Gibbons S, Bhakta S. 2012. Antimycobacterials from natural sources: ancient times, antibiotic era and novel scaffolds. Front Biosci (Landmark Ed) 17: 1861–1881. Hazra B, Das Sarma M, Sanyal U. 2004. Separation methods of quinonoid constituents of plants used in Oriental traditional medicines. J Chromatogr B Analyt Technol Biomed Life Sci 812: 259–275. Hazra B, Sarkar R, Bhattacharyya S, Ghosh PK, Chel G, Dinda B. 2002. Synthesis of plumbagin derivatives and their inhibitory activities against Ehrlich ascites carcinoma in vivo and Leishmania donovani promastigotes in vitro. Phytother Res 16: 133–137. Hur M, Moon H-W, Yun Y-M et al. 2011. Detection of tuberculosis using artus® M. tuberculosis PCR kit and COBAS® AMPLICORTM Mycobacterium tuberculosis test. Int J Tuberc Lung Dis 15: 795–798. Kanokmedhakul K, Kanokmedhakul S, Phatchana R. 2005. Biological activity of Anthraquinones and Triterpenoids from Prismatomeris fragrans. J Ethnopharmacol 100: 284–288. Karkare S, Chung TT, Collin F et al. 2013. The naphthoquinone diospyrin is an inhibitor of DNA gyrase with a novel mechanism of action. J Biol Chem 288: 5149–5156. Kouidhi B, Zmantar T, Jrah H et al. 2011. Antibacterial and resistance-modifying activities of thymoquinone against oral pathogens. Ann Clin Microbiol Antimicrob 10: 29. Kumarasamy KK, Toleman MA, Walsh TR et al. 2010. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis 10: 597–602.
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Lajubutu BA, Pinney RJ, Roberts MF, Odelola HA, Osog BA. 1995. Antibacterial activity of diosquinone and plumbagin from the root of Diospyros rnespiliformis (Hostch) (Ebenaceae). Phytother Res 9: 346–350. Lall N, Hazra B, Das Sarma M, Meyer JJM. 2003. In vitro activity of derivatives of diospyrin against drug-resistant and drugsensitive strains of Mycobacterium tuberculosis. J Antimicrob Chemother 51: 435–438. Mallavadhani UV, Panda AK, Rao YR. 1998. Pharmacology and chemotaxonomy of Diospyros. Phytochemistry 49: 901–951. Mathew R, Kruthiventi AK, Prasad JV, Kumar SP, Srinu G, Chatterji D. 2010. Inhibition of Mycobacterial Growth by Plumbagin Derivatives. Chem Biol Drug Des 76: 34–42. Mossa JS, El-Feraly FS, Muhammad I. 2004. Antimycobacterial constituents from Juniperus procera, Ferula communis and Plumbago zeylanica and their in vitro synergistic activity with isonicotinic acid hydrazide. Phytother Res 18: 934–937. Mukanganyama S, Chirisa E, Hazra B. 2012. Antimycobacterial activity of diospyrin and its derivatives against Mycobacterium aurum. Res Pharm 2: 01–13. Newton SM, Lau C, Wright CW. 2000. A review of antimycobacterial natural products. Phytother Res 14: 303–322. Nohl H, Jordan W, Youngman RJ. 1986. Quinones in biology: functions in electron transfer and oxygen activation. Adv Free Radic Biol Med 2: 211–279. Pullen C, Schmitz P, Meurer K et al. 2002. New and bioactive compounds from Streptomyces strains residing in the wood of Celastraceae. Planta 216: 162–167. Salem ML. 2005. Immunomodulatory and therapeutic properties of Nigella sativa L seed. Int Immunopharmacol 5: 1749–1770. Smolarz HD, Swatko-Ossor M, Ginalska G, Medyńska E. 2013. Antimycobacterial effect of extract and its components from Rheum rhaponticum. J AOAC Int 96: 155–160. Thorpe TC, Wilson ML, Turner JE et al. 1990. BacT/Alert: an automated colorimetric microbial detection system. J Clin Micro 28: 1608–1612. Tran T, Saheba E, Arcerio AV et al. 2004. Quinones as antimycobacterial agents. Bioorg Med Chem 12: 4809–4813. van Ingen J, van der Laan T, Dekhuijzen R, Boeree M, van Soolingen D. 2010. In vitro drug susceptibility of 2275 clinical nontuberculous Mycobacterium isolates of 49 species in the Netherlands. Int J Antimicrob Agents 35: 169–173. World Health Organization. 2010. Multidrug and extensively drugresistant TB (M/XDR-TB): global report on surveillance and response (WHO/HTM/TB/2010.3.). WHO: Geneva, Switzerland. (URL: http://whqlibdoc.who.int/publications/ 2010/9789241599191_eng.pdf) World Health Organization. 2011. World health statistics 2011. WHO: Geneva, Switzerland. (URL: http://www.who.int/ whosis/whostat/EN_WHS2011_Full.pdf) World Health Organization. 2012. Global tuberculosis report (WHO/HTM/TB/2012.6.). WHO: Geneva, Switzerland. (URL: http://www.who.int/tb/publications/global_report/ gtbr12_main.pdf) Zumla A, Grange J. 1998. Tuberculosis. BMJ 316: 1962–1964.
Phytother. Res. (2013)
Antitubercular and Antibacterial Activity of Quinonoid Natural Products Against Multi-Drug Resistant Clinical Isolates Diganta Dey,1,2 Ratnamala Ray2 and Banasri Hazra1* 1
Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India Department of Microbiology, Ashok Laboratory Clinical Testing Centre Private Limited, Kolkata 700068, India
2
Multi-drug resistant Mycobacterium tuberculosis and other bacterial pathogens represent a major threat to human health. In view of the critical need to augment the current drug regime, we have investigated therapeutic potential of five quinonoids, viz. emodin, diospyrin, plumbagin, menadione and thymoquinone, derived from natural products. The antimicrobial activity of quinonoids was evaluated against a broad panel of multi-drug and extensively drugresistant tuberculosis (M/XDR-TB) strains, rapid growing mycobacteria and other bacterial isolates, some of which were producers of β-lactamase, Extended-spectrum β-lactamase (ESBL), AmpC β-lactamase, metallo-betalactamase (MBL) enzymes, as well as their drug-sensitive ATCC counterparts. All the tested quinones exhibited antimycobacterial and broad spectrum antibacterial activity, particularly against M. tuberculosis (lowest MIC 0.25 μg/mL) and Gram-positive bacteria (lowest MIC emodin ~ menadione ~ thymoquinone > diospyrin, whereas their antibacterial efficacy was plumbagin > menadione ~ thymoquinone > diospyrin > emodin. Furthermore, this is the first evaluation performed on these quinonoids against a broad panel of drug-resistant and drug-sensitive clinical isolates, to the best of our knowledge. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: Mycobacterium tuberculosis; quinonoids; natural products; multi-drug resistance; clinical isolates.
INTRODUCTION Infectious diseases continue to be a leading cause of mortality around the world, particularly in developing countries. Bacterial diseases are rampant in India, and it is enlisted as one of the 22 countries with high tuberculosis (TB) burden (WHO, 2011, 2012). India, along with China, accounted for almost 40% of the total number of TB victims in the world, estimated as nine million new cases and 1.4 million deaths in 2011 (WHO, 2012). Moreover, clinical bacteria are becoming increasingly resistant due to antibiotic selection pressure. About a decade back, methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus spp. had raised a point of concern. Subsequently, multi-drug resistant (MDR) Gram-negative bacteria emerged, posing a greater threat to public health, since their increase in resistance would be faster than the Gram-positive ones, and there are fewer new antibiotics under development to provide sufficient therapeutic coverage in the near future (Kumarasamy et al., 2010). Again, the widespread appearance of MDR-TB further aggravated the scenario, particularly for those affected with the human immunodeficiency virus (HIV) infection (Zumla and Grange, 1998). In 2011, almost 60% of the notified pulmonary MDR-TB cases in the world were contributed by India, China and the Russian Federation (WHO, 2012).
* Correspondence to: Banasri Hazra, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India. E-mail: [email protected]
Copyright © 2013 John Wiley & Sons, Ltd.
Usually, the treatment of TB involves a combination regimen of four anti-TB drugs (ATDs), viz. isoniazid, rifampicin, ethambutol and pyrazinamide, administered over a period of six to eight months. Nevertheless, there is an urgent need to augment the current drug regime in view of the emerging drug resistance and co-infection with HIV (Mukanganyama et al., 2012; Guzman et al., 2012). In order to discover newer antibacterial therapeutics, it would be worthwhile to investigate selected plant products, as many antimicrobial agents, including ATDs like rifampicin, streptomycin, capreomycin and cycloserine, have been derived from natural products (Newton et al., 2000; Guzman et al., 2012). Worldwide exploration of certain medicinal plants generated many novel products, which, in turn, also validated the ethnomedical resources in terms of their key constituents possessing potent antimicrobial activity (Cowan, 1999). No wonder that the primary health care of 65–75% of the world’s population continue to be served by the age-old herbal formulations which are thriving even to this day (Newton et al., 2000). Plants have an amazing ability to synthesize secondary metabolites, mainly flavonoids, tannins and phenols or their oxygen-substituted quinonoid derivatives possessing unique structural features. In general, terpenoids endow the plants with distinctive odors, while quinones and tannins are responsible for the coloring pigments. Some such products also serve to defend their hosts from plant pathogens, signifying their capability to fight against infectious microbial diseases. In our laboratory, phytochemical screening and semi-synthetic derivatisation could unravel the pharmacological prospect of phenolics and quinones isolated from traditional medicinal Received 12 August 2013 Revised 28 October 2013 Accepted 06 November 2013
D. DEY ET AL.
plants (Hazra et al., 2002; Basu et al., 2005; Dey et al., 2012). Presently, we have evaluated five bioactive quinonoids (Fig. 1) against drug-resistant bacteria including Mycobacterium spp. characterized from clinical isolates. The plant-derived quinonoids, viz. emodin (1), diospyrin (2), plumbagin (3) and thymoquinone (5) have been identified as the active constituents in certain age-old Oriental medicinal formulations still popular in the Indian sub-continent (Hazra et al., 2004). However, menadione or vitamin K3 (4; a synthetic version of vitamin K2) was included due to its clinical implication and structural analogy with plumbagin (3). Preliminary findings on antimycobacterial (Newton et al., 2000; Guzman et al., 2012) and antibacterial (Cowan, 1999) activity of some of these compounds were reported by previous researchers. Here, we have performed a broad spectrum study on the inhibitory effect of the quinones against clinically significant drug-resistant strains sparsely investigated so far.
MATERIALS AND METHODS Test compounds. Emodin (1) was isolated from the stem bark of Ventilago madraspatana Gaertn. by the procedure described earlier (Basu et al., 2005). Plumbagin (3) was obtained from the root of Plumbago indica Linn. (syn. P. rosea Linn.), and diospyrin (2) from the stem-bark of Diospyros montana Roxb., following the respective methods described previously (Hazra et al., 2002). The products were characterized through spectroscopic analysis and direct comparison with previously authenticated samples. The plant materials were collected from Bolangir district, Orissa, and the voucher specimens were authenticated at the Botanical Survey of India, Kolkata. Menadione (4) and thymoquinone (5) were purchased from Sigma–Aldrich Co., MO, USA.
Isolation of mycobacteria. Detection of mycobacteria in clinical specimen was performed through enrichment in broth culture using BacT/ALERT 3D system (bioMérieux, Inc., Durham, NC, USA), after the sample was decontaminated by conventional N-acetyl-L-cysteine (NALC)-soduium hydroxide (NaOH) protocol (Collee et al., 1996). Decontaminated specimen (0.5 mL) was
Figure 1. Chemical structure of the studied quinones: emodin (1), diospyrin (2), plumbagin (3), menadione (4) and thymoquinone (5). Copyright © 2013 John Wiley & Sons, Ltd.
aseptically injected to the BacT/ALERT MP bottle containing 10 mL of aqueous media consisting of middlebrook 7H9 broth, pancreatic digest of casein, bovine serum albumin and catalase under vacuum. Lyophilized MB/BacT antibiotic supplement was reconstituted with 10 mL of MB/BacT reconstitution fluid and re-constituted solution (0.5 mL) was injected back into the preinoculated BacT/ALERT MP bottle for incubation within BacT/ALERT 3D Mycobacteria Detection System. The gas-permeable colorimetric sensor installed in the bottom of each culture bottle transmitted the increase of reflectance units proportional to the carbon dioxide produced by Mycobacterium growth, leading to a positive detection at approximately 106–107 colony forming units (CFUs) per mL (Thorpe et al., 1990).
Mycobacterial species identification. The culture broth from the BacT/ALERT MP bottle with positive growth of Mycobacterium was inoculated in Löwenstein–Jensen (LJ) slant (HiMedia, Mumbai), and identified by phenotypic tests and routine biochemical methods, viz., nitrate reduction, pyrazinamidase, catalase, urease and niacin accumulation tests using kits procured from Himedia, Mumbai (Collee et al., 1996). Additionally, genotypic confirmation of Mycobacterium tuberculosis complex (MTC) was accomplished by using artus® M. tuberculosis RG PCR kit (QIAGEN, Hilden). For this purpose, culture media (500 μL) collected from positive BacT/ALERT MP bottle was centrifuged at 13,000 rpm for 10 min. The resultant pellet was dissolved in 180 μL lysozyme (SRL, Mumbai; 20 mg/mL) and incubated at 37 °C for 45 min, followed by DNA extraction using QIAamp DNA Mini Kit (QIAGEN, Hilden), keeping appropriate internal control as per protocol. Real-time DNA detection was performed using the Rotor-Gene Q analyzer (QIAGEN, Hilden). The specific amplicons (a 159 base pair region of the mycobacterial genome) were detected directly using the 5′- nuclease (TaqMan) probe labelled with FAM dye (Hur et al., 2011).
Anti-tubercular drug (ATD) susceptibility. Susceptibility testing of isolated M. tuberculosis strains was carried out against first line (isoniazide, ethambutol, pyrazinamide, refampicin, streptomycin) and second line (kanamycin, amikacin, ethionamide, D-cycloserine, clarithromycin, p-amino salicylic acid, rifabutin) ATDs, following LJ proportion method on drug- incorporated LJ slants (HiMedia, Mumbai). The organism was considered to be resistant to a particular drug when the percentage ratio of the number of colonies on the drugcontaining slant and the control slant (without drug) was greater than 1%, sensitive in case of less than 1%, and intermediate when equal to 1% (Collee et al., 1996).
Antimycobacterial activity. Tetrazolium microplate assay with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, MO, USA) was done by following the method of Caviedes et al. (2002), with minor modification, for testing the susceptibility of five quinonoid test compounds against selected strains of Mycobacteria spp. Accordingly, mycobacterial suspension was taken from the tested BacT/ALERT MP Phytother. Res. (2013)
ANTIMICROBIAL ACTIVITY OF QUINONOID NATURAL PRODUCTS
bottle, turbidity was adjusted to 1 McFarland standard and subsequently diluted to 1:25 by adding Middlebrook 7H9 broth (bioMérieux BacT/ALERT MP), and used as inoculum in the microplate assay. In the 96-well plate, only sterile water (200 μL) was added to the upper and lower rows (A1-12, and H1-12, labelled as commercially stamped on the plates), and Middlebrook 7H9 broth (100 μL) was added to the rest of the wells. Serial 2× dilutions of each compound were prepared directly on the plate by adding 100 μL of the working solutions (1.024 mg/mL in dimethyl sulfoxide; DMSO) in five horizontal rows (B to F) of the microtitre plate. The residual row of 12 wells (G1-12) contained only DMSO as the ‘control’ serially diluted to the concentration corresponding to the respective wells used for MIC determination. Finally, the aforesaid inoculum (100 μL) was added to each well except those in A and H-rows, so that the final concentration range of the test compounds was 256 – 0.125 μg/mL. The microtitre plates were sealed with parafilm and incubated in plastic bags at 37 °C. The mycobacterial growth was detected by MTT solution (15 μL; 5 mg/mL MTT dissolved in ethanol: water =1:9), which was added to a single well in the ‘control’ row (G1-12). In case of positive growth, the colour turned purple after 24 h of incubation, when MTT was added to rest of the wells, and the colour change recorded after further 24 h of incubation. However, in case of incomplete growth, the ‘control’ well would remain yellow, and hence MTT was added to the next well for further incubation. In this way, the colour change was monitored until the tubercular growth appeared on days 8/9/10/14/16, depending on the nature of the particular strain. However, the rapid grower strains were monitored every 24 h starting from day 2, as these strains would grow faster (2 – 6 days) than M. tuberculosis (7 – 15 days; Collee et al., 1996). The lowest concentration, at which the mycobacterial growth was inhibited, as indicated by the colour change of MTT, was noted as the minimum inhibitory concentration (MIC) of the compound. To determine the minimum bactericidal concentration (MBC), a loop-full of culture from each well containing different dilutions of the tested compounds was streaked by a 4 mm loop (HiMedia, Mumbai; calibrated to 0.01 mL) on solid LJ medium base (HiMedia, Mumbai) containing glycerol. The lowest concentration of the tested compound that did not support the growth of visible mycobacterial colony on LJ medium was recorded as the MBC (Collee et al., 1996).
Isolation and characterization of the bacterial isolates. A total of 35 bacteria (13 Gram positive and 22 Gram negative), comprising of eight American Type Culture Collection (ATCC) strains and 27 pathogenic bacteria isolated from clinical specimen were included in this study. All the clinical isolates were identified by routine phenotypic and biochemical methods, as well as, agglutination with specific polyvalent and monovalent antisera (Collee et al., 1996). β-Lactamase production was identified by sharply demarcated zone edge around a penicillin G disc (10 U; HiMedia, Mumbai) (Collee et al., 1996; CLSI 2013) and inducible AmpC β-lactamase production was determined by blunting of cefotaxime (30 μg) zone adjacent Copyright © 2013 John Wiley & Sons, Ltd.
to the cefoxitin (30 μg) disc on a Mueller Hinton agar (MHA; HiMedia, Mumbai) plate as a result of their antagonism (Collee et al., 1996). Extended-spectrum β-lactamase (ESBL) production was identified by the enhancement of zone diameter of cephalosporin/ clavulanate disc in comparison to the cephalosporin disc alone. Metallo-β-lactamase (MBL) production was characterized by Imipenem-EDTA combined and double disc diffusion as well as by the modified Hodge test (Dey et al., 2012; CLSI 2013).
Antibacterial drug susceptibility. Antibacterial susceptibility studies were carried out by Kirby and Bauer disk diffusion technique using commercially available antibiotic discs (HiMedia, Mumbai) and interpretation of inhibition zone data were done by according CLSI guidelines (Dey et al., 2012; CLSI 2013).
Antibacterial activity. The broth micro-dilution assay was used for the determination of minimum inhibitory concentration (MIC) of five quinonoids against each of the selected bacteria (eight ATCC strains and 27 clinical isolates) to find the lowest concentration within the range of 512 – 4 μg/mL at which no growth was visible following the established procedure (Dey et al., 2012; CLSI 2013). To determine MBC, a loop-full of culture was taken from each well containing different dilutions of the tested compounds, and inoculated by a 4 mm loop (calibrated to 0.01 mL) on MHA plate. The lowest concentrations of the tested compounds that did not support the growth of visible bacterial colony were recorded as the MBC of the respective compounds (Collee et al., 1996).
RESULTS Antimycobacterial activity In the present study, five quinonoid compounds (1 – 5) were evaluated for antimycobacterial activity against one ATCC strain (H37Ra) and four clinical strains of M. tuberculosis, as well as two rapid-grower isolates of M. chelonei and M. fortuitum, in terms of MIC and MBC. The M. tuberculosis strains were isolated from pulmonary tuberculosis cases, while the two atypical rapid grower mycobacteria were taken from subcutaneous infections (Table 1). All these strains were selected from the assortment of patient samples routinely characterized in our clinic, and tested individually for resistance pattern against first- and second-line ATDs, using L-J proportional method (Collee et al., 1996). Thus, two out of the four M. tuberculosis strains selected for this study originated from MDR-TB cases, while the rest were from extensively drug resistant tuberculosis (XDR-TB; Table 1). According to WHO, MDR-TB is caused by strains of M. tuberculosis that are resistant to isoniazid and rifampicin, while additional resistance to a fluoroquinolone, such as, ciprofloxacin, and at least one second-line injectable agent, as for example, amikacin, kanamycin, signify XDR-TB (WHO, 2010). Phytother. Res. (2013)
D. DEY ET AL.
Table 1. Anti-tuberculous drug (ATD) resistance and minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) of emodin (1), diospyrin (2), plumbagin (3), menadione (4) and thymoquinone (5) against the tested mycobacterial strains MICa and MBC of tested quinonoids (μg/mL) Sl. no.
Species
Source
First line ATD resistance
Second line ATD resistance
1
2
3
5
None
4
16
EMB, INH, RMP
CS
8 4
32 4
M. tuberculosis (MDR) Pleural fluid
EMB, INH, PZA, RMP
CLR, RFB
8 16
16 64
1 8
8 16
8 8
4.
M. tuberculosis (XDR)
Sputum
INH, PZA, RMP, SM
AN, CIP, CS, KM
16 8
64 8
8 2
16 8
32 8
5.
M. tuberculosis (XDR)
Sputum
EMB, INH, RMP, SM
AN, CIP, ETA, KM
16 16
16 32
4 4
16 32
16 16
6.
M. chelonei (Rapid grower)
Wound discharge
EMB, INH, PZA, RMP
AN, CLR, CS, ETA, KM, PAS, RFB
32 16
64 128
8 16
32 32
32 16
7.
M. fortuitum (Rapid grower)
Pus from abscess
EMB, INH, PZA, RMP, SM CLR, CS, ETA, KM, PAS, RFB
64 32
128 256
64 32
64 64
64 32
128
>256
64
128
128
1.
M. tuberculosis (Reference)
H37Ra (ATCC 25177) None
2.
M. tuberculosis (MDR) Sputum
3.
8
4 8
8
16 16 0.25 4
16 4
a
Shaded row: MIC MDR: Multi-drug resistant; XDR: Extensively drug resistant; AN: Amikacin (700 μg/mL); CIP: Ciprofloxacin (12.5 μg/mL); CLR: Clarithromycin (8 μg/mL); CS: D-Cycloserine (30 μg/mL); ETA: Ethionamide (20 μg/mL); EMB: Ethambutol (2 μg/mL); INH: Isoniazide (0.2 μg/mL); KM: Kanamycin (30 μg/mL); PAS: p-Amino salicylic acid (2.5 μg/mL); PZA: Pyrazinamide (200 μg/mL); RFB: Rifabutin (0.5 μg/mL); RMP: Rifampicin (40 μg/mL); SM: Streptomycin (4 μg/mL)
The two rapid-grower strains were found to be resistant to most of the first-line and second-line ATDs (Table 1). All the tested quinones were found to show potential anti-TB activity with MIC values ranging between 0.25 and 64 μg/mL, although the MIC range was found to be comparatively higher (16 – 256 μg/mL) against the two rapid grower strains. Among all the compounds, plumbagin was found to be the most potent (MIC = 0.25 – 16 μg/mL), while menadione, emodin and thymoquinone showed more or less similar inhibitory activity against M. tuberculosis (MIC = 4 – 32 μg/mL). Diospyrin was found to be the least potent against M. tuberculosis as well as the rapid grower strains with the MIC range of 4 – 256 μg/mL. The other four quinonoids exhibited moderate activity against M. chelonei and M. fortuitum (MIC =16 – 32 μg/mL) (Table 1). Antibacterial activity The antibacterial efficacy of quinones (1 – 5) was evaluated against a broad spectrum of 35 bacteria, comprising 27 clinical isolates and eight ATCC strains (Table 2). The bacteria were isolated from different patients suffering from superficial wounds, urinary, enteric and pulmonary infections, and bacteremia (Table 2). All isolates were checked in vitro for antibiotic resistance property following CLSI guidelines (CLSI, 2013). Furthermore, the bacteria were selected in consideration of their clinical significance in the context of enzyme production related to their multi-drug resistant phenotypes. Hence, a vigorous screening of available clinical samples was undertaken to select one E. coli and one Pseudomonas aeruginosa as producers of AmpC β-lactamase and MBL enzymes, respectively. Similarly, a couple of Staphylococcus aureus and Morexella catarrhalis, in addition to one ATCC strain of Escherichia coli, were taken for their Copyright © 2013 John Wiley & Sons, Ltd.
ability to produce β-lactamase. Again, two clinical isolates and one ATCC strain of Klebsiella spp., and one Proteus mirabilis were chosen as ESBL producers (Table 2). In Table 2, the results of antibacterial evaluation, expressed as MIC and MBC, revealed the broadspectrum activity of the quinonoid compounds (1 – 5), albeit with variable efficacy. In general, the Grampositive bacteria exhibited greater susceptibility to the tested quinones in comparison to the Gram-negative bacteria. Among all the tested compounds, plumbagin was the most potent against both Gram-positive (MIC 512 μg/mL, and 8 – >512 μg/mL against the spectrum of tested bacteria.
DISCUSSION Antimycobacterial activity Naturally occurring quinones are ubiquitously distributed in living organisms. These compounds are characteristically capable of generating reactive oxygen species through redox cycling mechanism, easily undergoing either one-electron reduction to yield the semiquinone free radical, or two-electron reduction directly to the hydroquinone (Nohl et al., 1986). Thus, quinonoid natural Phytother. Res. (2013)
ANTIMICROBIAL ACTIVITY OF QUINONOID NATURAL PRODUCTS
Table 2. Antibiotic resistance pattern and minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) of emodin (1), diospyrin (2), plumbagin (3), menadione (4) and thymoquinone (5) against the bacterial strains tested in vitro MICa and MBC of tested quinonoids (μg/mL) Sl. no.
1.
Bacteria (source)
Enzyme produced
Gram positive bacteria Staphylococcus aureas (ATCC 25923)
–
Antibiotic resistance in vitro
1
2
3
4
5
None
>512
>512
16
32
32
>512 256
32 32
64 32
32 64
2.
Streptococcus pneumoniae (ATCC 27336)
–
None
>512 256
3.
S. pyogenes (ATCC 12384)
–
None
512 512
512 256
32 16
64 32
128 32
4.
Enterococcus faecalis (ATCC 29212)
–
None
>512 >512
512 256
32 32
32 256
64 16
5.
Staphylococcus aureus (Pus)
>512 256
>512 64
32 8
256 32
32 256
>512
128
16
64
256
6.
S. saprophyticus (Urine)
β-lactamase producer –
Cx, E, Cfx, Ca, Cd, Ox, As, Amc, Pt Ca, Cip, Nx
>512
64
64
128
128
128 128
64 32
128 64
256 128
7.
S. epidermidis (Wound swab)
–
None
>512 256
8.
Streptococcus pneumoniae (Sputum)
–
None
>512 128
256 256
32 64
64 64
256 64
9.
S. pyogenes (Throat swab)
–
None
128 256
512 512
64 32
64 64
128 128
10.
S. agalactiae (Pus)
–
None
>512 256
>512 64
64 32
128 64
128 64
11.
Bacillus subtilis (Faeces)
–
None
512 128
128 64
32 16
64 32
128 64
12.
B. cereus (Faeces)
–
P, Ca
256 8
128 64
16 512
128 128
8 64
32 512
32 128
>512
256
64
>512
256
14.
Gram negative bacteria Escherichia coli (ATCC 35218) β-lactamase producer
None
>512
256
16
64
256
Ao, As, Ca, Cfx, Do
>512 512
256 >512
16 128
64 128
512 128
None
>512 >512
>512 >512
128 128
256 128
256 128
None
>512 >512
>512 >512
256 32
256 32
512 64
>512 256
>512 >512
64 64
64 128
128 256
512 >512
>512 >512
128 64
256 128
256 256
>512 512
>512 >512
128 128
256 128
512 256
>512 128
>512 256
256 64
128 128
256 256
256 256
512 512
128 64
128 64
512 128
512 512
>512 256
64 128
128 128
256 256
>512 256
512 256
128 32
256 64
256 128
512 512
512 >512
64 64
128 128
256 128
>512
>512
64
128
256
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Klebsiella pneumoniae (ATCC 700603)
ESBL producer
Pseudomonas aeruginosa (ATCC 27853)
–
Proteus vulgaris (ATCC 6896)
–
Escherichia coli (Urine) Klebsiella pneumoniae (Sputum) K. oxytoca (Urine) Enterobacter aerogenes (Urine) Citrobacter freundii (Urine) Proteus mirabilis (Urine) P. vulgaris (Urine) Serratia marcescens (Blood)
AmpC β- lactamase producer
Amc, Cfx, Cl, Ao, Cip, Nx, Ca, Do, Nf, cfm
ESBL producer
As, Cfx, Cip, Cl, Lv, Mr, Nx, Of, Ca, Ao, Cfm
ESBL producer – – ESBL producer – –
As, Cfx, Cip, Cl, Do, Of, Nf, Ca, Ao, Cfm Cip, Nx, Ca, Gen Nx, Ca, Do Cfx, Cl, Ca, Ao, Cip, Gen, Pt, As Do Cfx
(Continues) Copyright © 2013 John Wiley & Sons, Ltd.
Phytother. Res. (2013)
D. DEY ET AL.
Table 2. (Continued) MICa and MBC of tested quinonoids (μg/mL) Sl. no. 26.
Bacteria (source) Pseudomonas aeruginosa (Urine)
Enzyme produced MBL producer –
Antibiotic resistance in vitro
1
2
3
4
5
Gen, Mr, Nx, Of, Ca
>512
256
128
128
128
Ca, Gen, Cip, Ak
>512 256
512 >512
128 128
256 128
256 256
Amp, Ca
512 256
>512 128
256 64
128 32
512 128
256 512
256 256
64 32
64 64
128 256
>512 >512
>512 >512
64 64
64 64
256 128
27.
Acinetobacter baumannii (Blood)
28.
Morexella catarrhalis (Throat swab)
29.
Vibrio cholerae (Faeces)
–
Ct
30.
Salmonella typhi (Blood)
–
As, Amc, Gen, Nx, Of, Ak, Ao, C
31.
S. paratyphi A (Blood)
–
None
>512 512
>512 >512
64 32
64 64
256 128
32.
Shigella flexneri (Faeces)
–
Amp, Nx, Ct
>512 256
>512 256
64 64
64 32
256 64
33.
Shigella sonnei (Faeces)
–
None
512 >512
512 512
64 32
32 64
64 256
34.
Burkholderia cepacia (Blood)
–
Ca, Ct
>512 256
>512 256
32 64
64 32
512 128
35.
Stenotrophomonas maltophilia (Pus)
–
Ca
512 256
512 >512
64 128
32 128
256 128
512
>512
128
256
128
β-lactamase producer
a
Shaded row: MIC Ak: Amikacin (30 μg), Amc: Amoxy-Clavulanic acid (20/10 μg), Amp: Ampicillin (10 μg), Ao: Aztreonam (30 μg), As: Ampicillin/Sulbactum (10/10 μg), C: Chloramphenicol (30 μg), Ca: Ceftazidime (30 μg), Cd: Clindamycin (2 μg), Cfm: Cefixime (5 μg), Cfx: Cefuroxime (30 μg), Cip: Ciprofloxacin (5 μg), Cl: Ceftriaxone (30 μg), Ct: Co-trimoxazole (Sulfamethoxazole/trimethoprim) (1.25/23.75 μg), Cx: Cefoxitin (100 μg), Do: Doxycycline (30 μg), E: Erythromycin (15 μg), Gen: Gentamicin (10 μg), Lv: Levofloxacin (5 μg), Mo: Moxifloxacin (5 μg), Mr: Meropenem (10 μg), Nf: Nitrofurantoin (300 μg), Nx: Norfloxacin (10 μg), Of: Ofloxacin (5 μg), Ox: Oxacillin (1 μg), P: Penicillin (100 μg), Pt: Piperacillin/Tazobactum (100/10 μg), Tp: Teicoplanin (30 μg)
products, particularly the anthracycline antibiotics, have been developed into anticancer agents and antimicrobials, as they could bind irreversibly with nucleophilic amino acids, often leading to inactivation of proteins and loss of function, with enormous toxicological and pharmacological implication. Again, ubiquinones (coenzyme Q) and plastoquinones are known to play vital roles in the biochemistry of energy production in mammalian and plant cells, respectively, by shuttling electrons between the membrane-bound proteins in the electron transport chain, while in bacteria, menaquinone (vitamin K2), too, is involved in the functioning of respiratory pathways. It was found that E. coli can utilize both ubiquinone and menaquinone, while only menaquinone is involved in mycobacteria. Therefore, menaquinone biosynthesis pathway is considered as an attractive target for developing novel inhibitors as antimycobacterial agents (Mathew et al., 2010). Menadione is a precursor of vitamin K2 as it undergoes alkylation to yield menaquinones. Thus, it would be worthwhile to study the selected quinonoids, representing benzo-, naphtho- and anthraquinones (Fig. 1), against a series of drug-resistant bacteria, both Gram-positive and negative, as well as Mycobacterium strains critically selected from clinical isolates. Emodin and its anthraquinonoid analogues are the ‘marker’ constituents in several herbal preparations popularly dispensed as Ayurveda and Unani medicines in India, Pen Ts’ao in China, Kampo in Japan and Copyright © 2013 John Wiley & Sons, Ltd.
numerous traditional systems in other Asian countries (Hazra et al., 2004). Diospyrin and plumbagin were identified to be the active principles found in medicinal plants traditionally used in ancient Ayurveda and Chinese medicines, and folklore systems in Africa and Latin America for treatment of miscellaneous ailments, such as skin lesions, febrile conditions, parasitic infestation, renal disease, dental problems, diarrhea, pneumonia, syphylis and tumors (Mallavadhani et al., 1998). Thymoquinone is an active ingredient isolated from Nigella sativa, or black cumin, used as a common spice in Mediterranean countries and Indian sub-continent. Historically, the use of its seed oil was recommended for the treatment of bronchitis, asthma, rheumatism, diarrhea and skin disorders, which had been mentioned in ancient Latin, Arabic and Sanskrit literature (Salem, 2005). Presently, multi-drug resistance has become a burning issue in the public health domain as the total number of XDR-TB in 84 countries comprises nearly 9.0% of the average proportion of MDR-TB cases (WHO, 2012). Therefore, a well-proven combination regimen for the treatment of MDR- and XDR-TB needs to be established. Previously, the antimycobacterial activity of some plant-derived quinonoids and derivatives have been demonstrated mostly against non-pathogenic reference and atypical strains of mycobacteria, but not in the clinical scenario (Pullen et al., 2002; Tran et al., 2004; Kanokmedhakul et al., 2005; Mathew et al., 2010; Mukanganyama et al., 2012). Therefore, it was Phytother. Res. (2013)
ANTIMICROBIAL ACTIVITY OF QUINONOID NATURAL PRODUCTS
important to extend our study to M. tuberculosis strains from MDR- and XDR-TB cases, as well as rapidly growing mycobacteria. The latter represented a wide variety of opportunistic pathogens responsible for pulmonary diseases, and primary skin and soft tissue infections usually found in patients with systemic impairment of immunity (Collee et al., 1996; van Ingen et al., 2010). Previously, the antimycobacterial activity of plumbagin against M. avium and M. smegmatis (MIC = 12.5 μg/mL) was reported by Tran et al. (2004). However, they found the response of plumbagin against M. tuberculosis H37Ra strain to be substantially lower (MIC = 400 μg/mL), which did not agree with our result (MIC = 8 μg/mL). Again, Mossa et al. (2004) and Mathew et al. (2010) found the MIC of plumbagin to be diospyrin > emodin. Furthermore, this is the first evaluation performed on these quinonoids against a broad panel of bacteria comprising drug-susceptible as well as resistant clinical bacteria in addition to their ATCC counterparts, to the best of our knowledge.
Acknowledgements D.D. acknowledges technical and management support provided by Ashok Laboratory Clinical Testing Centre Private Limited, Kolkata. Research Scientist Grant from the University Grants Commission, New Delhi, received by B.H.
Conflict of Interest The authors declare that there is no conflict of interest.
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