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Research Paper
Journal of Pharmacy And Pharmacology
Synergistic antibacterial activity of the combination of the alkaloid sanguinarine with EDTA and the antibiotic streptomycin against multidrug resistant bacteria Razan Hamoud, Jürgen Reichling and Michael Wink Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany
Keywords EDTA; multidrug resistance; sanguinarine; streptomycin; synergy Correspondence Michael Wink, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, INF 364, Heidelberg, 69120, Germany E-mail: [email protected] Received July 1, 2014 Accepted September 9, 2014 doi: 10.1111/jphp.12326
Abstract Objectives Drug combinations consisting of the DNA intercalating benzophenanthridine alkaloid sanguinarine, the chelator EDTA with the antibiotic streptomycin were tested against several Gram-positive and Gram-negative bacteria, including multi-resistant clinical isolates. Methods Microdilution, checkerboard and time kill curve methods were used to investigate the antibacterial activity of the individual drugs and the potential synergistic activity of combinations. Key findings Sanguinarine demonstrated a strong activity against Gram-positive and Gram-negative bacteria (minimum inhibitory concentrations, MIC = 0.5– 128 μg/ml), while streptomycin was active against Gram-negative strains (MIC = 2–128 μg/ml). EDTA showed only bacteriostatic activity. Indifference to synergistic activity was seen in the two-drug combinations sanguinarine + EDTA and sanguinarine + streptomycin (fractional inhibitory concentration index = 0.1– 1.5), while the three-drug combination of sanguinarine + EDTA + streptomycin showed synergistic activity against almost all the strains (except methicillinresistant Staphylococcus aureus), as well as a strong reduction in the effective doses (dose reduction index = 2–16 times) of sanguinarine, EDTA and streptomycin. In time kill studies, a substantial synergistic interaction of the three-drug combination was detected against Escherichia coli and Klebsiella pneumoniae. Conclusions The combination of drugs, which interfere with different molecular targets, can be an important strategy to combat multidrug resistant bacteria.
Introduction The worldwide occurrence of drug resistance against standard antibiotics and the lack of new antimicrobial drugs demand for new strategies to treat complicated infections. Drug combinations and multidrug therapy offer the possibility to overcome drug resistance.[1] It is less likely that pathogens develop a resistance if drugs, which affect different and multiple molecular targets, are used in combinations.[2] Multidrug therapy could include combinations of several antibiotics or of antibiotics with other antimicrobial agents. In our study, we performed two- and three-drug combinations consisting of an antibiotic (streptomycin) with a chelating agent (EDTA) and the DNA intercalating alkaloid (sanguinarine). Such a combination has not been studied before. The benzophenanthridine alkaloid sanguinarine, which can be isolated from Sanguinaria canadensis, Macleaya
cordata or Eschscholzia californica (all Papaveraceae), is a planar molecule that exhibits a strong DNA intercalating activity.[3] Sanguinarine exhibits pronounced antimicrobial, antifungal, antiviral, anti-inflammatory and anticancer activity.[4–6] Due to its antimicrobial properties and ability to inhibit bacterial attachment to solid substrates, sanguinarine has been used in toothpastes and mouthwashes.[7] The combination of sanguinarine with a chelating agent (EDTA) may increase its possibility to enter cells and reach its site of action. EDTA can disturb the permeability of the cell wall of bacteria by chelating of Ca++ and Mg++ cations, which are essential for the stability of the bacterial cell wall, especially of Gram-negative bacteria.[8] Both sanguinarine and EDTA have already been successfully used in combination with antibiotics and other antimicrobial drugs.[9,10] The third drug in our study is the aminoglycoside
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Razan Hamoud et al.
antibiotic streptomycin, which targets protein synthesis by direct interaction with the small ribosomal subunit.[11] Streptomycin was the first drug used in the treatment of Mycobacterium tuberculosis and is preferred for treatment of relapses.[12] In this study, two- and three-drug combinations of sanguinarine, EDTA and streptomycin were investigated against 34 standard bacterial strains and clinical isolates. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined for the individual drugs following checkerboard dilution and time kill curve analyses of two- and three-drug combinations. Using these methods, we demonstrated synergistic interactions, dose reduction and bactericidal effect for a combination of sanguinarine, EDTA and streptomycin.
Materials and Methods Microbial strains The following standard strains were studied: Grampositive bacteria: methicillin-resistant Staphylococcus aureus (MRSA NCTC 10442), vancomycin-resistant Enterococcus (VRE VanB ATCC 31299), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 14990), Streptococcus pyogenes (ATCC 12344), Streptococcus agalactiae (ATCC 27956) and Bacillus subtilis (ATCC 6051); and Gram-negative bacteria: Acinetobacter baumannii (ATCC BAA 747), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 700603), Escherichia coli (ATCC 25922) and E. coli O157:H7 (ATCC 35150). Ten clinical isolates of MRSA and 13 clinical isolates of Gram-negative bacteria were also included in this study. All microorganisms were supplied by the Medical Microbiology Laboratory, Hygiene Institute, Heidelberg University, Germany.
Culture media Columbia agar supplemented with 5% sheep blood (Becton Dickinson, Heidelberg, Germany) was used for bacterial cultivation and MBC determination, while broth medium like brain heart infusion (Merck, Darmstadt, Germany) and Mueller–Hinton broth (Fluka, Sigma-Aldrich, Seelze, Germany) were used for broth dilution analyses.
Inoculum preparation Bacteria were grown to a density of 0.5 McFarland, which is equal to 1 × 108 colony forming units per millilitre (CFU/ ml). A 1 : 100 broth dilution gave the final concentration of 1 × 106 CFU/ml, which was used in all assays. 2
Determination of minimum inhibitory concentrations and minimum bactericidal concentrations Twofold dilutions of drugs were used for MIC determination according to the Clinical and Laboratory Standards Institute.[13] Sanguinarine, EDTA and streptomycin were separately diluted in 96-well plates to obtain a range of concentrations between 128 μg/ml and 0.25 μg/ml for sanguinarine and streptomycin, and between 4000 μg/ml and 7.5 μg/ml for EDTA. Sanguinarine was dissolved in 5% Dimethyl Sulfoxide (DMSO). This DMSO concentration was included in the assays as a negative control. Streptomycin and EDTA were dissolved in sterile water. A bacterial suspension of 1 × 106 CFU/ml was added to each well of a 96-well plate, then the plates were incubated at 35 °C for 24 h. The concentration in the first clear well, which showed no bacterial growth (turbidity) compared with the negative control, was recorded as MIC. Then 3 μl from each clear well was inoculated onto agar plates. The lowest concentration, which consequently did not show a microbial growth, was considered as MBC.
Checkerboard dilution The checkerboard method evaluates the inhibitory activity of combinations; it is based on the MIC values of single drugs. Checkerboard was applied as described recently.[14] In two-drug combinations, sanguinarine and EDTA were diluted serially in a separate 96-well plate; after that, they were combined in the combination experiment. In the three-drug combination experiment, a fixed concentration of streptomycin (1/16 MIC) was added to the two-drug combination plate. A bacterial suspension of 1 × 106 CFU/ ml was added to each well of the plate, which was subsequently incubated at 35 °C for 24 h. The fractional inhibitory concentration index (FICI) describes the results of checkerboard, and it is calculated as follows:
FICI of two-drug combination = FIC A + FIC B FICI of three-drug combination = FIC A + FIC B + FIC C where FIC A is the MIC of drug A in the combination/ MIC of drug A alone, FIC B is the MIC of drug B in the combination/MIC of drug B alone, and FIC C is the MIC of drug C in the combination/MIC of drug C alone. The results indicate synergism when the corresponding FICI ≤ 0.5, additivity when 0.5 < FICI ≤ 1, indifference when 1 < FICI ≤ 4 and antagonism when the FICI > 4.[15]
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Razan Hamoud et al.
Drug combination toward resistant bacteria
Isobologram analysis The results of checkerboard experiments can be illustrated by means of isobolograms. The MIC of drug A is marked on the x-axis of an isobologram and the MIC of drug B on the y-axis. The line connecting these two marks is the indifference line (no interaction). The MIC values of the combination, which were located either below the indifference line, indicate additive (1 ≥ FICI > 0.5) or synergistic (FICI ≤ 0.5) interactions. Values that are found above the indifference line indicate indifferent (1 < FICI ≤ 4) or antagonistic (FICI > 4) interactions.[16]
The dose reduction index The dose reduction index (DRI) describes the difference between the effective doses in a combination in comparison with its individual dose. DRI is calculated as follows:
DRI = MIC of drug alone MIC of drug in combination DRI is of interest clinically when the dose reduction is associated with a toxicity reduction without efficacy changes.[17] Generally, a DRI > 1 is considered beneficial.[18]
Statistical analysis All experiments were carried out in triplicates and repeated at least three times in three independent days. Statistical analysis using a Student’s t-test was performed to investigate any significance between the MIC of the single drugs and their MIC in the two-drug and three-drug combinations.
Results
Time kill assay Time kill assays for single drug as well as drug combination were performed according to the National Committee For Clinical Laboratory Standard (NCCLS) guidelines.[19] In these assays, 1 × 106 CFU/ml of the test strains were incubated with different concentrations of the individual drugs Table 1
beside different two-drug and three-drug combinations in separate tubes. After several time intervals of 0, 1, 2, 4, 6 and 24 h, aliquots were removed from each tube and diluted serially (1 : 10) using sterile saline. From each dilution, 20 μl was inoculated onto agar plates, which were incubated for 24 h at 35 °C. After that, the number of viable colonies was counted only from the plates containing between 30 and 300 colony to calculate the CFU/ml.[20] According to NCCLS, an antimicrobial agent is considered bactericidal if it causes a ≥3 × log10 (99.9%) reduction in colony forming units per millilitre (CFU/ml) after 18–24 h of incubation, and the combination is considered synergistic when it causes a ≥2 × log10 reduction in CFU/ml.
Minimum inhibitory concentration and minimum bactericidal concentration values MIC and MBC values of the individual drugs against Gram-positive and Gram-negative are presented in Tables 1 and 2, respectively. Among the three drugs, sanguinarine
The MIC and MBC values of sanguinarine, EDTA and streptomycin against standard strains and clinical isolates of Gram-positive bacteria Sanguinarine μg/ml (μM)
EDTA μg/ml (μM)
Streptomycin μg/ml (μM)
Gram-positive bacteria
MIC
MBC
MIC
MBC
MIC
MBC
VRE VanB ATCC 31299 Staphylococcus aureus ATCC 25923 Staphylococcus epidermidis ATCC 14990 Streptococcus pyogenes ATCC 12344 Streptococcus agalactiae ATCC 27956 Bacillus subtilis ATCC 6051 MRSA NCTC 10442 MRSA KL 215947 MRSA KL 215790 MRSA BC 210301 MRSA UC 15127 MRSA MR 213561 MRSA MR 213652 MRSA MR 13165 MRSA MR 12921 MRSA MR 13023 MRSA MR 13037
8 (24) 1 (3) 0.5 (1.5) 4 (12) 8 (24) 1 (3) 4 (12) 2 (6) 2 (6) 2 (6) 2 (6) 2 (6) 2 (6) 1 (3) 2 (6) 1 (3) 2 (6)
>128 (>385) 64 (192.7) 32 (96.3) 8 (24) 16 (48.1) 16 (48.1) 16 (48.1) 32 (96.3) 32 (96.3) 32 (96.3) 32 (96.3) 32 (96.3) 32 (96.3) 16 (48.1) 32 (96.3) 32 (96.3) 32 (96.3)
250 (855.4) 60 (206) 60 (206) 125 (427.7) 250 (855.4) 60 (206) 125 (427.7) 60 (206) 60 (206) 60 (206) 60 (206) 125 (427.7) 60 (206) 60 (206) 60 (206) 60 (206) 60 (206)
>4000 >4000 >4000 250 (855.4) >4000 2000 (6843) 1000 (3421.8) >4000 >4000 4000 >4000 >4000 >4000 >4000 >4000 >4000 >4000
NA 4 (6.8) 4 (6.8) 32 (55) 128 (220) 32 (55) NA 4 (6.8) 4 (6.8) 2 (3.4) 4 (6.8) 4 (6.8) NA 2 (3.4) 8 (13.7) 2 (3.4) 2 (3.4)
NT 8 (13.7) 16 (27.5) 32 (55) >128 >128 NT 8 (13.7) 8 (13.7) 4 (6.8) 16 (27.5) 32 (55) NT 16 (27.5) 16 (27.5) 16 (27.5) 4 (6.8)
MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; NA, no activity; NT, not tested. All experiments were carried out in triplicates and repeated at least three times on three independent days. © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Table 2
Razan Hamoud et al.
The MIC and MBC values of sanguinarine, EDTA and streptomycin against standard strains and clinical isolates of Gram-negative bacteria Sanguinarine μg/ml (μM)
EDTA μg/ml (μM)
Streptomycin μg/ml (μM)
Gram-negative bacteria
MIC
MBC
MIC
MBC
MIC
MBC
Escherichia coli ATCC 25922 E. coli O157:H7 ATCC 35150 E. coli UL 300362/1 E. coli BL 300173/46 E. coli ML 300237/12 Klebsiella pneumoniae ATCC 700603 K. pneumoniae UL 300382/32 K. pneumoniae ML 300339/34 K. pneumoniae BL 300279/71 Pseudomonas aeruginosa ATCC 27853 P. aeruginosa ML 300258/20 P. aeruginosa ML 300245/21 P. aeruginosa AL 213370 Acinetobacter baumanii ATCC BAA 747 A. baumanii ML 301347/117 A. baumanii SR 201346 A. baumanii MS 202672
8 (24) 4 (12) 8 (24) 4 (12) 4 (12) 16 (48.1) 16 (48.1) 16 (48.1) 16 (48.1) 128 (385) NA NA 128 (385) 16 (48.1) 16 (48.1) 16 (48.1) 16 (48.1)
16 (48.1) 16 (48.1) 32 (96.3) 16 (48.1) 8 (24) >128 32 (96.3) 128 (385) 128 (385) >128 NT NT >128 64 (192.7) 32 (96.3) 64 (192.7) 64 (192.7)
2000 (6843) 2000 (6843) 4000 (13687) 4000 (13687) 2000 (6843) 4000 (13687) NA NA 4000 (13687) 4000 (13687) 125 (427.7) 1000 (3421.8) 125 (427.7) 125 (427.7) 125 (427.7) 125 (427.7) 125 (427.7)
>4000 >4000 >4000 >4000 >4000 >4000 NT NT >4000 >4000 >4000 >4000 >4000 >4000 >4000 >4000 >4000
8 (13.7) 4 (6.8) NA 32 (55) 8 (13.7) 2 (3.4) 2 (3.4) 128 (220) 128 (220) 4 (6.8) 16 (27.5) 16 (27.5) 32 (55) 2 (3.4) 16 (27.5) NA NA
16 (27.5) 8 (13.7) NT 64 (110) 16 (27.5) 4 (6.8) 4 (6.8) >128 >128 8 (13.7) 32 (55) 32 (55) 64 (110) 4 (6.8) 64 (110) NT NT
MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; NA, no activity; NT, not tested. All experiments were carried out in triplicates and repeated at least three times on three independent days.
Checkerboard analysis Isobolograms of two- and three-drug combinations against MRSA and Gram-negative bacteria are shown in Figures 1 and 2. FICI and DRI values of the strongest combinations against all the studied strains are presented in Table 3. A strong synergistic interaction was recorded against the standard strains and clinical isolates of E. coli in two-drug and three-drug combinations (FICI ≈ 0.1–0.4). Furthermore, a significant dose reduction of the three-drug combi4
MRSA
70 60
Sanguinarine + EDTA Sanguinarine + EDTA + Streptomycin
50 EDTA μg/ml
demonstrated the strongest antibacterial activity against Gram-positive bacteria, with MIC values ranging between 0.5 μg/ml against S. epidermidis and 8 μg/ml against VRE, while corresponding MBC values ranged between 8 μg/ml and 128 μg/ml. Next was streptomycin with MIC values between 2 μg/ml and 128 μg/ml. VRE and MRSA standard strains were resistant to streptomycin, while some clinical isolates of MRSA were still sensitive to streptomycin (MIC = 2 μg/ml). Streptomycin demonstrated the strongest activity against Gram-negative bacteria (MIC between 2 μg/ml and 128 μg/ ml); only some clinical isolates of A. baumannii, K. pneumoniae and E. coli were resistant to streptomycin. Sanguinarine also inhibited Gram-negative bacteria, with MIC values between 4 μg/ml against the strains of E. coli and 128 μg/ml against P. aeruginosa. EDTA showed only bacteriostatic activity against both Gram-positive (MIC between 60 μg/ml and 250 μg/ml) and Gram-negative bacteria (MIC between 125 μg/ml and 4000 μg/ml), while an MBC value was almost undetectable (MBC >4000 μg/ml).
40 30 20 10 0 0
1
2 3 Sanguinarine μg/ml
4
5
Figure 1 Isobologram analyses. Minimum inhibitory concentrations of sanguinarine are plotted on x-axis and minimum inhibitory concentration values of EDTA on y-axis. The curves represent the combinations with or without 1/16 minimum inhibitory concentration streptomycin against methicillin-resistant Staphylococcus aureus.
nation was recorded; the dose of sanguinarine could be reduced between four- and eightfold (P < 0.01). The DRI of EDTA increased between eight- and 16-fold in two-drug combinations with sanguinarine (P < 0.01) and reached a 32 times dose reduction in three-drug combination (P < 0.01). The dose of streptomycin was reduced around eight times in two-drug combinations with sanguinarine (P < 0.05) and around 16 times in three-drug combination (P < 0.01). Stronger synergistic interactions were recorded
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Razan Hamoud et al.
Drug combination toward resistant bacteria
Escherichia coli UL 300362
(a) 5000
2000 EDTA μg/ml
EDTA μg/ml
4000 3000 2000
1500 1000
1000 0
Escherichia coli ATCC 25922
2500
500
2
0
4 6 Sanguinarine μg/ml
8
0
10
0
2
4 6 Sanguinarine μg/ml
8
10
Sanguinarine + EDTA Sanguinarine + EDTA + Streptomycin Acinetobacter baumanii ATCC BAA 747
Acinetobacter baumanii ML 301347
140
120
120
100
100 EDTA μg/ml
EDTA μg/ml
140
80 60
80 60
40
40
20
20
0
0 0
2
4
6 10 12 8 Sanguinarine μg/ml
14
16
18
0
2
4
6 10 12 8 Sanguinarine μg/ml
14
16
18
Figure 2 (a,b) Isobologram analyses. Minimum inhibitory concentrations of sanguinarine are plotted on x-axis and minimum inhibitory concentration values of EDTA on y-axis. The curves represent the combinations with or without of 1/16 minimum inhibitory concentration streptomycin against different Gram-negative bacteria.
against the clinical isolates of K. pneumonia (FICI ≈ 0.3) by two-drug combinations of sanguinarine and EDTA or the three-drug combinations with streptomycin. The dose of sanguinarine was substantially and synergistically reduced in two-drug combinations with EDTA against the standard isolate of P. aeruginosa (DRI = 32 times; FICI ≈ 0.1) (P < 0.01). Synergistic activity was recorded in the three-drug combination against the standard strain of A. baumannii with FICI = 0.49 with a dose reduction of four, eight, and four times of sanguinarine, EDTA and streptomycin, respectively (P < 0.05). However, indifferent interactions were seen against multi-resistant clinical isolates of A. baumannii (FICI ≈ 0.6–1). The weakest activity was recorded for the combination sanguinarine + EDTA + streptomycin against the standard strains and the clinical isolates of MRSA with FICI = 1–1.5.
Nevertheless, the dose reduction was twofold for sanguinarine, fourfold for EDTA and twofold for streptomycin.
Time kill curve Time kill curves of different combinations of sanguinarine + EDTA + streptomycin against MRSA, E. coli and K. pneumoniae are presented in Figures 3–5. According to checkerboard results against MRSA, the studied combinations in time kill assay started from MIC of the individual drugs and the two-drug combinations (MIC sanguinarine + MIC EDTA, and MIC sanguinarine + MIC streptomycin), while the three-drug combination started from 1⁄2 MIC sanguinarine + 1⁄2 MIC EDTA + 1⁄2 MIC streptomycin. The two-drug combinations and the three-drug combination of 1⁄2 MIC could reduce the colony count of
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Drug combination toward resistant bacteria
(b)
Razan Hamoud et al.
Pseudomonas aeruginosa AL 213370
140
Pseudomonas aeruginosa ATCC 27853
5000
120
4000 EDTA μg/ml
EDTA μg/ml
100 80 60
3000 2000
40 1000 20 0
0 0
20
40 60 80 100 Sanguinarine μg/ml
120
140
0
20
40
60 80 100 Sanguinarine μg/ml
120
140
Klebsiella pneumoniae BL 300279
5000
EDTA μg/ml
4000
3000
Sanguinarine + EDTA Sanguinarine + EDTA + Streptomycin
2000
1000
0 0
Figure 2
2
4
6 8 10 12 Sanguinarine μg/ml
14
16
18
Continued.
MRSA during the 24-h incubation in an additive way (less than 2 log10 CFU/ml reduction). Only the three-drug combination MIC sanguinarine + MIC EDTA + MIC streptomycin could achieve the synergistic activity by the colony count reduction of 2 log10. Against E. coli, 1⁄4 MIC of the individual drugs, the twoand three-drug combinations beside the three-drug combination 1/8 MIC sanguinarine + 1/18 MIC EDTA + 1/8 MIC streptomycin were investigated for 24 h. The two-drug combination 1⁄4 MIC sanguinarine + 1⁄4 MIC EDTA beside the three-drug combinations resulted in a synergistic activity as compared with the drugs when studied alone, while the two-drug combination 1⁄4 MIC sanguinarine + 1⁄4 MIC streptomycin could additively reduce the colony count. According to the strong synergistic activity of the combinations by checkerboard analysis, three-drug interactions of 1/16 MIC sanguinarine + 1/16 MIC EDTA + 1/16 MIC streptomycin beside 1/8 MIC sanguinarine + 1/18 MIC 6
EDTA + 1/8 MIC streptomycin were investigated against K. pneumoniae BL 300279 for 24 h. Among the combinations, only the three-drug combinations of 1/8 MIC indicated synergistic activity as compared with the individual drugs; this activity started within 1 h of incubation and lasted for 6 h when the bacteria started to recover and grow again.
Discussion The main aim of this study was to find out if the treatment of resistant clinical isolates of pathogens was effective by combinations of drugs with different mode of actions. It was analysed whether the combinations could achieve additive or synergistic effects, reduce the toxic dose, and minimise or delay the induction of drug resistance.[21] Sanguinarine has already been combined with antibiotics and other antimicrobial natural products, and synergistic
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
16
8 2 4 16
>8
16
16 2 8 2
2
4
16 8 4 4
8
MRSA KL 215947
10
Log (CFU/ml)
8 16
0.37
–
4 16 16 32 8
–
–
1.5 0.31 0.37
Research Paper
Journal of Pharmacy And Pharmacology
Synergistic antibacterial activity of the combination of the alkaloid sanguinarine with EDTA and the antibiotic streptomycin against multidrug resistant bacteria Razan Hamoud, Jürgen Reichling and Michael Wink Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany
Keywords EDTA; multidrug resistance; sanguinarine; streptomycin; synergy Correspondence Michael Wink, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, INF 364, Heidelberg, 69120, Germany E-mail: [email protected] Received July 1, 2014 Accepted September 9, 2014 doi: 10.1111/jphp.12326
Abstract Objectives Drug combinations consisting of the DNA intercalating benzophenanthridine alkaloid sanguinarine, the chelator EDTA with the antibiotic streptomycin were tested against several Gram-positive and Gram-negative bacteria, including multi-resistant clinical isolates. Methods Microdilution, checkerboard and time kill curve methods were used to investigate the antibacterial activity of the individual drugs and the potential synergistic activity of combinations. Key findings Sanguinarine demonstrated a strong activity against Gram-positive and Gram-negative bacteria (minimum inhibitory concentrations, MIC = 0.5– 128 μg/ml), while streptomycin was active against Gram-negative strains (MIC = 2–128 μg/ml). EDTA showed only bacteriostatic activity. Indifference to synergistic activity was seen in the two-drug combinations sanguinarine + EDTA and sanguinarine + streptomycin (fractional inhibitory concentration index = 0.1– 1.5), while the three-drug combination of sanguinarine + EDTA + streptomycin showed synergistic activity against almost all the strains (except methicillinresistant Staphylococcus aureus), as well as a strong reduction in the effective doses (dose reduction index = 2–16 times) of sanguinarine, EDTA and streptomycin. In time kill studies, a substantial synergistic interaction of the three-drug combination was detected against Escherichia coli and Klebsiella pneumoniae. Conclusions The combination of drugs, which interfere with different molecular targets, can be an important strategy to combat multidrug resistant bacteria.
Introduction The worldwide occurrence of drug resistance against standard antibiotics and the lack of new antimicrobial drugs demand for new strategies to treat complicated infections. Drug combinations and multidrug therapy offer the possibility to overcome drug resistance.[1] It is less likely that pathogens develop a resistance if drugs, which affect different and multiple molecular targets, are used in combinations.[2] Multidrug therapy could include combinations of several antibiotics or of antibiotics with other antimicrobial agents. In our study, we performed two- and three-drug combinations consisting of an antibiotic (streptomycin) with a chelating agent (EDTA) and the DNA intercalating alkaloid (sanguinarine). Such a combination has not been studied before. The benzophenanthridine alkaloid sanguinarine, which can be isolated from Sanguinaria canadensis, Macleaya
cordata or Eschscholzia californica (all Papaveraceae), is a planar molecule that exhibits a strong DNA intercalating activity.[3] Sanguinarine exhibits pronounced antimicrobial, antifungal, antiviral, anti-inflammatory and anticancer activity.[4–6] Due to its antimicrobial properties and ability to inhibit bacterial attachment to solid substrates, sanguinarine has been used in toothpastes and mouthwashes.[7] The combination of sanguinarine with a chelating agent (EDTA) may increase its possibility to enter cells and reach its site of action. EDTA can disturb the permeability of the cell wall of bacteria by chelating of Ca++ and Mg++ cations, which are essential for the stability of the bacterial cell wall, especially of Gram-negative bacteria.[8] Both sanguinarine and EDTA have already been successfully used in combination with antibiotics and other antimicrobial drugs.[9,10] The third drug in our study is the aminoglycoside
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Drug combination toward resistant bacteria
Razan Hamoud et al.
antibiotic streptomycin, which targets protein synthesis by direct interaction with the small ribosomal subunit.[11] Streptomycin was the first drug used in the treatment of Mycobacterium tuberculosis and is preferred for treatment of relapses.[12] In this study, two- and three-drug combinations of sanguinarine, EDTA and streptomycin were investigated against 34 standard bacterial strains and clinical isolates. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined for the individual drugs following checkerboard dilution and time kill curve analyses of two- and three-drug combinations. Using these methods, we demonstrated synergistic interactions, dose reduction and bactericidal effect for a combination of sanguinarine, EDTA and streptomycin.
Materials and Methods Microbial strains The following standard strains were studied: Grampositive bacteria: methicillin-resistant Staphylococcus aureus (MRSA NCTC 10442), vancomycin-resistant Enterococcus (VRE VanB ATCC 31299), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 14990), Streptococcus pyogenes (ATCC 12344), Streptococcus agalactiae (ATCC 27956) and Bacillus subtilis (ATCC 6051); and Gram-negative bacteria: Acinetobacter baumannii (ATCC BAA 747), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 700603), Escherichia coli (ATCC 25922) and E. coli O157:H7 (ATCC 35150). Ten clinical isolates of MRSA and 13 clinical isolates of Gram-negative bacteria were also included in this study. All microorganisms were supplied by the Medical Microbiology Laboratory, Hygiene Institute, Heidelberg University, Germany.
Culture media Columbia agar supplemented with 5% sheep blood (Becton Dickinson, Heidelberg, Germany) was used for bacterial cultivation and MBC determination, while broth medium like brain heart infusion (Merck, Darmstadt, Germany) and Mueller–Hinton broth (Fluka, Sigma-Aldrich, Seelze, Germany) were used for broth dilution analyses.
Inoculum preparation Bacteria were grown to a density of 0.5 McFarland, which is equal to 1 × 108 colony forming units per millilitre (CFU/ ml). A 1 : 100 broth dilution gave the final concentration of 1 × 106 CFU/ml, which was used in all assays. 2
Determination of minimum inhibitory concentrations and minimum bactericidal concentrations Twofold dilutions of drugs were used for MIC determination according to the Clinical and Laboratory Standards Institute.[13] Sanguinarine, EDTA and streptomycin were separately diluted in 96-well plates to obtain a range of concentrations between 128 μg/ml and 0.25 μg/ml for sanguinarine and streptomycin, and between 4000 μg/ml and 7.5 μg/ml for EDTA. Sanguinarine was dissolved in 5% Dimethyl Sulfoxide (DMSO). This DMSO concentration was included in the assays as a negative control. Streptomycin and EDTA were dissolved in sterile water. A bacterial suspension of 1 × 106 CFU/ml was added to each well of a 96-well plate, then the plates were incubated at 35 °C for 24 h. The concentration in the first clear well, which showed no bacterial growth (turbidity) compared with the negative control, was recorded as MIC. Then 3 μl from each clear well was inoculated onto agar plates. The lowest concentration, which consequently did not show a microbial growth, was considered as MBC.
Checkerboard dilution The checkerboard method evaluates the inhibitory activity of combinations; it is based on the MIC values of single drugs. Checkerboard was applied as described recently.[14] In two-drug combinations, sanguinarine and EDTA were diluted serially in a separate 96-well plate; after that, they were combined in the combination experiment. In the three-drug combination experiment, a fixed concentration of streptomycin (1/16 MIC) was added to the two-drug combination plate. A bacterial suspension of 1 × 106 CFU/ ml was added to each well of the plate, which was subsequently incubated at 35 °C for 24 h. The fractional inhibitory concentration index (FICI) describes the results of checkerboard, and it is calculated as follows:
FICI of two-drug combination = FIC A + FIC B FICI of three-drug combination = FIC A + FIC B + FIC C where FIC A is the MIC of drug A in the combination/ MIC of drug A alone, FIC B is the MIC of drug B in the combination/MIC of drug B alone, and FIC C is the MIC of drug C in the combination/MIC of drug C alone. The results indicate synergism when the corresponding FICI ≤ 0.5, additivity when 0.5 < FICI ≤ 1, indifference when 1 < FICI ≤ 4 and antagonism when the FICI > 4.[15]
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Razan Hamoud et al.
Drug combination toward resistant bacteria
Isobologram analysis The results of checkerboard experiments can be illustrated by means of isobolograms. The MIC of drug A is marked on the x-axis of an isobologram and the MIC of drug B on the y-axis. The line connecting these two marks is the indifference line (no interaction). The MIC values of the combination, which were located either below the indifference line, indicate additive (1 ≥ FICI > 0.5) or synergistic (FICI ≤ 0.5) interactions. Values that are found above the indifference line indicate indifferent (1 < FICI ≤ 4) or antagonistic (FICI > 4) interactions.[16]
The dose reduction index The dose reduction index (DRI) describes the difference between the effective doses in a combination in comparison with its individual dose. DRI is calculated as follows:
DRI = MIC of drug alone MIC of drug in combination DRI is of interest clinically when the dose reduction is associated with a toxicity reduction without efficacy changes.[17] Generally, a DRI > 1 is considered beneficial.[18]
Statistical analysis All experiments were carried out in triplicates and repeated at least three times in three independent days. Statistical analysis using a Student’s t-test was performed to investigate any significance between the MIC of the single drugs and their MIC in the two-drug and three-drug combinations.
Results
Time kill assay Time kill assays for single drug as well as drug combination were performed according to the National Committee For Clinical Laboratory Standard (NCCLS) guidelines.[19] In these assays, 1 × 106 CFU/ml of the test strains were incubated with different concentrations of the individual drugs Table 1
beside different two-drug and three-drug combinations in separate tubes. After several time intervals of 0, 1, 2, 4, 6 and 24 h, aliquots were removed from each tube and diluted serially (1 : 10) using sterile saline. From each dilution, 20 μl was inoculated onto agar plates, which were incubated for 24 h at 35 °C. After that, the number of viable colonies was counted only from the plates containing between 30 and 300 colony to calculate the CFU/ml.[20] According to NCCLS, an antimicrobial agent is considered bactericidal if it causes a ≥3 × log10 (99.9%) reduction in colony forming units per millilitre (CFU/ml) after 18–24 h of incubation, and the combination is considered synergistic when it causes a ≥2 × log10 reduction in CFU/ml.
Minimum inhibitory concentration and minimum bactericidal concentration values MIC and MBC values of the individual drugs against Gram-positive and Gram-negative are presented in Tables 1 and 2, respectively. Among the three drugs, sanguinarine
The MIC and MBC values of sanguinarine, EDTA and streptomycin against standard strains and clinical isolates of Gram-positive bacteria Sanguinarine μg/ml (μM)
EDTA μg/ml (μM)
Streptomycin μg/ml (μM)
Gram-positive bacteria
MIC
MBC
MIC
MBC
MIC
MBC
VRE VanB ATCC 31299 Staphylococcus aureus ATCC 25923 Staphylococcus epidermidis ATCC 14990 Streptococcus pyogenes ATCC 12344 Streptococcus agalactiae ATCC 27956 Bacillus subtilis ATCC 6051 MRSA NCTC 10442 MRSA KL 215947 MRSA KL 215790 MRSA BC 210301 MRSA UC 15127 MRSA MR 213561 MRSA MR 213652 MRSA MR 13165 MRSA MR 12921 MRSA MR 13023 MRSA MR 13037
8 (24) 1 (3) 0.5 (1.5) 4 (12) 8 (24) 1 (3) 4 (12) 2 (6) 2 (6) 2 (6) 2 (6) 2 (6) 2 (6) 1 (3) 2 (6) 1 (3) 2 (6)
>128 (>385) 64 (192.7) 32 (96.3) 8 (24) 16 (48.1) 16 (48.1) 16 (48.1) 32 (96.3) 32 (96.3) 32 (96.3) 32 (96.3) 32 (96.3) 32 (96.3) 16 (48.1) 32 (96.3) 32 (96.3) 32 (96.3)
250 (855.4) 60 (206) 60 (206) 125 (427.7) 250 (855.4) 60 (206) 125 (427.7) 60 (206) 60 (206) 60 (206) 60 (206) 125 (427.7) 60 (206) 60 (206) 60 (206) 60 (206) 60 (206)
>4000 >4000 >4000 250 (855.4) >4000 2000 (6843) 1000 (3421.8) >4000 >4000 4000 >4000 >4000 >4000 >4000 >4000 >4000 >4000
NA 4 (6.8) 4 (6.8) 32 (55) 128 (220) 32 (55) NA 4 (6.8) 4 (6.8) 2 (3.4) 4 (6.8) 4 (6.8) NA 2 (3.4) 8 (13.7) 2 (3.4) 2 (3.4)
NT 8 (13.7) 16 (27.5) 32 (55) >128 >128 NT 8 (13.7) 8 (13.7) 4 (6.8) 16 (27.5) 32 (55) NT 16 (27.5) 16 (27.5) 16 (27.5) 4 (6.8)
MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; NA, no activity; NT, not tested. All experiments were carried out in triplicates and repeated at least three times on three independent days. © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Drug combination toward resistant bacteria
Table 2
Razan Hamoud et al.
The MIC and MBC values of sanguinarine, EDTA and streptomycin against standard strains and clinical isolates of Gram-negative bacteria Sanguinarine μg/ml (μM)
EDTA μg/ml (μM)
Streptomycin μg/ml (μM)
Gram-negative bacteria
MIC
MBC
MIC
MBC
MIC
MBC
Escherichia coli ATCC 25922 E. coli O157:H7 ATCC 35150 E. coli UL 300362/1 E. coli BL 300173/46 E. coli ML 300237/12 Klebsiella pneumoniae ATCC 700603 K. pneumoniae UL 300382/32 K. pneumoniae ML 300339/34 K. pneumoniae BL 300279/71 Pseudomonas aeruginosa ATCC 27853 P. aeruginosa ML 300258/20 P. aeruginosa ML 300245/21 P. aeruginosa AL 213370 Acinetobacter baumanii ATCC BAA 747 A. baumanii ML 301347/117 A. baumanii SR 201346 A. baumanii MS 202672
8 (24) 4 (12) 8 (24) 4 (12) 4 (12) 16 (48.1) 16 (48.1) 16 (48.1) 16 (48.1) 128 (385) NA NA 128 (385) 16 (48.1) 16 (48.1) 16 (48.1) 16 (48.1)
16 (48.1) 16 (48.1) 32 (96.3) 16 (48.1) 8 (24) >128 32 (96.3) 128 (385) 128 (385) >128 NT NT >128 64 (192.7) 32 (96.3) 64 (192.7) 64 (192.7)
2000 (6843) 2000 (6843) 4000 (13687) 4000 (13687) 2000 (6843) 4000 (13687) NA NA 4000 (13687) 4000 (13687) 125 (427.7) 1000 (3421.8) 125 (427.7) 125 (427.7) 125 (427.7) 125 (427.7) 125 (427.7)
>4000 >4000 >4000 >4000 >4000 >4000 NT NT >4000 >4000 >4000 >4000 >4000 >4000 >4000 >4000 >4000
8 (13.7) 4 (6.8) NA 32 (55) 8 (13.7) 2 (3.4) 2 (3.4) 128 (220) 128 (220) 4 (6.8) 16 (27.5) 16 (27.5) 32 (55) 2 (3.4) 16 (27.5) NA NA
16 (27.5) 8 (13.7) NT 64 (110) 16 (27.5) 4 (6.8) 4 (6.8) >128 >128 8 (13.7) 32 (55) 32 (55) 64 (110) 4 (6.8) 64 (110) NT NT
MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; NA, no activity; NT, not tested. All experiments were carried out in triplicates and repeated at least three times on three independent days.
Checkerboard analysis Isobolograms of two- and three-drug combinations against MRSA and Gram-negative bacteria are shown in Figures 1 and 2. FICI and DRI values of the strongest combinations against all the studied strains are presented in Table 3. A strong synergistic interaction was recorded against the standard strains and clinical isolates of E. coli in two-drug and three-drug combinations (FICI ≈ 0.1–0.4). Furthermore, a significant dose reduction of the three-drug combi4
MRSA
70 60
Sanguinarine + EDTA Sanguinarine + EDTA + Streptomycin
50 EDTA μg/ml
demonstrated the strongest antibacterial activity against Gram-positive bacteria, with MIC values ranging between 0.5 μg/ml against S. epidermidis and 8 μg/ml against VRE, while corresponding MBC values ranged between 8 μg/ml and 128 μg/ml. Next was streptomycin with MIC values between 2 μg/ml and 128 μg/ml. VRE and MRSA standard strains were resistant to streptomycin, while some clinical isolates of MRSA were still sensitive to streptomycin (MIC = 2 μg/ml). Streptomycin demonstrated the strongest activity against Gram-negative bacteria (MIC between 2 μg/ml and 128 μg/ ml); only some clinical isolates of A. baumannii, K. pneumoniae and E. coli were resistant to streptomycin. Sanguinarine also inhibited Gram-negative bacteria, with MIC values between 4 μg/ml against the strains of E. coli and 128 μg/ml against P. aeruginosa. EDTA showed only bacteriostatic activity against both Gram-positive (MIC between 60 μg/ml and 250 μg/ml) and Gram-negative bacteria (MIC between 125 μg/ml and 4000 μg/ml), while an MBC value was almost undetectable (MBC >4000 μg/ml).
40 30 20 10 0 0
1
2 3 Sanguinarine μg/ml
4
5
Figure 1 Isobologram analyses. Minimum inhibitory concentrations of sanguinarine are plotted on x-axis and minimum inhibitory concentration values of EDTA on y-axis. The curves represent the combinations with or without 1/16 minimum inhibitory concentration streptomycin against methicillin-resistant Staphylococcus aureus.
nation was recorded; the dose of sanguinarine could be reduced between four- and eightfold (P < 0.01). The DRI of EDTA increased between eight- and 16-fold in two-drug combinations with sanguinarine (P < 0.01) and reached a 32 times dose reduction in three-drug combination (P < 0.01). The dose of streptomycin was reduced around eight times in two-drug combinations with sanguinarine (P < 0.05) and around 16 times in three-drug combination (P < 0.01). Stronger synergistic interactions were recorded
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Razan Hamoud et al.
Drug combination toward resistant bacteria
Escherichia coli UL 300362
(a) 5000
2000 EDTA μg/ml
EDTA μg/ml
4000 3000 2000
1500 1000
1000 0
Escherichia coli ATCC 25922
2500
500
2
0
4 6 Sanguinarine μg/ml
8
0
10
0
2
4 6 Sanguinarine μg/ml
8
10
Sanguinarine + EDTA Sanguinarine + EDTA + Streptomycin Acinetobacter baumanii ATCC BAA 747
Acinetobacter baumanii ML 301347
140
120
120
100
100 EDTA μg/ml
EDTA μg/ml
140
80 60
80 60
40
40
20
20
0
0 0
2
4
6 10 12 8 Sanguinarine μg/ml
14
16
18
0
2
4
6 10 12 8 Sanguinarine μg/ml
14
16
18
Figure 2 (a,b) Isobologram analyses. Minimum inhibitory concentrations of sanguinarine are plotted on x-axis and minimum inhibitory concentration values of EDTA on y-axis. The curves represent the combinations with or without of 1/16 minimum inhibitory concentration streptomycin against different Gram-negative bacteria.
against the clinical isolates of K. pneumonia (FICI ≈ 0.3) by two-drug combinations of sanguinarine and EDTA or the three-drug combinations with streptomycin. The dose of sanguinarine was substantially and synergistically reduced in two-drug combinations with EDTA against the standard isolate of P. aeruginosa (DRI = 32 times; FICI ≈ 0.1) (P < 0.01). Synergistic activity was recorded in the three-drug combination against the standard strain of A. baumannii with FICI = 0.49 with a dose reduction of four, eight, and four times of sanguinarine, EDTA and streptomycin, respectively (P < 0.05). However, indifferent interactions were seen against multi-resistant clinical isolates of A. baumannii (FICI ≈ 0.6–1). The weakest activity was recorded for the combination sanguinarine + EDTA + streptomycin against the standard strains and the clinical isolates of MRSA with FICI = 1–1.5.
Nevertheless, the dose reduction was twofold for sanguinarine, fourfold for EDTA and twofold for streptomycin.
Time kill curve Time kill curves of different combinations of sanguinarine + EDTA + streptomycin against MRSA, E. coli and K. pneumoniae are presented in Figures 3–5. According to checkerboard results against MRSA, the studied combinations in time kill assay started from MIC of the individual drugs and the two-drug combinations (MIC sanguinarine + MIC EDTA, and MIC sanguinarine + MIC streptomycin), while the three-drug combination started from 1⁄2 MIC sanguinarine + 1⁄2 MIC EDTA + 1⁄2 MIC streptomycin. The two-drug combinations and the three-drug combination of 1⁄2 MIC could reduce the colony count of
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
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Drug combination toward resistant bacteria
(b)
Razan Hamoud et al.
Pseudomonas aeruginosa AL 213370
140
Pseudomonas aeruginosa ATCC 27853
5000
120
4000 EDTA μg/ml
EDTA μg/ml
100 80 60
3000 2000
40 1000 20 0
0 0
20
40 60 80 100 Sanguinarine μg/ml
120
140
0
20
40
60 80 100 Sanguinarine μg/ml
120
140
Klebsiella pneumoniae BL 300279
5000
EDTA μg/ml
4000
3000
Sanguinarine + EDTA Sanguinarine + EDTA + Streptomycin
2000
1000
0 0
Figure 2
2
4
6 8 10 12 Sanguinarine μg/ml
14
16
18
Continued.
MRSA during the 24-h incubation in an additive way (less than 2 log10 CFU/ml reduction). Only the three-drug combination MIC sanguinarine + MIC EDTA + MIC streptomycin could achieve the synergistic activity by the colony count reduction of 2 log10. Against E. coli, 1⁄4 MIC of the individual drugs, the twoand three-drug combinations beside the three-drug combination 1/8 MIC sanguinarine + 1/18 MIC EDTA + 1/8 MIC streptomycin were investigated for 24 h. The two-drug combination 1⁄4 MIC sanguinarine + 1⁄4 MIC EDTA beside the three-drug combinations resulted in a synergistic activity as compared with the drugs when studied alone, while the two-drug combination 1⁄4 MIC sanguinarine + 1⁄4 MIC streptomycin could additively reduce the colony count. According to the strong synergistic activity of the combinations by checkerboard analysis, three-drug interactions of 1/16 MIC sanguinarine + 1/16 MIC EDTA + 1/16 MIC streptomycin beside 1/8 MIC sanguinarine + 1/18 MIC 6
EDTA + 1/8 MIC streptomycin were investigated against K. pneumoniae BL 300279 for 24 h. Among the combinations, only the three-drug combinations of 1/8 MIC indicated synergistic activity as compared with the individual drugs; this activity started within 1 h of incubation and lasted for 6 h when the bacteria started to recover and grow again.
Discussion The main aim of this study was to find out if the treatment of resistant clinical isolates of pathogens was effective by combinations of drugs with different mode of actions. It was analysed whether the combinations could achieve additive or synergistic effects, reduce the toxic dose, and minimise or delay the induction of drug resistance.[21] Sanguinarine has already been combined with antibiotics and other antimicrobial natural products, and synergistic
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
16
8 2 4 16
>8
16
16 2 8 2
2
4
16 8 4 4
8
MRSA KL 215947
10
Log (CFU/ml)
8 16
0.37
–
4 16 16 32 8
–
–
1.5 0.31 0.37
