Printer Friendly

In vitro antimicrobial activity of pannarin alone and in combination with antibiotics against methicillin-resistant Staphylococcus aureus clinical isolates.

ARTICLE INFO

Keywords:

Pannarin

Lichen secondary metabolites

MRSA

Checkerboard assay

Antimicrobial activity

Membrane permeability

ABSTRACT

The in vitro antimicrobial activities of pannarin, a depsidone isolated from lichens, collected in several Southern regions of Chile (including Antarctica), was evaluated alone and in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. [MIC.sub.90], [MIC.sub.50], as well as [MBC.sub.50] and [MBC.sub.50], were evaluated. A moderate synergistic action was observed in combination with gentamicin, whilst antagonism was observed in combination with levofloxacin. All combinations with erythromycin were indifferent, whilst variability was observed for clindamycin and oxacillin combinations. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index and the response surface approach using the [DELTA] E model. Discrepancies were found between both methods for some combinations. In order to asses cellular lysis after exposure to pannarin, cell membrane permeability assay was performed. The treatment with pannarin produces bactericidal activity without significant calcein release, consistent with lack of lysis or even significant structural damage to the cytoplasmic membrane. Furthermore, pannarin shows low hemolytic activity and moderate cytotoxic effect on peripheral blood mononuclear cells. These findings suggest that the natural compound pannarin might be a good candidate for the individualization of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

[c] 2012 Elsevier GmbH. All rights reserved.

Introduction

Although medicinal plants have been used for centuries as sources of therapeutic agents worldwide, they cannot be classified as pure, efficient antimicrobial agents. However, in spite of the fact that plant-derived antibacterial compounds show a general low degree of activity, most plants, indeed, are successful in fighting infections (Hemaiswarya et al. 2008). Plants, in fact, adopt "synergy" as their peculiar different paradigm to fight pathogenic micro-organisms. Several studies have in fact demonstrated that a number of natural products, which failed as antimicrobials, are able to dramatically increase the effectiveness of chemotherapeutic agents against Gram-negative bacteria (Gibbons and Udo 2000; Hemaiswarya et al. 2008; Stavri et al. 2007; Tegos et al. 2002). Therefore, new sources for antimicrobials are tested. Amongst the new sources, wild organisms living in particular habitats are receiving great attention. At this purpose lichens produce a variety of secondary compounds that typically arise from the fungal component secondary metabolism, many of them exclusive of the lichen production. Chemotaxonomic studies have shown that the most unique lichen metabolites belong to the chemical classes of depsides, depsidones and dibenzofurans.

Pannarin is a secondary metabolite produced by lichens belonging to the chemical class of depsidones (Jackman et al. 1975). This molecule is usually found in Psoroma species (Piovano et al. 1985), whose use in traditional medicine is unknown. This substance was demonstrated to be active against prostate carcinoma (Russo et al. 2006), to induce apoptosis in human melanoma cells (Russo et al. 2008, p. 468) and to be active against cutaneous leishmaniasis (Fournet et al. 1997). At this time, nothing is known about the antimicrobial activity of pannarin. In this paper we demonstrated the antimicrobial activity of pannarin against multi-drug methicillin-resistant Staphylococcus aureus clinical isolates and its synergic activity in combination with therapeutically available antibiotics. Moreover, the hemolytic and cytotoxic activity was studied. Finally, we demonstrated that pannarin does not induce membrane permeabilization in S. aureus, thus indicating, a specific intracellular activity.

Materials and methods

Organisms

Twenty methicillin-resistant S. aureus strains were collected during a four years period, from 2006 to 2010, at the University Hospital "San Salvatore" of l'Aquila, Italy, from hospitalized patients. The clinical isolated were identified as MRSA organisms by Phoenix System (Becton Dickinson). The methicillin-resistant S. aureus ATCC 43300 from the American Type Culture Collection was used as control. Four strains, namely, AQ004, AQ006, AQ007 and AQ012 clinical isolates and the reference strain ATCC 43300 were used for the drug interaction assay. All those strains were resistant to clindamycin, erythromycin, gentamicin, levofloxacin, oxacillin, with the exception of ATCC 43300 that was sensible to levofloxacin.

Antibiotics and chemicals

All tested antibiotics, clindamycin (CLI), erythromycin (ERY), gentamicin (GEN), levofloxacin (LVX), oxacillin (OXA), were from Sigma-Aldrich (Milan, Italy). Calcein-AM and Triton X-100 were from Sigma-Aldrich (Milan-Italy).

Pannarin

Pannarin (Fig. 1) was purified and structurally characterized as previously described (Garbarino et al. 1991; Piovano et al. 1985), from Psoroma species (P. dimorphum Malme, P. pallidum (Mont.) Nyl., P. pulchrum Malme, P. reticulatum (Hue) Zalhbr), collected in Los Lagos y de Los Rios Regions, Southern Chile. The degree of purity for pannarin was > 98% as determined by thin layer chromatography (TLC) and [.sup.1]H NMR analyses.

In vitro susceptibility tests

The antimicrobial susceptibility pattern of the organisms used in this study was determined in accordance with the CLSI guidelines (CLSI 2010). In detail, 50 [micro]l of each bacterial suspension in 0.9% saline solution (NaCl) were added to the wells of a sterile 96-well microtitre plate already containing 50 [micro]l twofold serially diluted antibiotic or pannarin in cation-adjusted Mueller-Hinton, to reach a final volume in each well of 100 [micro]l. Positive control wells were prepared with culture medium and bacterial suspension. Negative control wells were prepared with culture medium and antibiotic or pannarin. The microtitre plates were incubated for 18h at 37 [degrees] C. The growth in each well was quantified spectrophotometrically at 595 nm by a microplate reader iMark, BioRad (Milan, Italy). The minimum inhibitory concentration (MIC) for drugs and pannarin was defined as the concentration of drug that reduced growth by 80% compared to that of organisms grown in the absence of drug. The MIC value was determined as the median of three independent experiments. [MIC.sub.50] and [MIC.sub.90] were defined as the MIC at which 50% and 90% respectively of the isolates were inhibited.

Bactericidal activity of pannarin was performed as previously described by Pearson et al. (1980) and Taylor et al. (1983). In detail, the viable cells were determined by performing tenfold serial dilutions of 100 [micro]l of the culture from the MIC test experiment in 0.9% saline solution. All dilutions were plated on Mueller-Hinton agar plates and incubated for 18 h at 37 [degrees] C. Percent viable was expressed as:

100 - [experimental viable count/control viable count] x 100.

The MBC was defined as the minimal amount of pannarin that results in a > 99.9% decrease in the initial inoculum. All experiments were performed in triplicate.

Checkerboard microdilution assay

The in vitro interactions between the antibiotics and pannarin were investigated by a two-dimensional checkerboard microdilution assay, using a 96-well microtitration plates. The range of concentrations tested for each drug was two to fourfold lower than the MIC calculated alone.

A stock solution of antibiotics and pannarin was prepared in the microbial growth medium used and serially diluted twofold in Mueller-Hinton medium. Drug dilutions were initially prepared in order to obtain four times the final concentration. In each well of the microplate 25 [micro]l of microbial growth medium were added. An aliquot of 25 [micro]l of a fourfold concentrated antibiotic was added to column 12. Then a twofold dilution was made from column 12 to column 2. A 25 [micro]l aliquot of each drug concentration of the compound was added to rows A-G. Row H contained only the antibiotic whilst column 1 only the compound. Well H1 was the drug free well used as growth control. Finally, 50 [micro]l of a 0.9% saline solution containing bacteria were added to each well of the microplate in order to obtain a final inoculum of 5 x 105 CFU/ml. The microtitre plates were incubated at 37 [degrees] C for 18h. The growth in each well was quantified spectrophotometrically at 595 nm by a microplate reader. The percentage of growth in each well was calculated as [([OD.sub.drug combination well] - [OD.sub.background)/([0D.sub.drug free well] - [OD.sub.background])], where the background was obtained from the microorganism-free plates, processed as the inoculated plates. The MICs for each combination of drugs were defined as the concentration of drug that reduced growth by 80% compared to that of organisms grown in the absence of drug. All experiments were performed in triplicate.

Drug interaction models

In order to assess the nature of the in vitro interactions between pannarin and antibiotics against each S. aureus, the data obtained from the checkerboard assay were analysed by nonparametric models based on the Loewe additivity model (LA) and the Bliss independence (BI) theory (Greco et al. 1995). In the Bliss models, the combined effects of the drugs, calculated from the effect of the individual drugs, are compared with those obtained experimentally.

Loewe additivity-based model

The nonparametric approach is based on the fractional inhibitory concentration index (FICI) model expressed as: [SIGMA] FIC = [FIC.sub.A] + [FIC.sub.B] = [FIC.sub.AB]/[FIC.sub.A] + [FIC.sub.BA]/[FIC.sub.B], where MICA and MICR are the MICs of drugs A and B when acting alone and [FIC.sub.AB] and [FIC.sub.BA] are the MICs of drugs A and B when acting in combination. Amongst all [SIGMA] FICs calculated for each microplate, the FICI was determined as the lowest [SIGMA] FIC ([SIGMA] [FIC.sub.min]) when the highest [SIGMA] FIC ([SIGMA] [FIC.sub.max]) was smaller then 4. In our experiments all FICIs where lower than 4, thus all AC index where calculated as [SIGMA] [FIC.sub.min].

Synergy was defined when FICI [less than or equal to] 0.5, whilst antagonism was defined when FICI > 4. A FIC index between 0.5 and 4 (0.5 < FICI [less than or equal to] 4) was considered indifferent. In order to visualize a deviation from the simply additivity, isobolograms were plotted.

Bliss independence-based model.

In the Bliss models, the combined effects of the drugs calculated from the effect of the individual drugs, are compared with those obtained experimentally. The BI theory is described by the equation [I.sub.i] = [I.sub.A] + [I.sub.B] - [I.sub.A] x [I.sub.B] where [I.sub.i] is the predicted percentage of inhibition of the theoretical non-interactive combination of drug A and B and [I.sub.A] and [I.sub.B] are the experimental percentages of inhibition of each drug acting alone. Since the percentage of inhibition is equal to one minus the percentage of growth (1=1 - E), the former equation can be expressed as: [E.sub.i] = [E.sub.A] x [E.sub.B], where [E.sub.i] is the calculated percentage of growth based on the theoretical non-interactive combination of drug A and B, and [E.sub.A] and [E.sub.B] are the experimental percentages of growth of each drug acting alone.

The experimental dose-response surface is subtracted from the calculated theoretical surface to reveal any significant deviation from the zero-plane. Thus the interaction is described by the difference ([DELTA] E) between the predicted and measured percentages of growth with drugs at various concentrations ([DELTA] E = [F.sub.predicted] - [E.sub.measured). To determine the significance of differences between the experimental and calculated additive effects, the upper and lower 95% confidence limits of the experimental data were compared to the calculated additive effects. If the lower confidence limit of a point was greater than the calculated additivity, the observed synergy was considered to be significant. Similarly, if the upper confidence limit was lower than the calculated additivity, the observed antagonism was considered to be significant (Prichard and Shipman 1990; Prichard et al. 1991). The AE values calculated on a point-by-point basis were subsequently plotted on the z axis. Points of the difference surface above zero (positive) indicate synergy, below zero (negative) antagonism. In order to summarize the interaction surface, the Bliss synergy and antagonism differences and all their combinations were added up to yield a summary measure, respectively of Bliss synergy ([DELTA] SYN) and Bliss antagonism ([DELTA] ANT). Interactions < 100% were considered weak, interactions between 100% and 200% were considered moderate, whilst interaction > 200% were considered strong (Meletiadis et al. 2005).

Membrane permeabilization assay

Membrane permeabilization of S. aureus ATCC43300 by pannarin was detected and quantified by fluorescence, via the release of the preloaded fluorophore calcein ([[lambda].sub.ex] 494 nm, [[lambda].sub.em] 517 nm). The nonfluorescent lipid soluble calcein acetoxymethyl ester (calcein AM), can readily diffuse across the membranes. Once within the cytoplasm, calcein AM is hydrolysed by esterases, yielding fluorescent calcein (EssodaIgui et al. 1998; Hollo et al. 1994).

S. aureus cells were first loaded with calcein AM as previously described with some modifications (Koo et al. 2001). Briefly, cells were grown in TSB (tryptic soy broth medium) at 37 [degrees] C up to a optical density at 600 nm of ([approximately equal to] [10.sup.9] CFU/ml). Bacteria were washed twice in PBS 1X (phosphate-buffered saline) pH 7.2. Then, bacteria were resuspended in a PBS solution containing 10% of TSB (vol/vol), 4 [micro] M of calcein AM and incubated at 37'.0 for 2 h. Bacteria were washed three-times in PBS 1X, pH 7.2 and resuspended to achieve a final concentration of [10.sub.8] CFU/ml. Calcein loaded S. aureus was incubated with three different concentration of pannarin: 16 [micro]g/ml, 4 [micro]g/ml and 1 [micro]g/ml. Fluorescence intensity ([I.sub.s]) for each treated samples was measured at 0, 60, 120 min and 12 h. Fluorescence intensity corresponding to 100% of intracellular calcein ([I.sub.100]) was determined by boiling the cells for 10 min. The background fluorescence ([I.sub.bkg]) of the untreated cells was also measured. The released of calcein was calculated as percent of total fluorescence released [([I.sub.s] - [I.sub.bkg])/(I.sub.100 - I.sub.bkg]) x 100. All experiments were performed in triplicate.

Hemolytic assay

Hemolytic activity of pannarin on human erythrocytes was determined as described earlier with some modifications (Young et al. 1986). Briefly, erythrocytes were isolated from heparinized human blood by centrifugation after washing three times with phosphate-buffered saline (PBS 5 mM phosphate buffer containing 150 mM NaCl pH 7.4). Red blood cells where diluted in PBS saline to obtain a final absorbance at 655 nm of 1.0 and an aliquot of 50 [micro]l of this solution was added to each wells of a sterile 96-well microtitre plate already containing 50 [micro]l of twofold serially diluted pannarin to reach a final volume in each well of 100 [micro]l The absence of hemolysis during the assay was ascertained preparing wells with PBS saline and erythrocytes (0% hemolysis). Complete hemolysis was obtained preparing wells with PBS saline containing 0.2% of Triton X-100 and erythrocytes (100% hemolysis). The microtitre plate was incubated at 37 [degrees] C with gentle mixing. The hemolysis in each well was quantified spectrophotometrically at 655 nm by a microplate reader iMark, BioRad (Milan, Italy). The hemolysis percentage was calculated using the following equation: hemolysis(%) = 100 - [([0D.sub.655 nm well containing pannarin] - [0D.sub.655 nm 0% hemolysis)/([0D.sub.655 nm 100% hemolysis) - ([0D.sub.655 nm 100% hemolysis])] x 100. All experiments were performed in triplicate.

Cell culture

Normal peripheral blood mononuclear cells (PBMCs) were isolated from heparinized human whole blood by Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA) density-gradient centrifugation. PBMCs were washed and resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 [micro] g/ml streptomycin, at 37 [degrees] C in a humidified atmosphere of 5% [CO.sub.2] in air. The effect of pannarin on resting PBMCs was tested by seeding 1.5 x [10.sup.5] cells per well in microplates. For testing the effect of lichen metabolite on dividing lymphocytes, PBMCs (1 x [10.sup.5]/well) were preactivated with 90 [micro] g/ml phytoemoagglutinin (Remel Europe Ltd., Dartford, Kent, UK) for 24h.

Cytotoxicity assay

The effect of pannarin on PBMCs viability was assayed by the MTT colorimetric method which measures viable cells by assessing metabolic integrity (Mosmann 1983).

Cells were treated with a twofold serially diluted of pannarin, from 128 to 1 [micro] g/ml. Negative controls received the same amount of DMSO. After 48 h Mert solution was added to each wells at a concentration of 1 mg/ml and incubated at 37 [degrees] C for 5 h.The amount of MTT-formazan product, dissolved in acidified isopropanol, was estimated by measuring the absorbance at 570 nm in a microplate reader (Biorad, iMark).

Results and discussion

In vitro antimicrobial susceptibility tests

The in vitro antibacterial effect of pannarin was tested alone in order to calculate the MIC values against methicillin-resistant S. aureus. As reported in Table 1, for all the twenty MRSA clinical isolates and the ATCC43300, [MIC.sub.50] and [MIC.sub.90] were calculated, as well as [MBC.sub.50] and [MBC.sub.90]. The growth of the 50% of the strains were inhibited by pannarin at a concentration of 4 [micro] g/ml whilst, a concentration of 8 [micro] g/ml of pannarin is enough to inhibit the growth of the 90% of the strains. Since, the MBCs calculated were close to the MICs, we hypothesize that pannarin, at least against MRSA, might act as bactericidal.

Table 1
MIC and MBC for pannarin calculated for all S. aureus strains.

 MIC                                MBC
([mu]                              ([mu]
g/ml)                              g/ml)

Range  [MIC.sub.50]  [MIC.sub.90]  Range  [MBC.sub.50]  [MBC.sub.90]

4-8              4             8             8-64   16            32


Checkerboard microdilution assay

All twenty S. aureus clinical isolates and the reference strain ATCC43300 were tested for their susceptibility to clindamycin, erythromycin, gentamicin, levofloxacin and oxacillin by microdilution test (data not shown). Amongst these strains, four (namely AQ004, AQ006, AQ007 and AQ012) and the reference strain ATCC4330 were chosen for the interaction assay, since they were resistant to all tested antibiotics (Table 2), with the exception of the control strains which was sensible to levofloxacin.

Table 2
MIC of antibiotics for the strains of S. aureus used in the
checkerboard assay.

Strain     Median MIC
           ([mu]
           g/ml)
           (range)

           Antibiotic

             CLI         ERY          GEN           LVX      OXA

ATCC43300        8192   (512-1024)  (64-256) 256     [less   (8-16) 8
                              1024                 than or
                                                     equal
                                                   to] 0.5

AQ004            8192   (512-1024)     (16-32) 32  (16-64)  (128-256)
                              1024                     16        128

AQ006        (64-128)          512  (128-256) 128   (8-32)       2048
                  128                                    8
AQ007      (32-64) 32   (16-64) 64            128  (16-64)  (256-512)
                                                        16        256

AQ012           16384  (1024-2048)     (32-64) 64       32  (256-512)
                              1024                                512


Clindamycin, erythromycin, gentamicin, levofloxacin and oxacillin were chosen for the drug-interaction assay, since they belong to different classes of antimicrobial agents, respectively, lincosamides, macrolides, aminoglycosides, quinolones and [beta]-lactams.

Table 3 summarizes the results of the broth microdilution checkerboard analysis interpreted by the FICI and [DELTA] E methods of the five MRSA strains for the combination of pannarin and antibiotics.

Table 3
In vitro interaction between pannarin and antibiotics
determined by nonparametric FICI and the [DELTA]E model. (a)

Antibiotic    Strain             FICI                  [DELTA]
                                                       E model
                                                         (b)

                             Median (range)     INT    [SIGMA]
                                                       SYN (n)

Clindamycin   ATCC43300  (1.0625-1.125) 1.0625  IND     80.0
                                                        (10)

              AQ004       (0.502-0.5078) 0.502  IND     90.0
                                                        (41)

              AQ006       (0.5625-0.625) 0.625  IND     67.4
                                                        (27)

              AQ007          (0.625-0.75) 0.75  IND     71.7
                                                        (28)

              AQ012      (1.0625-1.125) 1.0625  IND     62.8
                                                        (13)

Erythromycin  ATCC43300   (0.5625-0.625) 0.625  IND     82.6
                                                        (14)

              AQ004       (0.5625-0.625) 0.625  IND     55.3
                                                        (10)

              AQ006         (0.625-0.75) 0.625  IND     18.6
                                                         (5)

              AQ007          (0.625-0.75) 0.75  IND     18.7
                                                         (5)

              AQ012             (1.25-1.5) 1.5  IND  0.0 (0)

Gentamicin    ATCC43300    (0.5-0.5625) 0.5625  IND    162.6
                                                        (11)

              AQ004       (0.2813-0.375) 0.375  SYN   2639.7
                                                        (51)

              AQ006            (0.3125-0.3750)  SYN    841.3
                                        0.3125          (40)

              AQ007         (0.25-0.2578) 0.25  SYN    793.1
                                                        (41)

              AQ012                      0.375  SYN    396.7
                                                        (22)

Levofloxacin  ATCC43300                          ND       ND

              AQ004                (1.5-2) 1.5  IND     38.5
                                                         (6)

              AQ006                        2.5  IND     45.4
                                                        (20)

              AQ007                        1.5  IND     14.5
                                                         (2)

              AQ012            (2.25-2.5) 2.25  IND     48.8
                                                         (9)

Oxacillin     ATCC43300  (0.3125-0.375) 0.3125  SYN    889.5
                                                        (43)

              AQ004     (0.5156-0.5313) 0.5313  IND   1372.7
                                                        (50)

              AQ006                (1-1.125) 1  IND     94.7
                                                        (15)

              AQ007         (0.625-0.75) 0.625  IND    454.1
                                                        (21)

              AQ012      (0.5625-0.625) 0.5625  IND    651.7
                                                        (32)

Antibiotic    Strain


                           EANT     INT
                           (n)

Clindamycin   ATCC43300    -83.3  IND
                            (48)

              AQ004        -72.3  IND
                            (29)

              AQ006        -56.7  IND
                            (30)

              AQ007        -53.2  IND
                            (28)

              AQ012        -74.5  IND
                            (16)

Erythromycin  ATCC43300    -97.8  IND
                            (19)

              AQ004        -26.7  IND
                             (4)

              AQ006        -84.3  IND
                            (20)

              AQ007        -15.8  IND
                             (4)

              AQ012        -12.5  IND
                             (3)

Gentamicin    ATCC43300    -98.9  SYN
                            (18)

              AQ004        -79.1  SYN
                            (12)

              AQ006        -81.3  SYN
                            (15)

              AQ007        -98.5  SYN
                            (20)

              AQ012        -78.6  SYN
                             (8)

Levofloxacin  ATCC43300       ND

              AQ004       -989.7  ANT
                            (36)

              AQ006       -781.9  ANT
                            (57)

              AQ007      -1201.0  ANT
                            (46)

              AQ012       -510.6  ANT
                            (46)

Oxacillin     ATCC43300    -99.3  SYN
                            (14)

              AQ004        -75.1  SYN
                            (11)

              AQ006        -83.9  IND
                            (10)

              AQ007        -69.9  SYN
                            (11)

              AQ012      -15 (4)  SYN

(a.) INT, interpretation; IND, indifference; SYN, synergy; ANT,
antagonism. Synergy was defined as an FICI of [less than or equal to]
0.5, antagonism was defined as an FICI of > 4, and indifference was
defined as and FICI >0.5 and [less than or equal to] 4.
(b.) n, number of drug combinations (amongst the 77 drug combinations
for each strain) with statistically significant synergy or antagonism.


According to FICI interpretation, synergism was found in S. aureus AQ004, AQ006, AQ007 and AQ0012 (FICI < 0.5) in combination with gentamicin and ATCC43300 (FICI = 0.3125) in combination with oxacillin. Indifference (FICI > 0.5) was observed in all other combinations.

[DELTA]E model interpretation for gentamicin confirmed synergism for S. aureus AQ004, AQ006, AQ007 and AQ012. Moreover, the combinations of pannarin with gentamicin reported as indifference for the strain ATCC43300 by FICI interpretation (FICI = 0.5625) is interpreted by [DELTA] E model as synergism. The combinations with oxacillin, which FICI was approximately 0.5 and reported as indifference, were interpreted by the [DELTA] E model as synergism, with the exclusion of the strain AQ006 which FICI =1. The combination with levofloxacin was reported as indifferent by FICI methods. However, in accordance with [DELTA] E model interpretation, levofloxacin acts antagonistically. Our data seem to agree with several studies in which fluoroquinolones both Gram-positive and Gram-negative bacteria, in combination with ertapenem, rifampicin, fusidic acid and line-zolid act antagonistically (Hosgor-Limoncu et al. 2008; Murillo et al. 2008; Neu 1991; Sahuquillo Arce et al. 2006; Sweeney and Zurenko, 2003; Uri 1993).

The percentage of agreement in the interpretation of the FIC index and the response surface approach was highly variable amongst the combinations. It was ranging from 0% for the levofloxacin combinations, to 100% for the clindamycin and erythromycin combinations. For instance, most of the combinations interpreted as indifference by FIC index model, which value is more than 0.5 but less than 1, are interpreted as synergic using the [DELTA] E model. The same is found in levofloxacin combinations, in which, the interpretation of FIC index model as indifference (FICI > 1 but < 4) is interpreted by the AE model as antagonism.

The combination of pannarin with clyndamicin, erythromycin, levofloxacin, oxacillin and gentamicin, is highly synergic only in the last two cases, and, on the contrary, the combination with levofloxacin is not advantageous.

Membrane permeabilization assay

As shown in Fig. 2 treatment of S. aureus ATCC43300 with pannarin produces any significant calcein release in the first 2h. The data collected are consistent with a lack of lysis or even significant damage to the cytoplasmic membrane.

Hemolytic activity As shown in Fig. 3, pannarin exhibits a low hemolytic activity in the first 2 h of incubation, even at the highest concentration (16 [micro] g/ml). Moderate hemolytic activity was observed after 2 h of incubation at concentrations of pannarin below 2 [micro] g/ml, whilst about 50% of hemolysis was observed at concentrations of pannarin above 2 [micro] g/ml.

Cytotoxic assay

To verify the effect of pannarin on growth and viability on human normal cells, resting and dividing PBMCs were exposed to increasing concentrations, from 1 to 128 [micro] g/ml, of compound for 48 h. Then, cell survival, compared with untreated controls, was evaluated using the MIT assay. Non-stimulated and proliferating PBMC showed the same behaviour towards pannarin treatments.

Exposure from 1 to 2 [micro] g/ml pannarin did not exhibit cytotoxic effects, whilst an extensive and dose-dependent antiproliferative activity was detected in cells treated with concentrations ranging from 4 to 128 Kgiml. Resting and dividing PBMCs showed [IC.sub.50] values of 6 [micro] g/ml and 7.7 [micro] g/ml, respectively.

Conclusion

The antimicrobial activity of pannarin alone is comparable with those of therapeutically available antibiotics. The lack of lysis, as demonstrated by the permeabilization assay, strongly suggests a specific intracellular mechanism of action. In consideration of the low hemolytic activity and the moderate cytotoxic effect on peripheral blood mononuclear cells, pannarin seems to be a good candidate for further studies in order to understand the mechanism of action. Moreover, it might provide novel templates or leading structures in order to design new antimicrobial agents.

The presence of a chlorine atom makes the molecule interesting from a pharmacological point-of-view. In fact, although the presence of chlorine in secondary metabolites produced by plants are rare, this is not true for algae, fungi and other marine organisms. Highly functionalized depsidones can be synthesized, as previously demonstrated (Sala and Sargent 1978), in order to investigate the effect of chlorine atom or any other functional group on the antimicrobial activity.

Conflict of interest

There was no conflict of interest.

Acknowledgement

This work was partially supported by MIUR (Ministero dell'Istruzione, dell'Universita e della Ricerca) EX MURST 60%, Pr. Gianfranco Amicosante, Dr. Mariagrazia Perilli and Dr. Giuseppe Celenza.

References

Clinical Laboratory Standards Institute, 2010. Performance Standards for Antimicrobial Susceptibility Testing. In: Twenty-First Informational Supplement M100-S21.

Essodaigui, M., Broxterman, H J., Garnier-Suillerot, A., 1998. Kinetic analysis of calcein and calcein-acetoxymethylester efflux mediated by the multidrug resistance protein and P-glycoprotein. Biochemistry 8, 2243-2250.

Fournet, A., Ferreira, M.E., Rojas de Arias, A., Torres de Ortiz, S., Inchausti, A., Yaluff, G., Quilhot, W., Fernandez, E., Hidalgo, M.E., 1997. Activity of compounds isolated from Chilean lichens against experimental cutaneous leishmaniasis. Comp. Biochem. Physiol. C: Pharmacol. Toxicol. Endocrinol. 1, 51-54.

Garbarino, J.A., Piovano, M., Cespedes, E., Quilhot, W., 1991. Studies on Chilean lichens XI. Secondary metabolites from Antarctic lichens Institute Antarctic Chileno. Serie Cientifica INACH 41, 79-90.

Gibbons, S., Udo, E.E., 2000. The effect of reserpine, a modulator of multidrug efflux pumps, on the in vitro activity of tetracycline against clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) possessing the tet(K) determinant. Phytother. Res. 14, 139-140.

Greco, W.R., Bravo, G., Parsons, J.C., 1995. The search for synergy, a critical review from a response surface perspective. Pharmacol. Rev. 47, 331-385.

Hemaiswarya, S., Kruthiventi, A.K., Doble, M., 2008. Synergism between natural products and antibiotics against infectious diseases. Phytomedicine 15, 639-652.

Hollo Z., Homolya, L., Davis, C.W., Sarkadi, B., 1994. Calcein accumulation as a fluorometric functional assay of the multidrug transporter. Biochim. Biophys. Acta 2, 384-388.

Hosgor-Limoncu, M., Ermertcan, S., Tasli, H., Yurtman, A.N., 2008. Activity of amikacin, ertapenem, ciprofloxacin and levofloxacin alone and in combination against resistant nosocomial pathogens by time-kill. West Indian Med. J. 57, 106-111.

Jackman, D.A., Elix, J.A., Sargent, M.V., 1975. Structure of the lichen depsidone pannarin. J. Chem. Soc. Perkin Trans. 119, 1979-1985.

Koo, S.P., Bayer, AS., Yeaman, M.R., 2001. Diversity in antistaphylococcal mechanisms among membrane-targeting antimicrobial peptides. Infect. Immun. 8, 4916-4922.

Meletiadis, J., Verweij, P.E., TeDorsthorst, D.T., Meis, J.F., Mouton, J.W., 2005. Assessing in vitro combinations of antifungal drugs against yeasts and filamentous fungi, comparison of different drug interaction models. Med. Mycol. 43, 133-152.

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and

survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63.

Murillo, O., Pachon, M.E., Euba, G., Verdaguer, R., Tubau, F., Cabellos, C., Cabo, J., Gudiol, F., Ariza, J., 2008. Antagonistic effect of rifampicin on the efficacy of high-dose levofloxacin in staphylococcal experimental foreign-body infection. Antimicrob. Agents Chemother. 52, 3681-3686.

Neu, H.C., 1991. Synergy and antagonism of combinations with quinolones. Eur. J. Clin. Microbiol. Infect. Dis. 10, 255-261.

Pearson, R.D., Steigbigel, R.T., Davis, H.T., Chapman, S.W.. 1980. Method of reliable determination of minimal lethal antibiotic concentrations. Antimicrob. Agents Chemother. 18, 699-708.

Piovano, M., Garrido, M.I., Gambaro, V., Garbarino, J.A., Quilhot, W., 1985. Studies on Chilean Lichens VIII. Depsidones from Psoroma species. J. Nat. Prod. 48, 854-855.

Prichard, M.N., Shipman, C., 1990. A three-dimensional model to analyze drug-drug interactions. Antiviral Res. 14, 181-205.

Prichard, M.N., Prichard, LE., Baguley, W.A., Nassiri, M.R., Shipman, C., 1991. Three-dimensional analysis of the synergistic cytotoxicity of ganciclovir and zidovudine. Antimicrob. Agents Chemother. 35,1060-1065.

Russo, A., Piovano, M., Lombardo, L, Vanella, L, Cardile, V., Garbarino, J., 2006. Pannarin inhibits cell growth and induces cell death in human prostate carcinoma DU-145 cells. Anticancer Drugs 10, 1163-1169.

Russo, A., Piovano, M., Lombardo, L, Garbarino, J., Cardile, V., 2008. Lichen metabolites prevent UV light and nitric oxide-mediated plasmid DNA damage and induce apoptosis in human melanoma cells. Life Sri. 13-14, 468-474.

Sahuquillo Arce, J.M., Colombo Gainza, E., Gil Brusola, A., Ortiz Estevez, R., Canton, E., Gobernado, M., 2006. in vitro activity of linezolid in combination with doxycycline, fosfomycin, levofloxacin, rifampicin and vancomycin against methicillin-susceptible Staphylococcus aureus. Rev. Esp. Quimioter. 19, 252-257.

Sala, T., Sargent, M.V., 1978. Depsidone synthesis XII. Some exploratory synthetic routes to highly functionalized depsidones. Aust. J. Chem. 31, 1383-1389.

Stavri, M., Piddock, W., Gibbons, S., 2007. Bacterial efflux pump inhibitors from natural sources. J. Antimicrob. Chemother. 59, 1247-1260.

Sweeney, M.T., Zurenko, G.E., 2003. In vitro activities of linezolid combined with other antimicrobial agents against Staphylococci, Enterococci Pneumococci, and selected gram-negative organisms. Antimicrob. Agents Chemother. 47, 1902-1906.

Taylor, P.C.. Schoenknecht, ED., Sherris, J.C., Linner, E.C., 1983. Determination of minimum bactericidal concentrations of oxacillin for Staphylococcus aureus, influence and significance of technical factors. Antimicrob. Agents Chemother. 23,142-150.

Tegos, G., Stermitz, F.R., Lomovskaya, 0., Lewis, K., 2002, Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials. Antimicrob. Agents Chemother. 46, 3133-3141.

Uri, J.V., 1993. Antibacterial antagonism between fusidic acid and ciprofloxacin. Acta Microbiol. Hung. 40, 141-149.

Young, J.D., Leong, L.G., DiNome, M.A., Cohn, Z.A., 1986. A semiautomated hemolysis microassay for membrane lytic proteins. Anal. Biochem. 2, 649-654.

Giuseppe Celenza (a), *, Bernardetta Segatore (a), Domenico Setacci (a), Pierangelo Bellio (a), Fabrizia Brisdelli (a), Marisa Piovano (b), Juan A. Garbarino (b), Marcello Nicoletti (c), Mariagrazia Perilli (a), Gianfranco Amicosante (a)

(a.) Department of Biomedical Sciences and Technologies, University of I'Aquila. L'Aquila, Italy

(b.) Department of Chemistry, Universidad Tecnica F. Santa Maria, Casilla 110 V, Valparaiso, Chile

(c.) Department of Environmental Biology, University Sapienza, Rome, Italy

* Corresponding author at: Department of Biomedical Sciences and Technologies, University of I'Aquila, Via Vetoio. 1,67100 I'Aquila, Italy. Tel.: +39 0862433444.

E-mail address: celenza@univaq.it (G. Celenza).

0944-7113/$--see front matter [c] 2012 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2012.02.010
COPYRIGHT 2012 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2012 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Celenza, Giuseppe; Segatore, Bernardetta; Setacci, Domenico; Bellio, Pierangelo; Brisdelli, Fabrizia
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3CHIL
Date:May 15, 2012
Words:5234
Previous Article:Participation of cholinergic pathways in [alpha]-hederin-induced contraction of rat isolated stomach strips.
Next Article:Ardipusilloside inhibits survival, invasion and metastasis of human hepatocellular carcinoma cells.
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters