Ellagic Acid Derivatives from Rubus ulmifolius Inhibit Staphylococcus aureus Biofilm Formation and Improve Response to Antibiotics

Biofilms contribute to the pathogenesis of many forms of Staphylococcus aureus infection. Treatment of these infections is complicated by intrinsic resistance to conventional antibiotics, thus creating an urgent need for strategies that can be used for the prevention and treatment of biofilm-associated infections.This study demonstrates that a botanical natural product composition (220D-F2) rich in ellagic acid and its derivatives can limit S. aureus biofilm formation to a degree that can be correlated with increased antibiotic susceptibility. The source of this composition is Rubus ulmifolius Schott. (Rosaceae), a plant used in complementary and alternative medicine in southern Italy for the treatment of skin and soft tissue infections. All S. aureus clonal lineages tested exhibited a reduced capacity to form a biofilm at 220D-F2 concentrations ranging from 50-200 µg/mL, which were well below the concentrations required to limit bacterial growth (530-1040 µg/mL). This limitation was therapeutically relevant in that inclusion of 220D-F2 resulted in enhanced susceptibility to the functionally-distinct antibiotics daptomycin, clindamycin and oxacillin. Testing with kidney and liver cell lines also demonstrated a lack of host cell cytotoxicity at concentrations of 220D-F2 required to achieve these effects.These results demonstrate that extract 220D-F2 from the root of Rubus ulmifolius can be used to inhibit S. aureus biofilm formation to a degree that can be correlated with increased antibiotic susceptibility without toxic effects on normal mammalian cells. Hence, 220D-F2 is a strong candidate for development as a botanical drug for use in the prevention and treatment of S. aureus biofilm-associated infections

Biofilm inhibition of <i>R. ulmifolius</i> root extracts.

<p>A static microtiter plate biofilm assay which employed crystal violet as a biofilm matrix staining agent was used to assess the inhibitory activity of individual fractions from the <i>R. ulmifolius</i> root using UAMS-1 (<b>A</b>) or the USA300 isolate UAMS-1782 (<b>B</b>) as the wild-type (wt) test strains. Isogenic <i>sarA</i> mutants for each strains were included as controls. Other designations refer to the specific extract as defined by the fractionation scheme illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028737#pone-0028737-g001" target="_blank">Figure 1</a>. The MBICs for the crude EtoH extract (220), butanol partition (220D), and 40∶60 (220D-F2), 50∶50 (220D-F3), and 60∶40 (220D-F4) MeOH∶CHCl2 fractions from the butanol partition were 200, 100, 50, 50 and 50 µg/mL, respectively in both UAMS-1 and UAMS-1782. Statistical significance (*, <i>P</i><0.05; ‡, <i>P</i><0.001) refers to differences observed in each parent strain with and without the indicated extract at the indicated concentration.</p

LC-MS/MS analysis revealed a mixture of ellagic acid and glycosylated ellagic acid derivatives in 220D-F2.

<p>Corresponding ESI(+)-MS and MS/MS data is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028737#pone.0028737.s001" target="_blank">Table S1</a>. <b>Compound 1.</b> Ellagic Acid xylopyranoside or xylofuanoside <b>Compound 2.</b> Ellagic acid. <b>Compound 3.</b> Ellagic acid mannopyranoside. The configuration for each of the glycosylated ellagic acids could not be confirmed. Neutral loss of <i>m/z</i> 132 was used to confirm the presence of a pentose attached to ellagic acid.</p

Impact of 220D-F2 as assessed by confocal microscopy.

<p>Microtiter plate biofilm assays were undertaken with UAMS-1 (top) or UAMS-1782 (bottom) after the addition of either 220D-F2 at the indicated concentrations or excipient (DMSO) to the growth medium. Confocal images were obtained after 20 hours of incubation. An orthogonal view is included to illustrate overall biofilm architecture at a magnification of 10×. Isogenic <i>sarA</i> mutants grown in BM with DMSO were included as negative controls.</p

Compounds detected in extract 220D-F2 by accurate mass LC/UV/MS/MS.

†<p>Proposed structures corresponding to this data are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028737#pone-0028737-g004" target="_blank">Figure 4</a>. Of the major UV components identified, the most abundant was EA (#2, MW 302). The second most abundant UV component (#7) did not yield a clear MS signal suggestive of a single species or reasonable molecular formula, and thus no structure is proposed. The third most abundant UV component (#1, MW 434) appears to be EA plus a C₅H₈O₄ moiety. A fourth UV component (#3, MW 448) was found to be consistent with a glyosylated derivative of EA. Investigation of the possible formulae consistent with the mass measurement of the fifth UV component (#4) did not yield sufficient data for proposal of a structure. Of the major MS components, the most abundant (#8) did not yield enough information to support the proposal of a structure, however the molecular weights and mass defects suggest that they may be dimers of MW∼500 species (similar to #6). The second most abundant MS component (#6) is consistent with a sapogenin. Successive loss of water (m/z 18) is consistent with a poly-hydroxylated compound. The third most abundant MS component (#5, MW 534) appears to be similar to #6 and has MS/MS losses consistent with a multiply hydroxylated compound like a sapogenin. Losses consisted with neutral loss of a sugar were not observed.</p

Phytochemicals previously isolated from <i>Rubus ulmifolius</i>[31], [32], [36].

<p>*Individual compounds tested for anti-biofilm activity. Results are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028737#pone-0028737-t003" target="_blank">Table 3</a>.</p

Prophylactic use of 220D-F2 prior to concomitant antibiotic therapy.

<p>Biofilms were formed on plasma-coated catheters for 24 hours by growth of the test strain (UAMS-1) in BM containing 200 µg/mL 220D-F2. Catheters were then placed in fresh BM containing 200 µg/mL 220D-F2 with or without 10 µg/mL of daptomycin, which corresponds to 10× the CLSI-defined breakpoint MIC for a sensitive strain of <i>S. aureus</i>. Statistical significance (*, <i>P</i><0.05; ‡, <i>P</i><0.001) refers to differences between the untreated cultures and cultures exposed to the compounds. Results observed with the isogenic <i>sarA</i> mutant with and without the same concentration of daptomycin but without 220D-F2 are shown for comparison.</p

Anti-biofilm activity of 220D-F2 against genotypically- and phenotypically-diverse strains of <i>S. aureus</i>.

<p>A crystal violet microtiter plate biofilm assay was used to assess the impact of 220D-F2 on biofilm formation. Strain designations are given based on both the corresponding author's culture collection (UAMS) and the clonal lineage of each isolate (USA). Statistical significance (*, <i>P</i><0.05; ‡, <i>P</i><0.001) refers to differences between the untreated cultures and cultures exposed to the indicated concentrations. When available, the isogenic <i>sarA</i> mutant for each isolate in the absence of 220D-F2 was included as a control; results obtained with all 15 <i>sarA</i> mutants were significantly different from those obtained with the isogenic parent strain (<i>P</i><0.001).</p

Activity of 220D-F2 (µg/mL) against wild-type strains of <i>Staphylococcus aureus</i>.

<p>*A detailed description of the bacterial strains used in this study has been previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028737#pone.0028737-Beenken1" target="_blank">[52]</a>.</p