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This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology
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While bacteria play essential roles in many aspects of human health, as evidenced by the growing body of work on the human microbiome (
Formation of biofilms on native host tissues and indwelling medical devices leads to microbial infections that are recalcitrant to antimicrobials, even in the absence of issues related to acquired resistance. Biofilms increase cross-species gene transfer, lead to expression of more virulent phenotypes, and result in a much higher cell density (1011 CFU/mL) than their planktonic counterparts (108 CFU/mL) (
Biofilms are complex, three-dimensional bacterial communities that can lead to longer hospital stays, recurrent infection, and increased fatalities in the most recalcitrant infections [
Previously, anti-biofilm activity in
Extract 220D-F2 was prepared from wild harvested samples of
All of the chemically synthesized phenolic glycosides were subjected to HPLC LC–FTMS analysis using the same conditions described previously (Supplementary Figures
All test compounds and the 220D-F2 botanical extract control were examined for growth inhibitory and biofilm inhibitory activity following established methods. A well-characterized methicillin-sensitive
Growth curve experiments were also conducted, with OD600 nm readings taken at 0, 3, 5, 8, 10, 12, and 18 h post-inoculation. The number of colony forming units per mL of culture was measured by diluting and plating the culture on TSA at 18 h post-treatment and counting colonies following 22 h of incubation at 37°C.
Inhibition of biofilm formation was assessed in a static 96-well plate model with human plasma. Briefly, 20% human plasma diluted in carbonate buffer was added to the biofilm media [tryptic soy broth supplemented with 3.0% NaCl (wt/vol) and 0.5% dextrose (wt/vol)] to reach a final concentration of 2% human plasma in the media. Following addition of the test compounds and inoculation with UAMS-1, plates were incubated at 37°C for 22 h. Planktonic cells were then gently aspirated and the wells rinsed twice with phosphate-buffered saline (PBS) to remove non-adherent cells. Adherent biofilms were fixed with 200 μL of 100% ethanol prior to staining for 15 min with 50 μL of 2% (wt/vol) crystal violet in 20% ethanol (Hardy Diagnostics). The stain was washed with tap water, then after drying, 100 μL of 10% of [2.5% Tween 80(aq)] in ethanol (EtOH) was added to wells and incubated for 15 min. The eluate (20 μL) was transferred to a new plate containing 180 μL PBS/well and the OD595 nm measured by plate reader. The minimum biofilm-inhibiting concentration (MBIC) was defined as the lowest concentration of extract in which biofilm formation was limited to a level ≥90% (for MBIC90) or ≥50% (for MBIC50) by comparison to the vehicle-treated control (UAMS-1) strain.
Follow-up assays using the two-dimensional checkerboard method (
Parallel to the above described biofilm assay, biofilm architecture was assessed by confocal laser scanning microscopy (CLSM). Briefly, biofilms were formed as described above (including treatment and control groups). After 20 h, the well contents were aspirated and the wells gently washed three times with 0.85% (wt/vol) NaCl. The adherent biofilm was then stained with LIVE/DEAD stain (Invitrogen) at room temperature in the dark for 15 min, following manufacturer’s protocol. Then CLSM images were collected using an Olympus FluoView 1000 confocal scanning system and total internal reflection fluorescence (TIRF) inverted microscope. SYTO 9 fluorescence was detected by excitation at 488 nm and emission at 527 nm, fluo-3 bandpass filter. Propidium iodide fluorescence was detected by excitation/emission at 543/612 nm, Texas Red bandpass filter. All z-sections were collected at 4-μm intervals using a 10 × objective lens. A 1.27 × 1.27 mm section of biofilm was selected from the center of the well for each image. Image acquisition and processing was performed using Olympus Fluoview software. Identical acquisition settings were employed for all samples.
All assays performed were analyzed using a two-tailed Student’s
All reactions were performed using dry solvents in flame-dried glassware under a nitrogen atmosphere with magnetic stirring, unless otherwise noted. Reaction solvents were dried over 4 Å molecular sieves, according to a published procedure (
The compound was synthesized from
The compound was synthesized from
The compound was synthesized from
The appropriate phenol (1 eq) was dissolved in dichloromethane (DCM) containing 4 Å MS. The per-
The per-
The compound was synthesized according to the
The compound was synthesized according to the
The compound was synthesized according to the
The compound was synthesized according to the
The compound was synthesized according to the
The compound was synthesized according to the
The compound was synthesized by glycosylation of catechol with per-
The compound was synthesized by glycosylation of phenol with per-
The compound was synthesized by iodination of
The compound was synthesized by iodination of
The compound was synthesized in analogy to a published procedure with some modifications (
The synthesis was based on published glycosylation methodology (
The synthesis was based on published glycosylation methodology (
Deprotection of TBS ethers was performed in analogy to a published procedure (
The compound was prepared according to the
The compound was prepared according to the
Phenol and catechol were glycosylated using per-
As EA is extremely insoluble and quite unreactive, synthesis of EAGs employed per-
The mild fluoride source TASF (
The anomeric stereochemistry of the β-xylosides (
The synthetic compounds were used as standards for liquid chromatography–mass spectrometry (LC–MS) to probe for the occurrence of the various glycosylated aromatic compounds in the 220D-F2 extract. Both EA xyloside (
The panel of compounds was assayed to determine the effects on
Minimum concentrations of extract and synthetic compounds required to inhibit biofilm formation (MBIC) or growth (MIC) of
Compound | MBIC50 (μg/mL) | MBIC90 (μg/mL) | MIC50 (μg/mL) | MIC90 (μg/mL) |
---|---|---|---|---|
220D-F2 | 25 | 100 | 512 | ND |
Ellagic acid ( |
128 | ND | ND | ND |
Phenol ( |
64 | ND | ND | ND |
Catechol ( |
ND | ND | ND | ND |
Phenol mannoside ( |
ND | ND | 512 | ND |
Catechol mannoside ( |
ND | ND | ND | ND |
Phenol xyloside ( |
ND | ND | ND | ND |
EA xyloside ( |
64 | ND | 32 | ND |
Phenol rhamnoside ( |
64 | ND | ND | ND |
Catechol rhamnoside ( |
128 | ND | 512 | ND |
EA rhamnoside ( |
64 | 128 | ND | ND |
Assessment of combinations of single compounds identified in the extract 220D-F2 (EA, EA rhamnoside, and EA xyloside) by two-dimensional checkerboard assays resulted in lower MBIC50 values for all three. However, the combinations could not be confirmed as being strongly synergistic as all values were above the ΣFIC synergy cutoff of 0.5, and yet below the cutoff for additive effects (ΣFIC of 1) (
Fractional inhibitory concentration indices of biofilm inhibition for combinations of compounds identified in extract 220D-F2.
Compound combination | Lowest concentration with ≤50% biofilm formation (μg/mL) |
ΣFIC | ||
---|---|---|---|---|
Ellagic acid | EA rhamnoside | EA xyloside | ||
EA + EA rhamnoside | 64 | 8 | – | 0.625 |
EA + EA xyloside | 64 | – | 16 | 0.750 |
EA rhamnoside + EA xyloside | – | 32 | 16 | 0.750 |
As phenol and catechol are key structural components of EA (
The panel of compounds was synthesized by coupling the appropriate aromatic core to per-
Previous work demonstrated that
From the anti-biofilm studies, the identity of the sugar was identified as a key determinant of biological activity. Neither mannose nor xylose derivatives of phenol (
Of the synthetic compounds tested, only rhamnose derivatives were able to inhibit biofilm formation at concentrations lower than the growth inhibitory concentration. Rhamnose derivatives of phenol (
To determine if the synthetic compounds are components of the 220D-F2 extract, LC–MS/MS analysis of 220D-F2 and all of the synthetic compounds was performed (
The high anti-biofilm activity of EA rhamnoside, coupled with limited growth inhibition (Supplementary Figure
EA rhamnoside has been identified as an inhibitor of biofilm formation, without concomitant inhibition of bacterial growth. Glycosylation of phenolic compounds significantly modulates both biofilm and growth inhibitory activity, suggesting that there may be key interactions between the sugar and biological targets. EA rhamnosides and xyloside have been identified within the 220D-F2 extract, highlighting their roles in the previously observed anti-biofilm activity. Furthermore, identification of single compounds with anti-biofilm activity will allow for future studies to elucidate the mechanism of biofilm inhibition.
BF, KN, JL, CQ, and EW designed the study. BF, KN, JL, PJ, and JG-R performed all experiments. BF, KN, JL, CQ, and EW analyzed the data. BF, KN, JL, CQ, and EW wrote the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We are grateful to Dr. Fred Strobel of the Emory University Mass Spectrometry Center in Chemistry for assistance with LC–MS/MS experiments, the Emory Microscopy Core for support with the CLSM experiments, Dr. Shaoxiong Wu of the Emory NMR Research Center for assistance with NMR characterization, and members of the Weinert lab for helpful discussions. Thanks to Mark Smeltzer for provision of the bacterial strains and Donato Caputo for assistance with plant collections.
The Supplementary Material for this article can be found online at: