The leaves of Schinus terebinthifolia Raddi (Anacardiaceae), which were selected from a primary screen of the Quave Natural Product Library (QNPL), were collected from private property in DeSoto County, Florida, in November 2017. Plant samples were identified and catalogued by Dr. Cassandra Quave at the Emory Herbarium (GEO, Atlanta, Georgia) where voucher specimens were deposited for reference (CQ-651) and are accessible for view online via the SERNEC portal39. Fresh plant samples were dried in a dehumidified cabinet. Dried plant material was ground using a Wiley Mill Plant Grinder and filtered through a 0.5 mm mesh sieve. Powdered plant samples were subjected to two rounds of maceration in 80% aqueous ethanol for 72 h, filtered, and then dried via a rotary evaporator. Crude plant extracts were partitioned using a successive liquid–liquid partitioning scheme. Extraction solvents used were: hexanes, ethyl acetate, n-butanol, and water and were labelled B, C, D, and E according to solvent, respectively. Dried crude plant extracts were stored in – 20 °C until further use.
Isolation of bioactive compounds
Schinus terebinthifolia extract 429 underwent bioassay-guided fractionation as previously described40. The ethyl acetate partition 429C (13.56 g) was fractionated using a 330 g silica column (RediSep, Teledyne ISCO) via normal phase flash chromatography (Combi Flash Rf + Lumen, Teledyne ISCO) utilizing the following hexane:ethyl acetate gradient: 3 column volumes (CV) 100:0, 30 CV gradient to 0:100, and 32 CV isocratic 0:100. The bioactive fraction 429C–F8 (5.10 g, 37.6% yield), was eluted between 27.5 and 40.0 CV’s. All subsequent preparative high-performance liquid chromatography (Prep-HPLC) were carried out using an Agilent Technologies 1260 Infinity II LC System (CA, USA) equipped with an Agilent Technologies 1200 Infinity Series Diode Array Detector detecting at 214 nm and 254 nm. The column used for all subsequent Prep-HPLC purifications was an Eclipse XDB-C18 5 μm pore, 30 × 250 mm reverse phase column (Agilent). Fraction 429C–F8 was fractionated further via Prep-HPLC using a mobile phase of 0.1% (vol/vol) formic acid in water (A) and 0.1% (vol/vol) formic acid in acetonitrile (B) at a flow rate of 42.5 mL/min. To fractionate 429C–F8, the following gradient (A:B) was used: 0 min (98:2), 3 min (98:2), 11 min (90:10), 38 min (81:19), 58 min (81:19), 58.1 min (80:20), 68 min (80:20), 75.5 min (21:79). The bioactive fraction 429C–F8–PF11 (137.8 mg, 19.7% yield), was eluted between 36.75 and 41.17 min. Fraction 429C–F8–PF11 was fractionated further via Prep-HPLC using a mobile phase of 0.1% (vol/vol) formic acid in water (A) and 0.1% (vol/vol) formic acid in methanol (C) at a flow rate of 42.5 mL/min. To fractionate 429C–F8–PF11, the following gradient (A:C) was used: 0 min (85:15), 10 min (65:35), 25 min (65:35), 25.1 min (60:40), 35 min (60:40), 35.1 min (2:98), 47 min (2:98). The bioactive fraction 429C–F8–PF11–SF4 (53.6 mg, 36.0% yield), was eluted between 13.0 and 14.0 min.
A 14.1 T (600 MHz for 1H, 150 MHz for 13C) Bruker Ascend III HD NMR spectrometer with a 5 mm prodigy cryoprobe or an 18.8 T (800 MHz for 1H, 200 MHz for 13C) Bruker Avance III HD NMR spectrometer with a 3 mm triple resonance broadband cryoprobe were used to acquire NMR spectra (1H, 13C, COSY, HSQC, and HMBC). PGG was dissolved in either DMSO-d6 or CD3OD for NMR spectral acquisitions and referenced to solvent residual peaks (δH 2.50 or 3.31 for DMSO-d6; δC 39.52 or 49.00 for CD3OD). MestReNova 12.0.0 was used to process and analyze spectra.
A sample of 429C–F8–PF11–SF4 and a standard of pentagalloyl glucose (Sigma-Aldrich, St. Louis, MO) was analysed by liquid chromatography–Fourier transform mass spectrometry (LC-FTMS) using a Thermo Electron LTQ-FT Ultra MS (Thermo Scientific) coupled to a Shimadzu LC (Columbia, MD) equipped with a Shimadzu autosampler and Dionex (San Jose, CA, USA) HPLC pump and diode-array detector. The stationary phase was an Eclipse XDB-C18 5 μm pore, 30 × 250 mm reverse phase column (Agilent). A 10 μL injection of sample was applied for each run. Mobile phases consisted of 0.1% (vol/vol) formic acid in water (A) and 0.1% (vol/vol) formic acid in acetonitrile (B) at a flow rate of 1.0 mL/min. The following gradient (A:B) was used: 0 min (70:30), 7 min (65:35), 13 min (65:35), 13.01 min (60:40), 20 min (60:40), 20.01 min (0:100), 30 min (0:100), with a return to starting conditions at 30.01 min (70:30) until 40 min (70:30). Mass spectrometry samples were ionized by positive electrospray ionization at the following conditions: 5 kV voltage applied to needle, capillary voltage of 45 V, capillary temperature of 200.0 °C, and tube lens voltage of 100 V. Mass spectrometry data was processed with Freestyle 1.6 software (Thermo Scientific). The predicted formula and mass were taken for 429C–F8–PF11–SF4 and the PGG standard according to the peak compound signature as determined previously by analytical HPLC of 429C–F8–PF11–SF4 compared to a standard of PGG.
Bacterial growth conditions
Bacteria were maintained on tryptic soy agar (TSA) plates and grown in cation-adjusted Mueller Hinton broth (CAMHB) for experiments according to CLSI guidelines41. Experimental cultures were incubated at 37 °C in a humidified chamber. AR Bank strains were obtained from the FDA-CDC Antimicrobial Resistance Isolate Bank42; a full list of the strains tested is available in Table 1. A. baumannii AB5075, the primary strain used in the following assays, is a model strain that is more virulent and is a representative ST2 strain. ST2 is the dominant MLST-type clone worldwide, responsible for the majority of outbreaks in Europe, Middle East, South America, and Asia, and these strains are often the most antibiotic resistant as well43,44,45,46,47,48.
Bacterial growth inhibition was determined by microbroth dilution according to CLSI guidelines41. Bacteria were grown overnight in TSB and standardized to 5 × 105 CFU/mL in CAMHB to make the experimental culture. Treatments were added in triplicate and absorbance of experimental wells was measured at 600 nm with a BioTek Cytation3 plate reader before and after incubation (22 h for A. baumannii and 18 h for other ESKAPE species). Percent growth inhibition was calculated relative to vehicle control (DMSO); the minimum inhibitory concentration (MIC) was determined as the lowest treatment concentration with > 90% inhibition and the IC50 was the lowest concentration with > 50% inhibition. Data was analysed using Microsoft Excel and figures were created with GraphPad Prism version 8.3.1. Positive controls were meropenem and colistin for the 24-strain A. baumannii panel and meropenem and gentamicin for other growth inhibition experiments. A media blank was included in each experiment to test for contamination and each experiment was performed twice on separate days.
For supplementation experiments, oleic acid and polysorbate 80 were added to respective experimental cultures to a concentration of 0.02% vol/vol49, and iron (II) sulfate and iron (III) sulfate were dissolved in deionized water and added to a concentration of 1 mM12.
Time-kill methods were adapted from NCCLS guidelines50. PGG and meropenem were added at MIC concentrations (256 and 64 µg/mL, respectively) to A. baumannii AB5075 at 1 × 106 CFU/mL in CAMHB [alone or supplemented with 0.02% oleic acid or polysorbate 80 or 1 mM iron (II) sulphate]. At 0, 3, 6, and 24 h timepoints, 10 µL culture was removed from each of four replicates, serially diluted tenfold in sterile PBS, dispensed on TSA, and incubated to determine CFU/mL.
Biofilm inhibition and eradication
Methods for A. baumannii biofilm inhibition and eradication were adapted from Tipton et al.51. Briefly, A. baumannii AB5075 cells were grown in Luria–Bertani broth (LB) for 24 h in the presence of treatment in the case of biofilm inhibition and for 24 h pre-treatment and 24 h post-treatment in the case of biofilm eradication. After incubation, biofilms were fixed with ethanol, stained with crystal violet, gently rinsed with water, and eluted with 33% acetic acid. The absorbance of each well was measured at 595 nm.
Toxicity to immortalized human keratinocytes (HaCaTs) was assessed using an LDH cytotoxicity assay kit (G-Biosciences, St. Louis, MO, USA) as described previously52. Briefly, cells were standardized to 4 × 104 and incubated in 96-well plates for 48 h to allow for seeding, after which media was replaced and treatments were added in serial dilution from 256 to 32 µg/mL. Plates were subsequently incubated for 24 more hours and processed according to the manufacturer’s protocol for chemical induced cytotoxicity.
The effect of iron supplementation on PGG activity was determined for A. baumannii AB5075 by modifying Lin et al.’s iron restoration assays on S. aureus14. For the first half of the assay, PGG was mixed with solutions of cation-adjusted Mueller–Hinton Agar (CAMHA) and TSA at concentrations of 0.5 × MIC, 1 × MIC, and 2 × MIC (128, 256, and 512 µg/mL, respectively). A control condition with no PGG treatment was included. Each condition was tested in triplicate, along with media blank wells. Overnight culture of A. baumannii AB5075 was standardized to 5 × 105 CFU/mL in CAMHB media at OD600. AB5075 working culture was spread onto each treatment plate, statically incubated at 35 °C for 24 h, and observed for presence of bacterial growth. At the 24-h mark, 1 mM of iron (II) sulfate solution or 1 mM of iron (III) sulfate solution was spread onto the PGG-treated wells. The plates were visually observed for presence of bacterial growth at 24 and 48-h timepoints.
Development of resistance
Spontaneous development of resistance was tested using a method adapted from Ling et al.53. A. baumannii AB5075 culture was spread on TSA plates containing 0, 128, 256, and 512 µg/mL PGG and incubated for 24 h, after which colonies on each plate were counted.
Resistance over the course of serial passaging was tested using a method adapted from Maisuria et al.54. Briefly, a series of 21 microbroth dilution experiments for growth inhibition was carried with PGG or tetracycline against A. baumannii AB5075, using bacterial culture from the sub-MIC (0.5MIC) wells of each gradient to make the experimental culture for each subsequent experiment.
Material has been reviewed by the Walter Reed Army Institute of Research (WRAIR). There is no objection to its presentation and/or publication. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense, the Uniformed Services University of the Health Sciences, the Department of the Army, or any other agency of the U.S. Government.