Triterpenoid acids isolated from Schinus terebinthifolia fruits reduce Staphylococcus aureus virulence and abate dermonecrosis.

Staphylococcus aureus relies on quorum sensing to exert virulence to establish and maintain infection. Prior research demonstrated the potent quorum sensing inhibition effects of “430D-F5”, a refined extract derived from the fruits of Schinus terebinthifolia, a medicinal plant used for the traditional treatment of skin and soft tissue infections. We report the isolation and identification of three compounds from 430D-F5 that reduce virulence and abate dermonecrosis: 3-oxo-olean-12-en-28-oic acid (1), 3-oxotirucalla-7,24Z-dien-26-oic acid (2) and 3α-hydroxytirucalla-7,24 Z-dien-27-oic acid (3). Each compound inhibits all S. aureus accessory gene regulator (agr) alleles (IC50 2–70 μM). Dose-dependent responses were also observed in agr-regulated reporters for leucocidin A (lukA, IC50 0.4-25 μM) and glycerol ester hydrolase or lipase (gehB, IC50 1.5–25 μM). Surprisingly, dose-dependent activity against the nuclease reporter (nuc), which is under the control of the sae two-component system, was also observed (IC50 0.4–12.5 μM). Compounds 1-3 exhibited little to no effect on the agr-independent mgrA P2 reporter (a constitutive promoter from the mgrA two-component system) and the esxA reporter (under control of mgrA). Compounds 1-3 inhibited δ-toxin production in vitro and reduced dermonecrosis in a murine in vivo model. This is the first report of triterpenoid acids with potent anti-virulence effects against S. aureus.


Results
Bioassay-guided isolation of three bioactive triterpenoid acids. Bioassay-guided fractionation of an organic extract of S. terebinthifolia fruits (named extract 430) was directed by a set of reporter strain assays of MRSA agr::P3 activation for agr types I-IV 25 . Inhibition of YFP fluorescence in these strains was used as a preliminary indication for the inhibition of quorum sensing. An aqueous suspension of 430 was sequentially partitioned against hexanes (430B), then ethyl acetate (430 C). The final remaining water layer (430 F) was determined to be the most bioactive under these testing parameters and was selected for further fractionation with a flash chromatography system using a gradient of hexane, ethyl acetate and methanol. The most active fraction, 430F-F5, was chosen for compound isolation via reverse phase HPLC using a gradient system of water and methanol. A first round of HPLC yielded 12 fractions (430F-F5-PF1 through PF12). The most bioactive fractions, 430F-F5-PF11 and 430F-F5-PF12, were selected for a second round of HPLC fractionation, which led to the isolation of three active triterpenoid acids. An overview of the fractionation strategy, with percent yields of the active fractions and compounds is provided ( Supplementary Fig. 1).
Spectroscopic data for Compounds 1-3. 3-oxo-olean-12-en-28-oic acid (1). Crystal solid. 1  3-oxotirucalla-7,24Z-dien-26-oic acid (2). Crystal solid. 1  www.nature.com/scientificreports www.nature.com/scientificreports/ 3α-hydroxytirucalla-7,24Z-dien-27-oic acid (3). Amorphous solid. 1  Triterpenoid acids 1-3 exhibit limited growth inhibitory activity in S. aureus strains. Compounds 1-3 from S. terebinthifolia were screened against eight S. aureus strains at the test range of 4-563 µM in broth microdilution assays. Growth inhibition by dose response of the agr reporter strains is reported in Fig. 3, with a summary of MICs on all tested strains reported in Table 1. None of the three compounds exhibited an IC 50 (≥50% growth inhibition in comparison to vehicle control) and MIC (≥90% inhibition) values for MRSA agr group I (AH1677), II and IV reporter strains. With regards to MRSA agr group III, Compound 1 exhibited weak growth inhibition (~15% inhibition) at up to 80 µM, but reached an IC 50 by 141 µM and MIC at 281 µM, leaving a small gap between growth and quorum sensing inhibitory activity (QS IC 50 : 141 µM; IC 00 : 281 µM). Compound 3 exhibited stronger growth inhibitory activity against this agr group III reporter strain, reaching an MIC at 18 µM. Compounds 2 and 3 exhibited similar growth inhibitory activity against UAMS-929 and UAMS-1, IC 50 and MIC of 35 and 70 µM, respectively. Also, 2 and 3 inhibited the growth of MRSA agr group I (AH1263, NRS249) with MICs of 563 and 18 µM, respectively for 2 and MICs of 141 and 281 µM for 3. Importantly, MICs for AH1263 (LAC, the agr group I USA300 strain used in the murine model of dermonecrosis) were not detected at concentrations of up to 563 µM for compound 1, and were noted at the higher concentrations of 563 and 141 µM for compounds 2 and 3, respectively. www.nature.com/scientificreports www.nature.com/scientificreports/  . Schinus compounds 1-3 inhibit agr activity for all four agr alleles in a dose-dependent manner. Data are represented as percent agr activity or growth of the vehicle (DMSO) control at 24 hours against the following strains: AH1677 (agr I), AH430 (agr II), AH1747 (agr III), and AH1872 (agr IV). The dashed lines represent agr activity, measured by fluorescence, and the solid lines represent growth, measured using OD 600 . Significant differences (P < 0.05) in quorum sensing activity between the vehicle control and compounds 1-3 were observed at all doses tested in agr groups I, II and IV, with exception of the lowest concentration tested (2 µM) of 1 in agr III and 3 in agr IV. www.nature.com/scientificreports www.nature.com/scientificreports/ Triterpenoid acids (1-3) inhibit quorum sensing and toxin production. To identify small molecule inhibitors of S. aureus quorum sensing from the 430F-F5 fraction, we employed a bioassay-guided fractionation strategy using agr-fluorescent reporter stains that represent each of the four known agr allelic groups (Fig. 3, Supplementary Table 1). Strains were grown in the presence of the compounds and monitored for growth inhibition by optical density and agr activity by fluorescence.
Compounds 1-3 significantly inhibited quorum sensing in all four agr types, exhibiting IC 50 values ranging from 4-70 µM for 1, 2-9 µM for 2, and 4-9 µM for 3 ( Fig. 3, Table 2). Compound 3 showed the most potent quorum sensing inhibition activity in this model (IC 90 of 18 µM), but this was influenced by high growth inhibition of the agr III reporter by this compound. With the exception of this case (compound 3 growth inhibition of the agr III reporter strain), 1-3 exhibited limited growth inhibitory activity against the reporter strains.
To verify the observed quorum-inhibition effects of 1-3, we assessed their capacity to inhibit δ-toxin production at concentrations ranging from 9 to 141 µM. High level δ-toxin producing of strains of S. aureus were treated with the triterpenoid acids and the supernatants harvested for RP-HPLC and human keratinocyte cell (HaCaT) toxicity analysis. Each of compounds 1-3 were effective in significantly reducing δ-toxin production, with weak or no growth inhibition in the S. aureus strains examined (Fig. 4A).
To assess any remaining virulence factors linked to cytotoxicity (e.g., phenol soluble modulins, PSMs), sterile filtered supernatants of NRS249 cultures grown with compounds 1-3, were exposed to an immortalized line of human keratinocytes (HaCaTs) and assessed for damage by lactate dehydrogenase (LDH) assay. The toxicity of S. aureus supernatants to HaCaTs cells was inhibited in a dose dependent manner (Fig. 4B).
To examine the specificity of 1-3 against agr, we also chose reporter strains (promoter-GFP fusions in the Staphylococcus aureus AH1263 background) that are known to be either agr-regulated or independent of agr regulation.
The agr-regulated reporters (lukA: leukocidin A, gehB: glycerol ester hydrolase or lipase) showed dose-dependent inhibition by 1-3 as expected since we have shown that these compounds target the agr system ( Fig. 5A,B).
To our surprise, the nuc reporter (nuclease), which is primarily under the control of the sae two-component system, is also inhibited in a dose-dependent manner by all of the Schinus compounds (1-3), suggesting broader inhibitory effects against multiple MRSA virulence factors (Fig. 5C). Additionally, we tested the agr-independent mgrA P2 reporter (a constitutive promoter from the mgrA two-component system) and saw little effect of any Schinus compound (1-3), suggesting that these two components do not primarily target this system (Fig. 5D). We found for the agr-independent esxA reporter, which is a component of the MRSA type VII secretion system and generally thought to be under the control of the mgrA two-component system (but may be regulated by other systems as well), that there was modest inhibition by 1-3 (Fig. 5E).
Taken together, these results confirm that 1-3are potent agr inhibitors but are also broader MRSA virulence inhibitors that may also target the sae two-component system.

Triterpenoid acids 1-3 impact biofilm formation.
In microtiter plate biofilm assays, compound 1 increased biofilm formation at low dosages (2-70 µM), and reduced biofilm at higher concentrations   www.nature.com/scientificreports www.nature.com/scientificreports/ (141-281 µM) without growth inhibition. This is noteworthy as the neither the growth inhibitory IC 50 or MIC for 1 in the biofilm strain (UAMS-1) was detected at a maximum test concentration of 563 µM (Table 1). Compounds 2 and 3 caused significant, dose-dependent increases in biofilm biomass at concentrations ranging from 2-16 μM. In contrast, both biofilm formation and bacterial growth were strongly reduced at concentrations of 35 μM and above (Fig. 6). For compounds 2 and 3, these biofilm inhibitory concentrations are roughly 4-18 times higher than the IC 50 for quorum sensing inhibition in the four reporter strains ( Table 2).

Impact of compounds 1-3 on human keratinocytes.
To determine the potential toxicity to human cells, compounds 1-3 were tested on human keratinocyte cells (HaCaTs) using a lactate dehydrogenase (LDH) assay for cytotoxicity. The IC 50 values for cytotoxicity to HaCaTs and therapeutic indices for each reporter strain are reported in Fig. 7. The therapeutic index ranged from 2 to 35, depending on the reporter strain and compound combination.

Compounds 1-3 abate quorum sensing and dermonecrosis in vivo.
The antivirulence activity of compounds 1-3 observed in vitro led us to assess their efficacy as an in vivo anti-infective. Using a previously established murine model of dermo-necrosis 15,25,29 , BALB/c mice were intradermally challenged with MRSA (USA300) and 50 µg of each Schinus compound or DMSO vehicle control at the time of infection. Over the two-week course of infection, compounds 1 and 3 exhibited similar potency in reducing MRSA-mediated skin damage (Fig. 8). Compound 2 effectively reduced the dermonecrotic lesion area to nearly undetectable levels (Fig. 8A). All compounds significantly minimized animal weight loss, indicating decreased morbidity compared to wild-type infection (Fig. 8B). Additionally, each compound significantly reduced dermonecrotic lesion size throughout the course of MRSA infection (Fig. 8C). Taken together, these results demonstrate that all of the compounds protect mice from MRSA infection-associated dermonecrotic injury and morbidity. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
The emergence of antibiotic resistance, together with the lack of antibiotics based on novel molecular scaffolds, marks the entry to the 'post antibiotic era' . Interference with bacterial virulence has emerged as an attractive approach among the current strategies for developing new anti-infective drugs. Prior studies have reported the capacity of plant extracts and phytochemicals to interfere in intra and inter-species quorum sensing communication systems 30,31 . The ability of plants to interrupt quorum sensing systems may serve as a defense mechanism to fight against bacterial invasion. One of the keys factors of success of phytochemicals could be their similitude to what is considered the ideal quorum sensing inhibitor, which includes being chemically stable, highly effective low-molecular-mass molecules and harmless for human health 32 .
In a previous study, we confirmed the quorum sensing activity of a refined S. terebinthia fruit extract (430D-F5) against multiple MRSA strains 25 . Here, we successfully isolated three triterpenoid acids using a bioassay-guided fractionation approach. The structure of these compounds (1-3) was determined via NMR, MS, and X-ray crystallography, and matches with substances previously isolated from S. terebinthifolia and other plants [33][34][35] . The crystal structures of compounds 2 and 3 were previously reported 36,37 .
We found that the three triterpenoid acids (1-3) inhibited expression of all S. aureus agr types in a dose dependent manner with limited inhibition of bacterial growth in agr groups I, II, and IV, but moderate growth inhibition in the agr group III reporter. Compound 3 showed the most potent anti-virulence activity against S. aureus in vitro ( Table 2, Fig. 3), while compound 2 exhibited the greatest level of protection from dermonecrosis in vivo. To confirm the inhibitory activity of isolated compounds with a downstream protein level readout, we assessed their ability to inhibit the production of agr-regulated δ-toxin. These triterpenoid acids (1-3) notably decreased δ-toxin production in two S. aureus strains, and the general toxicity of pre-treated S. aureus supernatants to HaCaTs also confirmed these effects.
The impact on other agr-regulated reporters were also assessed, revealing dose-dependent inhibition of leucocidin A (lukA) and glycerol ester hydrolase or lipase (gehB). Little to no effect of any compound was noted for the agr-independent mgrA P2 or esxA reporters. Interestingly, a dose-dependent response was noted in tests with the nuclease reporter, which is under the control of the sae two-component system, suggesting a dual role of 1-3 in broader virulence inhibition across both agr and sae systems.
The agr operon is essential for virulence-factor production during MRSA skin-infection and reducing agr functionality could potentially mitigate acute tissue damage. This is supported by the results of our in vivo experiments, where 2 decreased MRSA-associated tissue damage to the limit of detection. Notably, 1 and 3 also exhibited significant anti-virulence effects, and mice were protected from severe dermonecrosis and morbidity. However, it is well-established that the absence of agr signaling drives MRSA biofilm formation under in vitro conditions 38 . Indeed, low concentrations of all compounds resulted in significant biomass accumulation while higher concentrations mitigated biomass accumulation in our in vitro assays. These results suggest that careful titration of quorum sensing inhibitory compound doses will certainly be necessary if administered in a human  (B) The therapeutic index for each compound is calculated as IC 50 for HaCaT cytotoxicity divided by the IC 50 for agr activity (see Table 2) in each reporter strain. www.nature.com/scientificreports www.nature.com/scientificreports/ infection scenario. Additionally, this study employed inhibitory compounds at the onset of infection in a prophylactic model, effectively reducing MRSA virulence during infection initiation and allowing for rapid clearance of bacteria by the immune system. The use of quorum sensing inhibitors at the onset of human infection is far less likely if disease progression is slow or access to healthcare providers is limited. Thus, we postulate that natural product quorum sensing inhibitors, such as those proposed in this study, could be paired with a secondary bactericidal or biofilm dispersal agents to fully abrogate MRSA infection. However, further research is needed to identify antibiotics that synergize with virulence inhibitory compounds while mitigating the risk of acquired MRSA resistance 39 .
Our results also suggest that the biological activity of these compounds may be particularly sensitive to chemical modifications of their terpenoid skeleton. In particular, this is the case for compounds 2 and 3, where the substituent at carbon 3 could play a key role in the inhibition of virulence factors. A medicinal chemistry approach to modification of the terpenoid skeleton could also be explored to improve the therapeutic indices of these compounds, with an optimal difference between activity and toxicity being several hundredfold. This will require study of a large number of related compounds in order to approach the minimum requirements necessary for reaching a more definitive conclusion with respect to structure-activity relationships (SAR).
Previous studies have reported related pentacyclic triterpenoids with anti-virulence and biofilm formation/ eradication activities against S. aureus and other strains, such as betulinic acid 40 , asiatic and corosolic acids 41 , ursolic acid and derivatives 42,43 , glycyrrhetinic acid 44 and oleanolic acid 40 . However, to the best of our knowledge, this is the first time that the anti-quorum sensing activity of tirucallane-type triterpenoids (2 and 3) is described.

Materials and Methods
Plant material. Schinus terebinthifolia Raddi, Anacardiaceae fruits were collected in bulk from private lands in DeSoto County, Florida in November of 2013 and 2014 after obtaining permission from the land owner. Vouchers were deposited at the Emory University Herbarium (GEO) (Voucher CQ-400, GEO Accession No. 020063) and were identified using the standard Flora for Florida. Plant material was then dried in a desiccating cabinet at low heat. Once dry, plant material was sealed in paper bags and stored at room temperature until further processing.
Extraction and bioassay guided fractionation. Fruits of S. terebinthifolia were dried in a dehumidifying chamber, ground into a powder with a Wiley Mill at 2 mm mesh and extracted using an ultrasonic bath at room temperature with methanol (ratio of 1 g dry material: 10 mL MeOH) for a total of three times. Filtered extracts were combined, concentrated at reduced pressure with rotary evaporators (<40 °C), and lyophilized. The crude extract (15.0 g) was suspended in 400 mL methanol-water (1:4) solution and underwent sequential liquid-liquid www.nature.com/scientificreports www.nature.com/scientificreports/ partitioning with hexane (3×500 mL), ethyl acetate (3×500 mL), and n-butanol (3×500 mL). The combined partitions were evaporated to dryness to yield 1.16 g (3.92%) of hexane residue, 1.15 g (2.98%) of ethyl acetate residue and 12.61 g (32.71%) of aqueous residue.
Following initial quorum sensing assays, the most active partition, the aqueous residue (430 F) was subjected to fractionation through flash chromatography. Fractionation was performed using a CombiFlash ® Rf+ Lumen (Teledyne ISCO) flash chromatography system with a RediSep Rf Gold silica column. The dry load column was prepared by binding 430 F to Celite 545 at a ratio of 1:4. Flash chromatography was performed using a three-solvent system of (A) hexane, (B) dichloromethane, and (C) methanol. The gradient began with 100% A for 6 column volumes (CV), then went to 100% B over 12 CV and was held for 18.2 CV. The gradient was then changed to 74.5:25.5 B:C over the course of 3.1 CV. These conditions were held for 6.8 CV, following which the gradient changed to 68.8:31.2 B:C over 0.7 CV and was held at these conditions for 7.5 CV. Finally, the gradient was adjusted to 100% C over 2. Analytical chemistry procedures. Nuclear magnetic resonance (NMR) data, 1 H NMR (600 MHz) were measured on a Bruker AVANCE III HD 600 (600 MHz for 1 H-NMR, 5 mm CryoProbe) spectrometer in CD 3 OD or CDCl 3 solvents. Chemical shifts (δ) are reported in ppm with the solvent peaks used as reference.
High resolution/accurate mass-Atmospheric pressure chemical ionization (HRMS-APCI) experiments were performed on a Thermo Exactive Plus using the Ion Max Source with APCI probe. The sample was placed on a melting point capillary and then placed in an Ion Sense ASAP (Atmospheric Solid Analysis Probe) probe, which was inserted into the source to enable gas from the APCI probe to blow on the capillary tube. The APCI probe uses nitrogen gas at arbitrary units of 50 and was heated to a temperature of 450 °C. A discharge on the needle with a discharge current of 5 µA was used to ionize the sample, with a capillary temperature of 320 °C, S-lens RF level 70 and an AGC setting of 1 E-6. The maximum injection time was 50 ms. Spectra were collected with 140,000 resolution at m/z 200 using Tune software and analyzed with Thermo's Freestyle software.
Single crystals of compounds 1-3 were recrystallized from methanol by placing them into a −20 °C freezer overnight. A suitable crystal was selected and the crystal was mounted on a Rigaku XtaLAB Synergy-S diffractometer. The crystal was kept at 107(8) K during data collection. Using Olex2, the structure was solved with the ShelXT structure solution program using Intrinsic Phasing and refined with the ShelXL refinement package using Least Squares minimization.
Bacterial strains. Quorum sensing reporter strains of S. aureus representing the four agr groups included AH1677, AH430, AH1747 and AH1872, were used. AH1677 is an agr I reporter from strain AH845 (CA-MRSA USA 300 LAC), AH430 is an agr II reporter from SA502A, AH1747 is an agr III reporter from strain CA-MRSA MW2, and AH1872 is an agr IV reporter from MN EV. All strains contained plasmid pDB59 that served as an agr fluorescence reporter and was maintained through culture in media containing chloramphenicol at a concentration of 10 μg mL −1 . Chloramphenicol (Sigma-Aldrich) was dissolved in 95% EtOH to a stock concentration of 10 mg mL −1 and stored at −20 °C until being added to the media. Cultures were grown on Tryptic Soy Agar (TSA) supplemented with 10 μg mL −1 chloramphenicol. Two S. aureus strains (UAMS-1, UAMS-929) served as biofilm test strains, with the latter being an isogenic Δsar mutant that has a limited capacity to produce a biofilm. One S. aureus strain (NRS385) was used for δ-toxin quantification experiments. UAMS-1, UAMS-929 and NRS385 were grown in Tryptic Soy Agar (TSA) and Tryptic Soy Broth (TSB). A summary of strain characteristics is provided in Supplementary Table 1.

Minimum inhibitory concentration. Fraction 430F-F5 and isolated compounds of S. terebinthifolia 1-3,
were evaluated for minimum inhibitory concentrations (MICs) against the four agr groups, the biofilm test strain (UAMS-1) and NRS249 (for δ-toxin quantification experiments) following the Clinical Laboratory Standards Institute (CLSI) M100-S23 guidelines for microtiter broth dilution testing 45 . Fraction, compounds 1-3 and vehicle were tested at a concentration range of 4 to 563 μM, using 2-fold serial dilution. The optical density (OD 600nm ) was measured with a BioTek Cytation 3 multimode plate reader, to calculate percent inhibition of growth. The IC 50 for growth was defined as the lowest concentration at which an extract displayed ≥ 50% inhibition and MIC (IC 90 ) at ≥ 90% inhibition. Control included the vehicle (DMSO) and antibiotic (Ampicillin, MP Biomedicals Inc). All tests were performed in triplicate and repeated using a new stock of bacteria on a separate day. www.nature.com/scientificreports www.nature.com/scientificreports/ chloramphenicol at 37 °C while shaking at 200 rpm. Cultures of S. aureus were standardized by optical density (OD 600nm ) matching to a 0.5 McFarland standard to reach a final inoculum density in the wells of 5 × 10 5 CFU mL −1 in TSB, supplemented with 10 μg/mL chloramphenicol. Compounds 1-3 were tested to escalating concentrations (2-281 μM) of each compound. Black sided, 96-well, clear bottom, tissue-culture treated plates (Costar 3603) with final well volume of 200 μL were used for all agr inhibition assays. Plates were incubated in a humidified chamber at 37 °C while shaking at 260 rpm. After 24 hours, plates were removed and OD 600 and fluorescence were measured by plate reader at an excitation of 493 nm and emission of 535 nm. All tests were performed in triplicate and repeated using a new stock of bacteria on a separate day.
HaCaT cytotoxicity assay. The HaCaT cell line was maintained and cytotoxicity of compounds (1-3) were assessed using the LDH cytotoxicity assay as previously described 49 . The Therapeutic Index (TI) was calculated as a ratio of the TD 50 (Toxic dose for cytotoxicity at IC 50 ) and ED 50 (effective dose for quorum sensing inhibiton IC 50 ): = TD50 ED50 . Cytotoxicity of supernatants was further evaluated using a viability/cytotoxicity assay and imaged with fluorescent microscopy as previously described 49 . All tests were performed in triplicate and the full experiment was repeated on a separate day using fresh cell stock.
Quantification of δ-toxin. Levels of δ-toxin present in the culture supernatant of treated and untreated samples was quantified using NRS249 and AH1263 strains of S. aureus and following a previously described protocol 50 . Experiments were conducted in quadruplicate in 14 mL snap-cap tubes with a final volume of 1.5 mL, and repeated in full with fresh bacterial stock on a separate day. All compounds were tested at concentrations from 9-141 µM. Tubes were incubated at a 45° angle at 37 °C while shaking (275 rpm) for 15 hours. After incubation, cultures were placed on ice, an aliquot was taken to determine OD, then centrifuged at 13,000 rcf for 5 min at a temperature of 4 °C. Supernatants were removed and 750 μL of each supernatant was placed in a vial for HPLC quantification of δ -toxin. The remaining volume of supernatant was sterile filtered with a 0.22 μm nylon syringe filter and stored at −20 °C until needed for later treatment of cells in the HaCaT cytotoxicity assay. Level of δ-toxin in the bacterial supernatant treated and untreated with the compounds was quantified by RP-HPLC (Reversed-Phase High Performance Liquid Chromatography) as previously described 50 . Biofilm formation. All compounds were examined (2-281 μM) for impact on S. aureus biofilm formation using a human plasma protein-coated assay as previously described 49,51,52 . Additionally, to monitor for any growth impact in this assay, the optical density of planktonic cells in the biofilm wells was calculated by transferring the supernatant to a new 96-well plate and reading the OD 600nm in a BioTek plate reader. S. aureus isolate UAMS-1 was used for the biofilm assay, and its isogenic sarA mutant (UAMS-929), which has a biofilm deficient phenotype, served as a positive control. All tests were performed in triplicate and repeated using a new stock of bacteria on a separate day.
Animal Studies. A previously described murine model of MRSA skin infection was used to determine the efficacy of each Schinus compound as an anti-infective 25 . All animal experiments described herein were approved by and conducted in accordance with the recommendations of the Animal Care and Use Committee at the University of Colorado Anschutz Medical Campus (IACUC protocol number 117217). One day prior to inoculation, the abdominal hair of 8 week old female BALB/c mice was shaved and chemically removed with topical application of Nair for 1 minute. LAC (USA300, AH1263) was grown in TSB media overnight at 37 °C in a shaking incubator (200 rpm). Overnight LAC culture was diluted 1:100 in fresh TSB media and allowed to grow to early log phase (~2 hr to OD 600nm 0.5). Cells were washed in sterile PBS and resuspended to achieve an inoculum of 1×10 8 CFU. 50 μL inoculum suspensions containing 1×10 8 CFU LAC and 50 μg of each compound 1-3 (diluted in DMSO) or DMSO vehicle control were injected intradermally into the abdominal skin of each BALB/c mouse. Baseline body weights for the mice were taken prior to injection and each day following. For determination of lesion size, digital photos of skin lesions were taken with an iPhone camera and analyzed with ImageJ software for Mac. Inoculum CFU was verified by serial dilution, plating, and colony counting after overnight incubation of the plate.
Statistical Analyses. All data were analyzed using a two-tailed Student's t-test as calculated by GraphPad Prism 7 software (GraphPad Software, La Jolla, CA). DMSO or dH 2 O treated (vehicle control) cultures were used as a vehicle control and were compared to those treated with extract for all statistical analyses. P < 0.05 were considered statistically significant. All assays and other experiments were performed in triplicate or quadruplicate to ensure sufficient technical replicates and repeated on two separate days to ensure sufficient biological replicates.