Antibacterial Anacardic Acid Derivatives

Article abstract of DOI:10.1021/acsinfecdis.9b00378

We report on the antibacterial activity of five phenolic lipids derived from anacardic acid characterized by increasing alkyl chain lengths with 6, 8, 10, 12, or 14 carbon atoms. The compounds were profiled for their physicochemical properties, transport across epithelial monolayers, cytotoxicity, and antibacterial activity as compared to common antibiotics. No cytotoxicity was reported in cell lines of fibroblast, hepatic, colorectal, or renal origin. C10 and C12 significantly increased the survival in a Galleria mellonella model infected with multi-drug-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococci (VRE) as compared to the untreated control group. Future studies are required to corroborate these findings in relevant animal model systems of infection.

Full text of DOI:10.1021/acsinfecdis.9b00378

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Article
Anti-bacterial anacardic acid derivatives
Marco Saedtler, Niclas Förtig, Knut Ohlsen, Franziska Faber, Nelson
Masota, Kristin Kowalick, Ulrike Holzgrabe, and Lorenz Meinel
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00378 • Publication Date (Web): 10 Jun 2020
Downloaded from pubs.acs.org on June 13, 2020
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Anti-bacterial anacardic acid derivatives
Marco Saedtler1, Niclas Förtig1, Knut Ohlsen2, Franziska Faber2, Nelson Masota1, Kristin Kowalick3,
Ulrike Holzgrabe1, Lorenz Meinel1,4,*
1 Institute for Pharmacy and Food Chemistry, Am Hubland, 97074 Würzburg, Germany
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2
Institute for Molecular Infection Biology (IMIB), Josef-Schneider-Straße 2, 97080 Würzburg,
Germany
3 Labor LS SE & Co. KG, Mangelsfeld 4-6, 97708 Bad Bocklet, Germany
4
Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Center for Infection
Research (HZI), 97080 Würzburg, Germany
* Corresponding author. Email: lorenz.meinel@uni-wuerzburg.de
We report on the antibacterial activity of five phenolic lipids derived from anacardic acid characterized
by increasing alkyl chain lengths with C6, C8, C10, C12, or C14 carbon atoms. The compounds were
profiled for their physicochemical properties, transport across epithelial monolayers, cytotoxicity and
antibacterial activity as compared to common antibiotics. No cytotoxicity was reported in cell lines of
fibroblast, hepatic, colorectal, or renal origin. C10 and C12 significantly increased survival in a Galleria
mellonella model infected with multi-drug resistant Staphylococcus aureus (MRSA) or Vancomycin-
resistant Enterococci (VRE) as compared to the untreated control group. Future studies are required to
corroborate these findings in relevant animal model systems of infection.
Keywords: Methicillin resistant Staphylococcus aureus; VRE; Galleria mellonella; biofilm inhibition;
anacardic acid
Sub-therapeutic doses of antibiotics selectively pressure bacteria towards resistance. Such sub-
therapeutic exposure is particularly probable for bacterial biofilms, within which antibiotics form a
decreasing concentration gradient hence lower (and then potentially sub-therapeutic) antibiotic
concentrations might be established in vicinity of the bacteria.1 It is for this among other effects that
multi-drug-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant Enterococci (VRE)
have formed.2 A Study from 2019 estimated that around 600,000 infections with antibiotic-resistant
bacteria (63.5% considered nosocomial infections) lead to 30,000 deaths annually and cause 800,000
disability-adjusted life-years in the European Union alone.3 Within this context, biofilm disrupting
molecules with antiseptic activity are of increasing interest.4 Although current disinfectants such as
Chlorhexidine, Octenidine or Polyhexanide have antiseptic activity and a low tendency to induce
bacterial resistances, their application is only limited to local administration as of cytotoxic effects and
tissue irritation. This limitation motivated us to screen for novel starting off a series of amphiphilic
compounds derived from anacardic acid. Anacardic acid is naturally found in the cashew nut shell
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(Anacardium occidentale), or the shell of the pistachio nut (Pistachia vera). Anacardic acid’s
antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Streptococcus mutans has been
reported and was attributed to its amphiphilic structure.5-7 Additionally, anacardic acid has several other
reported activities such as inhibition of histone acetyl transferase (HAT)8, inhibition of matrix
metalloproteinases (MMP)9 and the chelation of metal ions as well as antioxidant properties.10 We built
off these findings synthesizing derivatives of anacardic acid by elongating their lipophilic alkyl tail in
analogy to a previous study on derivatives of ortho-hydroxybenzoic acid.11 The resulting compounds
were physically and chemically characterized and their antiseptic activity, cytotoxicity and ability for
biofilm reduction was characterized in comparison to relevant antibiotics including amoxicillin,
ciprofloxacin, vancomycin, and linezolid and to octenidine, a locally administered anti-infective.12
Lastly, the two most promising candidates were further assessed in vivo using a Galleria mellonella
infection model. G. mellonella larvae demonstrate an innate immune response similar to vertebrates and
have proofed themselves as reliable in vivo model for novel antibiotic drug candidates and toxicological
studies.13
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Figure 1 (left) Fruit and plant of the cashew nut (Anacardium occidentale) and (right) the synthetic compounds derived from
anacardic acid and used in this study.
Materials and Methods
Materials
Dulbecco’s Modifed Eagle’s Medium (DMEM; #D6429-500mL), high glucose, Minimum Essential
Medium Eagle (MEM; #M2279-500mL), Hanks’ Balanced Salts powder (HBSS) without phenol red,
MEM Non-essential Amino Acid Solution (100x), L-Alanyl-L-glutamine solution (200 mM, sterile),
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sodium pyruvate solution (100 mM, sterile), Penicillin-Streptomycin solution (Penicillin 10000 units
mL-1 and Streptomycin 10 mg mL-1, sterile), Endo Agar powder, Fluorescein sodium salt, BioReagent,
suitable for fluorescence, Lucifer Yellow CH dilithium salt, Ginkgolic acid C13:0 and (15:0) Anacardic
acid were purchased from Sigma Aldrich (Schnelldorf, Germany). Trypticasein Soy Broth (TSB)
powder, Müller-Hinton-Bouillon (MH) powder and Brain Heart Infusion (BHI) powder were purchased
from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). Mowiol 4-88 for histology was purchased from
AppliChem GmbH (Darmstadt, Germany), 4’,6-diamino-2-phenylindole (DAPI) from Invitrogen
(Carlsbad, CA), mouse antibody against human e-cadherin MAb from R&D systems (Minneapolis, MN)
and antimouse antibody Alexa Fluor 488 from Life Technologies (Darmstadt, Germany) and
hexadeuterio-dimethyl sulfoxide (DMSO-d6, 99.8% D) from Euriso-top (Saarbrücken, Germany).
Ciprofloxacin hydrochloride and Vancomycin hydrochloride and Linezolid were purchased from
Sigma-Aldrich. Amoxicillin was a gift from TAD Pharma GmbH (Cuxhaven, Germany). Deionized
water was obtained from our in-house Merck Millipore water purification system (Darmstadt,
Germany). Other chemicals and reagents used within this study were purchased from Sigma Aldrich if
not noted otherwise. The cell lines used for cell culture were obtained from ATCC with HEK293 (ATCC
ID# CRL-1573), Hep G2 (ATCC ID# HB-8065), Caco-2 (ATCC ID# HTP-37, and NIH 3T3. The origin
of the bacterial strains is shown below (Table 1).
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Table 1 Tested bacterial strains, including information on species, source and culture conditions.
Species
Code
Source
Anaerobic
Medium
Comment
Staphylococcus
aureus
JE2
Clin. isolate No
Clin. isolate No
Clin. isolate No
Clin. isolate No
MH
MRSA
Staphylococcus
aureus
Lac*
MH
MRSA
Staphylococcus
aureus
ST228
RP62a
51229
MH/TSB
MH
Biofilm-
forming
Staphylococcus
epidermidis
-
Enterococcus
faecalis
ATCC
No
BHI
VRE
VRE
-
Enterococcus
faecium
UL602570
13813
Clin. isolate No
BHI
Streptococcus
agalactiae
ATCC
No
BHI
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Species
Code
Source
Anaerobic
Medium
Comment
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Clostridium
difficile
DSM27543
DSM
Yes
BHI
-
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Methods
Proton nuclear magnetic resonance spectroscopy
Proton nuclear magnetic resonance spectra (1H-NMR) of all compounds were recorded on a Bruker
Avance 400 MHz spectrometer (Karlsruhe, Germany) operating at 400.13 MHz (Figure S22-32) and
additionally 13C-NMR spectra of all final products (Figure S33-37). The spectra were referenced to the
residual solvent signal of DMSO-d6 at 2.54 ppm.14
Synthetic procedures
Synthetic procedures 1, 2 and 3 were obtained from previously described methods with small changes
to obtain higher yields (Scheme 1).11, 15, 16
Scheme 1 Schematic route of synthesis used within this study. a) Synthetic Procedure 1; SOCl2, DMAP, acetone, diethyl
ether, 0 °C, 1 h, 25 °C, 1 day. b) Synthetic Procedure 2; 1-bromoalkane, K2CO3, acetonitrile, 50 °C, 4 days. c) Synthetic
Procedure 3; KOH, tetrahydrofuran, 60 °C, 1 day.
Synthetic Procedure 1
2,6-Dihydroxybenzoic acid 1 (6.16 g, 40 mmol) and N,N-dimethylpyridin-4-amine (244 mg, 2 mmol)
were dissolved in 30 mL diethyl ether under nitrogen atmosphere. The resulting dispersion was cooled
to 0 °C. Acetone (3.02 g, 3.85 mL, 52 mmol) and thionyl chloride (6.18 g, 3.77 mL, 52 mmol) were
carefully added. The reaction was stirred for 1 h at 0 °C and for 24 h at room temperature. The reaction
mixture was diluted with 20 mL aqueous saturated NaHCO3-solution and extracted with diethyl ether
(3 x 20 mL). The combined organic phases were washed with brine (3 x 10 mL), dried over MgSO4 and
concentrated under reduced pressure.
Synthetic Procedure 2
5-Hydroxy-2,2-dimethyl-4H-benzo[d][1,3]dioxin-4-one 2 (776 mg, 4 mmol) and potassium carbonate
(1.10 g, 8 mmol) were suspended in 10 mL acetonitrile under argon atmosphere. The reaction was cooled
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to 0 °C and 1-bromoalkane (5.2 mmol) was slowly added. The mixture was stirred for 30 min at 0 °C
and for 96 h at 50 °C. The reaction mixture was diluted with 30 mL deionized water and extracted with
ethyl acetate (3 x 40 mL). The combined organic phases were washed with deionized water (2 x 30 mL),
1 M hydrochloric acid (1 x 30 mL), brine (2 x 20 mL) and dried over Mg2SO4. The solvent was
evaporated under reduced pressure.
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Synthetic Procedure 3
5-(Alkyloxy)-2,2-dimethyl-4H-benzo[d][1,3]dioxin-4-one 3a/3b/3c/3d/3e (2.3 – 3.3 mmol) were
dissolved in 10 mL tetrahydrofuran and 10 mL 5 M potassium hydroxide aqueous solution was added.
The reaction was stirred for 24 h at 70 °C. The mixture was acidified with 6 M hydrochloric acid and
extracted with ethyl acetate (3 x 30 mL). The combined organic phases were washed with deionized
water (1 x 30 mL), brine (2 x 30 mL) and were dried over Mg2SO4. The organic solution was
concentrated under reduced pressure.
High Resolution Mass Spectrometry (HRMS)
HRMS of all final products was measured by electrospray ionization mass spectrometry (ESI-MS) on a
micrOTOF-Q III (Bruker Daltonics, Billerica, MA) equipped with an Apollo II ESI Ion Source. The
instrument has a resolution of 20,000 and a mass accuracy of 5 ppm with external calibration. Briefly,
samples were injected with a constant flow of 4 µL min-1. The spectra were recorded continuously in
the range from 50 to 2500 m/z with capillary voltage set to -4.5 kV, end plate voltage -4 kV, nitrogen
nebulizer pressure 0.3 bar, dry gas flow 4 L min-1 and dry temperature 200 °C. Funnel 1 was 100 Vpp
and Funnel 2 RF 200 Vpp, Hexapole RF 200 Vpp; Quadrupole ion energy 5 eV, Quadrupole low mass
50 m/z; collision energy 10 eV, collision RF 300 Vpp, transfer time 70 µs, pre pulse storage 10 µs. The
instrument was calibrated with sodium formate which was measured directly after the sample
measurements. The spectra were recorded with the Compass Software from Bruker Daltonics containing
the OTOF Control 3.4 software for controlling the instrument. For data evaluation the Data Analysis
software DA 4.2 from Bruker Daltonics was used.
Critical Micelle Concentration (CMC)
CMC values were determined using a Krüss K12 Tensiometer (Krüss GmbH, Hamburg, Germany)
equipped with a heating device to maintain 25 °C. Surface tension was measured at different
concentrations, using the Wilhelmy plate method.17 Briefly, 70 mL deionized water and a magnetic
stirring bar were added to a 120 mL glass vial and 1 mL aliquot of an aqueous stock solution was added
and stirred for 30 sec between each measuring point. Concentrations of the used stock solutions were
500, 150, 50, 100 and 100 µmol L-1 for C6, C8, C10, C12 and C14, respectively. The surface tension (σ)
was calculated by the software, measuring the force applied to a platinum plate which was slowly
immersing from the aqueous solution. CMC values are presented as the concentration in µmol L-1 at the
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crossing-point of two trend lines obtained by linear regression of the two linear sections before and after
the CMC. Experiments were done in triplicate and results are displayed as mean ± standard deviation.
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Partition Coefficient (logDpH7.4)
9
Partition coefficients was determined following OECD Guidelines on partition coefficient, HPLC
method.18 Briefly, an Agilent 1200 series HPLC consisting of a G1322A degasser, G1311A quarternary
pump, G1329A autosampler, G1316A column oven and a G1314 VWD detector equipped with a
Synergi™ Max-RP, 80 Å, 150 x 4.6 mm, 4µm column from Phenomenex (Torrance, CA). Mobile phase
consisted of 10 mmol L-1 phosphate buffer pH 7.4 and methanol 30:70 and was used in an isocratic
method for a total run time of 20 min with a flow of 1.5 mL/min. Temperature was set to 30 °C, injection
volume to 40 µL and the detection wavelength to λ = 254 nm. Samples were prepared by dissolving 4
mg of the compound in 1 mL methanol, diluting it 1:1000 with the same solvent and filtering it through
0.2 µm, 4 mm PTFE filters from Whatman (Maidstone, UK). A calibration curve was prepared from
measuring thiourea (dead-time), acetanilide, benzene, biphenyl, anthracene and triphenylamine, using
their calculated capacity factor k (Equation 1) (R² = 0.99). Partition coefficients logP were calculated
from logDpH 7.4 (Equation 2).
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Equation 1
tR ―to
k =
t0
k
tR
t0
-
-
-
capacity factor
retention time
dead-time
Equation 2
1
logP = logDpH - log(
)
1 + 10pH - pKa
logP
logD
pKa
-
-
-
partition coefficient
distribution coefficient
acid dissociation constant
Solubility Determination
Solubility was determined using a shake flask method.19 5-10 mg of compound were placed in a 1.5 mL
reaction vial (Eppendorf, Hamburg, Germany). 1 mL of phosphate buffer saline (PBS) pH 7.4 ± 0.05
was added and placed in a ThermoMixer F1.5 (Eppendorf). The aliquots were shaken at 800 RPM for
48 h at 25 °C. After centrifugation at 17880 rcf for 10 min at 25 °C in a Universal 302 R centrifuge
(Hettich Zentrifugen, Tuttlingen, Germany), 0.25 mL were carefully withdrawn and immediately diluted
with 0.75 mL methanol. Absorbance was measured UV-metric at 254 nm with an Evolution 201 UV-
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Visible Spectrophotometer (Thermo Scientific, Waltham, MA), if necessary further dilution was done
with methanol. Standard curves were conducted in methanol from 100 µmol L-1 – 5 µmol L-1 on the
same instrument, all yielding coefficients of determination R² ≥ 0.99. Results were calculated as means
with standard deviation (n = 3).
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Dissociation Constant (pKa)
Dissociation constants were determined using a UV-metric titration assay on a Sirius T3 titration
platform from Pion Inc. (Woburn, MA) following the manufacturer instructions.20 Briefly, 0.001 mL of
a 150 mmol L-1 stock solution in dimethyl sulfoxide (DMSO) was placed into a reaction vial and 1.5
mL ionic strength adjusted water (ISA water) was added automatically. The starting pH 12 value was
automatically adjusted by addition of 0.5 mol L-1 potassium hydroxide solution and stirred for 2 min.
The solution was stepwise titrated to pH 2 by addition of 0.5 mol L-1 hydrochloric acid solution aliquots.
After each addition a UV-spectrum and the respective pH value was recorded by the UV-dip probe and
the Ag/Cl electrode. The titration from pH 2 - 12 was repeated three times per experiment and each
experiment was performed in triplicate. The spectrum between 250 nm and 450 nm was used in the
range of pH 2 – 6 to identify the protonated and deprotonated species and calculate the pKa value.
Analysis was done using the SiriusT3Refine software (Version 1.1.3.0) from Pion Inc.
High Pressure Liquid Chromatography (HPLC)
HPLC analysis was performed on a Hitachi LaChrom Ultra equipped with a UV Detector L-2400U,
Column Oven L-2300, Autosampler L-2200U and two pumps L-2160. ZORBAX Eclipse XDB-C18 5
µm 4.1x150 mm column from Agilent Technologies (Waldbronn, Germany) was used at 23 °C and 0.01
mL injection volume with a detection wavelength of 254 nm was set. An isocratic method was used with
acetonitrile and water (9:1) + 0.1% trifluoroacetic acid (TFA). Run time was up to 25 min.
Liquid Chromatography-Mass Spectrometry (LC-MS)
LC-MS analysis was performed on a Shimadzu LC-20A-Prominence HPLC instrument (Shimadzu,
Kyoto, Japan) coupled with a UV-detector set to λ = 254 nm, an electrospray ionization (ESI) and single
quadrupole mass analyzer (QMS) operating in positive mode. Samples were prepared by dissolving 1
mg of compound in 1 mL methanol (LC/MS grade) and further diluting it, resulting in a final
concentration of 0.1 mg mL-1. Separation after injection of 0.02 mL was achieved by using a
Phenomenex-Synergy-Fusion-RP, 4.6 x 150 mm, 5 µm, column (Phenomenex, Torrance, CA) and a
methanol/water gradient. In detail, mobile phase A was deionized water with 0.1% (v/v) formic acid and
mobile phase B was methanol with 0.1% (v/v) formic acid. For separation %B-content of the mobile
phase was continuously increased from 5 to 90% over 18 min.
Differential Scanning Calorimetry (DSC)
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Differential Scanning Calorimetry was measured with a DSC 8000 from Perkin Elmer (Waltham, MA).
Briefly, 3 – 4 mg substance were weighed into aluminum pans with a pinhole in the lid and analyzed at
a scanning rate (heating and cooling rate) of 20 °C min-1 from -50 °C to 100 °C. The mass of the crucible
was noted before and after the experiment to calculate any loss of mass during the process. Measurement
of three cycles was performed for every sample and the mean melting point was calculated.
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X-Ray Powder Diffraction (XRD)
X-Ray powder diffraction was measured on a silicon single crystal holder and analyzed with a Bruker
Discover D8 powder diffractometer (Karlsruhe, Germany) using Cu-Kα radiation (unsplit Kα1+Kα2
doublet, mean wavelength λ = 154.19 pm) at 40 mA and 40 kV, a focusing Goebel mirror and a 1.0 mm
microfocus alignment (1.0 mm pinhole with 1.0 mm snout). The scattered X-ray beam went through a
receiving slit with 7.5 mm opening, a 2.5° axial Soller slit and detection was performed with a LynxEye-
1D-Detector (Bruker AXS) using 192 channels. Measurements were done in reflection geometry in
coupled two theta/theta mode with a step size of 0.025° in 2θ and 0.25 s measurement time per step in
the range of 5 – 60° (2θ). Data collection and processing was carried out with the software packages
DIFFRAC.Suite (V2 2.2.69.0) and DIFFRAC.EVA 2.1 from Bruker.
Caco-2 Permeation Assay
The Caco-2 permeation assay was performed as described before with small variations.21 Transportation
was studied in apical-basolateral direction and cells were used in passage 51 – 54. In brief, cells were
incubated in culture medium (DMEM high glucose, 10% (v/v) FBS and 1% (v/v) non-essential Amino
acid (NEAA) solution) at 37 °C, 5% CO2. Passages were performed at 80% confluence in 1:2 - 1:4
ratios and three passages were necessary to develop the phenotype. Afterwards cells were seeded in the
trans-wells in a 2.6 x 105 cells cm-2 density and maintained for 27 days. Culture medium for maintenance
(DMEM high glucose, 10% (v/v) FBS, 1% (v/v) NEAA, 1% (v/v) penicillin G/streptomycin solution)
was changed every second to third day. Trans-epithelial electric resistance (TEER) values were
measured with an EVOM2 STX3 chopstick electrode connected to an EVOM2 epithelial voltammeter
from World Precision Instruments (Sarasota, Florida) to follow the monolayer development.
Transportation medium was HBSS at pH 7.4 altered by adding 25 mmol L-1 2-(4-(2-hydroxyethyl)-1-
piperazinyl)-ethansulfonic acid (HEPES) and 0.35 g L-1 NaHCO3. 100 mmol L1 stock solutions of all
substances were prepared in DMSO and diluted with transportation medium resulting in test solutions
of 100 µmol L-1 with a maximum of 0.1% (v/v) DMSO in all cases. Fluorescein- and Lucifer Yellow
solutions were used as controls. Trans-wells were washed with HBSS, removing residual culture
medium, and 0.5 mL of test solution was applied in the apical chamber (donor). The basolateral chamber
was filled with 1.2 ml HBSS (acceptor). Immediately after apical application, 0.1 mL was withdrawn
from the donor chamber to determine the initial concentration (c0). The plates were incubated at 37 °C,
0% CO2 while gently shaking at 150 RRM. 0.1 mL samples were withdrawn from the receiving chamber
after 30, 60, 90, and 120 min and immediately replaced with warm transportation buffer. Additionally,
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after 120 min a 0.1 mL sample was taken from the apical chamber as well to calculate the mass balance
(crest). TEER values were measured directly before and after the experiment to monitor the integrity of
the monolayer. Monolayers with a TEER value ≥ 165 Ω cm² were considered integer. Apical and
basolateral samples were stored at 4 °C and analyzed by HPLC on the next day. Fluorescein and Lucifer
Yellow (LY) were analyzed by fluorimetry. Experiments were performed in triplicate and results were
calculated and reported as apparent permeation coefficient (Papp). Representative cell monolayers were
cut out and stained to evaluate their integrity.
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Fluorimetric analysis of Fluorescein and LY
Analysis was performed using a LS50 B luminescence from Perkin Elmer (Waltham, MA) equipped
with a 96-well plate adapter as described before.22 Briefly, samples were sufficiently diluted with HBSS
buffer and 0.09 mL were pipetted into white Nunc™ F96 MicroWell™ plates from ThermoFisher
Scientific (Waltham, MA). Parameters for Fluorescein measurement were set to extinction wavelength
λ = 450 nm, extinction slit 2.5, emission wavelength λ = 514 nm, emission slit 15 and read time to 0.4
s. A standard curve was conducted in a range of 1 µmol L-1 to 0.05 µmol L-1 (R² = 0.99). LY analysis
was performed on the same LS50 B luminescence setting the parameters to extinction wavelength λ =
470 nm, extinction slit 2.5, emission wavelength λ = 535 nm, emission slit 2.5 and read time to 5 s. A
standard curve was conducted in a range of 10 µmol L-1 to 0.1 µmol L-1 (R² = 0.98).
Staining and Visualization of Caco-2 Monolayers
Preparation of the Caco-2 monolayers attached to the trans well-system was done by single placing
representative trans wells in a 6-well plate and staining with 4’,6-diamidino-2-phenylindole (DAPI) and
Alexa Fluor 488 as previously described.23 In detail, monolayers were washed two times apical and
basolateral with PBS pH 7.4 and exposed to cold methanol for 10 min (washing was always performed
with PBS pH 7.4). They were washed again three times and blocked using 0.5 mL Roti®-Block (Carl
Roth, Karlsruhe, Germany) in PBS for 1 h at room temperature. Inserts were washed three times and
mouse antibody against human e-cadherin, diluted 1:100 in PBS 4 °C, was added and incubated
overnight at 4 °C. On the next morning the cells were washed five times, followed by the secondary
antimouse antibody Alexa Fluor 488, diluted 1:200 in blocking solution and incubation 90 min at room
temperature in the dark. The cells were washed four times follow by DAPI, diluted 1:1000 in PBS. After
incubation for 10 min at room temperature in the dark, cells were washed three times. Stained
monolayers were cut out from the transwell-inserts using a scalpel and embedded with Mowiol 4-88 on
glass microscope slides. Samples were stored in the dark until further use. Representative pictures were
taken using a Leica DMIRE2 confocal microscope (Leica Camera AG, Wetzlar, Germany) equipped
with a HCX PL APO 63x/1.40 Oil lbd BL objective from Leica operated with the Leica confocal
software. Excitation was done by (blue channel) and (green channel). The average of four pictures in a
resolution of 1024x1024 pixels with a 2.0x zoom was taken and a series of 20 pictures per stack was
collected for every sample.
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Cell Proliferation Assay (WST-1)
Cytotoxicity was studied in human embryotic kidney cells (HEK 293), human liver cells (Hep G2),
human epithelial colorectal cells (Caco-2) and murine fibroblasts (NIH 3T3) in their respective culture
medium (Table 2).23 After thawing, cells were passaged at least three times before the experiments were
performed to develop their phenotype. Compound stock solutions were prepared in DMSO and diluted
with culture medium. For the experiments 0.1 mL cell suspension in the appropriate seeding density
(Table 2) was transferred into 96-well plates and incubated overnight at 5% CO2 and 37 °C. 0.1 mL
diluted substance solution was added and incubated for 24 h. Final DMSO concentrations did not exceed
0.5%. After incubation Cell Proliferation Reagent WST-1 (WST-1) was diluted (1:1 v/v) with culture
medium and 0.02 mL were added per well. The absorbance was measured after 2 h at 450 nm, using
630 nm as reference wavelength. The experiments on NIH 3T3 were performed in quadruplicate in three
separate replicates (n = 12) covering a concentration range from 100 µmol L-1 to 195 nmol L.1.
Cytotoxicity on HEK 293, Hep G2 and Caco-2 was screened in triplicate in one repetition (n = 3)
covering a concentration range from 100 µmol L-1 to 6.25 µmol L-1. The IC50 values were calculated if
possible, using controls without compound and cells without WST-1 Cell-types used in the cell
proliferation assay (WST-1). For overview, please see below.
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Table 2 Tested cell-types including information on culture medium and seeding density.
Cell-type
HEK 293
Culture medium
Seeding density
DMEM high glucose, +10% (v/v) FCS, +1% (v/v)
P/S
4 x 104 cells mL-1
Hep G2
MEM, +10% (v/v) FCS, +1% (v/v) P/S, +1% (v/v)
NEAA, +2 mmol L-1 glutamine, + 1 mmol L-1
sodium pyruvate, +1.5 g L-1 sodium bicarbonate
1.25 x 105 cells mL-1
Caco-2
DMEM high glucose, +10% (v/v) FCS, +1% (v/v)
NEAA, +1% (v/v) P/S
2 x 105 cells mL-1
5 x 104 cells mL-1
NIH 3T3
DMEM high glucose, +10% (v/v) FCS, +1% (v/v)
P/S
Reverse Mutation assay using Bacteria (Ames test)
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The potential to cause mutations was assessed in Salmonella typhimurium and Escherichia coli
following OECD Guidelines for the Testing of Chemicals and previous published protocols.24-26 Four
histidine-auxotrophic S. typhimurium strains (TA 98, TA 100, TA 1535 and TA 1537) and one
tryptophan-auxotrophic E. coli strain (WP2) were tested w/ or w/o addition of metabolic activity by S9
homogenate (Trinova Biochem GmbH, Giessen, Germany). Two different methods were tested, the
plate incorporation method and pre-incubation method. Prior the experiments bacteria were cultured in
broth containing 25 g L-1 Nutrient medium No.2 for 10 h at 37 °C while gently shaking up to a bacterial
density of approximately 109 cells ml-1 in late exponential phase of growth. The final concentrations of
the tested compounds were 500.7, 158.5, 50.1, 15.9, and 5.0 µg per agar plate. Briefly, for the plate
incorporation method, 2000 µL top agar, 100 µL test solution (dissolved test compound or control) 100
µL bacterial suspension and 500 µL S9 Mix (w/ metabolic activity) or S9 Mix substitution buffer were
mixed at 45 °C and poured on minimal-agar plates supplemented with 0.05 mmol L-1 histidine (for S.
typhimurium strains) or 0.05 mmol L-1 L-tryptophan (for E. coli strain). After solidification agar plates
were incubated at 37 °C for 40 – 72 h. For the pre-incubation method, 100 µL test solution, 100 µL
bacterial suspension and 500 µL S9 Mix or S9 Mix substitution buffer were mixed and incubated at 37
°C for 20 min. After this pre-incubation 2000 µL top agar were added, the solution was mixed, poured
on minimal-agar plates and after solidification incubated at 37 °C for 40 – 72 h. Negative control was
DI water, solvent control was DMSO. Positive controls w/o metabolic activity were 5 µg 2-nitrofluorene
for TA 98, 10 µg sodium azide for TA 100, 1.5 µg sodium azide for TA 1535, 50 µg 9-aminoacridine
for TA 1537 and 0.05 µg methyl methanesulfonate for WP2. Positive controls w/ metabolic activity was
2-aminoanthracene: 1 µg, 1 µg, 3 µg, 3 µg and 20 µg for TA 98, TA 100, TA 1535, TA 1537, and WP2,
respectively. Revertant colonies were counted and the mutation factor (MF) was calculated (Equation
3). The test compound was considered mutagenic if there was a dose dependent increase in number of
revertants and/or a biologically relevant positive response for one or more concentrations in at least one
strain. Biologically relevant increase was defined as a revenant count at least twice as high for test strains
TA 98, TA 100 and E. coli WP2 and at least three times higher for test strains TA 1535 and TA 1537
compared to the solvent control.27 Experiments were performed in triplicate. For an overview of used
solutions see below (Table 3).
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Equation 3
Mean value of revertant counts (test item)
MF =
Mean value of revertant counts (solvent control)
MF
-
mutation factor
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Table 3 Solutions used for the Ames test.
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Solution
Composition
Treatment
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Top agar
6.0 g L-1 bacto agar, 5.0 g L-1 sodium chloride in DI Sterile; autoclaved
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water
for 20 min at 121 °C
S9 Mix
Prepared from S9 liver homogenate as specified by
Trinova Biochem GmbH.
-
5% S9 Mix for S. typhimurium strains
10% S9 Mix for E. coli strain
S9 Mix
120 mL 0.2 mmol L-1 NaH2PO4 x H2O, 880 mL 0.2
mmol L-1 Na2HPO4, pH adjusted to 7.4
Sterile, autoclaved
substitution
buffer
for 20 min at 121 °C
Vogel-Bonner-
salts
10 g L-1 MgSO4 x 7 H2O, 100 g L-1 citric acid, 175 g Sterile; autoclaved
L-1 NaNH4HPO4 x 4 H2O, 500 g L-1 K2HPO4 in DI
water
for 20 min at 121 °C
Vogel Bonner
15 g L-1 agar agar, 20 mL L-1 Vogel-Bonner salts
Sterile; autoclaved
Medium E agar solution, 50 mL L-1 glucose solution (40%) in DI
plates water
for 20 min at 121 °C
Biofilm-inhibition Assay
Biofilm-formation was quantified by crystal violet staining.28 Staphylococcus aureus (ST228 – clinical
isolate) was plated on Mueller-Hinton agar plates and stored at 4 °C until usage. Bacteria were cultured
overnight in tryptic soy broth (TSB) at 37 °C and adjusted to an optical density (OD600) of 0.05 on the
next day. Compound stock solutions were prepared in DMSO and diluted with TSB resulting in a
maximum DMSO concentration of 1% (v/v), which did not affect bacterial growth or biofilm-formation
(data not shown). 0.2 mL bacterial solution and 0.05 mL compound solution were added into each well
of a 96-well plate and cultured overnight at 37 °C. Controls were 0.2 mL bacterial solution plus 0.05
mL TSB (negative control) and 0.25 mL TSB without bacteria (positive control). On the next day,
optical density at 595 nm (OD595) was measured using a Multiskan Ascent (Thermo Electron
Corporation, Waltham, MA) plate reader, afterwards all wells were carefully decanted and washed three
times with phosphate buffer saline (PBS) pH 7.4. Plates were placed on a heating device at 60 °C for 1
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h and afterwards stained with 0.1 mL crystal violet 0.1% (m/v) in water. After 15 min at room
temperature (RT) the crystal violet solution was decanted, the plates washed with DI water and dried
overnight at RT. 0.1 mL ethanol was added into each well and gently shaken in an orbital shaker for 20
min. Absorbance at 492 nm was measured in the same UV plate reader as above and used for calculation
of the relative biofilm formation. All experiments were performed three times with four replicates.
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Minimal Inhibitory Concentration (MIC)
MIC values were determined according to CLSI guidelines.29 Briefly, for aerobic bacteria these were
frozen in the respective growth medium containing 20% (v/v) glycerol. These were streaked and
incubated on agar-plates before testing and used within two weeks to prevent auto-lysis or mutation.
The day before the experiment, a colony was picked and cultured overnight at 37 °C in 3 mL of the
respective growth medium on an orbital shaker. On the next morning 0.1 mL of this bacterial suspension
was withdrawn and added to 30 mL of growth medium and cultured at 37 °C on an orbital shaker
operating at 150 RPM. After 2 – 5 hours depending on the bacterial growth, optical density at a
wavelength of 600 nm (OD600) was measured using a Eppendorf Biophotometer plus (Eppendorf,
Hamburg, Germany), adjusted to OD600 = 0.05 with growth medium and used within 30 min. 100 or 50
mmol L-1 stock solutions in DMSO were prepared and diluted to 200 µmol L-1 with growth medium. A
serial dilution was prepared in sterile, transparent U-bottom 96-well plates (VWR, Darmstadt,
Germany), each well containing 0.1 mL of the respective concentration. 0.1 mL of the bacterial
suspension (OD600 = 0.05) was added resulting in a tested concentration range of 100 µmol L-1 to 195
nmol L-1 where the maximum DMSO concentration did not exceed 0.2% (v/v). Negative control was
medium w/o compound and bacteria while growth control was bacteria w/o compound. The 96-well
plates were cultured overnight at 37 °C and visually evaluated for bacterial growth on the next morning.
The MIC values were defined as the lowest concentration w/o visible growth and were reported as
cautious as possible by stating the highest observed value from the total of six repetitions in three
independent experiments. All experiments were performed three times in duplicate. Representative
dilutions were plated on agar-plates for every bacterial strain.
MIC values in anaerobic bacteria were determined inside an Anaerobic Chamber (Coy Laboratory
Products Inc, Grass Lake, MI) with an anaerobic atmosphere composed of 85% N2, 10% H2 and 5%
CO2. Briefly, 96-well plates containing compound solutions, media and agar-plates were put into the
chamber the day before the experiment to remove any residual oxygen. Bacteria were grown under static
conditions at 37 °C within the chamber. Preparation and dilution of the solutions as well as evaluation
of the MIC values was done as described above.
MIC plating on Agar-plates
According to CLSI guidelines MIC values were confirmed after visual detection.30 0.01 mL of the well
with the detected MIC value and the next two concentrations above and below that value were
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withdrawn and diluted with 0.09 mL of the respective growth medium (1:10). 0.01 mL droplets of these
five dilutions were separately placed side by side on an agar-plate and the plate was carefully inclined
to allow the droplet to flow across it in a straight line. Plates were cultured overnight at 37 °C and
bacterial growth was evaluated on the next day by counting the colonies, if any were present.
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Galleria mellonella assay
Galleria mellonella were purchased (Feeders & more GmbH, Au in der Hallertau, Germany) with an
overall weight of 0.45 ± 0.10 g for the E. faecium UL602570-group and 0.31 ± 0.07 g for the S. aureus
Lac*-group. All G. mellonella larvae were used within 4 days post-delivery to avoid maturing and
pupating. Each group contained 20 G. mellonella larvae which were housed separately in petri dishes
with small amounts of wooden chips. Control groups were non-infected animals treated with phosphate
buffer saline pH 7.4 (PBS) and infected animals treated with PBS. Bacteria were streaked on Agar-
plates of the respective medium and used within one week. Cultures in 3 mL growth medium were
incubated at 37 °C on the evening before the experiment and 0.2 mL of these cultures were transferred
into 30 mL growth medium on the next morning and incubated at 37 °C (under gentle shaking). Optical
density was adjusted in PBS and the resulting bacterial suspension was mildly centrifuged at 3500 rcf
for 5 min. The supernatant was carefully withdrawn, and the bacteria resuspended in PBS. The
suspension was again centrifuged at 3500 rcf for 5min, supernatant removed and resuspended in PBS to
remove any residual bacterial toxins. Resulting bacterial concentrations were 2.5 x 108 CFU mL-1 for
ST228 and 1.25 x 107 CFU mL-1 for UL602570, respectively. Galleria were infected by injection of 0.02
mL bacterial suspension (or 0.02 mL PBS for the non-infected group) into the lower part of their body
using a BD Micro-Fine™ + Demi 0.3 mL, 0.30 mm (30G) x 8 mm syringe. Compound stock solutions
were prepared in DMSO and further dilutions were done in PBS resulting in final concentrations of 200
µmol L-1 for all compounds. All PBS media and compound dilutions in PBS used for treatment contained
residual 5% DMSO (v/v). 1 hour post infection, 0.02 mL of compound solution was injected in the same
body part of the G. mellonella and the animals were placed at 37 °C. Observation period was set to 94
h and dead animals were counted (darkened color and no visible movement) and removed from the petri
dish every 6 h in the first 48 hours and after that every 12 h until 96 hours post infection. Results for all
groups are presented as a survival plot.
Statistics
Data evaluation and statistics was done using GraphPad Prism version 6.04 from GraphPad Software,
Inc. (San Diego, CA). Robust regression and Outlier removal (ROUT) identification was performed for
all data containing 10 or more data sets (n ≥ 10) using a ROUT coefficient Q of 1%. Data sets were
compared using a One-way ANOVA test, differences among groups were calculated using Dunnett’s
multiple comparison test and represented by asterisks (* p-value ≤ 0.05; ** p-value ≤ 0.01; *** p-value
≤ 0.001).
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Results
A series of 5 compounds, inspired by the naturally occurring anacardic acid, were synthesized
systematically extending the aliphatic alkane residue from 6 to 14 carbon atoms (Figure 1). Namely, 2-
(hexyloxy)- (C6), 2-(octyloxy)- (C8), 2-(decyloxy)- (C10), 2-(dodecyloxy)- (C12) and 2-(tetradecyloxy)-
6-hydroxybenzoic acid (C14) were obtained and fully characterized concerning their physicochemical
properties (Figure 2). The purity of all products exceeded ≥ 95% as assessed by high-pressure liquid
chromatography coupled uv metric (HPLC-UV) analysis (Figure S2-6). All solid compounds were
crystalline, except for C8 which was liquid at room temperature. The endothermic melting point was at
32 °C (C6), 20 °C (C8), 33 °C (C10), 45 °C (C12) and 51 °C (C14) (Figure S12-16). Dissociation constants
(pKa) were similar for all substances with 3.32 ± 0.07 (C6), 3.24 ± 0.04 (C8), 3.21 ± 0.07 (C10), 3.21 ±
0.06 (C12) and 3.34 ± 0.05 (C14), respectively. The apparent solubility was assessed in phosphate buffer
saline (PBS; pH 7.4) with 8.40 ± 0.18 mmol L-1 (C6; 2 mg mL-1), 3.19 ± 1.02 mmol L-1 (C8; 0.85 mg
mL-1), 0.48 ± 0.07 mmol L-1 (C10; 0.14 mg mL-1), 0.45 ± 0.02 mmol L-1 (C12; 0.15 mg mL-1) and 0.18
± 0.24 mmol L-1 (C14; 0.06 mg mL-1), respectively (Figure 2A). LogP increased linearly with increasing
alkyl-chain length (R² = 0.99) with logP values of 4.42 ± 0.06 (C6), 6.00 ± 0.02 (C8), 7.29 ± 0.02 (C10),
8.47 ± 0.03 (C12) and 9.43 ± 0.02 (C14) (Figure 2B). The Critical Micelle Concentration (CMC) was the
lowest for C10 (6.5 ± 0.4 µmol L-1), and increasing with to 31.3 ± 1.2 µmol L-1 and 176.3 ± 6.1 for C8
and C6 as well as 16.9 ± 1.7 µmol L-1 for C12 , respectively (Figure 2C). As of its low aqueous solubility
we were unable to collect reliable data for the CMC of C14.
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Figure 2 Physicochemical properties of the synthesized compounds. (A) Solubility in phosphate buffer saline (PBS) pH 7.4
at 25 °C, (B) Octanol/Water partition coefficient log D at pH 7.4 and (C) Critical Micelle Concentration of C6, C8, C10, C12
and C14. CMC determination of C14 was not possible, due to low water solubility. All results are displayed as mean ±
standard deviation (n = 3).
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Epithelial permeability was recorded across Caco-2 cell layers only after a trans epithelial electric
resistance (TEER) ≥ 200 Ω cm² was reached before and maintained throughout the experiment,
respectively, and an intact state was confirmed histologically. Recovery rates were 104.9 ± 2.2% (C6),
78.8 ± 2.4% (C8) and 5.4 ± 2.2% (C10) (Figure 3A). The apparent permeation coefficients (Papp) was
9.84 ± 0.20 × 10-5 cm s-1 (C6) and 7.95 ± 0.34 × 10-5 cm s-1 (C8) (Figure 3B). We classified the data
obtained for C10 as inconclusive (reflecting the low substance recovery rate) as we did for C12 and C14
(no compound was found in basolateral or initial apical samples). In order to characterized unspecific
adsorption, the assay was repeated without cells (in a single experiment) with recovery of 110% (C6),
85% (C8), 79% (C10), 69% (C12) and 58% (C14; Figure S39).
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Figure 3 (A) Compound recovery and (B) apparent permeability coefficient (Papp) of C6, C8 and C10 (mean ± standard
deviation; n=3).
All compounds did have IC50 values exceeding 100 µmol L-1 (the upper limit of compound solubility)
hence demonstrating a desirable profile as assessed in cell lines of murine fibroblasts (NIH 3T3), human
kidney cells (HEK 293), human liver cells (Hep G2) or human colorectal cells (Caco-2; Figure S18-21)
In general, the fibroblasts (Figure 4A) and liver cells (Figure 4C) responded more sensitively than the
colorectal and kidney cells to the highest applied concentration for C10, C12 and C14. Liquid/liquid phase
separation was observed for C14 during solubility and CMC experiments resulting in a high standard
deviation and no observable surface activity. C14 was excluded from further investigations as poor water-
solubility limit its potential as antibiotic drug candidate as well as antibiotic activity was not observed
in preliminary experiments.
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Figure 4 Cytotoxicity assay C6, C8, C10, C12 and C14 in (A) NIH 3T3 fibroblasts (n = 12), (B) HEK 293 kidney cells (n = 3),
(C) Hep G2 liver cells (n = 3) and (D) Caco-2 epithelial colorectal cell line. Data was buffer-normalized (n = 3; mean ±
standard deviation; significance represented by asterisks with * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001).
The assessment of the minimal inhibitory concentration (MIC) values (Table 4, Table S1-9) was limited
to C8, C10 and C12 in comparison to selected antibiotics but not for C6 and C14 as these compounds did
not show activity in pilot experiments. C8 was not antibacterial up to the highest concentration tested
(100 µmol L-1) in any bacterial strain. C10 and C12 showed antibacterial activity in all strains with MIC
values ranging from 100 – 50 µmol L-1 (C10) and 50 – 12.5 µmol L-1 (C12; except for C. difficile). S.
aureus strains ST228 and Lac* (MRSA), S. epidermidis RP62 as well as E. faecalis (VRE) and the
Streptococcus agalactiae strain were most susceptible to C10 and C12 among all strains tested.
Table 4 Minimal inhibitory concentrations (MIC) for different bacterial strains in µmol L-1 (tested concentration range 100 –
0.2 µmol L-1). The highest observed MIC value of three independent experiments is reported (for full data set refer to Tables
S1-9).
Staphylococci
Enterococci
GBS
C. diff.
Compound
Octenidine
ST228
JE2
Lac* RP62a Faecium Faecalis
6.25
6.25
6.25
3.125
6.25
12.5
12.5
12.5
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Staphylococci
Enterococci
GBS
C. diff.
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Compound
ST228
JE2
1.563
> 100
> 100
n.d.
Lac* RP62a Faecium Faecalis
Vancomycin
1.563
1.563
> 100
100
3.125
25
> 100
> 100
> 100
> 100
> 100
100
25
6.25
3.125
100
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Ciprofloxacin > 100
3.125
3.125
> 100
> 100
50
3.125
12.5
50
Amoxicillin
Linezolid
C8
> 100
n.d.
> 100
n.d.
> 100
50
25
n.d.
> 100
> 100
100
> 100
100
> 100
100
> 100
50
> 100
50
C10
C12
25
50
25
25
50
12.5
6.25
25
12.5
6.25
6.25
100
GA (C13)
AA (C15)
25
25
25
25
6.25
n.d.
> 100
> 100
> 100
> 100
12.5
n.d.
GA = Ginkgolic acid
AA = Anacardic acid
n.d. = not determined
GBS = group B streptococci
C. diff = Clostridium difficile
We further assessed the impact of the compounds on bacterial biofilm. While no effect on bacterial
biofilm formation was observed with C6, C8, C12 or C14, compound C10 inhibited biofilm formation in
sub-inhibitory concentrations in S. aureus ST228 (Figure 5). C10 reduced the growth of ST228 (Figure
5A) and reduced the biofilm mass significantly to 74% ± 13% when exposed to 0.39 µmol L-1 (Figure
5B).
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Figure 5 (A) Optical density or bacterial growth and (B) biofilm formation of S. aureus (ST228) after exposure to C10 for 24
hours. Data was buffer-normalized (n = 12; mean ± standard deviation, one-way ANOVA with Tukey post hoc comparison
among groups; significance represented by asterisks with * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001).
C10 and C12 were selected for a first assessment of in vivo activity in a Galleria mellonella larvae model
infected with the MRSA strain LAC* or Vancomycin-resistant E. faecium strain UL602570 (VRE)
against the antibiotics amoxicillin, ciprofloxacin, vancomycin, and linezolid and the disinfectant
octenidine as controls. Injection of 20 µL (200 µmol L-1) compound solution to the VRE infected group
(bodyweight 0.45 ± 0.10 g) and the MRSA infected group (bodyweight 0.31 ± 0.07 g) resulted in an
applied dose of 9.10 ± 0.56 µmol kg-1 in the VRE group and 12.43 ± 0.77 µmol kg-1 in the MRSA group,
respectively (Table S9-12).
Figure 6 Survival of Galleria mellonella when infected with vancomycin resistant E. faecium UL602570 and following
treatment (Kaplan-Meier plot). Survival under C10 and C12 treatment was significantly better as compared to the PBS control
group and not different as compared to (Vancomycin and Linezolid. Amoxicillin, ciprofloxacin or octenidine did not
significantly differ from the PBS control group (n = 20).
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Larvae infected with Vancomycin-resistant E. faecium showed an overall survival rate of 35% in
contrast to 90% observed in non-infected controls, respectively (Figure 6). Treatment with C10 or C12
resulted in a significantly better survival of 70% as compared to the infected control group (p ≤ 0.05).
Vancomycin and Linezolid treatment insignificantly increased the survival to 65% and neither
Amoxicillin, nor ciprofloxacin or octenidine improved survival statistically. The compounds were
further assessed in another model using multi-drug resistant S. aureus (Figure 7). Whereas C12 (5%)
failed to improve survival, C10 (25%) was significantly better as compared to the infected control (5%
survival; p ≤ 0.05) or amoxicillin (0% survival; p ≤ 0.01) but inferior to vancomycin (45% survival; p ≤
0.01).
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Figure 7 Survival of Galleria mellonella infected with multi-drug resistant S. aureus strain LAC* (Kaplan-Meier plot; n =
20).
Discussion
The anacardic acid derivatives C10 and C12 showed good antibacterial activity as well as promising
toxicological profiles and both compounds challenged multi resistant bacterial strains in vitro and in
vivo. Additionally, biofilm inhibition in S. aureus was found at sub-therapeutic doses for C10.
Encouraging physicochemical results were obtained for solubility and lipophilicity. The found pKa
values suggested that the compounds will be charged/ionized in blood (pH 7.4) or the intestine (pH 6.8)
thereby exhibiting amphiphilic properties resulting in interaction with biological barriers. Except for C14
- which was not surface active in the tested concentration range - all compounds decreased the surface
tension in water with C10 resulting in the lowest observed CMC value as compared to all other tested
compounds (Figure 2C). Permeation through human colorectal adenocarcinoma cell monolayers (Caco-
2) reflected the importance of hydrophilic/lipophilic balance as the apparent permeability coefficient
(Papp) correlated with the alkyl chain length (Figure 3B). While C6 and C8 had Papp values larger than
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2-5 × 10-6 cm s-1 – suggesting complete absorption in human intestine - the assessment of Papp values
for the more lipophilic compounds C10, C12 and C14 suffered from a poor recovery rate and as previously
reported for lipophilic compounds tested in Caco-2 permeation assays.21, 31 The poor recovery rate was
not a result of unspecific absorption as repeating the experiment without cells revealed higher recovery
rates for all compounds with a trend of decreasing recovery with increasing alkyl chain length (Figure
S39). These experiments suggested that the overall poor recovery of C10, C12 and C14 in CaCo-2
monolayers was mainly driven by effective absorption onto/within Caco-2 cells an effect which was
previously linked to prokaryotic and eukaryotic cytotoxicity of surfactants on the one hand and the
reason for antimicrobial activity on the other.32, 33 For example, octenidine displays similar surfactant
like properties with a broad antiseptic activity which is why it finds application for topical treatment of
skin and wound infections but prevents other administration routes as of cytotoxic effects which in fact
have also been reported for skin keratinocytes.4, 34 All tested compounds demonstrated encouraging
cytotoxicity data and no IC50 values were obtained in the tested concentration range. Additionally, Ames
test for C10 showed no increase in revenant colony numbers, neither in presence or absence of metabolic
activity, indicating non-mutagenicity of this compound concerning base pair changes or frameshift in
the genome (Table S13-22). However, decrease in proliferation was observed in higher concentrations
for C12 and C14 (Figure 4). This observation is in accordance to recent publications linking the cytotoxic
behavior of lipophilic entities with their ability to interact with cellular membranes.32, 35 Compound C10
and C12 show similar antibacterial activity when compared to their natural precursors Anacardic acid
(C15) and Ginkgolic acid (C13; Table 4). While the activity in Staphylococci strains is unaffected by
introducing an ether function, the activity in Enterococci- and Streptococcus strains is slightly lower.
However, C10 and particularly C12 reflected a broad antibacterial activity similar to octenidine and
contrasting the antibiotics for which the antibacterial activity depended on the selected strain and its
resistance profile. Therefore, C10 and C12 were further tested in infection models in Galleria mellonella
(Figure 6, 7), a model in which pathogen virulence compares to mice.36-39- Findings in this study
correlated with previously measured MIC values for amoxicillin, ciprofloxacin, vancomycin, and
linezolid in E. faecium UL602570 (Table 4). One surprising finding for octenidine was that in spite of
having higher antibacterial activity compared to C10 and C12 in vitro, this did not translate into better
survival in vivo. We hypothesize this might reflect the amphiphilic nature of C10, C12 and octenidine 4
alike, possibly reducing the available compound in vivo as of strong cellular absorption hence little
availability at sites of infection. Additionally, low survival was observed for the uninfected control group
in the MRSA G. mellonella experiment (Figure 7). A retro perspective evaluation of our laboratory
procedure revealed survival for uninfected, PBS treated groups of 88% ± 3% (n=7) and in line of what
was observed for the VRE infected G. mellonella experiment presented in this study (Figure 6). The
poor survival in the uninfected group within the MRSA infected G. mellonella experiment was likely
linked to longer transportation of the larvae and possibly starving of this particular G. mellonella larvae
batch. Despite the resulting overall low survival, the data indicated non-inferiority to vancomycin,
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linezolid and PBS treated in the MRSA infected group. It is for these aspects and general differences in
bacterial virulence in different hosts why the Galleria data reported here should be corroborated in
mammalian hosts before final conclusions are drawn. The demonstrated in vivo superiority of C10 and
C12, while statistically significant in the MRSA strain, has to be interpreted cautiously and is encouraging
further investigations in mammals to evaluate a potential benefit. In addition and from a cytotoxicity
perspective, the compounds reported here within are promising candidates for further testing and much
safer than commonly used antibiotics and antiseptics including erythromycin, dicloxacillin or
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cefuroxime as well as ciprofloxacin, povidone-iodine and octenidine dihydrochloride 34, 41, 42. As
mentioned, experimental data in other animal model systems is required before drawing final
conclusions on the promising risk-benefit profile of these anacardic acid derivatives.
Supporting Information
This information is available free of charge on the ACS Publications website. Full characterization data
of new compounds, plots of surface tension for CMC calculation, HPLC chromatograms for purity
calculation, DSC diagrams for determination of melting point, X-Ray Powder Diffraction patters, full
1
13
cytotoxicity data sets, H and C NMR spectra of new compounds, full Caco-2 permeation data sets,
full MIC data sets including µg mL-1 values, full Galleria mellonella data sets, full Reverse Mutation
assay using Bacteria (Ames test) data sets.
Acknowledgments
We gratefully acknowledge the financial support by the Bayerische Forschungsstiftung (Grant # AZ-
1204-16). Furthermore, we thank Dr. Gerd Vogg from the botanical garden in Würzburg for providing
pictures of Anacardium occidentale fruits.
References
(1)
Flemming, H. C.; Wingender, J. (2010) The biofilm matrix. Nat. Rev. Microbiol. 8 (9), 623-
33. DOI: 10.1038/nrmicro2415.
(2)
Warnke, P. H.; Lott, A. J.; Sherry, E. (2013) Wiltfang, J.; Podschun, R., The ongoing battle
against multi-resistant strains: in-vitro inhibition of hospital-acquired MRSA, VRE, Pseudomonas,
ESBL E. coli and Klebsiella species in the presence of plant-derived antiseptic oils. J.
Craniomaxillofac. Surg. 41 (4), 321-6. DOI: 10.1016/j.jcms.2012.10.012.
(3)
Cassini, A.; Högberg, L. D.; Plachouras, D.; Quattrocchi, A.; Hoxha, A.; Simonsen, G. S.;
Colomb-Cotinat, M.; Kretzschmar, M. E.; Devleesschauwer, B.; Cecchini, M.; Ouakrim, D. A.;
Oliveira, T. C.; Struelens, M. J.; Suetens, C.; Monnet, D. L.; Strauss, R.; Mertens, K.; Struyf, T.;
Catry, B.; Latour, K.; Ivanov, I. N.; Dobreva, E. G.; Tambic Andraševic, A.; Soprek, S.; Budimir, A.;
Paphitou, N.; Žemlicková, H.; Schytte Olsen, S.; Wolff Sönksen, U.; Märtin, P.; Ivanova, M.;
Lyytikäinen, O.; Jalava, J.; Coignard, B.; Eckmanns, T.; Abu Sin, M.; Haller, S.; Daikos, G. L.; Gikas,
A.; Tsiodras, S.; Kontopidou, F.; Tóth, Á.; Hajdu, Á.; Guólaugsson, Ó.; Kristinsson, K. G.; Murchan,
S.; Burns, K.; Pezzotti, P.; Gagliotti, C.; Dumpis, U.; Liuimiene, A.; Perrin, M.; Borg, M. A.; de
Greeff, S. C.; Monen, J. C. M.; Koek, M. B. G.; Elstrøm, P.; Zabicka, D.; Deptula, A.; Hryniewicz,
W.; Caniça, M.; Nogueira, P. J.; Fernandes, P. A.; Manageiro, V.; Popescu, G. A.; Serban, R. I.;
Schréterová, E.; Litvová, S.; Štefkovicová, M.; Kolman, J.; Klavs, I.; Korošec, A.; Aracil, B.; Asensio,
A.; Pérez-Vázquez, M.; Billström, H.; Larsson, S.; Reilly, J. S.; Johnson, A.; Hopkins, S. (2019)
Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant
ACS Paragon Plus Environment

Page 23 of 26
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
bacteria in the EU and the European Economic Area in 2015: a population-level modelling analysis.
The Lancet Infect. Dis. 19 (1), 56-66. DOI: 10.1016/s1473-3099(18)30605-4.
(4)
Hubner, N. O.; Siebert, J.; Kramer, A. (2010) Octenidine dihydrochloride, a modern antiseptic
for skin, mucous membranes and wounds. Skin Pharmacol. Physiol. 23 (5), 244-58. DOI:
10.1159/000314699.
(5)
Kubo, I.; Muroi, H.; Himejima, M.; Yamagiwa, Y.; Mera, H.; Tokushima, K.; Ohta, S.;
Kamikawa, T. (1993) Structure Antibacterial Activity Relationships of Anacardic Acids. J. Agric.
Food Chem. 41 (6), 1016-1019. DOI: 10.1021/jf00030a036.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(6)
Muroi, H.; Kubo, I. (1996) Antibacterial activity of anacardic acid and totarol, alone and in
combination with methicillin, against methicillinresistantStaphylococcus aureus. J. Appl. Bacteriol. 80
(4), 387-394. DOI: 10.1111/j.1365-2672.1996.tb03233.x.
(7)
(10), 2263-2266. DOI: 10.1016/s0031-9422(00)80158-2.
(8) Balasubramanyam, K.; Swaminathan, V.; Ranganathan, A.; Kundu, T. K. (2003) Small
molecule modulators of histone acetyltransferase p300. J. Biol. Chem. 278 (21), 19134-40. DOI:
10.1074/jbc.M301580200.
(9)
Kumar, G. B.; Tainer, J. A.; Banerji, A.; Perry, J. J.; Nair, B. G. (2012) Anacardic acid inhibits the
catalytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. Mol. Pharmacol. 82
(4), 614-22. DOI: 10.1124/mol.112.079020.
(10)
acids and their derivatives. Int. J. Mol. Sci. 16 (4), 8569-90. DOI: 10.3390/ijms16048569.
(11) Wisastra, R.; Kok, P. A.; Eleftheriadis, N.; Baumgartner, M. P.; Camacho, C. J.; Haisma, H.
J.; Dekker, F. J. (2013) Discovery of a novel activator of 5-lipoxygenase from an anacardic acid
derived compound collection. Bioorg. Med. Chem. 21 (24), 7763-78. DOI:
10.1016/j.bmc.2013.10.015.
Yalpani, M.; H.P. Tyman, J. (1983) The phenolic acids of Pistachia vera. Phytochemistry 22
Omanakuttan, A.; Nambiar, J.; Harris, R. M.; Bose, C.; Pandurangan, N.; Varghese, R. K.;
Hamad, F. B.; Mubofu, E. B. (2015) Potential biological applications of bio-based anacardic
(12)
Kapoor, G.; Saigal, S.; Elongavan, A. (2017) Action and resistance mechanisms of antibiotics:
A guide for clinicians. J. Anaesthesiol., Clin. Pharmacol. 33 (3), 300-305. DOI:
10.4103/joacp.JOACP_349_15.
(13)
Di Marco, R. (2019) Galleria mellonella as a consolidated in vivo model hosts: New developments in
antibacterial strategies and novel drug testing. Virulence 10 (1), 527-541. DOI:
10.1080/21505594.2019.1621649.
(14)
solvents as trace impurities. J. Org. Chem. 62 (21), 7512-7515. DOI: 10.1021/jo971176v.
(15) Ghizzoni, M.; Boltjes, A.; Graaf, C.; Haisma, H. J.; Dekker, F. J. (2010) Improved inhibition
of the histone acetyltransferase PCAF by an anacardic acid derivative. Bioorg. Med. Chem. 18 (16),
5826-34. DOI: 10.1016/j.bmc.2010.06.089.
(16)
T. (2006) Regiocontrolled intramolecular cyclizations of carboxylic acids to carbon-carbon triple
bonds promoted by acid or base catalyst. Org. Lett. 8 (24), 5517-20. DOI: 10.1021/ol062190+.
(17) Yoon, J. K.; Burgess, D. J. (1992) Comparison of dynamic and static interfacial tension at
aqueous/perfluorocarbon interfaces. J. Colloid Interface Sci. 151 (2), 402-409. DOI: 10.1016/0021-
9797(92)90489-9.
Cutuli, M. A.; Petronio Petronio, G.; Vergalito, F.; Magnifico, I.; Pietrangelo, L.; Venditti, N.;
Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. (1997) NMR chemical shifts of common laboratory
Uchiyama, M.; Ozawa, H.; Takuma, K.; Matsumoto, Y.; Yonehara, M.; Hiroya, K.; Sakamoto,
(18)
OECD (2004) Test No. 117: Partition Coefficient (n-octanol/water), HPLC Method. DOI:
10.1787/9789264069824-en.
(19)
dependent solubility of organic bases. Revisit of Henderson-Hasselbalch relationship. Anal. Chim.
Acta 673 (1), 40-6. DOI: 10.1016/j.aca.2010.05.022.
(20)
Beato, S.; Becker, D.; Weitschies, W. (2015) Characterisation of selected active agents regarding pKa
values, solubility concentrations and pH profiles by SiriusT3. Eur. J. Pharm. Biopharm. 92, 155-70.
DOI: 10.1016/j.ejpb.2015.02.028.
Volgyi, G.; Baka, E.; Box, K. J.; Comer, J. E.; Takacs-Novak, K. (2010) Study of pH-
Schonherr, D.; Wollatz, U.; Haznar-Garbacz, D.; Hanke, U.; Box, K. J.; Taylor, R.; Ruiz, R.;
(21)
Hubatsch, I.; Ragnarsson, E. G.; Artursson, P. (2007) Determination of drug permeability and
prediction of drug absorption in Caco-2 monolayers. Nat. Protoc. 2 (9), 2111-9. DOI:
10.1038/nprot.2007.303.
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7
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(22)
Reggane, M.; Wiest, J.; Saedtler, M.; Harlacher, C.; Gutmann, M.; Zottnick, S. H.; Piechon,
P.; Dix, I.; Muller-Buschbaum, K.; Holzgrabe, U.; Meinel, L.; Galli, B. (2018) Bioinspired co-crystals
of Imatinib providing enhanced kinetic solubility. Eur. J. Pharm. Biopharm. 128, 290-299. DOI:
10.1016/j.ejpb.2018.05.012.
(23)
Balk, A.; Widmer, T.; Wiest, J.; Bruhn, H.; Rybak, J. C.; Matthes, P.; Muller-Buschbaum, K.;
Sakalis, A.; Luhmann, T.; Berghausen, J.; Holzgrabe, U.; Galli, B.; Meinel, L. (2015) Ionic liquid
versus prodrug strategy to address formulation challenges. Pharm. Res. 32 (6), 2154-67. DOI:
10.1007/s11095-014-1607-9.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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32
33
34
35
36
37
38
39
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(24)
471. Bacterial Reverse Mutation Test, Adopted July 21, 1997.
(25) Mortelmans, K.; Zeiger, E. (2000) The Ames Salmonella/microsome mutagenicity assay.
Mutat. Res., Fundam. Mol. Mech. Mutagen. 455 (1-2), 29-60. DOI: 10.1016/s0027-5107(00)00064-6.
(26) Green, M. H. L.; Muriel, W. J. (1976) Mutagen testing using TRP+ reversion in Escherichia
coli. Mutat. Res., Environ. Mutagen. Relat. Subj. 38 (1), 3-32. DOI: 10.1016/0165-1161(76)90076-5.
(27) Kier, L. E.; Brusick, D. J.; Auletta, A. E.; Von Halle, E. S.; Brown, M. M.; Simmon, V. F.;
Dunkel, V.; McCann, J.; Mortelmans, K.; Prival, M.; Rao, T. K.; Ray, V. (1986) The Salmonella
typhimurium/mammalian microsomal assay. Mutat. Res., Rev. Genet. Toxicol. 168 (2), 69-240. DOI:
10.1016/0165-1110(86)90002-3.
OECD (1997) OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects. No.
(28)
Lerch, M. F.; Schoenfelder, S. M. K.; Marincola, G.; Wencker, F. D. R.; Eckart, M.; Forstner,
K. U.; Sharma, C. M.; Thormann, K. M.; Kucklick, M.; Engelmann, S.; Ziebuhr, W. (2019) A non-
coding RNA from the intercellular adhesion (ica) locus of Staphylococcus epidermidis controls
polysaccharide intercellular adhesion (PIA)-mediated biofilm formation. Mol. Microbiol. 111 (6),
1571-1591. DOI: 10.1111/mmi.14238.
(29)
Clinical and Laboratory Standards Institute (2015) Methods for Dilution Antimicrobial
Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard - Tenth Edition. CLSI
document M07-A10. Wayne, PA.
(30)
Activity of Antimicrobial Agents. Approved Guideline, CLSI document M26-A. Wayne, PA.
(31) Cai, X.; Walker, A.; Cheng, C.; Paiva, A.; Li, Y.; Kolb, J.; Herbst, J.; Shou, W.; Weller, H.
Clinical and Laboratory Standards Institute (1999) Methods for Determining Bactericidal
(2012) Approach to improve compound recovery in a high-throughput Caco-2 permeability assay
supported by liquid chromatography-tandem mass spectrometry. J. Pharm. Sci. 101 (8), 2755-62.
DOI: 10.1002/jps.23194.
(32)
Yoo, B.; Shah, J. K.; Zhu, Y.; Maginn, E. J. (2014) Amphiphilic interactions of ionic liquids
with lipid biomembranes: a molecular simulation study. Soft Matter 10 (43), 8641-51. DOI:
10.1039/c4sm01528b.
(33)
Pernak, J.; Chwała, P. (2003) Synthesis and anti-microbial activities of choline-like quaternary
ammonium chlorides. Eur. J. Med. Chem. 38 (11-12), 1035-1042. DOI:
10.1016/j.ejmech.2003.09.004.
(34)
Hirsch, T.; Jacobsen, F.; Rittig, A.; Goertz, O.; Niederbichler, A.; Steinau, H. U.; Seipp, H.
M.; Steinstraesser, L. (2009) [A comparative in vitro study of cell toxicity of clinically used
antiseptics]. Hautarzt 60 (12), 984-91. DOI: 10.1007/s00105-009-1842-x.
(35)
Stolte, S.; Matzke, M.; Arning, J.; Böschen, A.; Pitner, W.-R.; Welz-Biermann, U.; Jastorff,
B.; Ranke, J. (2007) Effects of different head groups and functionalised side chains on the aquatic
toxicity of ionic liquids. Green Chem. 9 (11), 1170. DOI: 10.1039/b711119c.
(36)
Brennan, M.; Thomas, D. Y.; Whiteway, M.; Kavanagh, K. (2002) Correlation between
virulence of Candida albicans mutants in mice and Galleria mellonella larvae. FEMS Immunol. Med.
Microbiol. 34 (2), 153-7. DOI: 10.1111/j.1574-695X.2002.tb00617.x.
(37)
1313-1318. DOI: 10.1126/science.284.5418.1313.
(38) Mylonakis, E.; Moreno, R.; El Khoury, J. B.; Idnurm, A.; Heitman, J.; Calderwood, S. B.;
Hoffmann, J. A. (1999) Phylogenetic Perspectives in Innate Immunity. Science 284 (5418),
Ausubel, F. M.; Diener, A. (2005) Galleria mellonella as a model system to study Cryptococcus
neoformans pathogenesis. Infect. Immun. 73 (7), 3842-50. DOI: 10.1128/IAI.73.7.3842-3850.2005.
(39)
Jander, G.; Rahme, L. G.; Ausubel, F. M. (2000) Positive correlation between virulence of
Pseudomonas aeruginosa mutants in mice and insects. J. Bacteriol. 182 (13), 3843-5. DOI:
10.1128/jb.182.13.3843-3845.2000.
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Page 25 of 26
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1
2
3
4
5
6
7
8
9
(40)
Lanbeck, P.; Paulsen, O. (1995) Cytotoxic Effects of Four Antibiotics on Endothelial Cells.
Basic Clin. Pharmacol. Toxicol. 77 (6), 365-370. DOI: 10.1111/j.1600-0773.1995.tb01043.x.
(41) Sharma, P. C.; Jain, A.; Jain, S.; Pahwa, R.; Yar, M. S. (2010) Ciprofloxacin: review on
developments in synthetic, analytical, and medicinal aspects. J. Enzyme Inhib. Med. Chem. 25 (4),
577-89. DOI: 10.3109/14756360903373350.
(42)
Gurbay, A.; Garrel, C.; Osman, M.; Richard, M. J.; Favier, A.; Hincal, F. (2002) Cytotoxicity
in ciprofloxacin-treated human fibroblast cells and protection by vitamin E. Hum. Exp. Toxicol. 21
10
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47
48
49
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52
53
54
55
56
57
58
59
60
(12), 635-41. DOI: 10.1191/0960327102ht305oa.
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