N4-benzyl-N2-phenylquinazoline-2,4-diamine compound presents antibacterial and antibiofilm effect against Staphylococcus aureus and Staphylococcus epidermidis

Article abstract of DOI:10.1111/cbdd.13745

Staphylococcus aureus and Staphylococcus epidermidis are the main agents involved with implant-related infections. Their ability to adhere to medical devices with subsequent biofilm formation is crucial to the development of these infections. Herein, we described the antibacterial and antibiofilm activities of a quinazoline-based compound, N₄-benzyl-N₂-phenylquinazoline-2,4-diamine, against both biofilm-forming pathogens. The minimum inhibitory concentrations (MIC) were determined as 25?μM for S.?aureus and 15?μM for S.?epidermidis. At sub-MIC concentrations (20?μM for S.?aureus and 10?μM for S.?epidermidis), the compound was able to inhibit biofilm formation without interfere with bacterial growth, confirmed by scanning electron microscopy. Moreover, surfaces coated with the quinazoline-based compound were able to prevent bacterial adherence. In addition, this compound presented no toxicity to human red blood cells at highest MIC 25?μM and in vivo toxicity assay using Galleria mellonella larvae resulted in 82% survival with a high dose of 500?mg/kg body weight. These features evidence quinazoline-based compound as interesting entities to promising applications in biomedical fields, such as antimicrobial and in anti-infective approaches.

Full text of DOI:10.1111/cbdd.13745

MISS ISADORA SERRAGLIO FORTES (Orcid ID : 0000-0002-3260-4242)
PROFESSOR SAULO FERNANDES ANDRADE (Orcid ID : 0000-0003-2873-7939)
DR ALEXANDRE JOSE MACEDO (Orcid ID : 0000-0002-8951-4029)
Article type : Research Article
Corresponding author email id: alexandre.macedo@ufrgs.br
N4-benzyl-N2-phenylquinazoline-2,4-diamine compound presents antibacterial and
antibiofilm effect against Staphylococcus aureus and Staphylococcus epidermidis
Sharon Vieira dos Reis1, Nicole Sartori Ribeiro1, Débora Assumpção Rocha2, Isadora
Serraglio Fortes2, Danielle da Silva Trentin3, Saulo Fernandes de Andrade2, Alexandre
José Macedo1,2*
1Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio
Grande do Sul, Brazil
2Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio
Grande do Sul, Brazil
3Departamento de Ciências Básicas da Saúde, Programa de Pós-Graduação em
Biociências, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA),
Porto Alegre, Rio Grande do Sul, Brazil
* Corresponding author
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13745
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Abstract
Staphylococcus aureus and Staphylococcus epidermidis are the main agents
involved with implant-related infections. Their ability to adhere to medical devices with
subsequent biofilm formation is crucial to the development of these infections. Herein we
described the antibacterial and antibiofilm activities of a quinazoline-based compound,
N4-benzyl-N2-phenylquinazoline-2,4-diamine, against both biofilm-forming pathogens.
The minimum inhibitory concentrations (MIC) were determined as 25 µM for S. aureus
and 15 µM for S. epidermidis. At sub-MIC concentrations (20 µM for S. aureus and 10 µM
for S. epidermidis), the compound was able to inhibit biofilm formation without interfere
with bacterial growth, confirmed by scanning electron microscopy. Moreover, surfaces
coated with the quinazoline-based compound were able to prevent bacterial adherence.
In addition, this compound presented no toxicity to human red blood cells at highest MIC
25 µM and in vivo toxicity assay using Galleria mellonella larvae resulted in 82% survival
with a high-dose of 500 mg/kg body weight. These features evidence quinazoline-based
compound as interesting entities to promising applications in biomedical fields, such as
antimicrobial and in anti-infective approaches.
Key words: Staphylococcus aureus, Staphylococcus epidermidis, antibiofilm,
antibacterial, coating, quinazoline
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It is well known that biofilm formation is an important virulence factor to develop
many chronic infections (Koo, 2017), accounting for more than 80% of microbial
infections in humans (Davies, 2003). Biofilms are formed by multiple microbial cells
attached to a surface or suspended in interfaces, which are organized in a complex
tertiary structure embedded in an extracellular matrix of polymeric substances
(Blackledge, 2013). Bacteria in biofilm are phenotypically distinct of their planktonic
counterparts, especially regarding gene expression and growth rates (Del Pozo, 2017),
being more tolerant to clearance by antibiotics and the host immune system (Blackledge,
2013). Since biofilms can serve as reservoirs for bacterial pathogens, leading to
persistent infections and allow resistant gene transfer, biofilm-related diseases represent
a considerable economic burden, contributing to patient morbidity and increased mortality
rates (Del Pozo, 2017).
Medical devices can improve the quality of life and longevity of patients, and are
frequently used in almost all fields of medicine for diagnostic and therapeutic procedures.
The average rates of infection related to surgical implants are 0.5-5% for joint prosthesis,
7-15% for mechanical and bioprosthetic heart valves, 3.1-7.5% for urinary devices, and
40% for ventricular assist devices (VanEpps, 2016). Biomaterials are prone to be infected
during its implantation by microorganisms from patient's skin or from the hands of the
surgical or clinical staff, and the infected implants often need to be removed to complete
elimination of infection (VanEpps, 2016). Staphylococcus aureus and Staphylococcus
epidermidis account together for two thirds of etiological agents related to implant-
associated infections (Campoccia, 2016), evidencing their importance in this type of
disease. S. epidermidis and S. aureus are opportunistic pathogens that only can cause
infection in healthy patients when disrupting epithelial barrier (Otto, 2012). Biofilm
formation is an essential virulence factor that contributes to establish chronic infections.
S. aureus biofilm-related infections require intensive care, and infected devices need to
be removed more frequently than those infected by S. epidermidis.
Due to the difficulty in treating biofilm-related infections, efforts are made to find
novel active compounds able to treat or prevent biofilm formation. Quinazolines are an
emerging class of compounds, and many appropriate substituted quinazolines have
shown a wide range of activities for medication purpose, such as antimalarial, antiviral,
anticancer, antifungal, antiprotozoan, anti-inflammatory, besides other biological activities
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(Asif, 2014). Quinazoline ring is part of many approved drugs in the market such as
prazosin hydrochloride, doxazosine mesylate and terazosine hydrochloride, all of them
being used to treat high blood pressure and benign prostatic hyperplasia. Also, there are
numerous reports describing antibacterial activity of quinazoline derivatives against both
Gram-positive and negative strains (Fleeman, 2017; Jafari, 2016; Van Horn, 2014) .
There are still few studies focused on antibiofilm activity of this class of compounds,
although they demonstrate its promising potential for this purpose (Bessa, 2016;
Bisacchi, 2015; Chandrasekera, 2017; Fleeman, 2017; Kant, 2017; Rasmussen, 2011;
Zhang, 2015). In this work we evaluated the antibacterial and antibiofilm activities of a
quinazoline-based compound, N4-benzyl-N2-phenylquinazoline-2,4-diamine, denominated
PH100, against S. aureus and S. epidermidis bacteria.
Results and Discussion
The antibacterial activity of the substituted quinazoline PH100 (Figure 1A) was
firstly assessed by determination of MIC and MBC against S. aureus ATCC 25904 and S.
epidermidis ATCC 35984 after 24 h incubation (Table S1). The MIC values of PH100 for
S. aureus and S. epidermidis were 25 µM and 15 µM, and the MBC values were 400 µM
and 100 µM, respectively. The ratios MBC/MIC were 16 for S. aureus and 6.7 for S.
epidermidis (Table S1), which characterize bacteriostatic compounds since those
numbers are greater than 4 (Pankey, 2014).
Growth curves for both microorganisms (Figure 1B and C) show that at MIC and
higher concentrations of PH100 (25 and 50 µM for S. aureus and 15, 20 and 25 µM for S.
epidermidis), an initial growth was observed within the first 3-4 h, but it decreased
afterwards. However, at sub-inhibitory concentrations (15 and 20 µM for S. aureus and 5
and 10 µM for S. epidermidis), PH100 had no statistically significant inhibitory effect on
bacterial growth when compared with the vehicle control during the stationary growth
phase. The bacteriostatic profile of PH100 against these microorganisms was also
noticed through growth curves, where both bacteria were able to restore growth after 24
h incubation when treated at MIC. Trentin et al. (2013) also observed this pattern on
kinetic analysis of antibacterial activity with plant extracts, which demonstrated to be
bacteriostatic against Pseudomonas aeruginosa (Trentin, 2013).
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Figure 1. Bacterial kinetic growth curve in presence of N4-benzyl-N2-phenylquinazoline-
2,4-diamine (PH100) structure (A) of S. aureus (B) and S. epidermidis (C). Bacterial
growth evaluated at different concentrations of PH100 during 48 h incubation. DMSO
(2%) was used as vehicle control, and vancomycin was used as bactericide control. Error
bars indicate the standard deviations of three independent experiments.
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Once biofilms are established, they are difficult to eradicate due to their inherent
reduced susceptibility to antibacterial, often resulting in failure of antimicrobial treatment
and mechanical removal requirement of the biofilm (Del POzo, 2017). Bacterial adhesion
on medical devices surfaces is the first and crucial step for established device-related
infections (Campoccia, 2006). Therefore, prevention of bacterial attachment still
represents the best approach to suppress infectious process development. As an attempt
to eliminate or reduce bacterial adhesion and biofilm formation, intensive efforts have
been focused on the development of antibacterial surfaces by application of coatings,
modification or alteration its architecture (Hasan, 2013). In this sense, numerous works
focus on coating surfaces with one or two antimicrobials or entrapping these chemicals
within the material, aiming to obtain medical devices with different antimicrobial spectra
and span of the antimicrobial effect (Francolini, 2010).
Treatments with sub-inhibitory concentrations of PH100 significantly reduced S.
aureus and S. epidermidis biofilm formation (Figure 2A and B, respectively). The
production of biofilm biomass was reduced approximately 51% when S. aureus was
treated with 20 µM PH100, and 94% when S. epidermidis was treated with 10 µM PH100,
in comparison to the untreated control. Interestingly, these concentrations under MIC had
no impact on bacterial growth rates, as demonstrated by kinetics curve and confirmed by
concomitant spectrophotometric evaluation at OD
during biofilm assays (Figure 1
600 nm
and 2). As expected, treatments at MIC and upper concentrations, as well as
vancomycin, were able to inhibit biofilm formation by impairing bacterial growth (Figure
2). Both strains of S. aureus and S. epidermidis tested are strong biofilm formers.
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Figure 2. Evaluation of S. aureus (A) and S. epidermidis (B) biofilm formation and
bacterial growth in the presence of PH100. Biofilm biomass was quantified using crystal
violet staining and bacterial growth was assessed by spectrophotometric measurements
at 600 nm. Results are presented as percentage compared to vehicle controls, which are
considered as 100% of bacterial growth and biofilm formation. The error bars indicate the
standard deviations of three experiments. *p < 0.01 vs. untreated vehicle control groups.
An ideal approach to fight biofilms would be the prevention of bacterial adhesion or
the interruption of the regulatory systems involved in biofilm formation and maintenance,
without killing bacteria, avoiding higher pressure for the selection of resistant bacterial
population (Blackledge, 2013). In this regard, PH100 at sub-inhibitory concentrations
could be a possible candidate for this purpose. It is important to mention that many
studies have demonstrated that some antibacterials, such as kanamycin, erythromycin
and azithromycin, when used at sub-MIC concentrations, can enhance biofilm formation
(Jones, 2013; Kuehl, 2009; Wang, 2010), evidencing that inappropriate antibacterial
choice or usage would improve bacterial biofilm formation during infectious disease,
leading to a worsening clinical outcome. Otherwise, the compound evaluated in this study
did not stimulate this response in any strain tested and can be useful as antibacterial on
higher concentrations or antibiofilm on sub-MIC concentrations. The significant reduction
in S. aureus and S. epidermidis biofilm development observed in crystal violet assay
when cells were treated those sub-MIC concentrations of PH100 (Figure 2) was
confirmed by scanning electron microscopy images (Figure 3). Several bacterial
aggregates can be visualized in control sample, which did not receive PH100 treatment
(Figure 3A and D). S. epidermidis formed thicker biofilm than S. aureus, as observed in
the controls. As expected, in the presence of PH100, the adhesion of bacterial cells
decreased in number and in size of clusters in concentration-dependent manner (Figure
3B, C, E and F). Even at the lowest concentration tested (PH100 15 µM for S. aureus,
and PH100 5 µM for S. epidermidis), a reduction of adhered cells can be observed for
both bacteria (Figure 3B and E), however this effect was not observed using only the
crystal violet technique.
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Figure 3. SEM images of S. aureus and S. epidermidis biofilm in the absence or
presence of sub-MIC concentrations of PH100. S. aureus biofilms formed in the
presence of 2% DMSO vehicle (A); S. aureus biofilms formed in the presence of PH100
15 µM (B), and in the presence of PH100 20 µM (C); S. epidermidis formed in the
presence of 2% DMSO vehicle (D); S. epidermidis biofilms formed in the presence of
PH100 5 µM (E), and in the presence of PH100 10 µM (F). Scale bars: 50 µm at the main
images, 5 µm at the inserts.
Based on these results, we prepared PH100-coated surfaces by using spin-
coating technique, in order to verify its ability to prevent bacterial adherence and biofilm
growth. SEM images of PH100-coated surface samples showed that acetone-treated
surface (vehicle control) allowed bacteria attachment and biofilm development by S.
aureus (Figure 4A) and S. epidermidis (Figure 4C). As previously observed in SEM, S.
epidermidis formed thicker biofilms than S. aureus, presenting many aggregated cells in
the control condition. However, a lower number of bacterial attached cells were
presented on PH100-treated surface in comparison to acetone-treated surface for both
bacteria, which means that the adhesion was inhibited in PH100- surface presence
(Figure 4B and D), with no bacterial clusters observed. Despite of these results, this
assay did not measure the bacterial viability, therefore this effect could be reached by
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action on bacteria growth or on virulence factor related to attachment. The compound has
low aqueous solubility (Van Horn, 2014), which may contribute to a strong interaction with
the PermanoxTM material used as model surface, avoiding its removal even after several
washes. This feature might be important not only for a long maintenance of biofilm
inhibition, but also to prevent the compound detachment from the material surface.
Figure 4. SEM images of S. aureus and S. epidermidis biofilm formation on PH100-
coated surface. S. aureus (A) and S. epidermidis (C) acetone-treated surface used as
vehicle control; S. aureus (B) and S. epidermidis (D) biofilm in PH100-coated surface.
Scale bars: 100 µm at the main images, 10 µm at the inserts.
In order to investigate the potential toxicity of PH100, a preliminary study using
human red blood cells and an invertebrate host model were carried out. Hemolytic effect
in human red blood cells was conducted as indicative of cytotoxicity (Figure S1). For this,
triton X-100 was used as a positive control and was considered to cause 100% hemolysis
while vehicle (2% DMSO) was used as negative control and considered to cause no
hemolysis. PH100 concentrations, ranging from 10 to 25 µM, did not present a significant
hemolytic activity. Only the highest concentration tested (50 µM) presented low
(approximately 3.5%), but significant hemolysis when compared to negative control.
Fleeman et al. (2017) also tested the hemolytic capacity of six substituted quinazolines
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using whole human blood. They registered average hemolysis below 1% at 10 µM, which
was in accordance with the present study. Additionally, Galleria mellonella larvae was
used as an alternative invertebrate model to evaluate in vivo toxicity of PH100. This
model has been used over the last years for toxicity evaluation, demonstrating data
correlation to cell culture and mammalian models (Maguire, 2016; Megaw, 2015; Sardi,
2017). A single high-dose of 500 mg/kg of larvae was tested in groups of 10 larvae. Since
PH100 was only soluble in DMSO, it was injected at a final volume of 1 µL to larvae
hemocoel. As control, 1 µL of DMSO was administered. All larvae groups were incubated
at 37 °C during 5 days and survival was evaluated at each 24 h through touch stimuli.
PH100-treated group presented approximately 82% survival (Table S2), which was not
statistically different from the negative control, indicating that LD50 for PH100 (minimum
dose able to kill 50% of the larvae over time) is higher that dose of 500 mg/kg.
In conclusion, this work presented a promising quinazoline compound displaying
both antibiofilm and antibacterial activity, depending on employed concentration. PH100
presented low toxicity and was able to inhibit biofilm formation under sub-inhibitory
concentrations without interfering with bacterial growth. The bioactivity of PH100 is kept
even when adsorbed in a material surface since PH100 avoids bacterial adhesion,
leading to the generation of an anti-staphylococcal surface. Since staphylococci comprise
the major cause of implant-related biofilms, PH100 would have potential uses as anti-
infective strategy such as for the coating of medical devices surfaces.
Methods
PH100 (N4-benzyl-N2-phenylquinazoline-2,4-diamine) synthesis
PH100 is a quinazoline derivative, denominated N4-benzyl-N2-phenylquinazoline-
2,4-diamine. The synthesis procedure is represented in Figure S2. First, to a stirred
solution of 2.4-dichloroquinazoline 1 (2.51 mmol) in THF/H2O (3:1) (15 mL), were added
sodium acetate (2.76 mmol) and benzilamine (2.76 mmol). The mixture was stirred for 6
h at 65 °C. After, it was cooled and diluted with ethyl acetate (40 mL) and water (40 mL)
and the organic layer was washed with water (2x40 mL), dried over Na2SO4, filtered and
concentrated under reduced pressure. The residue was purified by silica gel column
chromatography eluting with cyclohexane: ethyl acetate (85:15) to give 2 in 62% yield
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which was used immediately in the next step. To a solution of 2 (0.0926 mmol) in ethanol
(0.5 mL), aniline was added (0.139 mmol). The correspondent mixture was stirred for 3 h
at 120 °C. After, the mixture was cooled, diluted with ethyl acetate (20 mL) and washed
with a solution of sodium bicarbonate 10% (p/v) (20 mL) and water (20 mL). The organic
layer was dried with Na2SO4 anhydrous, filtered and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography eluting with
cyclohexane: ethyl acetate (7:3) to give PH100 as a yellow solid in 81% yield. After this
1
purification we proceeded with spectroscopy analyses using infrared (IR), H and 13C
Nuclear Magnetic Resonance (NMR) and mass spectrometry (Figures and description
available in the supporting information) to verify identity and purity of PH100 which is in
accordance with the work of Van Horn (2014)25. M.p. 142-145 ºC. IR (v /cm-1): 3431,
3027, 1568, 1485, 1415, 1326. 1H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.68 (t, 1H,
J = 5.4 Hz), 8.16 (d, 1H, J = 8.0 Hz), 7.84 - 7.82 (m, 2H), 7.60 (t, 1H, J = 8.0 Hz), 7.44-
7.40 (m, 3H), 7.33 (t, 2H, J = 7.4 Hz), 7.25- 7.18 (m, 4H), 6.86 (t, 1HJ = 7.2 Hz), 4.83 (d,
2H, J = 5.4 Hz). 13C NMR (100 MHz, DMSO-d6) δ 160.1, 156.9, 151.4, 141.4, 139.7,
132.6, 128.3, 127.1, 126.7, 125.3, 122.8, 121.5, 120.4, 118.5, 111.6, 43.5. HRMS (m/z):
[MH+] calc for C21H19N4, 326,1604; found, 327,1604 (Fig. S3 – S5).
Bacterial strains and culture conditions
Staphylococcus aureus ATCC 25904 and Staphylococcus epidermidis ATCC
35984 were grown in Muller Hinton (MH) agar (Himedia, India) overnight at 37 ºC.
Bacterial suspensions used in the assays were prepared in sterile 0.9% NaCl at optical
density at 600 nm (OD600) of 0.150 (corresponding to a concentration of 3x108 UFC/mL
and 1 McFarland scale). PH100 stock solutions were prepared in DMSO (Sigma-Aldrich,
USA). As routine of the laboratory the quality control of the strains were performed by
disk diffusion following procedures and interpretation criteria indicated by CLSI.
Particularly in this case, we used the gram-positive reference bacteria S. aureus ATCC
25923 and all parameters fitted with CLSI (2018).
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
determination
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The MIC was evaluated as described by Trentin et al. (2013) with slight
modifications. Final concentrations ranging from 5 to 400 µM of PH100 were evaluated in
96-well microtiter plates (Costar 3599, Corning, Inc., USA). Each well was inoculated with
80 µL of bacterial suspension, 40 µL of TSB (Oxoid Ltd., England) (supplemented with
1% glucose for S. aureus), 76 µL of deionized water and 4 µL of PH100 in DMSO. Wells
without addition of bacteria were used as negative controls. The plates were incubated
statically at 37 ºC for 24 h, and bacterial growth was evaluated by measuring turbidity at
600 nm. MIC values were reported as the lowest concentration of PH100 at which no
increase in OD600 measure was observed after 24 h incubation. The MBC was
determined by dropping 10 µL of bacterial culture from wells with concentrations greater
or equal to the MIC value on MH agar plates. The agar plates were incubated overnight
at 37 °C and MBC was defined as the concentration at which no bacterial colony growth
was observed after 24 h incubation.
Antibacterial activity kinetics
The effect of PH100 on bacterial growth was evaluated at MIC and at
concentrations below and above the MIC. The assay was prepared as described for MIC
determination. OD600 was measured at 0, 1, 2, 4, 6, 9, 12, 24, 36 and 48 hours of
incubation at 37 °C. Controls were prepared with DMSO replacing PH100 treatment.
Vancomycin (8 µg/ mL) was used as bactericide control.
Biofilm formation assay
The antibiofilm activity of PH100 was evaluated using the crystal violet assay
adapted from Trentin et al. (2011). Briefly, 40 µL of TSB (supplemented with 1% glucose
for S. aureus), 80 µL of bacterial suspension, 76 µL of deionized water and 4 µL of
PH100 in DMSO at various concentrations were added to 96-well microtiter plates and
incubated for 24 h at 37 ºC. After the incubation period, the wells were washed 3 times
with sterile saline, heat-fixed at 60 ºC for 1 hour, and stained with 0.4% crystal violet for
15 minutes. The plates were washed, the remaining dye were solubilized with 99%
ethanol and the absorbance was measured at 570 nm. PH100 was tested at final
concentrations ranging from 5 to 50 µM, and it was replaced by DMSO in the untreated
control, which represented 100% biofilm formation.
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Scanning electron microscopy (SEM)
Biofilms of S. aureus ATCC 25904 and S. epidermidis ATCC 35984 were formed
on pieces of PermanoxTM slide (Nalge Nunc International, USA) placedin 96-well
microtiter plates, in the same conditions as described for biofilm formation assays. After
24 h incubation at 37 ºC, the PermanoxTM pieces were washed with sterile saline and
fixed with 2.5% glutaraldehyde. The samples were prepared for analysis by washing with
100 mM cacodylate buffer (pH 7.2), dehydrating in increasing concentrations of acetone
(10%, 30%, 50%, 70%, 90% and 100%), and drying using the CO2 critical point
technique. The PermanoxTM pieces were fixed in aluminum stubs, covered with gold film,
and visualized in a JEOL JSM-6060 scanning electron microscope at 5 kV. Sub-inhibitory
concentrations of PH100 (15µM and 20µM for S. aureus and S. epidermidis, respectively)
were evaluated, and untreated samples were used as controls for biofilm formation.
PH100 coated-surface
The coating surface protocol was adapted from Trentin et al. (2015). Briefly, 300
µL of a 4.0 mg ml-1 P100 solution in 70% aqueous acetone (Merk, Germany) was spin
coated on pieces of PermanoxTM slide (30 x 25 mm²) first with a 500 rpm (5 s) cycle and
then accelerated to 5000 rpm for 40 s in a spin coater (Laurell Model WS-650MZ-
23NPP/LITE). After this step, the pieces were heat-treated at 80°C for 2 h to fix the
compound and remove solvent excess. The slides were sterilized with UV light during
20 minutes, cut in 10 x 25 mm² coated pieces and then washed 10 times with sterile
saline to analyze compound adhesion. As control, Permanox slides were spin coated
with 300 µL of 70% acetone solution and submitted to the same procedures as
described above. Samples were then analyzed to evaluate biofilm growth by SEM as
described at 2.6 section.
Hemolysis assay
Human venous blood (Universidade Federal do Rio Grande do Sul Ethics
Committee under number 1.202.565) was collected in tubes containing EDTA, and
centrifuged at 1200 g for 10 min. The supernatant was discarded; the erythrocytes were
washed three times with 10 mM phosphate buffer saline (pH 7.4/ 0.9% NaCl) and
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resuspended in the same buffer at 3% hematocrit. For hemolytic assay, 500 µL of
erythrocytes suspension, 480 µL of PBS and 20 µL of PH100 (final concentration ranging
from 10 to 50 µM) were incubated for 1 h at 37 ºC. The negative control (NC) was
prepared replacing treatment by DMSO, and 1% Triton X-100 was used for positive
control (PC). The supernatants were collected by centrifugation at 3000 rpm for 5 min,
and their hemoglobin content were assessed spectrophotometrically at 540 nm. The
percentage of hemolysis was calculated as follows: %Hemolysis= Abs Treatment ×100/
Abs PC – Abs NC
In vivo toxicity in Galleria mellonella larvae
For toxicity assay, groups of ten larvae of G. mellonella in the final instar larval
stage weighing 225–275 mg were used. Larvae were injected using a 10 µL Hamilton
syringe via the last right proleg with 500 mg/kg of PH100 diluted in 1 µL DMSO. As a
control of vehicle, a group of larvae received 1 µL DMSO. The larvae were incubated at
37 °C for 5 days and daily the survival was recorded as response to touch stimuli.
Statistical analysis
These assays were performed at least in triplicate. Data were analyzed using the
software GraphPad Prism version 6.01 by two-tailed t test comparing with the untreated
controls. A p < 0.01 was considered statistically significant and the results are expressed
as mean ± standard deviation. Statistical significance in G. mellonella survival analyses
was determined by the log-rank test (GraphPad Prism 6.01).
Acknowledgments
We thankfully acknowledge CME/UFRGS for technical assistance in scanning
electron microscopy and Viviane Nunes da Silva Anselmo for technical support. This
work was supported by FAPERGS-PRONEM (16/2551-0000244-4), MCTI/CNPq
(303353/2016-3, 312351/2017-8), Universal/CNPq (443150/2014-1 and 425970/2018-3),
CAPES (88887.130212/2017-01). and CAPES (Finance Code 001).
Conflict of Interest
The authors declare no competing financial interest.
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Data Availability Statement
The data that support the findings of this study are available from the corresponding
author upon reasonable request.
References
Asif, M. (2014). Chemical Characteristics, Synthetic Methods, and Biological Potential of
Quinazoline and Quinazolinone Derivatives. International Journal Medical Chemistry,
395637. doi:10.1155/2014/395637
Bessa, L. J., Buttachon, S., Dethoup, T., Martins R., Vasconcelos, V., Kijjoa, A., Martins
da Costa, P. (2016). Neofiscalin A and fiscalin C are potential novel indole alkaloid
alternatives for the treatment of multidrug-resistant Gram-positive bacterial infections.
FEMS Microbiology Letters, 363, fnw150. doi:10.1093/femsle/fnw150
Bisacchi, G. S. & Manchester, J. I. (2015). A New-Class Antibacterial-Almost. Lessons in
Drug Discovery and Development: A Critical Analysis of More than 50 Years of Effort
toward ATPase Inhibitors of DNA Gyrase and Topoisomerase IV. ACS Infectious
Disease, 1, 4−41. doi: 10.1021/id500013t
Blackledge, M. S., Worthington, R. J., Melander, C. (2013). Biologically inspired
strategies for combating bacterial biofilms. Current Opinion Pharmacology, 13, 699–706.
doi:10.1016/j.coph.2013.07.004
Campoccia, D., Montanaro, L., Arciola, C. R. (2006). The significance of infection related
to orthopedic devices and issues of antibiotic resistance. Biomaterials, 27, 2331–2339.
doi:10.1016/j.biomaterials.2005.11.044
Chandrasekera, N. S., Berube, B. J., Shetye, G., Chettiar, S., O’Malley, T., Manning, A.,
Flint, L., Awasthi, D., Ioerger, T. R., Sacchettini, J., Masquelin, T., Hipskind, P. A.,
Odingo, J., Parish, T. (2017). Improved Phenoxyalkylbenzimidazoles with Activity against
Mycobacterium tuberculosis Appear to Target QcrB. ACS Infectious Disease, 3,
This article is protected by copyright. All rights reserved

898−916. doi: 10.1021/acsinfecdis.7b00112
Clinical and Laboratory Standards Institute (CLSI) (2018). M100: Performance Standards
for Antimicrobial Susceptibility Testing. 28th ed.
Davies D. (2003). Understanding biofilm resistance to antibacterial agents. Nature
Reviews Drug Discovery, 2, 114–122. doi:10.1038/nrd1008
Del Pozo, J. L. (2017). Biofilm-related disease. Expert Review of Anti-infective Therapy,
1–15. doi:10.1080/14787210.2018.1417036
Fleeman, R., Van Horn, K. S., Barber, M. M., Burda, W. N., Flanigan, D. L., Manetsch,
R., Shaw, L. N. (2017). Characterizing the antimicrobial activity of N2,N4-disubstituted
quinazoline-2,4-diamines toward multidrug-resistant Acinetobacter baumannii.
Antimicrobial Agents Chemotherapy, 61, 1–11. doi: 10.1128/AAC.00059-17
Francolini I. & Donelli, G. (2010). Prevention and control of biofilm-based medical-device-
related infections. FEMS Immunology Medical Microbiology, 59, 227–238.
doi:10.1111/j.1574-695X.2010.00665.x
Hasan , J., Crawford, R. J., Ivanova , E.P. (2013). Antibacterial surfaces: the quest for a
new generation of biomaterials. Trends Biotechnology, 31, 295–304.
doi:10.1016/j.tibtech.2013.01.017
Jafari, E., Khajouei, M. R., Hassanzadeh, F., Hakimelahi, G. H., Khodarahmi, G. A.
(2016). Quinazolinone and quinazoline derivatives: Recent structures with potent
antimicrobial and cytotoxic activities. Research in Pharmaceutical Sciences, 11, 1–14.
Jones, C., Allsopp, L., Horlick , J., Kulasekara, H., Filloux A. (2013). Subinhibitory
concentration of kanamycin induces the Pseudomonas aeruginosa type VI secretion
system. PLoS One, 8, e81132. doi:10.1371/journal.pone.0081132
This article is protected by copyright. All rights reserved

Kant, S., Asthana, S., Missiakas, D., Pancholi, V. (2017). A novel STK1-targeted small-
molecule as an antibiotic resistance breaker against multidrug-resistant Staphylococcus
aureus. Scientific Reports, 7, 5067. doi:10.1038/s41598-017-05314-z
Koo, H., R.N. Allan, Howlin, R. P., Stoodley, P., Hall-stoodley, L. (2017). Targeting
microbial biofilms: current and prospective therapeutic strategies. Nature Reviews
Microbiology, 15, 740–755. doi:10.1038/nrmicro.2017.99
Kuehl, R., Al-Bataineh, S., Gordon, O., Luginbuehl, R., Otto, M., Textor, M., Landmann,
R. (2009). Furanone at subinhibitory concentrations enhances staphylococcal biofilm
formation by luxS repression. Antimicrobial Agents Chemotherapy, 53, 4159–4166.
doi:10.1128/AAC.01704-08
Maguire, R., Duggan, O., Kavanagh K. (2016). Evaluation of Galleria mellonella larvae as
an in vivo model for assessing the relative toxicity of food preservative agents. Cell
Bioogy Toxicology, 32, 209–216. doi:10.1007/s10565-016-9329-x.
Megaw, J., Thompson, T. P., Lafferty, R. A., Gilmore, B. F. (2015). Galleria mellonella as
a novel in vivo model for assessment of the toxicity of 1-alkyl-3-methylimidazolium
chloride ionic liquids, Chemosphere, 139, 197–201.
doi:10.1016/j.chemosphere.2015.06.026
Otto, M. (2012) Molecular basis of Staphylococcus epidermidis infections. Semin.
Immunopathol. 34, 201–214. doi:10.1007/s00281-011-0296-2.
Pankey G. A. & Sabath, L. D. (2014). Clinical Relevance of Bacteriostatic versus
Bactericidal Mechanisms of Action in the Treatment of Gram‐Positive Bacterial Infections,
Clinical Infectious Disease, 38, 864–870. doi:10.1086/381972
Rasmussen L., White, E. L., Pathak, A., Ayala, J. C., Wang, H., J.-H. Wu, Benitez, J. A.,
Silva, A. J. (2011). A high-throughput screening assay for inhibitors of bacterial motility
identifies a novel inhibitor of the Na+-driven flagellar motor and virulence gene expression
in Vibrio cholerae. Antimicrobial Agents Chemotherapy, 55, 4134–43.
This article is protected by copyright. All rights reserved

doi:10.1128/AAC.00482-11
Sardi, J. C. O., Freires, I. A., Lazarini, J. G., Infante, J., Alencar, S. M., Rosalen, P. L.
(2017). Unexplored endemic fruit species from Brazil: Antibiofilm properties, insights into
mode of action, and systemic toxicity of four Eugenia spp. Microbial Pathogenesis, 105,
280–287. doi:10.1016/j.micpath.2017.02.044
Trentin, D. S., Giordani, R. B., Zimmer, K. R., Silva, A. G., Silva, M. V., Correia, M. T. S.,
Baumvol, I. J. R., Macedo, A. J. (2011). Potential of medicinal plants from the Brazilian
semi-arid region (Caatinga) against Staphylococcus epidermidis planktonic and biofilm
lifestyles. Journal of Ethnopharmacology, 137, 327–335. doi:10.1016/j.jep.2011.05.030
Trentin, D. S., Silva, D. B. Amaral, M.W., Zimmer, K.R., Silva, M. V, Lopes , N.P.,
Giordani, R.B., Macedo, A.J. (2013). Tannins Possessing Bacteriostatic Effect Impair
Pseudomonas aeruginosa Adhesion and Biofilm Formation. Plos One, 8, e66257.
doi:10.1371/journal.pone.0066257
Trentin, D. S., Silva, D. B., Frasson, A. P., Rzhepishevska, O., Silva, M. V., Pulcini, E. L.,
James, G., Soares G. V., Tasca, T., Ramstedt, M., Giordani, R. B., Lopes, N.P., Macedo,
A. J. (2015). Natural Green coating inhibits adhesion of clinically important bacteria.
Scientific Reports, Feb 6;5:8287. doi: 10.1038/srep08287
Van Horn, K. S., Burda, W. N., Fleeman, R., Shaw, L.N., Manetsch, R. (2014).
Antibacterial activity of a series of N2,N4-disubstituted quinazoline-2,4-diamines. Journal
of Medicinal Chemistry, 57, 3075–3093. doi:10.1021/jm5000408
Van Horn, K.S., Zhu, X., Pandharkar, T., Yang, S., Vesely, Vanaerschot, B., Dujardin, M.
J.-C., Rijal, S., Kyle, D. E., Wang, M. Z., Werbovetz, K.A., Manetsch, R. (2014).
Antileishmanial activity of a series of N 2,N 4-disubstituted quinazoline-2,4-diamines,
Journal Medicinal Chemistry, 57, 5141–5156. doi:10.1021/jm5000408
VanEpps, J. S. & Younger, J. G (2016). Implantable Device-Related Infection,
This article is protected by copyright. All rights reserved

Shock, 46(6), 597–608. https://doi.org/10.1097/SHK.0000000000000692
Wang, Q., Sun, F.J., Liu, Y., Xiong, L. R., Xie, L. L., Xia, P. Y. (2010). Enhancement of
biofilm formation by subinhibitory concentrations of macrolides in icaADBC-positive and -
negative clinical isolates of Staphylococcus epidermidis. Antimicrobial Agents
Chemotherapy, 54, 2707–2711. doi:10.1128/AAC.01565-09
Zhang, Q., Nguyen, T., McMichael, M., Velu, S. E., Zou, J., Zhou, X., Wu, H. (2015). New
small-molecule inhibitors of dihydrofolate reductase inhibit Streptococcus mutans.
International Journal Antimicrobial Agents, 46, 174–82.
doi:10.1016/j.ijantimicag.2015.03.015
This article is protected by copyright. All rights reserved