Synthetic Flavonoid BrCl-Flav—An Alternative Solution to Combat ESKAPE Pathogens

ESKAPE pathogens are considered as global threats to human health. The discovery of new molecules for which these pathogens have not yet developed resistance is a high medical priority. Synthetic flavonoids are good candidates for developing new antimicrobials. Therefore, we report here the potent in vitro antibacterial activity of BrCl-flav, a representative of a new class of synthetic tricyclic flavonoids. Minimum inhibitory/bactericidal concentration, time kill and biofilm formation assays were employed to evaluate the antibacterial potential of BrCl-flav. The mechanism of action was investigated using fluorescence and scanning electron microscopy. A checkerboard assay was used to study the effect of the tested compound in combination with antibiotics. Our results showed that BrCl-flav displayed important inhibitory activity against all tested clinical isolates, with MICs ranging between 0.24 and 125 µg/mL. A total kill effect was recorded after only 1 h of exposing Enterococcus faecium cells to BrCl-flav. Additionally, BrCl-flav displayed important biofilm disruption potential against Acinetobacter baumannii. Those effects were induced by membrane integrity damage. BrCl-flav expressed synergistic activity in combination with penicillin against a MRSA strain. Based on the potent antibacterial activity, low cytotoxicity and pro-inflammatory effect, BrCl-flav has good potential for developing new effective drugs against ESKAPE pathogens.


Introduction
Antimicrobial resistance (AMR) remains a major public concern, posing a serious threat to human health and economic development around the world. According to the World Health Organization, antimicrobial resistance is one of the 10 global public health menaces facing humanity today, which increases mortality and morbidity and strains healthcare systems [1]. AMR is defined by the European Centre for Disease Prevention and Control (ECDC) as the ability of microorganisms (viruses, bacteria, fungi and parasites) to resist the action of one or more antimicrobial agents [2]. It may occur when antimicrobial drugs used to treat infections become less effective or inefficient due to changes in pathogenic microorganisms, especially bacteria. Extensive previous studies have estimated the AMR consequences in terms of deaths, hospital length of stay and healthcare costs [1][2][3][4][5]. New data from a 2022 study show the true AMR burden, with an estimated 4.95 million deaths associated with bacterial AMR in 2019, including 1.27 million deaths as a direct result of antibiotic-resistant bacterial infections [6]. One pathogen alone-methicillin-resistant Staphylococcus aureus (MRSA), a member of the ESKAPE group-was responsible for more than 100,000 deaths attributable to AMR in 2019 [6].

BrCl-flav Exhibits Potent Antibacterial Activity against ESKAPE Pathogens
The recorded MIC and MBC values showed that BrCl-flav poses important antibac-

BrCl-flav Exhibits Potent Antibacterial Activity against ESKAPE Pathogens
The recorded MIC and MBC values showed that BrCl-flav poses important antibacterial activity against all clinical isolates used in this study ( Table 1). The MICs for Grampositive bacteria ranged between 0.24 µg/mL (recorded for two MRSA strains-S. aureus prxbio4 and S. aureus prxbio5) and 31.3 µg/mL (registered for E. faecalis prxbio8). The lowest MIC evidenced for Gram-negative bacteria was 0.48 µg/mL (Haemophillus spp. prxbio13), and the highest MIC value (125 µg/mL) was recorded for several strains-K. pneumoniae, E. cloacae and S. enterica. Regarding MBCs, lower values were also recorded for Gram-positive bacteria (0.48 µg/mL-MRSA strain) compared with Gram-negative bacteria (1.95 µg/mL-Haemophillus spp. prxbio13). Additionally, we need to emphasize that BrCl-flav showed more potent antibacterial activity (up to 16-fold higher) compared to chloramphenicol for several S. aureus strains, as well as for S. pneumoniae prxbio10 and Haemophillus spp. prxbio13. Comparable activity to gentamicin was recorded against K. pneumoniae medbio6-2013. However, for most of the Gram-negative isolates tested, BrCl-flav showed lower activity compared with gentamicin. Based on antibiotic resistance profile and MIC/MBC values, several strains were selected to perform further tests using BrCl-flav as an antibacterial agent.

BrCl-flav Induced a Concentration-and Time-Dependent Bacteriostatic Effect
The activity of BrCl-flav on the growth of selected clinical isolates over time was evaluated using concentrations equivalent to 1 2 × MIC, MIC and 2 × MIC ( Figure 2). Compared to the control, BrCl-flav displayed dose-dependent and time-dependent bacteriostatic effects on all tested bacterial strains, as the growth curve analyses revealed. Thus, concentrations corresponding to 1 2 × MIC induced a significant growth delay (p ≤ 0.0056) up to Antibiotics 2022, 11, 1389 4 of 23 7 h (recorded for penicillin-resistant S. aureus prxbio1). Increasing the concentrations to the corresponding MIC values revealed a progressive inhibition of the growth of all tested strains up to 12 h compared with control. We must emphasize that no growth was detected by spectrophotometric measurements for S. aureus prxbio1 and A. baumannii medbio3-2013 cells exposed for more than 12 h to BrCl-flav at concentrations equivalent to 2 × MIC. Moreover, the analysis of the bacterial growth dynamics also revealed that the growth of the S. aureus prxbio1 strain was suppressed up to 24 h at 2 × MIC, showing that BrCl-flav has important bacteriostatic activity.

BrCl-flav Induced a Concentration-and Time-Dependent Bacteriostatic Effect
The activity of BrCl-flav on the growth of selected clinical isolates over time was evaluated using concentrations equivalent to ½ × MIC, MIC and 2 × MIC ( Figure 2). Compared to the control, BrCl-flav displayed dose-dependent and time-dependent bacteriostatic effects on all tested bacterial strains, as the growth curve analyses revealed. Thus, concentrations corresponding to ½ × MIC induced a significant growth delay (p ≤ 0.0056) up to 7 h (recorded for penicillin-resistant S. aureus prxbio1). Increasing the concentrations to the corresponding MIC values revealed a progressive inhibition of the growth of all tested strains up to 12 h compared with control. We must emphasize that no growth was detected by spectrophotometric measurements for S. aureus prxbio1 and A. baumannii med-bio3-2013 cells exposed for more than 12 h to BrCl-flav at concentrations equivalent to 2 × MIC. Moreover, the analysis of the bacterial growth dynamics also revealed that the growth of the S. aureus prxbio1 strain was suppressed up to 24 h at 2 × MIC, showing that BrCl-flav has important bacteriostatic activity.

BrCl-flav Possesses Important Bactericidal Activity against Selected Clinical Isolates
Time-killing curves of BrCl-flav were performed using the MBC values as reference. A total kill effect (no viable cells) was recorded for all selected strains at different exposure times ( Figure 3). This effect was evidenced after only 30 min of incubation of A. baumannii medbio3-2013 cells in the presence of the tested antibacterial agent. We must emphasize that no viable cells were evidenced up to 24 h of exposure, showing the significant bactericidal potency of BrCl-flav against all tested resistant bacterial strains.
Time-killing curves of BrCl-flav were performed using the MBC values as reference. A total kill effect (no viable cells) was recorded for all selected strains at different exposure times ( Figure 3). This effect was evidenced after only 30 min of incubation of A. baumannii medbio3-2013 cells in the presence of the tested antibacterial agent. We must emphasize that no viable cells were evidenced up to 24 h of exposure, showing the significant bactericidal potency of BrCl-flav against all tested resistant bacterial strains. The bacterial cells were exposed to BrCl-flav at concentrations equivalent to MBC. Untreated cells served as control. The data were presented as the means of three independent experiments. Bars indicate SEM.

The Anti-Biofilm Activity of BrCl-Flav
A biofilm formation assay was employed to determine the capability of the clinical isolates to form biofilms in vitro. The results are presented in Figure S1 (Supplementary Files). By far, Acinetobacter baumannii medbio3-2013 exhibited the best biofilm-forming capacity, being classified as a strongly adherent strain. Based on this ability, the strain was selected as a representative ESKAPE pathogen for further tests regarding the anti-biofilm activity of BrCl-flav.

Bacterial Biofilm Formation was Inhibited by BrCl-flav
The formation of A. baumannii medbio3-2013 biofilms in the presence of BrCl-flav was significantly inhibited at different concentrations compared to the biofilms formed The bacterial cells were exposed to BrCl-flav at concentrations equivalent to MBC. Untreated cells served as control. The data were presented as the means of three independent experiments. Bars indicate SEM.

The Anti-Biofilm Activity of BrCl-flav
A biofilm formation assay was employed to determine the capability of the clinical isolates to form biofilms in vitro. The results are presented in Figure S1 (Supplementary Files). By far, Acinetobacter baumannii medbio3-2013 exhibited the best biofilm-forming capacity, being classified as a strongly adherent strain. Based on this ability, the strain was selected as a representative ESKAPE pathogen for further tests regarding the anti-biofilm activity of BrCl-flav.

Bacterial Biofilm Formation Was Inhibited by BrCl-flav
The formation of A. baumannii medbio3-2013 biofilms in the presence of BrCl-flav was significantly inhibited at different concentrations compared to the biofilms formed by unexposed cells (Figure 4a). BrCl-flav at concentrations between 3.9 and 62.5 µg/mL inhibited the biofilm formation more than 95% compared to the control. Lower and nonsignificant biofilm inhibition was recorded for 1 2 MIC (29.73%).
Because BrCl-flav exhibited encouraging inhibitory activity against biofilm formation, we proceeded with further experiments designed to assess the possible disruptive potential of the mature biofilms. Thus, A. baumannii medbio3-2013 biofilms were allowed to develop for 24 h prior to BrCl-flav exposure. Our data revealed that BrCl-flav displayed an important biofilm disruption potential (Figure 4b). Concentrations equivalent to MIC (3.9 µg/mL) and ½ MIC (1.95 µg/mL) induced a significant reduction of biofilm biomass of 57.71% and 39.91%, respectively.

Mode of Action
The mechanism of activity against representative Gram-positive and Gram-negative bacteria was investigated using fluorescence microscopy and SEM.

BrCl-flav Impair the Cellular Membrane Integrity
Fluorescence microscopy was used to assess the uptake of PI fluorescent dye into S. aureus medbio1-2012 and E. coli medbio4-2013 cells with injured membranes after exposure to BrCl-flav at concentrations equivalent to MBC (Figure 5a,b). The low levels of fluorescence detected in control cells during the entire experiment confirmed the inability of PI to penetrate viable cells with intact membranes [25]. On the contrary, exposing S. aureus and E. coli cells to BrCl-flav significantly increased over time the number of fluorescent cells compared to control, most likely due to the increased permeability of the cellular membrane to PI. After only 25 min of incubation in the presence of the antibacterial agent, the percentage of S. aureus and E. coli fluorescent cells was 75.02% and 87.93%, respectively. A 100% fluorescent cell percentage was recorded after approximatively 1 h (E. coli) and 2 h (S. aureus) of BrCl-flav exposure ( Figure 5).

BrCl-flav Showed Important Biofilm Disruption Potential
Because BrCl-flav exhibited encouraging inhibitory activity against biofilm formation, we proceeded with further experiments designed to assess the possible disruptive potential of the mature biofilms. Thus, A. baumannii medbio3-2013 biofilms were allowed to develop for 24 h prior to BrCl-flav exposure. Our data revealed that BrCl-flav displayed an important biofilm disruption potential (Figure 4b). Concentrations equivalent to MIC (3.9 µg/mL) and 1 2 MIC (1.95 µg/mL) induced a significant reduction of biofilm biomass of 57.71% and 39.91%, respectively.

Mode of Action
The mechanism of activity against representative Gram-positive and Gram-negative bacteria was investigated using fluorescence microscopy and SEM.

BrCl-flav Impair the Cellular Membrane Integrity
Fluorescence microscopy was used to assess the uptake of PI fluorescent dye into S. aureus medbio1-2012 and E. coli medbio4-2013 cells with injured membranes after exposure to BrCl-flav at concentrations equivalent to MBC (Figure 5a,b). The low levels of fluorescence detected in control cells during the entire experiment confirmed the inability of PI to penetrate viable cells with intact membranes [25]. On the contrary, exposing S. aureus and E. coli cells to BrCl-flav significantly increased over time the number of fluorescent cells compared to control, most likely due to the increased permeability of the cellular membrane to PI. After only 25 min of incubation in the presence of the antibacterial agent, the percentage of S. aureus and E. coli fluorescent cells was 75.02% and 87.93%, respectively. A 100% fluorescent cell percentage was recorded after approximatively 1 h (E. coli) and 2 h (S. aureus) of BrCl-flav exposure ( Figure 5). As the fluorescence dynamics depicted in Figure 6 shows, the first fluorescent cells were detected after only 2 and 3 min of exposing S. aureus and E. coli cells, respectively, to BrCl-flav. As the fluorescence dynamics depicted in Figure 6 shows, the first fluorescent cells were detected after only 2 and 3 min of exposing S. aureus and E. coli cells, respectively, to BrCl-flav.   (Figure 7). S. aureus medbio1-2012-and E. coli medbio4-2013-exposed cells were deformed, collapsed and had wrinkled surfaces ( Figure 7). Moreover, the cell shrinkage was evidenced along with cellular debris, most likely resulting from the lysis of the cells, confirming the impairment of cellular membrane integrity. SEM analysis revealed significant cell morphological damages of the BrCl-flav exposed cells. Control groups presented cells with normal morphologies (spheric coccus and short rods), clear boundaries and smooth surfaces ( Figure 7). S. aureus medbio1-2012-and E. coli medbio4-2013-exposed cells were deformed, collapsed and had wrinkled surfaces ( Figure 7). Moreover, the cell shrinkage was evidenced along with cellular debris, most likely resulting from the lysis of the cells, confirming the impairment of cellular membrane integrity.

Effect of BrCl-flav in Combination with Antibiotics against a MRSA Strain
FICIs were evaluated to investigate whether BrCl-flav used in combination with penicillin, ciprofloxacin and tetracycline provided synergistic or additive effects against an MRSA clinical isolate. The results showed additive responses in the case of BrCl-flav in combination with all three tested antibiotics (Table 2). Penicillin is the only antibiotic for which synergistic effects were recorded in combination with the tested antibacterial agent (FICI values: 0.5-0.264). When used in combination, the MICs of the two agents were reduced 68-fold for BrCl-flav and up to 4-fold for penicillin.
The antibacterial activity of one synergistic combination, BrCl-flav-penicillin (0.03/32 µg/mL), was further evaluated using a time-kill assay. No significant reduction of the viable cell number was recorded when BrCl-flav and penicillin were used alone, compared with the control (Figure 8). However, significant bactericidal activity was evidenced when the two agents were used in combination, with a total kill (no viable cells) effect recorded after 24 h (Figure 8).

Effect of BrCl-flav in Combination with Antibiotics against a MRSA Strain
FICIs were evaluated to investigate whether BrCl-flav used in combination with penicillin, ciprofloxacin and tetracycline provided synergistic or additive effects against an MRSA clinical isolate. The results showed additive responses in the case of BrCl-flav in combination with all three tested antibiotics (Table 2). Penicillin is the only antibiotic for which synergistic effects were recorded in combination with the tested antibacterial agent (FICI values: 0.5-0.264). When used in combination, the MICs of the two agents were reduced 68-fold for BrCl-flav and up to 4-fold for penicillin. The antibacterial activity of one synergistic combination, BrCl-flav-penicillin (0.03/32 µg/mL), was further evaluated using a time-kill assay. No significant reduction of the viable cell number was recorded when BrCl-flav and penicillin were used alone, compared with the control (Figure 8). However, significant bactericidal activity was evi-denced when the two agents were used in combination, with a total kill (no viable cells) effect recorded after 24 h (Figure 8).
Antibiotics 2022, 11, x FOR PEER REVIEW

BrCl-flav Effect on Human Cell Viability
Four cell lines were chosen for their different phenotypes (intestinal epithelial mucous-producing cells: Caco-2 and HT29-MTX; hepatocytes: He rophages: U937) to evaluate the relative cytotoxicity of BrCl-flav by deter ward human cells (Figure 9).

BrCl-flav Effect on Human Cell Viability
Four cell lines were chosen for their different phenotypes (intestinal epithelial and epithelial mucous-producing cells: Caco-2 and HT29-MTX; hepatocytes: HepG2; and macrophages: U937) to evaluate the relative cytotoxicity of BrCl-flav by determining IC 50 toward human cells (Figure 9).
The results obtained showed that the monocytes differentiated in macrophages appeared to be the most sensitive cell line to BrCl-flav (IC 50 = 5.30 µg/mL), followed by hepatocytes (IC 50 = 13.16 µg/mL). Epithelial cells appeared to be much more tolerant, as the calculated IC 50 for the goblet-like cells (HT29-MTX) was 31.86 µg/mL. The IC 50 calculation for Caco-2 was not possible regarding the increasing viability effect of BrCl-flav at concentrations ranging from 5 to 25 µg/mL.

Inflammation Study
To evaluate the pro-or anti-inflammatory effects of BrCl-flav, the secretion of a proinflammatory cytokine (TNF-α) and an anti-inflammatory cytokine (IL10) were quantified on LPS-induced macrophages. As shown in Figure 10, the secretion of the two cytokines studied was increased by exposure to LPS alone compared to the non-inflamed control.
isolate. Values are the means of three replicates. Bars indicate SEM.

BrCl-flav Effect on Human Cell Viability
Four cell lines were chosen for their different phenotypes (intestinal epithelial and epithelial mucous-producing cells: Caco-2 and HT29-MTX; hepatocytes: HepG2; and macrophages: U937) to evaluate the relative cytotoxicity of BrCl-flav by determining IC50 toward human cells (Figure 9). Means are presented ± SD (N = 2, n = 6). The molecule concentration required to cause 50% inhibition The results obtained showed that the monocytes differentiated in macrophages appeared to be the most sensitive cell line to BrCl-flav (IC50 = 5.30 µg/mL), followed by hepatocytes (IC50 = 13.16 µg/mL). Epithelial cells appeared to be much more tolerant, as the calculated IC50 for the goblet-like cells (HT29-MTX) was 31.86 µg/mL. The IC50 calculation for Caco-2 was not possible regarding the increasing viability effect of BrCl-flav at concentrations ranging from 5 to 25 µg/mL.

Inflammation Study
To evaluate the pro-or anti-inflammatory effects of BrCl-flav, the secretion of a proinflammatory cytokine (TNF-α) and an anti-inflammatory cytokine (IL10) were quantified on LPS-induced macrophages. As shown in Figure 10, the secretion of the two cytokines studied was increased by exposure to LPS alone compared to the non-inflamed control. The mean concentration of cytokines increased from 82.50 ± 165.00 pg/mL and 118.10 ± 3.06 pg/mL for the non-inflamed control to 2121 ± 281.7 pg/mL and 1707 ± 176.1 pg/mL for the LPS control, for TNF-α and IL10, respectively. The two cytokines, whether pro-or anti-inflammatory, were downregulated by the addition of glucocorticoid dexamethasone (positive control of inflammation inhibition) at the concentration of 20 µM. Indeed, TNF- The mean concentration of cytokines increased from 82.50 ± 165.00 pg/mL and 118.10 ± 3.06 pg/mL for the non-inflamed control to 2121 ± 281.7 pg/mL and 1707 ± 176.1 pg/mL for the LPS control, for TNF-α and IL10, respectively. The two cytokines, whether pro-or anti-inflammatory, were downregulated by the addition of glucocorticoid dexamethasone (positive control of inflammation inhibition) at the concentration of 20 µM. Indeed, TNF-α and IL10 concentrations decreased to 136.80 ± 23.55 pg/mL and 155.80 ± 30.00 pg/mL, corresponding to 95.55% and 90.87% inhibition, respectively.
BrCl-flav enhanced the secretion of TNF-α by LPS-stimulated macrophages by approximately 44% and decreased the IL10 anti-inflammatory cytokine secretion by 59%, both at all tested concentrations (not in a dose-dependent manner) ( Figure 10).

Discussion
Antibiotic-resistant bacteria, particularly ESKAPE pathogens, are considered a global threat to human health. The acquisition of antimicrobial-resistance genes by ESKAPE pathogens has reduced the treatment options, increasing death rates due to treatment failure and stimulating the interest in the development of new antimicrobial therapies [26]. A possible solution could be the discovery of new molecules, not used until now in clinical therapy, for which the pathogens have not yet developed resistance. In this context, we hypothesize that BrCl-flav-a representative of a new class of synthetic sulfur containing tricyclic flavonoids with different halogen substituent at the benzopyran core-could be a reliable candidate for the formulation of new effective antimicrobials. We previously reported the important bactericidal and fungicidal effects of BrCl-flav [23,24,27]. However, no investigations were carried out on clinical bacterial isolates with different antibiotic resistance profiles. Additionally, no information is available on the cytotoxicity and antiinflammatory activity. Here, we report the potent antibacterial activity against antibioticresistant bacteria from the ESKAPE group, low cytotoxicity and pro-inflammatory effect of BrCl-flav.
Determination of minimum inhibitory concentration revealed the important antibacterial activity of BrCl-flav against all bacterial strains tested in vitro. The most sensitive clinical isolates to BrCl-flav were MRSA strains, for which the lowest MICs values (0.24 µg/mL) were recorded. Moreover, our compound showed a more pronounced antibacterial activity against several S. aureus, S. pneumoniae and Haemophillus spp. strains compared to chloramphenicol, and comparable activity against one K. pneumoniae strain compared to gentamicin. Our data analysis also showed that Gram-negative bacteria were less susceptible to BrCl-flav, with the highest MIC of 125 µg/mL (registered for ESKAPE pathogens such as K. pneumoniae or Enterobacter spp.), compared to Gram-positive bacteria for which the highest MIC value recorded was 31.25 µg/mL (registered for an E. faecalis strain). This different susceptibility between Gram-positive and Gram-negative bacteria could be explained by the different cell wall structure and composition. Thus, the presence of the outer membrane in the Gram-negative cell wall provides protection against different antimicrobials, explaining the milder effect of BrCl-flav [28]. In this case, due to the strong ionization and the pronounced hydrophilic character, the tricyclic flavonoid presents a lower capability to penetrate the external hydrophobic membrane of the Gram-negative bacteria.
The antibacterial activity of BrCl-flav against representative ESKAPE pathogens (S. aureus, E. faecium and A. baumannii) was also assessed using growth kinetic studies. A significant decrease in the growth rate of all tested strains was detected when BrCl-flav was used at different concentrations, starting with the corresponding 1 2 × MIC. Exposing bacterial cells to concentrations equivalent to MIC induced a significant growth delay compared with control cells up to 12 h, as the growth curves of S. aureus prxbio1 show.
When BrCl-flav was tested at concentrations corresponding to 2 × MIC, the growth of all tested pathogens was progressively inhibited, implying a significant dose-dependent inhibitory effect. MRSA and A. baumannii strains were most affected by BrCl-flav exposure at concentrations equivalent to 2 × MIC by diminishing the growth rate and reducing the final cellular density. We must highlight that no turbidity was revealed by the spectrophotometric measurements for all strains up to 12 h or up to 24 h for S. aureus prxbio1, denoting important bacteriostatic activity.
Biofilm formation is related to the virulence potential of many bacterial strains, including ESKAPE pathogens. Infections caused by biofilm forming bacteria are very difficult to treat with current antibiotics; therefore, prevention of early-stage biofilm formation is essential for the treatment of these infections [13]. BrCl-flav showed important dose-dependent anti-biofilm activity, significantly inhibiting biofilm formation of A. baumannii with more than 95% at concentrations equivalent to the MIC. Because biofilms pose a serious medical challenge that is difficult to control, it is essential to find new agents that are able to eradicate biofilms [43]. Therefore, we examined the capability of BrCl-flav to disrupt mature biofilms of A. baumannii. Our compound showed important concentration-dependent biofilm-disruptive activity. At a low concentration of 1.95 µg/mL (corresponding to 1 2 MIC), BrCl-flav disrupted more than 39.91% of the biofilm mass and more than 50% at concentrations equivalent to 2 × MIC. The inhibition of biofilm formation by flavonoids was previously reported [42]. However, BrCl-flav showed higher anti-biofilm activity against A. baumanni compared with some natural flavonoids such as fisetin, phloretin and curcumin, reported to decrease biofilm formation of MDR A. baumannii strains with 46 and 93% at concentrations of 20 or 100 µg/mL [44]. Altogether, our results suggest that BrCl-flav possesses important anti-biofilm activity against A. baumannii and could represent a reliable solution to treat bacterial biofilm-dependent infections.
It has been shown so far that BrCl-flav has important antibacterial potential against ESKAPE pathogens. For the development of new therapeutic solutions, it is important to know the mechanism of action. We previously showed that BrCl-flav interferes with the cell membrane integrity of non-pathogenic bacterial strains [24]. Therefore, we investigated the effect of BrCl-flav on two multidrug-resistant S. aureus and E. coli strains using fluorescence microscopy. Cells with a damaged membrane considered to be dead or dying will appear stained red, while cells with an intact membrane will stain green when a Live/Dead BacLight bacterial viability kit is used. Exposing the Gram-positive and Gram-negative bacterial cells to concentrations of BrCl-flav equivalent to MBC revealed that the number of red fluorescent cells increased in a time-dependent manner. A percentage of 100% red fluorescent cells was reported for E. coli after approximatively 1 h and for S. aureus after 2 h of exposure to BrCl-flav, suggesting massive uptake of PI fluorescent dye and severe cell membrane damage. To verify the membrane type mechanism of action, SEM was employed to determine morphological damage induced by BrCl-flav exposure. Severe morphological alterations of S. aureus and E. coli cells were revealed by SEM analysis, together with cellular debris, sustaining the hypothesis that BrCl-flav targets the cellular membrane, inducing membrane structure alteration and cell lysis. Those effects could be a consequence of other mechanisms of action [19]. For instance, BrCl-flav could interact with purinic bases from bacterial DNA with the electrophilic C(2) atom of the 1,3-dithiolium ring after a Maxam-Gilbert mechanism, causing cell death followed by cell lysis [45]. However, our data support the hypothesis of a primary membrane-type mechanism of action. Thus, fluorescence dynamics tests showed that the S. aureus cell membrane is permeabilized for PI after only 2 min of exposure to BrCl-flav, while the fluorescent dye penetrates E. coli cell membranes within the first 3 min of exposure (for technical reasons it was not possible to obtain relevant pictures before 3 min). In addition, our previous investigations revealed that the antibacterial activity of BrCl-flav compared with the precursor 3-N,Ndiethylaminodithiocarbamates flavanone is the consequence of the appearance of the third fused cycle, the 1,3-dithiolium ring [22]. 1,3-Dithiolium systems are well known for the reactivity of the C(2)-position towards nucleophiles [46,47]. Thus, the excellent antibacterial properties of BrCl-flav could be the result of the interaction between nucleophilic moieties of membrane constituents with the electrophilic C(2) atom of the 1,3-dithiolium ring.
The treatment of infections caused by bacteria that are resistant to multiple antibiotics (e.g., MRSA) is a real medical challenge, and very few therapeutic options are available. One option lies in the combination of antibiotics with new compounds to exploit potential synergistic effects [48]. Therefore, we used in our study a MRSA strain (S. aureus medbio1-2012) resistant to penicillin, ciprofloxacin and tetracycline. Combinations of those three antibiotics with BrCl-flav were tested to evidence possible synergistic effects. Our results showed that additive effects were recorded for all three tested antimicrobials. However, synergistic combinations were identified only for penicillin (FICI values: 0.5-0.264). To verify the synergistic effect, one combination, BrCl-flav-penicillin (0.03/32 µg/mL), was further used in a time-kill assay. The results revealed a significant bactericidal effect of penicillin in combination with BrCl-flav with no viable cells recorded after 24 h, while the two agents used separately showed no effects on S. aureus medbio1-2012 viability. These findings agree with previously reported data that showed synergistic effects of natural and synthetic flavonoids such as rutin, morin, quercetin, galangin, phenolic compound, substituted chalcones or pentacyclic triterpenoids with different antibiotics against S. aureus [49][50][51][52][53]. The synergistic effects could be explained by different cell structure targeted by the two antimicrobials used in our study; penicillin inhibits cell wall synthesis, while BrCl-flav induces membrane alterations, enhancing penicillin uptake. Our results suggest that BrCl-flav could be a potential solution to solve a serious problem caused by bacterial resistance to β-lactam antibiotics.
Our data showed up to this point that BrCl-flav could be a reliable alternative to develop effective drugs used to combat ESKAPE pathogens. Therefore, a cytotoxicity study was performed with hepatocytes, macrophages, epithelial and epithelial-mucusproducing cells to assess the effect of BrCl-flav on the cell phenotypes that would be in contact with the compound after being orally absorbed. Results revealed that macrophages and hepatocytes are more sensitive to BrCl-flav when compared to intestinal epithelial and goblet-like cells. Moreover, the MICs recorded for different tested bacterial strains such as MRSA, Streptococcus spp. prxbio9, S. pneumoniae prxbio10, E. faecium medbio2-2012, E. coli medbio4-2013 and Haemophillus spp. prxbio13 were relatively low compared to IC 50 values registered for the tested cell lines. For Caco-2 cells, the compound exerted very low toxicity with an estimated IC 50 value of around 80 µg/mL. Surprisingly, BrCl-flav increased cell viability at concentrations ranging from 5 to 25 µg/mL. This result is surprising but reproducible and may be due to BrCl-flav metabolization by the cells. Altogether, our findings suggest that BrCl-flav could be a reliable candidate for the formulation of new effective antimicrobials.
The effects of BrCl-flav (at non-cytotoxic concentrations of 0.1, 0.5 and 1 µg/mL) on inflammation were studied by measuring two cytokines, TNF-α and IL10, on U937 cells differentiated into macrophages and stimulated by LPS. TNF-α is a pro-inflammatory cytokine, necessary for host defense against infectious agents [54]. However, excessive inflammatory cytokine production results in tissue damage, toxicity and cell death. Proinflammatory cytokine synthesis by macrophages can also be modulated and inhibited by cytokines such as IL10 (anti-inflammatory cytokine) [55]. In our study, dexamethasone, a molecule belonging to the glucocorticoid family, was used as a positive control of inhibition inflammation. Indeed, glucocorticoids are considered anti-inflammatory and protective molecules due to their capacity to inhibit gene expression of pro-inflammatory cytokines and are widely used for the treatment of inflammation [56]. The results obtained showed that BrCl-flav exerts a pro-inflammatory effect due to the enhancement of TNF-α secretion and the reduction of IL10 production. Inflammation is a normal protective response to kill infectious agents. For example, Flynn et al. demonstrated that TNF-α plays an important role in host resistance to mycobacterial infection [57]. Moreover, aza-alkyl lysophospholipids, just like BrCl-flav, can induce TNF-α production and IL10 inhibition in peripheral blood-derived monocytes and therefore have a beneficial action in fighting microbial infections [58].

Antibacterial Agents
The synthesis of tricyclic flavonoid BrCl-flav has been described in detail in our previous report [59]. NMR, MS, IR and elemental analysis were used to determine the structure and purity (>99%) of the final compound. The stability of BrCl-flav towards Mueller-Hinton broth (Scharlau, Barcelona, Spain) and phosphate buffer saline (PBS) was monitored by UV-Vis spectroscopy. The tricyclic flavonoid was stable during the performed antimicrobial tests.
The antibiotics used in this study were purchased from local suppliers (Carl Roth and Sigma-Aldrich, Darmstadt, Germany, Scharlau, Barcelona, Spain); chloramphenicol and gentamicin were used as reference antibiotics for minimum inhibitory concentration assays, while ciprofloxacin, penicillin and tetracycline were used for combination tests; different antibiotics (Oxoid, Basingstoke, UK) were used for the antimicrobial susceptibility assay according to CLSI guidelines [60].

Disk Diffusion Method
The antibiotic resistance profile of the clinical isolates used in this study was determined by Kirby-Bauer's disk diffusion method on Muller-Hinton agar, following the guidelines of Clinical and Laboratory Standards Institute [60]. The results are presented in Table S1 (Supplementary Files).

Determination of Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the broth microdilution method as we previously described [24]. Briefly, BrCl-flav was serially diluted in MHB (a concentration range from 0.12 to 250 µg/mL) using 96-well plates and DMSO (Merck, Darmstadt, Germany) as a solvent. Inoculum, represented by bacterial suspensions with a cell density adjusted to approximately 2 × 10 6 CFU/mL (CFU = colony-forming units) was added into each well of the microplate. DMSO at concentrations ranging from 0.006 to 12.5% (v/v), MHB medium and inoculum served as the control. MHB medium and inoculum were used as growth control. S. aureus ATCC 43300 and K. pneumoniae ATCC BAA-1705 were used as reference strains. Chloramphenicol and gentamicin were used as reference antibiotics. The lowest concentration with no visible growth after 24 h at 37 • C was considered as the MIC. To evaluate MBC, a volume of 15 µL taken from each well with no visible growth was inoculated on MHA plates. The MBC was considered as the lowest concentration at which bacteria failed to grow after plating onto MHA.

Growth Inhibition Assay
The effect of BrCl-flav on bacterial growth was assessed using the method described by Babii et al. [24], with some modifications. A volume of 250 µL from an overnight preculture was added in 25 mL MHB supplemented with BrCl-flav at final concentrations equivalent to 1 2 MIC, MIC and 2 × MIC (final cell density of approximately 10 6 CFU/mL). Inoculated MHB medium supplemented with DMSO was used as a control. All flasks were incubated at 37 • C for 24 h under shaking conditions (190 rpm). The growth was monitored by measuring the optical density (OD) at 600 nm of samples taken at each hour up to 12 h and at 24 h, using a Beckman Coulter DU 730 spectrophotometer (Danaher Corporation, Washington, DC, USA).

Time-Kill Kinetic Assay
The killing rate of BrCl-flav was determined by measuring the reduction in the number of CFU per mL, following the procedure adapted after Aqil et al. [61]. The bacterial cells were harvested by centrifugation (4000 rpm, 20 min) and washed twice with PBS. Cell density was adjusted to obtain a bacterial suspension of approximately 10 8 CFU/mL. A volume of 100 µL from this cell suspension served as inoculum and was added to 10 mL PBS supplemented with a concentration of BrCl-flav equivalent to MBC value (final cell density of approximately 10 6 CFU/mL). The control was prepared similarly using DMSO at the appropriate concentration. All flasks were incubated at 37 • C for 24 h under shaking conditions (190 rpm). Samples were removed at each hour up to 12 h and at 24 h, serially diluted in PBS, plated onto MHA and incubated at 37 • C for 24 h. Afterwards, the CFU per mL was calculated by colony counting and transformed into log10 values. Bactericidal activity was defined as a ≥3 log10 reduction in the total CFU/mL from the original inoculum. Time-kill curves were constructed by plotting mean colony counts versus time [24].

In Vitro Anti-Biofilm Activity Assay
The clinical isolates ability to form biofilms was assessed using the method proposed by Onsare et al. [62] with some modifications. Static biofilm formation was evaluated in 96-well plates with lids (Becton Dickinson, Franklin Lakes, NJ, USA). A volume of 200 µL bacterial cells in MHB (approximately 10 6 CFU/mL) was added into each well of a microtiter plate and cultured for 24 h at 37 • C. Uninoculated MHB medium served as control. The quantification of the bacterial biofilms was performed using a crystal violet assay [63]. A Beckman Coulter spectrophotometer was used to determine the ODs at a wavelength of 595 nm. To identify the strains with biofilm-forming ability, the following formula was used: ODc = average OD of negative control + (3 × standard deviation of negative control) The clinical isolates were classified as follows: OD ≤ ODc = non-adherent strains, ODc < OD ≤ 2 × ODc = weakly adherent strains, 2 × ODc < OD ≤ 4 × ODc = moderately adherent strains, 4 × ODc < OD = strongly adherent strains [64].
The same method as presented above was used to assess the anti-biofilm activity of BrCl-flav. For the inhibition of biofilm formation, MHB supplemented with different concentrations of antibacterial agent (samples) was inoculated with bacterial suspensions (final density adjusted to approximately 1 × 10 6 CFU/mL). Inoculated MHB supplemented with DMSO served as a control. After incubation, the developed biofilm was assessed using crystal violet staining. For biofilm disruption assay, inoculated MHB was incubated for 24 h; following incubation, the culture medium was carefully discarded, and the wells were washed three times with PBS to remove non-attached cells. The same amount of MHB supplemented with different concentrations of BrCl-flav (samples) and DMSO (control) were added in each well, and the plate was incubated for another 24 h at 37 • C.
Biofilm formation as well as biofilm disruption in the presence of BrCl-flav were expressed as a percentage of the control biofilm formed in the absence of tested antimicrobial agent (considered as 100%), according to the following formula: Bio f ilm inhibition (%) = OD595 nm control well with DMSO − OD595 nm experimental well with BrCl − flav OD595 nm control well with DMSO

Evaluation of Cell Membrane Integrity
The integrity of cell membranes was assessed using fluorescence microscopy and the Live/Dead BacLight Bacterial Viability Kit (Invitrogen, Waltham, MA, USA), following the manufacturer's instructions. Bacterial cells were harvested by centrifugation (4000 rpm, 20 min) and washed twice with PBS. Cell density was adjusted to approximately 10 8 CFU/mL in PBS. A volume of 2 mL from the bacterial suspension was incubated in the presence of BrCl-flav at a concentration equivalent to the MBC value (125 µg/mL for E. coli medbio4-2013 and 62.5 µg/mL for S. aureus medbio1-2012) at 37 • C for 4 h under shaking conditions (190 rpm). Cells in PBS supplemented with DMSO served as control. Samples was taken at 30 min, 1, 1.5, 2, 3 and 4 h, washed twice with PBS and stained with SYTO 9 and propidium iodide (PI) for 15 min in the dark. The fluorescent cells were counted using a DM100 LED fluorescence microscope (Leica, Solms, Germany) and an I3 blue excitation range filter cube (BP 450 ± 490 nm band-pass filter). At least five random, independent images were captured per sample, and the ratio between green/red fluorescent cells and total cells was calculated as percentage. The dynamics of dye penetration into cells exposed to BrCl-flav at concentration equivalent to MBC was performed following the same protocol. Photographs were taken every minute, up to 10 min using a DM100 LED fluorescence microscope.

Scanning Electron Microscopy
Suspensions in PBS of the logarithmic growth phase of S. aureus medbio1-2012 and E. coli medbio4-2013 cells (approximately 1 × 10 8 CFU/mL) were incubated in PBS for 6 h in the presence of BrCl-flav (final concentrations equivalent to MBC values). DMSO served as a control. Samples (untreated and treated bacterial cells) were prepared for SEM analysis following the protocol previously described [23] and examined with a Tescan Vega II SBH microscope using the secondary electron detector at an acceleration voltage of 30 kV.

Checkerboard Assay
The effect of BrCl-flav in combination with different antibiotics against S. aureus methicillin-resistant strain medbio1-2012 was assessed using the checkerboard microdilution method [65], with some modifications. Briefly, the synthetic flavonoid and the antibiotics were serial two-fold diluted in Eppendorf microtubes containing MHB medium. The concentrations of the antibiotics and BrCl-flav were selected based on previously determined MIC values. A volume of 50 µL of each compound dilution was added to each well of a microplate to obtain antibiotics and BrCl-flav concentration ranges of 0.125-256 µg/mL (penicillin), 1.22-156.25 µg/mL (ciprofloxacin), 0.15-19.53 µg/mL (tetracyclin) and 0.0002-0.96 µg/mL (BrCl-flav). Next, 100 µL of bacterial suspension was added to each well to reach a final cell density of approximatively 10 6 CFU/mL. Inoculated MHB medium was used as a control. After 24 h of incubation at 37 • C, the bacterial growth was assessed visually in the presence of resazurin (0.05%) (Difco, Tucker, GA, USA). The MIC of the combined antibacterial agents was considered the lowest concentration at which no visible growth was observed.

Inflammation Study
The purpose of this study was to investigate the effects of BrCl-flav on the inflammatory responses to lipopolysaccharide (LPS)-induced U937 macrophage cells. For this, U937 cells were differentiated into macrophages as previously described, plated into 12-well plates at a density of 2 × 10 6 viable cells/well and incubated with LPS from E. coli O26:B6 (L2654, Sigma-Aldrich, St. Louis, MO, USA) at 50 µg/mL and BrCl-flav at the concentrations 0.1, 0.5 and 1 µg/mL for 4 h. RPMI was used as a negative control for inflammation, LPS alone as an inflammation control and 50 µg/mL-LPS/20 µM-dexamethasone as a positive inflammation inhibition control. The cell culture supernatants were then collected for further analysis of the secreted cytokines and stored at −20 • C. The levels of TNF-α and IL10 in macrophage culture media were measured by commercially available enzymelinked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA), by comparing the obtained optical densities (microplate spectrophotometer SpectraMax ® iD3) to the standard curve.

Statistical Analysis
Experiments were performed in triplicate. The statistical evaluation of the results was carried out by Dunnett's multiple comparisons test, the data being presented as mean (n = 3) ± SEM. For the inflammation study, a Mann-Whitney test to compare the LPS control to the dexamethasone control and sample was used, and data are presented as mean (n = 4) ± SD. All data were analyzed using GraphPad Prim 9 software (GraphPad Software, Inc., La Jolla, CA, USA). Differences between groups were considered significant when p < 0.05.

Conclusions
BrCl-flav showed important in vitro inhibitory activity against antibiotic-resistant "priority pathogens" such as S. aureus, A. baumannii, P. aeruginosa and E. faecium. Additionally, the synthetic flavonoid expressed strong bactericidal activity with total kill in a very short time. Our compound inhibited biofilm formation and displayed important biofilm disruption potential against A. baumannii. Those effects are induced most likely by membrane integrity damage and cell lysis. BrCl-flav expressed synergistic antibacterial activity in combination with penicillin against an MRSA clinical isolate. Additionally, BrCl-flav showed low cytotoxicity and pro-inflammatory effects. These very promising results suggests that BrCl-flav is in fact a compound with potent antibacterial activity against representative ESKAPE pathogens, which may be used to develop new effective antimicrobial agents able to bypass bacterial multidrug resistance.