Biofilm Eradication by Symmetrical Selenoesters for Food-Borne Pathogens.

Infections caused by Salmonella species and Staphylococcus aureus represent major health and food industry problems. Bacteria have developed many strategies to resist the antibacterial activity of antibiotics, leading to multidrug resistance (MDR). The over-expression of drug efflux pumps and the formation of biofilms based on quorum sensing (QS) can contribute the emergence of MDR. For this reason, the development of novel effective compounds to overcome resistance is urgently needed. This study focused on the antibacterial activity of nine symmetrical selenoesters (Se-esters) containing additional functional groups including oxygen esters, ketones, and nitriles against Gram-positive and Gram-negative bacteria. Firstly, the minimum inhibitory concentrations of the compounds were determined. Secondly, the interaction of compounds with reference antibiotics was examined. The efflux pump (EP) inhibitory properties of the compounds were assessed using real-time fluorimetry. Finally, the anti-biofilm and quorum sensing inhibiting effects of selenocompounds were determined. The methylketone and methyloxycarbonyl selenoesters were the more effective antibacterials compared to cyano selenoesters. The methyloxycarbonyl selenoesters (Se-E2 and Se-E3) showed significant biofilm and efflux pump inhibition, and a methyloxycarbonyl selenoester (Se-E1) exerted strong QS inhibiting effect. Based on results selenoesters could be promising compounds to overcome bacterial MDR.


Introduction
The emergence of multidrug resistant pathogens is a major problem, leading to a progressive reduction in the efficiency of many antibacterial agents. This phenomenon is a serious challenge in public healthcare and medicine [1].
The most frequent multidrug resistance (MDR) mechanisms enable the resistant bacteria to achieve one or several of the following effects: (a) limited uptake of drug; (b) target modification; (c) drug inactivation; and (d) active efflux mediated by efflux pumps. Some efflux pumps are expressed
Gram-positive Staphylococcus aureus American Type Culture Collection (ATCC) 25923 was used as the methicillin-susceptible reference bacterial strain, and the methicillin and ofloxacin-resistant S. aureus 272123 clinical isolate (MRSA), which was kindly provided by Prof. Dr. Leonard Amaral (Institute of Hygiene and Tropical Medicine, Lisbon, Portugal), was used in the assays.
For QS tests we used Chromobacterium violaceum 026 (CV026) as a sensor strain and Enterobacter cloacae 31298 as a N-acyl-homoserine lactone (AHL) producer clinical bacterial isolate. If C. violaceum reaches a high cell density, it produces violacein, which is a purple pigment [26,27].

Cell Line
MRC-5 human embryonal lung fibroblast cell line (ATCC CCL-171) was purchased from LGC Promochem, Teddington, UK. The cells were cultured in Eagle's Minimal Essential Medium (EMEM, containing 4.5 g/L glucose) supplemented with a non-essential amino acid mixture, a selection of Scheme 1. Chemical structure of the symmetrical selenoesters evaluated.
Gram-positive Staphylococcus aureus American Type Culture Collection (ATCC) 25923 was used as the methicillin-susceptible reference bacterial strain, and the methicillin and ofloxacin-resistant S. aureus 272123 clinical isolate (MRSA), which was kindly provided by Prof. Dr. Leonard Amaral (Institute of Hygiene and Tropical Medicine, Lisbon, Portugal), was used in the assays.
For QS tests we used Chromobacterium violaceum 026 (CV026) as a sensor strain and Enterobacter cloacae 31298 as a N-acyl-homoserine lactone (AHL) producer clinical bacterial isolate. If C. violaceum reaches a high cell density, it produces violacein, which is a purple pigment [26,27].

Cell Line
MRC-5 human embryonal lung fibroblast cell line (ATCC CCL-171) was purchased from LGC Promochem, Teddington, UK. The cells were cultured in Eagle's Minimal Essential Medium (EMEM, containing 4.5 g/L glucose) supplemented with a non-essential amino acid mixture, a selection of vitamins, and 10% heat-inactivated fetal bovine serum. The cell lines were incubated at 37 • C, in a 5% CO 2 , 95% air atmosphere.

Determination of Minimum Inhibitory Concentrations by Microdilution Method
The minimum inhibitory concentrations (MICs) of compounds were determined according to the Clinical and Laboratory Standard Institute guidelines (CLSI) [28]. MIC values of the compounds were determined by visual inspection. The solvent was also assayed to ensure there was no antibacterial effect and the concentration (1 v/v%) applied in the assays had no antibacterial activity. DMSO was used at subinhibitory concentration (1 v/v%) in the assays.

Cytotoxicity Assay
The adherent MRC-5 human embryonal lung fibroblast cells were cultured in 96-well flat-bottomed microtiter plates, using EMEM supplemented with 10% heat-inactivated fetal bovine serum. The density of the cells was adjusted to 1 × 10 4 cells in 100 µL per well, the cells were seeded overnight at 37 • C, 5% CO 2 , then the medium was removed from the plates containing the cells, and the dilutions of selenocompounds previously made in a separate plate were added to the cells in 200 µL.
The culture plates were incubated at 37 • C for 24 h; at the end of the incubation period, 20 µL of MTT (thiazolyl blue tetrazolium bromide, Sigma) solution (from a stock solution of 5 mg/mL) was added to each well. After incubation at 37 • C for 4 h, 100 µL of sodium dodecyl sulfate (SDS; Sigma) solution (10% in 0.01 M HCI) was added to each well and the plates were further incubated at 37 • C overnight. Cell growth was determined by measuring the optical density (OD) at 540/630 nm with Multiscan EX ELISA reader (Thermo Labsystems, Cheshire, WA, USA). Inhibition of the cell growth was determined according to the formula below: Results are expressed in terms of IC 50 , defined as the inhibitory dose that reduces the growth of the cells exposed to the tested compounds by 50%.

Resistance Modulation Assay
The resistance modulation effect of compounds with ciprofloxacin (CIP) and tetracycline (TET) antibiotics were evaluated by the checkerboard method on S. aureus strains. Briefly, CIP or TET was diluted in a 96-well microtiter plate by two-fold serial dilution in MH broth and then the compounds were added at subinhibitory concentrations ( 1 2 MIC). In this assay, only the tested compounds with well-defined MIC values were tested. Finally, 10 −4 dilution of the overnight bacterial culture in MH was added to each well. The final volume was 200 µL in each well. The microtiter plates were incubated at 37 • C for 18 h. MIC values in the presence of the antibiotics alone and in combination with Se-compounds were determined by visual inspection.

Real-Time Ethidium Bromide Accumulation Assay
The impact of compounds on EB accumulation was determined by the automated EB method using a CLARIOstar Plus plate reader (BMG Labtech, UK). Firstly, the bacterial strain was incubated until it reached an optical density (OD) of 0.6 at 600 nm. The culture was washed with phosphate buffered saline (PBS; pH 7.4) and centrifuged at 13,000× g for 3 min, the cell pellet was re-suspended in PBS. The compounds were added at 1 2 MIC concentration to PBS containing a non-toxic concentration of EB (1 µg/mL). Then, 50 µL of the EB solution containing the compound were transferred into 96-well black microtiter plate (Greiner Bio-One Hungary Kft, Hungary), and 50 µL of bacterial suspension (OD 600 0.6) were added to the each well. Then, the plates were placed into the CLARIOstar plate reader, and the fluorescence was monitored at excitation and emission wavelengths of 530 nm and 600 nm every minute for one hour on a real-time basis. From the real-time data, the activity of the compounds, namely the relative fluorescence index (RFI) of the last time point (minute 60) of the EB accumulation assay, was calculated according to the following formula: where RF treated is the relative fluorescence (RF) at the last time point of EB retention curve in the presence of an inhibitor, and RF untreated is the RF at the last time point of the EB retention curve of the untreated control having the solvent control (DMSO).

Measuring Biofilm Formation Using Crystal Violet
The anti-biofilm effect of the tested compounds against S. aureus strains and wild-type S. Typhimurium SE01 was measured using crystal violet (CV; 0.1% (v/v)). This dye is used to detect the total biofilm biomass formed. Overnight cultures were diluted to OD of 0.1 at 600 nm in TSB medium. Then, the bacterial cultures were added to 96-well microtiter plates and the compounds were added at 1 2 MIC concentration. The final volume was 200 µL in each well. The microtiter plates were incubated at 30 • C for 48 h with gentle agitation (100 rpm). After the incubation period, TSB medium was discarded, and the plates were washed with tap water to remove unattached cells. Then 200 µL crystal violet was added to the wells and incubated for 15 min at room temperature. Then, CV was removed from the wells and the plates were washed again with tap water, and 200 µL of 70% ethanol was added to the wells. Finally, the biofilm formation was determined by measuring the OD at 600 nm using Multiscan EX ELISA plate reader (Thermo Labsystems, Cheshire, WA, USA). The anti-biofilm effect of compounds was expressed in the percentage (%) of decrease in biofilm formation.

Quorum Sensing (QS) Assay
The QS inhibitory effect of selenocompounds was examined on the AHL producer E. cloacae strain and C. violaceum sensor bacterial strain. These strains were inoculated in parallel. The QS inhibition was monitored by agar diffusion method on LB* agar plate as described previously [29]. Filter paper discs (7.0 mm in diameter) were placed between the parallel inoculated strains and impregnated with 10 µL compounds. Starting concentration of the compounds was 1 2 MIC. The agar plates were incubated at room temperature (20 • C) for 24-48 h and the inhibition of violacein production was measured.

Statistical Analysis
The values are given as the mean ± standard deviation (SD) determined for three replicates from three independent experiments. The analysis of data was performed using SigmaPlot for Windows Version 12.0 software (Systat Software Inc, San Jose, CA, USA), applying the two-tailed t-test.

Determination of Minimum Inhibitory Concentrations by Microdilution Method
Based on the MIC values, the Se-compounds were more effective against S. aureus strains. The most effective compounds were the ketone selenoesters Se-K1, Se-K2, and Se-K3 on the reference S. aureus ATCC 25923, showing an MIC of 0.39 µM. Interestingly, these three derivatives share a common moiety, namely a methylketone group in the alkyl moiety bound to the selenium atom. The replacement of this methylketone by a cyano or by a methyloxycarbonyl moiety reduced the activity dramatically, as the MICs were 16-and 32-fold higher against S. aureus ATCC 25923, respectively; with the exception of the trisubstituted derivative Se-C3, as its MIC was only 4-fold higher than the MIC of the trisubstituted methylketone Se-K3. The same tendency, but accentuated, was observed in S. aureus MRSA 272123, where the MIC values of the methylketone derivatives were in the range of 64-to 128-fold lower than the equivalent methyloxycarbonyl derivatives and in the range of 16-to 32-fold lower than the equivalent nitrile-containing selenoesters. The compounds showed a slight antibacterial effect on Salmonella strains. The most effective compound was Se-C3 on SE01, SE02, and SE03 strains, showing an MIC of 12.5 µM (Table 1). Importantly, the MIC to the efflux knockout strains was unchanged suggesting that the compounds were not substrates of the AcrAB-TolC efflux pump.

Resistance Modulation Assay
As the Se-compounds were more effective on S. aureus strains, these strains were selected for combination studies with reference antibiotics. Selenocompound Se-E3 showed synergism with TET on the methicillin-susceptible S. aureus ATCC 25923.
Surprisingly, all selenocompounds showed synergism with TET on the methicillin-resistant S. aureus strain. Se-E3 and Se-C2 were the most effective compounds in combination with TET, as they reduced the MIC value of TET against this MRSA strain to a value 32-fold lower. Additionally, compounds Se-E1 and Se-C1 also exerted a noteworthy reduction of the MIC value, of 16-fold in this case. On the other hand, Se-K1 and Se-E3 showed synergism with CIP on the MRSA strain, achieving a 2-fold reduction of the MIC value (Table 2).

Ethidium Bromide Accumulation Assay
The activity of the selenocompounds on EB accumulation was determined by the automated EB method on sensitive and resistant S. aureus and S. Typhimurium SE01, -02, -03, and -39 strains. The relative fluorescence index was calculated based on the means of relative fluorescence units (RFUs ;  Table 3). In case of Salmonella strains, the Se-compounds increased the intracellular EB accumulation more efficiently on the tolC gene inactivated mutant S. Typhimurium SE39 after 60 min. In contrast, RFUs obtained in the presence of Se-compounds were the lowest on the wild-type S. Typhimurium SE01. CCCP, the reference efflux pump inhibitor (EPI) was the positive control in case of Salmonella and reference S. aureus strain. In addition, verapamil was applied as reference EPI on S. aureus MRSA. The solvent DMSO served as a negative control in the experiments. Se-E2 significantly increased the intracellular EB accumulation on S. Typhimurium SE02, -03, and -39. In addition, a significant EB accumulation was observed for Se-K3 on S. Typhimurium SE39 (Figure 1).  In case of the reference S. aureus and resistant MRSA strain the highest RFUs were recorded in the presence of Se-E3, for this reason this compound exerted the most prominent EPI activity. In addition, methylcarbonyl selenoesters Se-E1 and Se-E2 were proven to be effective in both S. aureus strains ( Figure 2).

Microorganisms 2020, 8, x FOR PEER REVIEW 9 of 15
In case of the reference S. aureus and resistant MRSA strain the highest RFUs were recorded in the presence of Se-E3, for this reason this compound exerted the most prominent EPI activity. In addition, methylcarbonyl selenoesters Se-E1 and Se-E2 were proven to be effective in both S. aureus strains (Figure 2).

Measuring Biofilm Formation Using Crystal Violet
The effect of selenocompounds on biofilm formation of sensitive and resistant S. aureus strains and wild-type S. Typhimurium SE01 was evaluated. The biofilm inhibition (%) was calculated based on the mean of absorbance units (AUs). The absorbance expressed in AUs was the following on non-treated samples: reference S. aureus showed an absorbance of 2.4 ± 0.1, the resistant S. aureus

Measuring Biofilm Formation Using Crystal Violet
The effect of selenocompounds on biofilm formation of sensitive and resistant S. aureus strains and wild-type S. Typhimurium SE01 was evaluated. The biofilm inhibition (%) was calculated based on the mean of absorbance units (AUs). The absorbance expressed in AUs was the following on nontreated samples: reference S. aureus showed an absorbance of 2.4 ± 0.1, the resistant S. aureus exhibited 1.3 ± 0.1 AU, and the wild-type S. Typhimurium presented 2.

Quorum Sensing (QS) Assay
The sensor strain C. violaceum 026 and the AHL producer strains E. cloacae 31298 were inoculated as parallel lines. Interactions between the strains and compounds were evaluated for the reduction in the size of the zone of pigment production and the zone of growth inhibition of the affected strains, in millimeters. Promethazine (PMZ) was applied as a QS inhibitor and its zone of inhibition was 46 mm. Selenocompounds Se-K1, Se-K2, and Se-E1 had QS inhibitory effect. In addition, Se-K1 and Se-K2 showed inhibition zones of 37 mm and 40 mm, respectively, whereas the methyloxycarbonyl selenoester Se-E1 was the most effective QS inhibitor with an inhibition zone of 41 mm (Figure 4).

Quorum Sensing (QS) Assay
The sensor strain C. violaceum 026 and the AHL producer strains E. cloacae 31298 were inoculated as parallel lines. Interactions between the strains and compounds were evaluated for the reduction in the size of the zone of pigment production and the zone of growth inhibition of the affected strains, in millimeters. Promethazine (PMZ) was applied as a QS inhibitor and its zone of inhibition was 46 mm. Selenocompounds Se-K1, Se-K2, and Se-E1 had QS inhibitory effect. In addition, Se-K1 and Se-K2 showed inhibition zones of 37 mm and 40 mm, respectively, whereas the methyloxycarbonyl selenoester Se-E1 was the most effective QS inhibitor with an inhibition zone of 41 mm (Figure 4).

Cytotoxicity Assay on Normal Human Fibroblasts
In order to determine the toxicity and safety of the selenocompounds on human cells, a cytotoxicity assay was performed using normal MRC-5 human embryonal lung fibroblast cells (Table 4).

Discussion
In case of MIC determination, the symmetrical selenoesters evaluated herein (whose seleniumbound alkyl moiety contains functional groups as a ketone, oxygen ester or nitrile) were more effective on sensitive and resistant S. aureus strains compared to the four S. Typhimurium bacterial strains. This suggests that these symmetrical selenoesters are more active against Gram-positive

Cytotoxicity Assay on Normal Human Fibroblasts
In order to determine the toxicity and safety of the selenocompounds on human cells, a cytotoxicity assay was performed using normal MRC-5 human embryonal lung fibroblast cells (Table 4).

Discussion
In case of MIC determination, the symmetrical selenoesters evaluated herein (whose selenium-bound alkyl moiety contains functional groups as a ketone, oxygen ester or nitrile) were more effective on sensitive and resistant S. aureus strains compared to the four S. Typhimurium bacterial strains. This suggests that these symmetrical selenoesters are more active against Gram-positive bacteria (as Staphylococcus aureus) than against Gram-negative bacteria (as Salmonella enterica serovar Typhimurium). This fact is in accordance with the antibacterial activity of non-symmetrical selenoesters, which were evaluated in a previous work of the group [27]; only three non-symmetrical ketone selenoesters (9-11 in [27]) were active against S. aureus, whereas none of them were active against Escherichia coli. Interestingly, all of them were active against Chlamydia trachomatis (Gram-negative), but since Chlamydia is an intracellular bacterium this may affect its sensitivity to the compounds [27].
The methylketone selenoesters Se-K1, Se-K2, and Se-K3 were the most potent antibacterials on reference S. aureus. In contrast, the methyloxycarbonyl selenoesters Se-E1, Se-E2, and Se-E3 and the cyano selenoesters Se-C1 and Se-C2 showed strong resistance modulating activity with tetracycline against the MRSA strain. Comparing the antibacterial activity with the previously reported data [27], two observations are of interests. First, the symmetrical selenoesters are more potent antibacterials against S. aureus ATCC 25923 than the respective asymmetrical derivatives. This is observed when we compare the 0.39 µM MIC values of Se-K1, Se-K2, and Se-K3 with the 3.12 µM MIC value of 9 in [27] (methylketone selenoesters), and the 12.5 µM MIC values of Se-E1, Se-E2 and Se-E3 with 7 in [27], which was not active at concentrations below 100 µM (methyloxycarbonyl selenoesters). Second, the symmetrical methyl selenoesters 2-5 in [27] were not active against S. aureus ATCC 25923 (MIC > 100 µM), whereas all the functionalized selenoesters evaluated in this work (-CH 2 COCH 3 , -CH 2 COOCH 3 , -CH 2 CN) showed MIC values against this strain at 12.5 µM or lower. This indicates that these second-generation selenoesters have improved antibacterial activity compared with those that have been previously reported.
If we compare the antibacterial activity of the symmetrical selenocompounds with its toxicity against MRC-5 normal embryonal lung fibroblast cell line, we observe that the MIC values of the compounds against S. aureus ATCC 25923 were lower than the IC 50 values against this cell line.
In the resistance modulation assay, the selenocompounds were tested at 1 2 of their MIC in combination with tetracycline and ciprofloxacin in the two S. aureus strains (ATCC 25923 and MRSA 272123). As mentioned previously, all compounds were able to modulate the activity of tetracycline against S. aureus MRSA 272123. The results were somehow comparable with the antibacterial activity. Interestingly, the -CH 2 COOCH 3 and -CN containing symmetrical selenoesters were more potent modulators than the -CH 2 COCH 3 selenoesters (X-fold reductions of 2-4, 8-32, and 4-32, respectively). However, as MIC values of the selenocompounds were higher against this S. aureus strain, only Se-C1 and Se-C2 could be used at a safe concentration (25 µM, non-toxic in MRC-5 cells) with a noteworthy effect (16-and 32-fold reduction of MIC value of tetracycline).
Real-time EB accumulation was applied in order to monitor the EPI activity of the compounds. The intracellular EB accumulation was the highest on the tolC gene inactivated mutant S. Typhimurium SE39, and the lowest EB accumulation was obtained in the wild-type S. Typhimurium SE01 in the presence of methyloxycarbonyl selenoester Se-E2. This compound significantly increased the EB accumulation in the efflux pump gene inactivated (∆acrA, ∆acrB, and ∆tolC) mutant S. Typhimurium strains due to efflux independent mechanisms, e.g., membrane destabilizing effect. In addition, methyloxycarbonyl selenoester Se-E3 showed significantly effective pump inhibition on sensitive (p < 0.001) and resistant (p = 0.001) S. aureus strains. Unfortunately, these two Se-compounds have to be applied at a high concentration (50 µM, which is 1 2 of their MIC) against S. Typhimurium (Se-E2) or S. aureus MRSA 272123 (Se-E3), respectively. Compound Se-E3 could be used in this application against S. aureus ATCC 25923, as in this case its concentration would be 6.25 µM, much lower.
Regarding the anti-biofilm effect, the methyloxycarbonyl selenoester Se-E3 showed significant biofilm inhibition on both of sensitive and resistant S. aureus strains. Furthermore, the methylketone selenoester Se-K3 was the most effective anti-biofilm agent on resistant S. aureus MRSA. In addition, Se-K1 was also interesting, as it showed a biofilm inhibiting effect higher than 50% against MRSA. It was surprising that Se-K2 promoted the biofilm formation of S. aureus MRSA, because it has the same chemical formula as Se-K1 (both are 2-oxopropyl selenodiesters); they only differ in the substitution pattern at the phenyl ring, such that Se-K1 has a para substitution (1,4) and Se-K2 has a meta substitution (1,3). It is interesting to see how such a small change in the substitution pattern at the core phenyl ring leads to completely different activities. What is more, in Se-K2 the inclusion of a third -COSeCH 2 COCH 3 at the position five of the core phenyl ring led to Se-K3, recovering the biofilm inhibition in respect to Se-K2 and enhancing it in respect to Se-K1. In the case of the methyloxycarbonyl selenoesters, only the trisubstituted derivative Se-E3 was capable of significantly inhibiting the biofilm formation in both strains of S. aureus (reference and MRSA), whereas the two disubstituted ones were inactive. Methylcyano selenoesters showed a lower inhibition than the other two families of compounds, however, one of them (the para-disubstituted (Se-C1)) was close to exerting a 50% inhibition of S. aureus MRSA.
Finally, QS inhibiting effect of compounds was evaluated based on the inhibition of violacein production. The methylketone selenoester Se-K1 and Se-K2 and the methyloxycarbonyl selenoester Se-E1 were potent QS-inhibitors, with Se-E1 being the most effective QS inhibitor of these three derivatives by showing an inhibition close to the reference promethazine (positive control).
All these findings reveal that the symmetrical selenoesters have a potent antibacterial activity, mainly against S. aureus strains. Furthermore, the methylcyano selenoesters could be used as potential novel antibiotics. Additional studies to evaluate the ADME-Tox properties of these compounds is needed to evaluate their applicability in medicine more in-depth. Besides, the methylketone selenoesters, which are less selective, still could be used, for example, in disinfection of surfaces or in the coating of surfaces to prevent biofilm formation.

Conclusions
It can be concluded that all the symmetrical selenoesters evaluated have a potent antibacterial activity against S. aureus ATCC 25923. The most potent derivatives were the methylketone selenoesters (Se-K1, Se-K2, and Se-K3), followed by the cyano selenoesters (Se-C1, Se-C2, and Se-C3), and at the end by the methyloxycarbonyl selenoesters (Se-E1, Se-E2, and Se-E3). After determining the toxicity on normal fibroblasts, the more selective ones were the cyano selenoesters, followed by the methyloxycarbonyl selenoesters, and the ones by the methylketone selenoesters. Combining both the antibacterial activity and the cytotoxic activity, the most promising compound against S. aureus ATCC 25923 was Se-C3. The tested selenocompounds also showed antibacterial activity against S. aureus MRSA 272123 and against different strains of S. Typhimurium, although with higher MIC values.
In addition to the antibacterial activity, the methyloxycarbonyl selenoesters and two cyano selenoesters showed strong resistance reversing activity in the presence of tetracycline against the MRSA strain. Additionally, the methyloxycarbonyl selenoester Se-E3 was the most effective compound concerning the reversal of resistance, efflux pump inhibition, and anti-biofilm activity on S. aureus strains.

Patents
This work explores the antibacterial activity of compounds covered by the patent EP18382693 [21] (filed on 28 September 2018 by Enrique Domínguez-Álvarez, Gabriella Spengler, Claus Jacob and Carmen Sanmartín) more in-depth.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.