Antibiofilm Activity and Synergistic Effects of Thymol-Loaded Poly (Lactic-Co-Glycolic Acid) Nanoparticles with Amikacin against Four Salmonella enterica Serovars

Background Salmonella species are frequently linked to biofilm-associated infections. Biofilm formation intensively reduces the efficacy of antibiotics and the host immune system. Therefore, new therapeutic strategies are needed. Thymol, the main monoterpene phenol found in Thymus vulgaris, has been shown to possess potent antibiofilm activity. Our previous findings showed that thymol enhanced the antibiofilm activity of aminoglycosides against Salmonella enterica serovars. However, the clinical potential of thymol has not yet been realized due to its low aqueous solubility and high volatility. Nano-based drug delivery systems have emerged as a novel strategy to resolve these problems. This study aimed to investigate the antibiofilm activity of thymol-loaded poly (lactic-co-glycolic acid) nanoparticles (TH-NPs) and their synergism when used in combination with amikacin antibiotics. Methods The antibacterial activity of TH-NPs was evaluated using the broth microdilution method. Biofilm formation and antibiofilm assays were performed by the miniaturized microtiter plate method. Interaction studies between TH-NPs and amikacin against biofilm were determined using the checkerboard method. Results TH-NPs exhibited antibacterial activity against planktonic cells of S. enterica serovars that were more efficient (8 to 32 times) than free thymol alone. S. Typhimurium and S. Choleraesuis isolates were considered strong biofilm producers. The combination of TH-NPs with amikacin showed synergistic activity in the inhibition and eradication of S. enterica serovar biofilm. The minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) of amikacin were reduced by 32 to 128-fold when used in combination with TH-NPs. Time-kill kinetic studies showed that the combination of TH-NPs with amikacin possesses bactericidal action. Conclusion This study suggests that the combination of TH-NPs with amikacin can be an alternative to overcome biofilm-associatedSalmonella diseases and therefore should be further explored as a model to search for new antibiofilm drugs.


Introduction
Salmonella is a genus of the family Enterobacteriaceae. Taxonomically, this genus is separated into two species: Salmonella bongori and Salmonella enterica, which contain six subspecies with serovars like Salmonella Typhi, Salmonella Enteritidis, Salmonella Paratyphi, Salmonella Typhimurium, and Salmonella Choleraesuis [1].Salmonella enterica causes typhoidal and nontyphoidal salmonellosis (NTS) [2]. Te WHO estimates the global typhoid fever disease burden at 11-20 million cases annually, resulting in about 128 000-161 000 deaths per year [3]. On the other hand, the global human health impact of nontyphoidal Salmonella is high, with an estimated 93.8 million cases, of which an estimated 80.3 million are food-borne, and 155.000 deaths each year [4]. Salmonellosis is an important source of morbidity [5]. Teir clinical picture varies from a common gastroenteric to enteric fevers, which are life-threatening, requiring prompt and correct antibiotherapy [6]. However, resistance to antibiotics is increasing. One of the mechanisms of resistance used by Salmonella is bioflm formation, which also appears as a potent virulence factor [7]. A bioflm is defned as a sessile microbial community in which cells are attached to a surface or other cells and embedded in a protective extracellular polymeric matrix. Tis mode of growth exhibits altered characteristics, especially regarding gene expression and protein production [8]. Te infections associated with bioflms are difcult to treat because of the slow penetration and/or sequestration of antimicrobial agents into the bioflm through the extracellular matrix, the presence of multidrug-resistant persister cells, and the low metabolic state at the base of the bioflm [9]. Tereby, bioflms formed by bacteria are more resistant to antimicrobials than nonadherent cells with minimum eradication concentrations 10 to 1000 times higher than planktonic cells [10]. Terefore, there is an urgent need for the identifcation of new approaches and therapeutic formulations that target the bioflm mode of growth to prevent or treat bioflmrelated infections. Drug combinations have been used as an alternative to efectively combat bioflm. Antibioticantibiotic combination and association of an antibiotic with bioactive natural products are interesting approaches to fght bioflms.
Natural products, such as thymol and their structural analogs, have historically made a major contribution to pharmacotherapy [11]. Tymol has been applied as a spice and antibacterial product. In our recent study, thymol has shown antibioflm activity and synergistic efects with aminoglycoside antibiotics against Salmonella spp and Klebsiella spp [12,13]. However, the use of thymol in the pharmaceutical feld has recently been limited by its poor water solubility, stability, and high volatility [14]. To overcome this limitation, thymol can be smartly encapsulated into a nanocarrier [14,15]. Among the diferent polymers used for nanoparticle preparation, copolymers of poly (lactic-co-glycolic acid) (PLGA) are frequently used for their good properties as used in this study. PLGA is a US Food and Drug Administration (FDA)-approved copolymer for human use. It is widely exploited in the design of nanoparticles because of its biodegradability and biocompatibility.
Furthermore, as nanoparticles, it protects drug molecules from degradation and aids in producing sustained and targeted delivery [16]. A previous study reported a strong antibacterial activity of TH-NPs against planktonic cells of Escherichia coli and Staphylococcus aureus [17]. We have recently demonstrated a remarkable synergistic antibioflm efect of thymol in combination with aminoglycoside antibiotics against S. enterica serovars. Tymol was shown to potentiate the antibioflm activity of amikacin, kanamycin, and streptomycin, and we recorded the amikacin-thymol combination as the most efective one [12,13]. Terefore, we hypothesized that the previously observed antibioflm and synergistic efects of thymol might be improved by the encapsulation of thymol into nanoparticles. Te present study aimed to investigate the combination of TH-NPs with amikacin against the bioflm of S. enterica serovars.

Nanoparticle Preparation.
TH-NPs were prepared using the single emulsion-solvent evaporation method previously described with slight modifcations [15]. Briefy, 75 mg of PLGA and 7.5 mg of thymol were dissolved in 1.5 mL of dichloromethane. Ten, 3 mL of polyvinyl alcohol solution prepared in distilled water at 2% (W/V) was added dropwise into the organic phase and magnetically stirred for 3 h to evaporate dichloromethane. Te solution was centrifuged at 65,000 ×g for 20 min, and the obtained suspension was washed thrice with distilled water. Te TH-NP sample suspension obtained was freeze-dried using a Christ Alpha 2-4 LDplus freeze dryer (Christ, Osterode am Harz, Germany) and stored at −20°C for further analysis.
Dynamic light scattering (DLS) was used to measure the particle size, polydispersity index (PDI), and zeta potential of the obtained nanoparticles.

Microorganisms and Growth
Conditions. Te S. Typhi (ATCC 6539) reference strain used in this study was purchased from the American Type Culture Collection (ATCC). Four clinical isolates of Salmonella enterica serovars, S. Enteritidis, S. Typhi, S. Typhimurium, and S. Choleraesuis, were all provided by "Centre Pasteur du Cameroun." All of them were plated from cultures, which were stored at -80°C onto Salmonella-Shigella agar (SSA) (Condalab) for 18-24h at 37°C. Cultures were subsequently subcultured and maintained onto Muller-Hinton agar (MHA, Sigma-Aldrich) plates at 4°C until needed for further bioassay.

Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of TH-NPs.
Te MIC test was performed in a sterile 96wellround-bottom microtiter plate using standard broth microdilution methods, while the MBC test was performed on MHA plates. For the MIC test, frst, the 96-well microtiter plates were only flled with 100 μL of MHB. After that, 100 μL of TH-NP stock solution (4096 μg/mL) was added and diluted twofold. Concentrations of TH-NPs ranged from 0.5 to 1024 μg/mL. 100 μL of bacteria at 1.5 × 10 6 CFU/mL (with 7.5 × 10 5 as the fnal concentration) were added into the wells. Te selected wells flled with MHB only and MHB and organism, to serve as negative and positive controls, respectively. Te microtiter plates were then incubated at 37°C for 24 h, after which 40 μL of p-iodonitrotetrazolium (INT) chloride at 0.2 mg/mL was introduced into each well, and observations were made for the yellow dye to pink coloration after 30 min of incubation at 37°C, which signifes growth. Te MIC was recorded as the lowest concentration that prevented the color change in the medium. Te experiment was carried out in triplicate [18].
Te minimum bactericidal concentration (MBC) was defned as the lowest concentration of antibacterial agents that completely kill organisms. Tis test was performed by plating the suspension from each well of the microtiter plates into an MHA plate. After that, the plates were incubated at 37°C for 24 h. Te lowest concentration with no visible growths on the MHA plate was taken as the MBC value [19].

Ability of Bioflm Formation.
A microtiter plate (MtP) assay was used to determine and quantify bioflm production by using a microplate reader. Based on the kinetic metabolic activity in our previous work, the bioflm biomass of S. enterica serovars was determined with a colorimetric method based on safranin dye. 48 hours has been chosen as the training time [12]. Briefy, 100 μL of a bacterial suspension at 1.5 × 10 6 CFU/mL was added to 100 μL of MHB supplemented with 2% glucose into the 96-wellfatbottomed sterile polystyrene microplates and incubated at 37°C for 48 h [20]. 2% glucose was added to MHB to increase bioflm formation [21]. After incubation, planktonic cells in the well of the microplate were discharged by washing twice with 300 μL of phosphate-bufered saline (PBS) at 7.2 pH. After that, step bioflms were fxed with 150 μL of methanol for 20 min at room temperature. To perform bioflm formation, 150 μL of 1% (w/v) safranin 1% was used to stain bioflms. After 15 min of incubation at room temperature, excess safranin was removed, and the dye bound to the adherent cells was solubilized with 150 μL of 95% ethanol. Optical density was measured spectrophotometrically at 570 nm by using a microplate (SpectraMax ® ix 3, Molecular Devices). Te studies were repeated in triplicate. Uninoculated wells containing sterile MHB supplemented with 2% glucose were considered negative controls and were used as blanks. Te blank absorbance values were used to identify whether bioflm formation of S. enterica serovars exists or not. Te wells of isolates whose ODi (OD isolate) values were higher than those of blank wells were all considered to be bioflm producers. Te cutof value (ODc) can provide categorization of isolates as bioflm producers or not. ODc � average OD of the negative control + (3 × standard deviation (SD) of the negative control); ODi � average OD of the isolate − ODc. A negative value obtained from this formula and represented as zero indicates a lack of bioflm production, whereas a positive value indicates bioflm production. Based on OD of the isolate (ODi), interpretations can be performed as follows: ODi ≤ ODc no bioflm production (NBP), ODc ˂ ODi ≤2 × ODc weak bioflm production (WBP), 2 × ODc ˂ OD ≤ 4 × ODc moderate bioflm production (MBP), and 4 × ODc ˂ ODi strong bioflm production (SBP) [22].  [23]. Te assay was performed in a sterile 96-well polystyrene plate. Briefy, 100 μL of bacterial inoculum (1.5 × 10 6 CFU/mL) was inoculated and cultured with or without 100 μL of TH-NPs (at concentrations ranging from 0.5 to 1024 μg/mL), without shaking at 37°C. After 24 h incubation, planktonic cells were removed by washing each sample three times in sterile phosphate bufer solution (PBS) at pH 7.2, and adherent cells were fxed with methanol for 20 min at room temperature. Ten, the plates were treated as described above in the bioflm formation assay [12]. Wells containing MHB supplemented with glucose 2% without bacteria were used as the negative control and taken as the blank, whereas wells containing MHB supplemented with glucose 2% with bacteria were used as the positive control. Te experiment was carried out in triplicate and repeated three times. Te percentage inhibition of bioflm biomass is calculated as follows: Te minimum bioflm inhibitory concentration (MBIC) was defned as the lowest concentration of TH-NPs that inhibit bioflm biomass by 100%.

Bioflm Eradication Assay.
Eradication of bioflm by TH-NPs was carried out according to the protocol described by Kirmusaoaylu and Kalşkçl [20]. After bioflm formation for 48 h, the medium was gently discarded. To remove nonadherent bacterial cells, the wells were carefully washed with PBS. Ten, the plates were flled with 100 μL of MHB supplemented with 2% glucose and 100 μL of TH-NPs at concentrations ranging from 1 to 2048 μg/ mL. Te plates were incubated at 37°C for 24 h. After that, the plates were treated as previously described for the bioflm inhibition assay. Te minimum bioflm eradication concentration (MBEC) was defned as the lowest concentration of TH-NPs reducing bioflm biomass in the preformed bioflm by 100% and calculated as previously described. Te experiment was performed in triplicate and repeated three times.

TH-NPs in Combination with Amikacin against Bioflm
Formation. Te checkerboard method was performed to determine the efect of combining TH-NPs and amikacin on the bioflm formation of S. enterica serovars. By using MHB supplemented with 2% glucose, TH-NPs were serially diluted across each row of the 96-well microtiter tray, and serial dilutions of antibiotics were then added to each column. Terefore, each row and column contained a fxed concentration of one agent and decreasing concentration of the other agent. Each tray contained one row and one column, with serial dilution of each agent alone. Te remainder of the assay was performed as described for the broth microdilution assay. Ten, 100 μL of the bacterial inoculum (1.5 × 10 6 CFU/mL) was added to each well, and the plates were incubated at 37°C for 24 h. Te fnal concentrations ranged from 1 to 64 μg/mL for TH-NPs and from 15625 × 10 −6 to 16 μg/mL for amikacin. After incubation, the broth in the wells was gently removed, and the plates were washed three times with PBS. Untreated wells were used as the positive control, and wells containing MHB without bacteria were used as the blank. Te minimum bioflm inhibitory concentration (MBIC) of each compound was determined. Te efect of combinations was determined by calculating the fractional inhibitory concentration index (FICI) by using the following equation: FICI � (MBIC of TH-NPs in the combination/MBIC of TH-NPs alone) + (MBIC of amikacin in combination/MBIC of amikacin alone). Te type of interaction was defned based on the FICI value as follows: synergy when FICI ≤ 0.5, additivity when 0.5 ˂ FICI≤1, indiference when 1 ˂ FICI ≤ 4, and antagonism when FICI ˃ 4 [24].

TH-NPs in Combination with Amikacin against Preformed Bioflm.
To evaluate the efect of the combination of TH-NPs with amikacin to eradicate the bioflm of the same strains previously used, the checkerboard method was used as described by Hu et al., with slight modifcations [25]. After bioflm formation for 48 h, planktonic cells were gently removed, and the plate was washed three times with PBS at pH 7.2. Ten, 100 μL of MHB supplemented with 2% glucose and 50 μL of each substance as described above were added to adherent cells into the wells at a fnal concentration of 1 to 64 μg/mL for TH-NPs and 15625 × 10 −6 to 16 μg/mL for amikacin. Te wells containing medium and bacteria were used as negative and positive controls, respectively.
After incubation at 37°C for 24 h, the minimum bioflm eradication concentration (MBEC) was then determined. Te FICI was calculated and interpreted as described above.

Kinetic Studies of Bioflm Biomass.
Te combination of TH-NPs with amikacin was used to assess the prevention of bioflm biomass and disruption of mature bioflm biomass. Tis assay was examined by using a 96-well plate (Flat bottom, Sterile; Corning, USA) as previously described with slight modifcations [26]. Concerning kinetics of the inhibition of bioflm formation, frst, bioflm was incubated and treated at 37°C at six-time points 4,8,12,16,20, and 24 h under static conditions. After that, planktonic cells were removed, and the wells were gently washed thrice with PBS at 7.2 pH. Ten, the plates were treated as previously described. Note that untreated wells were used as the positive control, and the wells containing MHB broth without bacteria were used as the blank. Te microplate was measured spectrophotometrically at 570 nm by using a microplate reader.
Kinetic bioflm biomass disruption of the preformed bioflm was carried out as described by Kamble and Pardesi with some modifcations [27]. 100 μL of a bacterial suspension at 1.5 × 10 6 CFU/mL of the 18 h old culture was added to wells of a fat-bottom96-well microplate containing 100 μL of MHB supplemented with 2% glucose. Te plates were incubated further at 37°C for 48 h to allow for bioflm formation. Ten, media containing planktonic cells were removed from the wells, and the bioflm was washed thrice with PBS. Untreated wells containing bacteria and wells with MHB only were used as positive and negative controls, respectively. After treatment, TH-NPs and amikacin were added alone or in combination and incubated at six-time points 1.5, 3.0, 4.5, 6.0, 7.5, and 24 h at 37°C. After the plates were treated as mentioned above, absorbance was measured at 570 nm. In both cases, the experiments were conducted three times in triplicate.

Time-Kill Kinetic Assay.
Te time-kill kinetic assay was used to study the activity of combination of TH-NPs with amikacin against Salmonella enterica serovars and determine the bactericidal, bacteriostatic, or synergistic activity of this combination over time [28]. Tis assay was carried out by estimating surviving bioflm cells using the viable cell plate-counting method [29]. Te kinetic against inhibition of bioflm biomass was conducted over a range of seven diferent times 0, 4, 8, 12, 16, 20, and 24 h, whereas the time-kill kinetic assay against disruption of preformed bioflm was performed at 0, 1.5, 3.0, 4.5, 6, 7.5, and 24 h. After each incubation period, media were gently discarded, and the plates were washed three times with PBS. Ten, individual components of each combination, either TH-NPs and amikacin alone or in combination at a synergistic concentration efect, were introduced, and the plates were incubated at diferent time points mentioned above at 37°C. After that, adherent cells were suspended with PBS via scraping followed by serial dilution in sterile normal saline. Ten, 100 μL of each dilution sample was plated on MHA.
After incubation of the MHA plate at 37°C for 24 h, colonies were counted, and results were expressed as log 10 CFU/mL. Bactericidal activity was defned as greater than or equal to 3 log 10 − fold decrease in colony-forming units (surviving bacteria) compared with the positive control, whereas synergy was defned as ≥2 log 10 decrease in colony-forming units at 24 h by combination of TH-NPs with amikacin in comparison with TH-NPs or amikacin alone [28].

Statistical Analysis.
GraphPad Prism software version 8.0.1 was used to analyse the data obtained. Te results were presented as means ± standard deviation (SD) of three independent experiments. Statistical signifcance between the treated and control groups was analysed through the oneway analysis of variance (ANOVA) at p value ˂0.05.

Nanoparticle Preparation.
We obtained nanoparticles of size around 192 nm with a polydispersity index of 0.09 and a zeta potential of −5.39 mV [15].  Table 1. TH-NPs were more active (4 to 16 times) against S. enterica, showing MIC and MBC values ranging from 4 to 8 μg/mL and 4 to 16 μg/mL, respectively, compared to free thymol where MIC values ranged from 64 to 128 μg/mL and 128 to 256 μg/mL for MBC values. It should be noted that MIC and MBC of thymol are data taken from the previous study and were used in this study only for comparison purposes [12].

Antibioflm Activity of TH-NPs Alone and in Combination with Amikacin against Bioflm Formation.
Te results of antibioflm activity of TH-NPs and amikacin alone, and in combination against bioflm formation, are shown in Table 2. It was observed that MBIC values of TH-NPs in combination ranged from 4 to 8 μg/mL. TH-NPs combined with amikacin exhibited synergistic (FICI � 0.13-0.28) antibioflm activity against all isolates of S. enterica tested and reduced from 4 to 128 times the MBIC of amikacin.

Antibioflm Activity of TH-NPs Alone and in Combination with Amikacin against Preformed Bioflm.
Te result of antibioflm activity of TH-NPs alone and in combination with amikacin against preformed bioflm is   Table 3. It was observed that the MBEC values of TH-NPs ranged from 64 to 128 μg/mL. TH-NPs showed synergistic action (FICI � 0.06-0.27) with amikacin against all mature bioflm of S. enterica serovars. MBEC of amikacin and TH-NPs was reduced 4 to 32 times and 32 to 128 times, respectively. Note that the results of amikacin alone recorded in this table come from our previous study [12].

Kinetic Study of Bioflm Biomass.
Te efects of TH-NPs and amikacin alone or in combination against the prevention of bioflm biomass formation of S. enterica serovars are shown in Figure 2. All curves have an upward trend. On the one hand, it appears that TH-NPs are more efective than antibiotics, and on the other hand, the combination of TH-NPs with amikacin completely inhibits bioflm formation in all strains. Figure 3 shows the kinetic results of the bioflm biomass destruction of Salmonella enterica serovars treated with TH-NPs and amikacin alone or in combination. Compared to inhibition, where curves were ascending, here, curves are descending due to preformed bioflm. It should be noted that the combination efectively destroys the bioflm biomass of isolates at point times (4.5, 6, and 7.5 h). Te bioflm disruption of S. Typhi was the most marked at 1.5 h with an optical density of 0.65.

Time-Kill Kinetic Assays.
Te time-kill kinetic curves of TH-NPs and amikacin alone or in combination against bioflm biomass formation are shown in Figure 4. It was observed that, between 0 and 4 h, there was no growth. From 4 h, all the curves were ascending with log 10 CFU/mL, ranging from 0 to 4.84. Taken individually, TH-NPs or amikacin did not signifcantly reduce the number of cells, while their combination induced signifcant (p ˂ 0.05) cell death.
Te results of the time-kill kinetic assay of TH-NPs and amikacin alone or in combination against disruption of preformed bioflm are plotted in Figure 5.
A perusal of Figure 5 reveals that the combination showed better activity than TH-NPs and amikacin only taken. At 3.0 h, we obtained a drop of at least 2 log 10 CFU/ mL compared to controls and compounds taken alone. Note that similar results were obtained during the kinetic study against bioflm biomass production. Here, at 6.0 h, cell death was ˃3 log 10 CFU/mL reduction. Based on statistical analysis, (p ˂ 0.05), the combination compared to TH-NPs showed signifcant cell destruction.

Discussion
Antibiotic resistance is a serious challenge to the healthcare community both in developed and developing countries. Te emergence and spread of multidrug-resistant pathogens have substantially threatened the current antibacterial therapy efcacy [30]. Indeed, the causes of antibiotic resistance are numerous, and several mechanisms have been proposed to explain the phenomenon of resistance within bioflms [31]. Recent studies have shown the ability of  bacterial bioflms to survive high concentrations of antibiotics [32]. In addition, it is estimated that about 65% of all bacterial infections are associated with bioflms, and these include both device and nondevice-associated infections [32]. Tis necessitates a search for a new therapeutic agent with antibioflm properties. In the last two decades, new approaches in preventing and eradicating bioflm have been widely developed and reported, including the potentiation of antibiotic activity by combination with bioactive natural products from plants [33]. Tymol (2-isopropyl-5-methylphenol) is a versatile molecule with a wide variety of practical applications such as medical, dentistry, veterinary, food, and agrochemicals. Its pharmacological applications have been most investigated and reported, focusing on its prominent antimicrobial, antioxidant, anti-infammatory, and cicatrizing activities [34]. About antibacterial activities with MIC ranging from 64-128 μg/mL and 4-8 μg/mL, respectively, for thymol unloaded and TH-NPs on all four planktonic Salmonella enterica serovars and S. Typhi ATCC 6539, it is clear that TH-NPs are more efcient than free thymol. Indeed, the hydrophobic nature of thymol presents a challenge for microbial inhibition in aqueous media. However, PLGA produces acid by-products during the degradation process, which increases its hydrophobicity and enables it to partition into the lipids of the microbial cell membrane or to bind the hydrophobic regions of proteins [35]. Terefore, thymol could easily reach microorganisms after encapsulation in PLGA [36]. Moreover, recently, antibacterial activities of thymol-loaded PLGA microparticles have been tested against Gram-negative and Gram-positive bacteria via the colony-counting method, and it has been observed that antibacterial activity of thymol-loaded microparticles for both bacteria increased as the number of microparticles increased [17]. Tis can also be justifed by the size (192.21 nm) and solubility of TH-NPs which can easily infltrate the matrix, which acts as a barrier for many antibiotics [23], and pass through cell membranes [35]. Tese results are in agreement with those of Folle and collaborators, where the MICs of thymol nanoparticles were lower  than those of nonencapsulated thymol on Cutibacterium acnes [37], and are also similar to those of Zhu et al., where TH-NPs had strong antibacterial activity against Escherichia coli and Staphylococcus aureus [17].
Concerning antibioflm activity, with MBIC and MBEC ranging from 16 to 64 μg/mL and 64 to 128 μg/mL, respectively, for TH-NPs and unloaded thymol, they showed their ability to inhibit bioflm formation and eradicate Salmonella species mature bioflms.
Once again, the higher antibioflm activity of TH-NPs may be due to their size and surface charge. TH-NPs' size substantially impacts their difusion into the bioflm matrix, leading to more damage to bioflm cells. Te nanoparticle's surface can be responsible for specifc bioflm targeting by electrostatic interactions with the bioflm matrix, which favors the attachment of nanoparticles to the bioflm matrix and results in the release of the drug inside the bioflm [38].
In addition, the high surface area to volume ratio of nanoparticles allows for drug loading, which can result in strong antibioflm efcacy [39]. Te research found that thymol could inhibit bioflm formation and remove mature bioflms by inhibiting the production of polysaccharide intracellular adhesin (PIA) and the release of extracellular DNA (eDNA) [40]. In addition, previous studies have indicated that essential oils' components could inhibit the quorum-sensing system [41]. Yuan et al. reported antibioflm activity of thymol toward methicillin-resistantStaphylococcus aureus [40]. Given the interesting activities of thymol, for clinical use, it would be important to pay attention to its toxicity. Note that, recently, a study has been performed to evaluate the acute and repeated 28-day oral dose toxicity of thyme essential oil in rats. Studies revealed moderate oral toxicity of this oil and suggested that the no-observed-adverse-efect level (NOAEL) is greater than 250 mg/kg/day [42]. Tis can sufciently justify that thymol, a major component of thyme essential oil [43], is toxic at high concentrations. Nevertheless, natural compounds like thymol also present challenges for drug discovery, such as technical barriers to screening, isolation, solubility, stability, and volatility [44]. To overcome these problems, nanoparticles of natural products can be used. Nanoparticles of PLGA have been chosen in this study for their properties. PLGA is one of the most efective biodegradable polymeric nanoparticles. It has been approved by the United States Food and Drug Administration for use in drug delivery systems due to its controlled and sustainedrelease properties, low toxicity, biocompatibility with tissue [45], and biodegradability into lactic acid and glycolic acid, two monomers that are naturally produced under physiological conditions by several metabolic pathways [46]. In the previous study, based on the results obtained with aminoglycosides, amikacin was specially chosen for this study. Terefore, the synergistic efect of TH-NPs in combination with amikacin was investigated against bioflm formation by S. enterica serovars. All combinations give synergetic efects on all strains. Some studies have explored the combination of synthetic drugs with essential oils, intending to evaluate and enhance antimicrobial efcacy [47]. For example, a synergistic efect was observed between norfoxacin and essential oil (and its main components) from Pelargonium graveolens [48], between gentamicin and the essential oil from Croton zehntneri [49], and between eugenol with antibiotics. To our knowledge, the antibioflm capacity of TH-NPs and amikacin against microbial bioflms remains largely unknown. However, the antibacterial efect of thymol-loaded chitosan nanoparticles (TCNPs) has been reported against a broad spectrum of Gram-positive and Gram-negative pathogens. It is proposed that the charged groups in the polymer of nanoparticles and the moieties in thymol possibly interact with the negatively charged bacterial membrane, enhancing the killing efciency of pathogens [45]. Note that the  phenolic hydroxyl group of thymol increased its hydrophilic ability, which could help thymol dissolve in microbial membranes and damage them [17]. However, it is known that the ability of nanoparticles to attach and penetrate bioflm cells depends on their physicochemical properties such as size and morphology. TH-NPs would penetrate the bioflm and afect the quorum-sensing gene cascade, thereby hampering the cell-to-cell communication mechanism, which inhibits bioflm synthesis [44].
Moreover, it was shown that, at subinhibitory concentrations, thymol also reduces bioflm formation [40]. Te results strongly suggest that a combination of TH-NPs with amikacin may be successfully used as an antibioflm agent.
Te synergistic antibioflm activity and bactericidal action of TH-NPs in combination with amikacin were confrmed by the kinetic and time-kill assay of inhibition of bioflm biomass formation and eradication of preformed bioflm. Concerning the time-kill assay, a reduction greater than 3 log UFC/mL, compared to the control, which is equivalent to 99.9% killing of the inoculum bactericidal activity, was obtained [50].
Tis sufciently shows the bactericidal activity of a combination of amikacin with TH-NPs. Tis study confrms that thymol is a potential efective source for medicinal chemists, which will take natural product-based antibiotic research to the next level [51].

Conclusion
Tis study showed that TH-NPs potentiate the antibioflm activity of amikacin against the prevention and removal of S. enterica serovar bioflms. Synergistic and bactericidal efects  obtained indicate that the combination of TH-NPs with amikacin is a promising approach for the development of novel antibacterial combination therapies against bioflmassociated infections. Further studies are under consideration to investigate the antibioflm mechanism of action of TH-NPs and their combinations with antibiotics, especially the efect on quorum sensing. TH-NPs in combination with amikacin can be a promising alternative for the management of Salmonella bioflm.

Data Availability
Te data used to support the fndings of this study are available upon reasonable request from the corresponding author.

Conflicts of Interest
Te authors declare that they have no conficts of interest.