Antimicrobial Activity of Sertraline on Listeria monocytogenes

We explored the antimicrobial activity of sertraline on Listeria monocytogenes and further investigated the effects of sertraline on biofilm formation and the virulence gene expression of L. monocytogenes. The minimum inhibitory concentration and minimum bactericidal concentration for sertraline against L. monocytogenes were in the range of 16–32 μg/mL and 64 μg/mL, respectively. Sertraline-dependent damage of the cell membrane and a decrease in intracellular ATP and pHin in L. monocytogenes were observed. In addition, sertraline reduced the biofilm formation efficiency of the L. monocytogenes strains. Importantly, low concentrations (0.1 μg/mL and 1 μg/mL) of sertraline significantly down-regulated the expression levels of various L. monocytogens virulence genes (prfA, actA, degU, flaA, sigB, ltrC and sufS). These results collectively suggest a role of sertraline for the control of L. monocytogenes in the food industry.


Introduction
Listeria monocytogenes (L. monocytogenes) is a Gram-positive pathogen and the causative agent of listeriosis [1] in which the typical clinical symptoms include meningitis, septicemia and stillbirth with a mortality rate of up to 30%, especially in immunocompromised hosts [2]. L. monocytogenes inhabits a broad ecologic niche and can contaminate various food products and food processing environments [3]. As this bacterium is psychrophilic; resistant to desiccation, acid and heat; and tolerant to increased sub-lethal concentrations of disinfectants or resistant to lethal concentrations [4], it can persist in food processing environments and poses a challenge to the food production industry.
L. monocytogenes can adhere to food-processing surfaces and form a biofilm on these surfaces [5]. The biofilm formation ability is essential for L. monocytogenes to survive and persist in food processing environments [5]. Biofilm is composed of microbial cells and self-produced extracellular polymeric substances (EPS), including polysaccharides, nucleus acids, proteins and lipids [6]. The EPS forms unique three-dimensional (3D) spatial structures and provides mechanical stability to the biofilm [6]. Notably, biofilm is closely related to functional properties, such as decreasing the efficiency of cleaning treatments and providing in-biofilm located bacteria with high resistance to antimicrobial agents [6]; for these reasons, bacterial biofilm is a severe threat to food safety. Thus, to secure public health, it is important for the food industry to find effective approaches to control the biofilm formation of L. monocytogenes.
Sertraline, a selective serotonin reuptake inhibitor originally commercialized as an antidepressant drug, is reported to possess antimicrobial activity against a wide range of bacteria, such as Salmonella spp., Shigella spp., Staphylococcus spp., Staphylococcus epidermidis, Streptococcus spp., Vibrio spp., etc. [7,8]. Although the antimicrobial activity of sertraline has attracted attention, it is not fully elucidated how this compound performs its functions, and little is known about its effects on biofilm and virulence gene expression.
In this study, we examined the antimicrobial activity of sertraline against L. monocytogenes and further analyzed its effects on biofilm formation and the virulence gene expression of L. monocytogenes strains.

Antimicrobial Activity of Sertraline
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of sertraline for the L. monocytogenes strains were 16-32 µg/mL and 64 µg/mL, respectively (Table 1). Interestingly, the treatment of 64 µg/mL sertraline sharply decreased the bacterial concentration of both L. monocytogenes strains, which reached 0 CFU/mL at 12 h (Figure 1), indicating the bactericidal effect of sertraline on L. monocytogenes. antidepressant drug, is reported to possess antimicrobial activity against a wide range of bacteria, such as Salmonella spp., Shigella spp., Staphylococcus spp., Staphylococcus epidermidis, Streptococcus spp., Vibrio spp., etc. [7,8]. Although the antimicrobial activity of sertraline has attracted attention, it is not fully elucidated how this compound performs its functions, and little is known about its effects on biofilm and virulence gene expression. In this study, we examined the antimicrobial activity of sertraline against L. monocytogenes and further analyzed its effects on biofilm formation and the virulence gene expression of L. monocytogenes strains.

Antimicrobial Activity of Sertraline
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of sertraline for the L. monocytogenes strains were 16-32 μg/mL and 64 μg/mL, respectively (Table 1). Interestingly, the treatment of 64 μg/mL sertraline sharply decreased the bacterial concentration of both L. monocytogenes strains, which reached 0 CFU/mL at 12 h (Figure 1), indicating the bactericidal effect of sertraline on L. monocytogenes.

LIVE/DEAD Assay
Fluorescent staining can distinguish living, damaged and dead cells, which can indicate the viability of the bacteria [9]. The results depicted by the standard curves

LIVE/DEAD Assay
Fluorescent staining can distinguish living, damaged and dead cells, which can indicate the viability of the bacteria [9]. The results depicted by the standard curves illustrated a good linear relationship (R 2 = 0.99) between the green fluorescence intensity at 530 nm and the percentage of viable bacteria of L. monocytogenes ATCC 11915 and L. monocytogenes 001 ( Figure S1). When exposed to 16, 32 and 64 µg/mL of sertraline, the green fluorescence intensity of L. monocytogenes 11915 decreased by 29.1%, 38.7% and 46.9%, respectively, and the green fluorescence intensity of L. monocytogenes 001 decreased by 45%, 47.9% and 50.9%, respectively ( Figure 2). at 530 nm and the percentage of viable bacteria of L. monocytogenes ATCC 11915 and L. monocytogenes 001 ( Figure S1). When exposed to 16, 32 and 64 μg/mL of sertraline, the green fluorescence intensity of L. monocytogenes 11915 decreased by 29.1%, 38.7% and 46.9%, respectively, and the green fluorescence intensity of L. monocytogenes 001 decreased by 45%, 47.9% and 50.9%, respectively ( Figure 2).

Effect of Sertraline on the Bacterial Cell Membrane
ATP is essential for many cell functions including substance transportation across the cell membrane [10]. When the cell membrane is impaired, the intracellular ATP decreases as a result of the decreased synthesis and increased hydrolysis of the ATP and the loss of inorganic phosphate through the highly compromised permeable cell membrane [11]. Compared with the control group, the intracellular ATP concentrations ( Figure 3) and the intracellular pH (pHin) (Figure 4) of L. monocytogenes were significantly reduced when the concentration of sertraline increased from 16 to 64 μg/mL. The pHin of normal L. monocytogenes ATCC 11915 was 7.87. After exposure to 16, 32 and 64 μg/mL sertraline, the pHin decreased to 7.55, 7.46 and 6.95, respectively. The pHin of normal L. monocytogenes 001 was 7.13. After adding 16, 32 and 64 μg/mL sertraline, the pHin decreased to 7.09, 6.91 and 6.82, respectively.

Effect of Sertraline on the Bacterial Cell Membrane
ATP is essential for many cell functions including substance transportation across the cell membrane [10]. When the cell membrane is impaired, the intracellular ATP decreases as a result of the decreased synthesis and increased hydrolysis of the ATP and the loss of inorganic phosphate through the highly compromised permeable cell membrane [11]. Compared with the control group, the intracellular ATP concentrations ( Figure 3) and the intracellular pH (pHin) (Figure 4) of L. monocytogenes were significantly reduced when the concentration of sertraline increased from 16 to 64 µg/mL. The pH in of normal L. monocytogenes ATCC 11915 was 7.87. After exposure to 16, 32 and 64 µg/mL sertraline, the pH in decreased to 7.55, 7.46 and 6.95, respectively. The pH in of normal L. monocytogenes 001 was 7.13. After adding 16, 32 and 64 µg/mL sertraline, the pH in decreased to 7.09, 6.91 and 6.82, respectively.

FESEM Observation
Normal L. monocytogenes cells have a smooth surface, a complete structure and an elongated rod shape ( Figure 6). After treatment with sertraline concentrations higher than 16 μg/mL, the cell surface of L. monocytogenes became irregular and showed varying degrees of contraction and intercellular aggregation. The percentage of damaged cells and the degree of damage increased as the concentration of sertraline increased. The microstructural observations were in accordance with the findings on the impaired cell membrane integrity, decreased intracellular ATP concentration and reduced pHin under

FESEM Observation
Normal L. monocytogenes cells have a smooth surface, a complete structure and an elongated rod shape ( Figure 6). After treatment with sertraline concentrations higher than 16 µg/mL, the cell surface of L. monocytogenes became irregular and showed varying degrees of contraction and intercellular aggregation. The percentage of damaged cells and the degree of damage increased as the concentration of sertraline increased. The microstructural observations were in accordance with the findings on the impaired cell membrane integrity, decreased intracellular ATP concentration and reduced pH in under the application of sertraline as observed in this study.

FESEM Observation
Normal L. monocytogenes cells have a smooth surface, a complete structure and an elongated rod shape ( Figure 6). After treatment with sertraline concentrations higher than 16 μg/mL, the cell surface of L. monocytogenes became irregular and showed varying degrees of contraction and intercellular aggregation. The percentage of damaged cells and the degree of damage increased as the concentration of sertraline increased. The microstructural observations were in accordance with the findings on the impaired cell membrane integrity, decreased intracellular ATP concentration and reduced pHin under the application of sertraline as observed in this study.

Virulence Gene Expression
The results showed that sertraline had different effects on the 10 virulence genes (hly, argA, prfA, degU, actA, flaA, sigB, ltrC, sufS and sufU) of L. monocytogenes. Compared with the control group, sertraline at 0.1 and 1 µg/mL significantly inhibited the expression of these virulence genes in the L. monocytogenes ATCC 11915 strain ( Figure 7). However, when the concentration was higher at 2-8 µg/mL, sertraline exposure weakened the inhibition effect on actA, flaA, sigB, ltrC, sufS and sufU gene expression. Especially when the sertraline concentration exceeded 4 µg/mL, the expression of hly, argA, prfA and degU genes was up-regulated.
Sertraline had slightly different inhibition effects on the virulence gene expression of L. monocytogenes 001 (Figure 7). Sertraline at 0.1 µg/mL resulted in down-regulation in the expression of prfA, degU, actA, flaA, sigB, ltrC and sufS genes. When the concentration was higher than 1 µg/mL, the inhibitory effect of sertraline on virulence gene expression was weakened. Sertraline at 4 and 8 µg/mL increased the expression of all evaluated virulence genes.
Sertraline had slightly different inhibition effects on the virulence gene express L. monocytogenes 001 (Figure 7). Sertraline at 0.1 μg/mL resulted in down-regulation expression of prfA, degU, actA, flaA, sigB, ltrC and sufS genes. When the concentratio higher than 1 μg/mL, the inhibitory effect of sertraline on virulence gene expressio weakened. Sertraline at 4 and 8 μg/mL increased the expression of all evaluated vir genes.

Discussion
Previous studies have reported on the antimicrobial activity of sertraline with a MIC value of 4-128 µg/mL for S. aureus, 8-128 µg/mL for B. subtilis, 128 µg/mL for Candida albicans and 4-256 µg/mL for E. coli [12,13]. The antimicrobial activity of sertraline is highly species dependent. In this study, the intrinsic antibacterial activity of sertraline against the L. monocytogenes strains was similar to the activity reported in S. aureus and B. subtilis [14]. In addition, consistent with previous studies, sertraline exhibited a bactericidal effect against L. monocytogenes at 2 × MIC concentrations. Although not completely elucidated, the intrinsic antimicrobial activity of sertraline may be due to the benzene rings in the structure [12].
The bacterial cell membrane keeps the internal environment of the cell stable and maintains the normal metabolic function and energy transfer of the cell [10,15]. When the cell membrane is damaged, phosphate bonds and ion gradients as well as the energy (such as pH and ATP) transfer will change [16]. Thus, intracellular ATP and pH in are good indicators of the integrity of the cell membrane. In the present study, we observed that sertraline can interact with the cell membrane of L. monocytogenes as reflected by the decreased intracellular ATP and pH in . These findings are in accordance with previous studies, which demonstrated that sertraline can destroy the cell membrane of H. pylori [17] and, when combined with polymyxin, can significantly affect the ability of Acinetobacter baumannii, K. pneumoniae and P. aeruginosa to reshape their outer membranes [18]. In addition, sertraline is reported to cause Candida cell death by blocking the mitochondrial respiration and significantly decreasing transmembrane potential [19]. These results together indicate that sertraline most likely interacts with the bacterial cell membrane.
L. monocytogenes is a consistent source of cross-contamination, both in housing storage and food processing environments, and biofilm is easily formed on contaminated surfaces [5,20]. Previously, sertraline was shown to reduce the biofilm formation in different species, e.g., in Candida spp. [21]. In the present study, sertraline was found to have strong inhibitory effects on the biofilm formation ability of L. monocytogenes in food processing environments.
The virulence gene expression of L. monocytogenes involves several key steps: host cell adhesion and invasion, intracellular proliferation and motility and intercellular diffusion [22]. Specific bacterial factors are involved in each stage. For example, the hly gene, an important pathogenic factor, can help the bacteria escape the vacuole and interact with the host [23,24]. The prfA gene can regulate the expression of other virulent genes and control the biofilm formation [25]. The actA gene is required for the intracellular movement of L. monocytogenes in host cells [26]. The agrA gene affects biofilm formation and assists bacteria in intracellular invasion [1,27]. The degU and flaA genes are thought to stimulate the synthesis of bacterial flagella [28]. The sigB and ltrC genes have been shown to be involved in the low temperature adaptation of L. monocytogenes [29,30]. The sufS and sufU genes are associated with bacterial pathogenicity and virulence [31]. In this study, we found sertraline regulated the expression of the virulence genes of L. monocytogene; however, the regulation was not in a dosedependent manner. In concentrations as low as 0.1 and 1 µg/mL, the expression levels of most virulence genes were down-regulated. However, high concentrations of sertraline increased the virulence gene expression. In a previous in vivo study, high concentrations of sertraline exacerbated pathological outcomes in chickens infected with resistant E. coli [30]. These results suggest sertraline might have multi-effects on virulence gene expression. Nonetheless, the inhibitory effect of a low concentration of sertraline on virulence gene expression indicates the potential application of sertraline on modulating the virulence of pathogens. Further modification of sertraline or synthesis of its structural analogue is expected to improve its inhibitory effect on pathogens.
The L. monocytogenes strains that were used are shown in Table 1. The strains were inoculated with brain heart infusion broth (BHI) and cultured at 37 • C for 16 h.

MIC and MBC
The MIC values of sertraline against the L. monocytogenes strains were determined with the broth microdilution method [32]. Briefly, the L. monocytogenes strains were grown aerobically overnight at 37 • C on BHI broth. Then, the colonies were suspended in 0.9% NaCl and adjusted to 0.5 McFarland standard with a Sensititre TM nephelometer (Thermo-Fisher Scientific, Eugene, OR, USA). Subsequently, the suspensions were diluted 100-fold in Mueller-Hinton broth (MHB), and 100 µL of the dilution was transferred to the wells of a sterile 96-well plate that had different concentrations of sertraline in MHB (100 µL). The final concentrations ranged from 2 to 128 µg/mL. The positive controls contained bacteria inoculum only, whereas the negative controls contained MHB only. The lowest concentration of the compounds that resulted in no visible growth of the test organisms was determined as the MIC. The MBC was the lowest concentration at which microbial growth could not be observed on the medium [33]. All experiments were determined in biological triplicates.

Growth and Viability Assays
The growth and viability assays were performed as previously described [9]. Briefly, L. monocytogenes ATCC 11915 and a selected L. monocytogenes isolate (L. monocytogenes 001), previously obtained from a food product, were applied as test strains.
The assay was prepared at 37 • C for 12 h with continuous shaking. The strains were grown overnight, resuspended in BHI broth to reach an OD 600 = 0.1 and then exposed to 0, 16, 32 and 64 µg/mL of sertraline. The samples (100 µL) were collected at seven different time points during the 12 h period. Then, the samples were serially diluted, spread on MHA plates and incubated at 37 • C for 18 h followed by successive counting. The experiment was performed in triplicate, and the results were expressed as the average Log 10 CFU/mL.

Assessment of Membrane Integrity
The influence of the sertraline treatments on the membrane integrity of L. monocytogenes ATCC 11915 and L. monocytogenes 001 was assessed using the LIVE/DEAD BacLight TM Bacterial Vitality Kit (Thermo-Fisher Scientific, Eugene, OR, USA) as previously reported [34]. Briefly, standard samples were first prepared to construct a standard curve. The strain cultures were grown to the late exponential phase and then centrifuged, washed two times and re-suspended in 0.85% NaCl or 70% isopropyl alcohol (for the killed bacteria). Subsequently, the suspensions were incubated at room temperature for 1 h with mixing every 15 min. After incubation, the samples were pelleted two times with centrifugation (10,000× g, 10 min) and resuspended in NaCl to reach an OD 600 = 0.5. Different viable cell proportions (0%, 10%, 50%, 90% and 100%) were utilized as the standard samples. A working stain solution (2×) was prepared by adding 6 µL of SYTO 9 and 6 µL of propidium iodide (PI) to 2 mL of filter-sterilized water.
The cultures of the strains were grown overnight and then adjusted to an OD 600 = 0.5 followed by treatment with sertraline at 0, 16, 32 and 64 µg/mL for 15 min at 37 • C. After treatment, each culture was centrifuged and resuspended in 0.85% NaCl. Then, an equal volume of 100 µL of cell suspension and working stain solution (2×) was added to the black opaque 96-well microtitration plates (Corning, New York, NY, USA) and mixed thoroughly. The mixture was cultured in darkness at 25 • C for 15 min. The fluorescence was determined using a multifunctional enzyme marker (BioTek, Winooski, VT, USA). The green (530 nm) and red (630 nm) emission integral intensities of the suspension excited at 485 nm were obtained three times via wavelength measurement.

Measurement of Intracellular ATP Concentrations
The influence of sertraline on the intracellular ATP concentrations of L. monocytogenes ATCC 11915 and L. monocytogenes 001 was assessed as described previously [10]. Briefly, the overnight cultures of the strains were harvested with centrifugation (5000× g, 5 min). Then, the cells were washed three times with 0.1 mol/L of phosphate-buffered saline (PBS, pH 7.0) and resuspended in PBS to achieve an OD 600 = 0.5. Subsequently, sertraline was added to each tube to achieve the final concentrations of 0, 16, 32 and 64 µg/mL and cultured at 37 • C for 30 min. The ATP was ultrasonically extracted on ice and centrifuged (5000× g, 5 min). The supernatant was kept on ice to avoid loss of ATP. The ATP content was determined using an adenosine triphosphate detection kit with a multifunctional enzyme marker (BioTek, Winooski, VT, USA) following the manual's instructions (Beyotime Biotechnology, Shanghai, China). A good linearity was found between intracellular ATP content and luminescence (R 2 = 0.99).

PH in Measurements
The influence of sertraline on the pHin of L. monocytogenes ATCC 11915 and L. monocytogenes 001 was determined according to a modified method of Wang et al. [35]. First, the cal-ibration curve was constructed by measuring a series of fluorescence intensities of the pH buffers with values in the range of 3 to 10. The buffers consisted of 50 mmol/L KCl, 50 mmol/L Na 2 HPO 4 ·2H 2 O, 50 mmol/L glycine and 50 mmol/L citric acid, and they were adjusted with either NaOH or HCl. The pH in and pH out were equilibrated by adding 10 µmol/L valinomycin and 10 µmol/L nigericin.
A total of 300 µL of the overnight-cultured strains was transferred into 30 mL BHI broth and cultured at 37 • C for 8 h. After centrifugation (5000× g, 10 min), the cells were washed two times with 50 mmol/L HEPES buffer (containing 5 mmol/L EDTA, pH = 8) and resuspended in 20 mL buffer. Then, 3 µmol/L of the probe (carboxyfluorescein diacetate succinimidyl ester; cFDA-SE) (Meilunbio, Dalian, China) was added and cultured at 37 • C for 20 min. The cells were subsequently washed with 50 mmol/L potassium phosphate buffer added with 10 mmol/L MgCl 2 (pH = 7.0), resuspended in 10 mL buffer and subsequently added with 10 mmol/L glucose and cultured at 37 • C for 30 min to eliminate the unbound cFDA-SE. The obtained particles were washed two times using the above mentioned method and suspended in 50 mmol/L potassium phosphate buffer on ice. Sertraline was added to the treated cell suspension to obtain the final concentrations of 0, 16, 32 and 64 µg/mL. Then, the mixture was transferred into a black opaque 96-well microtiter plate. After treatment for 20 min, the fluorescence intensity was measured under the excitation wavelengths of 440 and 490 nm with an emission wavelength of 520 nm at 25 • C by using a multifunctional enzyme marker (BioTek, Winooski, VT, USA). The pH in was determined as the ratio of the fluorescence signals at the pH sensitive wavelength of 490 nm and pH insensitive wavelength of 440 nm. The fluorescence of the cell-free controls was measured and deducted from the fluorescence of the samples.

Biofilm Formation
The effect of sertraline on biofilm formation was investigated according to the method described previously [6,36]. Briefly, the concentration of the bacterial solution was adjusted to an OD 600 = 0.5. Then, 100 µL of the bacterial solution was added into a 96-well plate and incubated at 37 • C for 6 h to form biofilms. Sertraline was added to the treated cell suspension to obtain the final concentrations of 0, 16, 32 and 64 µg/mL. MH broth and the bacterial solution were used as the negative and positive controls, respectively. After treatment for 16 h at 37 • C, the plate was washed 3 times with normal saline and dyed with 125 µL 1% crystal violet solution. The optical density at 595 nm was determined with a multifunctional microplate analyzer (BioTek, Winooski, VT, USA). The biofilm formation ability was the value after deducting background staining.

Virulence Gene Expression
The effect of sertraline at different concentrations on the expression of 10 virulence genes (hly, argA, prfA, actA, degU, flaA, sigB, ltrC, sufS and sufU) of L. monocytogenes was detected with real-time quantitative PCR using the primers listed in Supplementary Table S1 [23].
First, the total RNA of L. monocytogenes cultures treated with sertraline at 0, 16, 32 and 64 µg/mL for 4 h was extracted with a commercial kit (Magen, GuangZhou, China) following the manufacturer's instructions. The concentration and quality of the RNA were assessed with agarose gel electrophoresis and a Nanodrop 2000 ® spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA), respectively. Then, the cDNA was synthesized with a commercial kit (Vazyme Biotech Co., Ltd., Nanjing, China) following the manufacturer's instructions.
The real-time PCR system was performed with the 2×Ultra SYBR Green qPCR Mix kit (CISTRO, GuangZhou, China) with the CFX96 TM Real-Time System (Bio-rad, Hercules, CA, USA). The 16S rRNA gene was used as the reference gene [14]. The relative expression levels of the virulence genes were calculated according to the Ct values.

Statistical Analysis
All experiments were conducted in triplicate. The statistical analyses were performed in GraphPad Prism ver. 8.0.1 (San Diego, CA, USA). The data were presented as the mean ± standard deviation (n = 3), and the differences between the mean values were tested via the one-way ANOVA. The differences were considered to be significant at p < 0.05.

Conclusions
This study investigated the antimicrobial activity of sertraline against L. monocytogenes. Sertraline caused damage of the cell membrane and decreased the intracellular ATP and pH in of L. monocytogenes. Moreover, sertraline significantly inhibited biofilm formation and regulated the virulence gene expression of L. monocytogenes. These results suggest that sertraline may potentially be used to control L. monocytogenes in food-processing environments, thereby reducing the risk of food contamination.