Pectolinarin Inhibits the Bacterial Biofilm Formation and Thereby Reduces Bacterial Pathogenicity

Bacterial biofilms are a growing problem as it is a major cause of nosocomial infection from urinary catheters to chronic tissue infections and provide resistance to a variety of antibiotics and the host’s immune system. The effect of pectolinarin on the biofilm formation in Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Streptococcus mutans, Streptococcus sobrinus, Staphylococcus aureus, Pseudomonas aeruginosa, Cutibacterium acnes, and Porphyromonas gingivalis was studied in TSBg (tryptic soy broth supplemented with 1% glucose). Pectolinarin inhibited biofilm formation of E. faecalis (IC50 = 0.39 μg/mL), E. faecium (IC50 = 0.19 μg/mL), E. coli (IC50 = 0.25 μg/mL), S. mutans (IC50 = 1.2 μg/mL), S. sobrinus (IC50 = 1.4 μg/mL), S. aureus (IC50 = 0.39 μg/mL), P. aeruginosa (IC50 = 0.9 μg/mL), P. acnes (IC50 = 12.5 μg/mL), and P. gingivalis (IC50 = 9.0 μg/mL) without inhibiting the bacterial growth. Pectolinarin also showed increased susceptibility of antibacterial activity with commercially available antibiotics including ampicillin, vancomycin, streptomycin, and oxytetracyclin against E. faecalis and E. faecium. Finally, pectolinarin dose-dependently reduced the expression of genes including cytolysin genes (cylLS, cylR2 and cylM), quorum sensing (QS) genes (fsrB, fsrC, gelE, ebpA, ebpB, acm, scm and bps), and biofilm virulence genes (esp) of E. faecalis and E. faecium. Pectolinarin reduced the bacterial biofilm formation, activated the antibacterial susceptibility, and reduced the bacterial adherence. These results suggest that bacterial biofilm formation is a good target to develop the antibacterial agents against biofilm-related infections.


Introduction
Bacterial biofilms are microbial communities encased within a complex matrix and capable of colonizing natural body surfaces such as the epithelium, lungs, and heart, as well as implanted medical devices such as central venous and urinary catheters, intrauterine devices, and prosthetic heart valves [1]. The biofilm offers many advantages to bacteria, including the ability to acquire increased resistance toward antibiotics, which is of particular importance. This resistance leads to complications in the management of biofilm infections and limits therapeutic options [2][3][4]. In addition, bacterial biofilms pose a challenge to the host immune system [5]. During the past few decades, Enterococcus spp. have emerged as important healthcare-associated pathogens. The continuing progress of modern medical care with the overuse of antibiotics has undoubtedly contributed to increase the emergence of antibiotic resistance among clinical Enterococcus spp. isolates including E. faecium, which is essentially more drug-resistant than E. faecalis, with more than half of the isolates appearing resistant to the antibiotics [6]. Healthcare-associated infections due to E. faecalis and E. faecium more frequently showed resistance to high-level vancomycin and ampicillin, and unsusceptibility to antibiotics [7,8]. Therefore, it is necessary to control bacterial biofilm formation to control the bacterial infection [1,8,9].
Pectolinarin is a flavonoid group, which is present in Cirsium spp. that is commonly known as plume thistle (Figure 1). Cirsium spp. has been reported with bioactive potential In this work, pectolinarin was tested for anti-biofilm formation of pathogenic bacteria for the candidate as an antibiotic's adjuvant. Pectolinarin showed inhibitory effect of the biofilm formation and thereby increasing the susceptibility of antibiotics. These results indicate that pectolinarin has the potential as an antibacterial adjuvant to treat the biofilmrelated infections.
Pectolinarin exhibited the strongest inhibitory effect of biofilm formation against E. faecalis and E. faecium, so those bacteria were used for further studies.  In this work, pectolinarin was tested for anti-biofilm formation of pathogenic bacteria for the candidate as an antibiotic's adjuvant. Pectolinarin showed inhibitory effect of the biofilm formation and thereby increasing the susceptibility of antibiotics. These results indicate that pectolinarin has the potential as an antibacterial adjuvant to treat the biofilmrelated infections.

Pectolinarin Increased the Susceptibility of E. faecalis and E. faecium to Commercialized Antibiotics
Biofilm formation was performed as described above, and the bacterial growth in the presence or absence of pectolinarin (PEC) with ampicillin (AMP), vancomycin (VAN), oxytetracycline (OXY), or streptomycin (STR) was tested after an additional 24 h. Pectolinarin increased the bacterial susceptibility to antibiotics regardless of whether the bacteria showed resistance against the antibiotics ( Figure 3). An amount of 1.56 μg/mL of pectolinarin treatment significantly activated the antibacterial activity of ampicillin, which reduced the viable cells to 0.9% compared with 9% of the only-ampicillin treatment condition. Pectolinarin treatment also reduced the bacterial viability by approximately 10% to 20% compared with only oxytetracycline, streptomycin, or vancomycin treatment ( Figure  3a). A total of 0.39 μg/mL of pectolinarin treatment also approximately increased by 10% to 20% the antibacterial activity of ampicillin, oxytetracycline, streptomycin, and vancomycin against E. faecium ( Figure 3b).

Pectolinarin Increased the Susceptibility of E. faecalis and E. faecium to Commercialized Antibiotics
Biofilm formation was performed as described above, and the bacterial growth in the presence or absence of pectolinarin (PEC) with ampicillin (AMP), vancomycin (VAN), oxytetracycline (OXY), or streptomycin (STR) was tested after an additional 24 h. Pectolinarin increased the bacterial susceptibility to antibiotics regardless of whether the bacteria showed resistance against the antibiotics ( Figure 3). An amount of 1.56 µg/mL of pectolinarin treatment significantly activated the antibacterial activity of ampicillin, which reduced the viable cells to 0.9% compared with 9% of the only-ampicillin treatment condition. Pectolinarin treatment also reduced the bacterial viability by approximately 10% to 20% compared with only oxytetracycline, streptomycin, or vancomycin treatment ( Figure 3a). A total of 0.39 µg/mL of pectolinarin treatment also approximately increased by 10% to 20% the antibacterial activity of ampicillin, oxytetracycline, streptomycin, and vancomycin against E. faecium (Figure 3b).

Pectolinarin Reduced Bacterial Adherence to T24 Cells in a Dose-Dependent Manner
Pectolinarin showed significant inhibition of biofilm formation at 0.01-50 µg/mL. Microscopic analysis revealed that pectolinarin inhibited adhesion of the bacteria to the human urinary bladder cancer T24 cells ( Figure 4) without inhibition of the growth of T24 cells (data not shown). Especially, 0.01 µg/mL of pectolinarin treatment decreased E. faecium adherence by 26% compared to planktonic bacteria.

Pectolinarin Did Not Affect Bacterial Growth
Several antimicrobial compounds also inhibit bacterial biofilm formation because antibiotics kill the bacteria and indirectly decrease biofilm formation [12]. To test whether the biofilm formation inhibition of pectolinarin was due to the antibacterial activity, E. faecalis and E. faecium were treated with 100 µg/mL of pectolinarin for 24 h. Pectolinarin, despite its notable inhibition of biofilm formation, did not show any effect on the bacterial growth ( Figure 5).

Pectolinarin Reduced Bacterial Adherence to T24 Cells in a Dose-Dependent Manner
Pectolinarin showed significant inhibition of biofilm formation at 0.01-50 μg/mL. Microscopic analysis revealed that pectolinarin inhibited adhesion of the bacteria to the human urinary bladder cancer T24 cells (Figure 4) without inhibition of the growth of T24 cells (data not shown). Especially, 0.01 μg/mL of pectolinarin treatment decreased E. faecium adherence by 26% compared to planktonic bacteria.

Pectolinarin Did Not Affect Bacterial Growth
Several antimicrobial compounds also inhibit bacterial biofilm formation because a tibiotics kill the bacteria and indirectly decrease biofilm formation [12]. To test wheth the biofilm formation inhibition of pectolinarin was due to the antibacterial activity, faecalis and E. faecium were treated with 100 μg/mL of pectolinarin for 24 h. Pectolinari despite its notable inhibition of biofilm formation, did not show any effect on the bacteri growth ( Figure 5).

Discussion
Pathogenic bacteria such as E. faecalis, E. faecium, E. coli, P. gingivalis, S. muta sobrinus, S. aureus, C. acnes, and P. aeruginosa contribute to the important global cau nosocomial infections including community-and hospital-acquired infections [7] causes community spreading of pathogenic bacteria that leads to a large increase at-risk and high cost to controlling pathogens [7]. The bacterial biofilm reduced the bacterial susceptibility that make it difficult to eradicate pathogenic bacteria. Entero spp. in biofilms are more resistant to antibiotics than planktonic enterococci are [8,13

Discussion
Pathogenic bacteria such as E. faecalis, E. faecium, E. coli, P. gingivalis, S. mutans, S. sobrinus, S. aureus, C. acnes, and P. aeruginosa contribute to the important global cause of nosocomial infections including community-and hospital-acquired infections [7]. This causes community spreading of pathogenic bacteria that leads to a large increase in the at-risk and high cost to controlling pathogens [7]. The bacterial biofilm reduced the antibacterial susceptibility that make it difficult to eradicate pathogenic bacteria. Enterococcus spp. in biofilms are more resistant to antibiotics than planktonic enterococci are [8,13]. The development of natural products with bioactive ingredients will, therefore, help overcome the issue of drug resistance in bacteria [14]. Cirsium species are considered edible plants and are used as various ailments including hemorrhaging, jaundice, and gastrointestinal disorders [10,11]. Pectolinarin is a secondary metabolite of Cirsium spp. with demonstrated biological activities including antimicrobial, antioxidant, antidiabetic, and anti-inflammatory activity [10,11]. In this study, pectolinarin was identified as an inhibitor of biofilm formation caused by bacteria including E. faecalis, E. faecium, E. coli, P. gingivalis, S. mutans, S. sobrinus, S. aureus, C. acnes, and P. aeruginosa (Figure 2). Pectolinarin also Antibiotics 2022, 11, 598 9 of 12 showed increasing susceptibility of an antibacterial effect in combination with antibiotics. Even though E. faecalis and E. faecium showed resistance against vancomycin, pectolinarin decreased their growth by approximately 20%. Pectolinarin dose-dependently decreased bacterial adherence to T24 cells (Figure 4). These results suggest that pectolinarin should be useful as the inhibitor of infection of E. faecalis and E. faecium. Pectolinarin dose-dependently inhibited the QS system and virulence factor expression (Figures 6 and 7). Virulence factors contribute to the pathogenesis of E. faecalis and E. faecium and many of them were involved in bacterial adhesion to host cells or abiotic surfaces, leading to biofilm formation. In addition, genes for biofilm-associated factors and virulence factors including acm, scm, ebpA, ebpB, esp, bps, gelE, cylR2, cylLS, and cylM were significantly down-regulated by the treatment of pectolinarin (Figures 6 and 7). Therefore, the QS system, virulence factor expression, bacterial adherence, and antibiotic resistance were connected with each other, and thus, biofilm inhibition is for not only the biofilm inhibition but also the reduction in the bacterial survival and infection.
In conclusion, pectolinarin inhibited the bacterial QS system (Figures 6 and 7) to block biofilm formation (Figure 2), enhanced the antibiotic susceptibility (Figure 3), and reduced bacterial adherence to the human host cell (Figure 4). Additional studies including the determination of the molecular mechanism, the relation between adherence, biofilm formation, and virulence, and preclinical animal experiments increase the proof for pectolinarin as an agent for a new antibacterial agent or adjuvant.

Bacterial Strains
Strains used in this study are listed in Table 1. E. faecalis, E. faecium, E. coli, and S. aureus were maintained in tryptic soy broth (TSB) and tryptic soy broth supplemented with 1% glucose (TSBg). S. mutans and S. sobrinus were maintained in Brain Heart Infusion (BHI) broth. P. gingivalis was maintained in tryptic soy broth supplemented with 10% defibrinated horse blood agar. P. aeruginosa was maintained in nutrient agar, and C. acnes was maintained in Reinforced Clostridial agar. All cultures were incubated at 37 • C [13,15,16].

Biofilm Formation Assay
Biofilm formation was performed using TSBg as previously described [12,15,[17][18][19][20][21]. E. faecalis, E. faecium, E. coli, S. mutans, S. sobrinus, S. aureus, P. aeruginosa, C. acnes, and P. gingivalis were added (OD 600 = 0.1) to individual wells of 96-well flat-bottomed plates. Pectolinarin with concentrations ranging from 0.01 to 100 µg/mL was added to respective wells, and the cells were incubated at 37 • C for 24 h. The inhibition of biofilm formation by compound was detected by the crystal violet staining method. Briefly, after 24 h of treatment, the supernatant was removed and the wells were rinsed with physiological saline. In addition, 1% crystal violet (CV) solution was added to each well and incubated for 30 min. The excess of dye was removed by washing the plates under running water. The bound CV was released by adding 125 µL of 30% acetic acid followed by an incubation for 15 min at room temperature. The absorbance was measured at 595 nm using a microplate reader (Bio Tek Instruments, CA, USA) [15,22].

The Combinatorial Antibacterial Effects of Pectolinarin with Commercialized Antibiotics
The antibacterial effects by the combinatorial treatment of pectolinarin with antibiotics were evaluated using the plate-counting method. The bacterial culture at an absorbance of OD 600 = 0.1 was prepared, and 1 mL of the bacterial suspension with pectolinarin (final concentrations of 1.56, 0.39, 0.09, and 0.01 µg/mL) was added. Planktonic bacteria were removed using sterile saline and the medium (TSBg and Pectolinarin) was incubated for 24 h. The bacteria in the biofilm were enumerated using the plate-counting method [12].

Bacterial Adherence Assays
The human urinary bladder carcinoma T24 cell was purchased from the Korean Cell Line Bank (Seoul, Korea). T24 cells were maintained in RPMI 1640 media supplemented with 10% fatal bovine serum in a 5% CO 2 incubator at 37 • C [12,23,24]. T24 cells were added to 24-well plates (0.5 × 10 6 cells per well) for 24 h prior and co-incubated with 0.01-50 µg/mL of pectolinarin-treated E. faecium or E. faecalis (100 MOI, 5 × 10 7 CFU per well) for 24 h. Cells were then washed three times with PBS. Gram iodine mordant was applied for 1 min and briefly washed with PBS. To remove any nonspecific crystal violet, a Gram decolorizer solvent was added to the plate for 30 s. After washing with PBS until the PBS ran clear, safranin was applied for 30 min. Samples were assessed under a microscope [25,26].

Time Kill Assay
Growth curves were obtained as previously described with slight modification [22]. E. faecalis and E. faecium cultures were prepared with tryptic soy broth at an OD 600 = 0.1. Pectolinarin (100 µg/mL) was added and then the cells were incubated at 37 • C. Growth was evaluated by measuring the optical density of OD 600 using a microplate reader after 0, 1, 2, 4, 8, 12, 24, and 72 h [11].

Quantitative RT-PCR Analysis
Bacterial cultures and the pectolinarin treatment condition was the same as the method to check the biofilm formation inhibition. Total RNA was isolated using TRIzol reagent (Life Technology, Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions, and the reverse transcriptase (NanoHelix, Daejeon, Korea) reaction was prepared using 1 µg of RNA to obtain cDNA. qRT-PCR was carried out using the 2X Sybr Green qPCR Mater Mix (CellSafe, Yongin, Korea). Primer sets for the fsrB, fsrC, gelE, acm, scm, ebpA, ebpB, esp, bps, cylM, cylR2, and cylLS genes are listed in Table 2. Genes encoding 23 sRNA and tufA were used as endogenous controls. Table 2. Gene-specific primers used for real-time RT-PCR.

Statistical Analysis
All experiments were performed at least three times, and all data are represented as the mean ± S.D.

Conclusions
Pectolinarin showed the reduced bacterial biofilm formation, activated the susceptibility of commercially available antibacterial agents, and reduced the bacterial adherence to host cells. These results recommend the bacterial biofilm formation as a good target to develop the antibacterial agents against biofilm-related infections with low cytotoxicity, and pectolinarin should be a promising candidate.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.