Evaluation of the Bactericidal and Fungicidal Activities of Poly([2-(methacryloyloxy)ethyl]trimethyl Ammonium Chloride)(Poly (METAC))-Based Materials

Poly([2-(methacryloyloxy)ethyl]trimethyl ammonium chloride) (METAC) and the gels were prepared and evaluated for their bactericidal and fungicidal activities. The antimicrobial properties of poly(METAC) were tested against Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Saccharomyces cerevisiae (Sa. cerevisiae), methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (P. aeruginosa), and Candida albicans (C. albicans). Moreover, the structural forms of the linear and cross-linked poly(METAC) were investigated for their influences on bacterial aggregation, precipitation, and cell-death. To our knowledge, this is the first report on the comparison of the antimicrobial properties of poly(METAC) and poly(METAC)-gels. The bactericidal and fungicidal activities were evaluated by determining minimum inhibitory concentrations (MICs), UV–Vis spectroscopy, and fluorescence and confocal microscopies. The MICs were found to be 123 (MSSA), 123 (MRSA), 123 (P. aeruginosa), 370 (E. coli), 123 (B. subtilis), 370 (C. albicans), and 370 μg/mL (Sa. cerevisiae), as determined by broth dilution, and 370 (MSSA), 370 (MRSA), 370 (P. aeruginosa), 3300 (E. coli), 370 (B. subtilis), 1100 (C. albicans), and >10,000 μg/mL (Sa. cerevisiae), as determined by paper disc diffusion (on solid medium). The poly(METAC)-gels achieved rapid adsorption/precipitation of bacteria via the cationic surface charge. Thus, these poly(METAC)-based polymers can potentially be used as antibacterial materials.


Introduction
Antimicrobial polymers have been receiving substantial attention owing to their multiple advantages, such as long-term activity, limited residual toxicity, chemical stability, non-volatility, and non-penetration via skin [1]. Cationic polymers are one of the most studied antimicrobial polymers [2,3]. The accepted mechanism by which cationic polymers lead to bacterial death is as and non-penetration via skin [1]. Cationic polymers are one of the most studied antimicrobial polymers [2,3]. The accepted mechanism by which cationic polymers lead to bacterial death is as follows: 1. adsorption onto the bacterial cell surface, 2. diffusion through the cell wall, 3. binding to the cytoplasmic membrane, and 4. disruption of the bacterial membrane [4]. This mechanism is effective regardless of the types of bacteria. Therefore, it is expected that the cationic polymers can also show an antimicrobial effect against drug-resistant bacteria. Recently, the World Health Organization (WHO) has listed the drug-resistant bacteria that could shortly become a menace to humans [5]. For example, in the US, the methicillin-resistant Staphylococcus aureus (MRSA) causes more deaths than human immunodeficiency virus (HIV) [6]. Very recently, Su et al. prepared cationic polypeptide-based polymers that present antimicrobial properties when administered in MRSAinfected mice [7,8]. The poly(hexamethylene biguanide), another type of cationic polymer, is approved by the Food and Drug Administration (FDA), and there are no reports on emergent drugresistant bacteria [9,10]. Poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride) (poly(METAC)) is one of the methacrylate types of cationic polymer, consisting of quaternary ammonium cations. The methacrylate monomer can be polymerized by (living/controlled) radical polymerizations and can also be copolymerized with other types of typical (meth)acrylate monomers [11][12][13]. The development of polymeric synthesis has facilitated access to functional polymers, allowing not only the control of molecular weights and molecular weight distribution, but also the control of the physicochemical properties of the polymeric structures [14,15]. Stopiglia et al. showed that the METAC monomer presents antimicrobial properties against 31 kinds of Candida albicans (C. albicans) [16]. Prijick et al. succeeded in preventing the formation of a C. albicans biofilm on a surface coated with copolymers of poly(METAC) [17]. The poly(METAC) has also been conjugated with natural polymers. The cotton grafting poly(METAC) shows antimicrobial properties against Grampositive and Gram-negative bacteria, and this effect increases with the amount of grafted poly(METAC) [18]. A wool-modified surface was combined with the chitosan grafting poly(METAC), and the conjugated materials shows efficient antimicrobial properties [19]. In this study, the antimicrobial properties of poly(METAC) were tested against Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Saccharomyces cerevisiae (Sa. cerevisiae), methicillin-susceptible Staphylococcus aureus (MSSA), MRSA, Pseudomonas aeruginosa (P. aeruginosa), and C. albicans. In addition, poly(METAC)gel was prepared to allow a comparison of the two different structural forms, linear and cross-linked poly(METAC), on their ability to induce bacterial aggregation, precipitation, and cell-death. To our knowledge, this is the first report on a comparison of antimicrobial properties of poly(METAC) and poly(METAC)-gel ( Figure 1). The minimum inhibitory concentration (MIC) was determined by broth dilution and paper disc diffusion methods, and the bacterial aggregation, precipitation, and celldeath were measured by UV-Vis spectroscopy, and fluorescence and confocal microscopies.

Determination of Minimum Inhibitory Concentration (MIC) of Poly(METAC)
MICs were determined by broth dilution and paper disc diffusion methods. Luria broth was used for E. coli and B. subtillis, Müller Hinton broth (MHB) for S. aureus, cation-adjusted MHB for P. aeruginosa, potato-dextrose broth for S. cerevisiae, and MHB containing glucose (2% (w/v)) and methylene blue (0.5 µg/mg) for C. albicans. Except for P. aeruginosa, corresponding agar media were used for the paper disc diffusion method. Müller Hinton agar medium was used for P. aeruginosa. The suitable broths for S. aureus, P. aeruginosa, and C. albicans were selected using the performance standards for antimicrobial susceptibility testing of the medical field. Other bacteria were incubated using the typical broths. Bacterial suspensions were prepared via the turbidity (McFarland standards) in accordance with each bacterial performance standards. For the broth dilution method, microbial cells (150 µL of the overnight culture fluid) cultivated overnight at 37 • C were inoculated into fresh liquid medium supplemented with poly(METAC) and subsequently incubated for 24 h at 37 • C with shaking at 150 rpm. MIC was defined as the minimum poly(METAC) concentration that completely inhibited microbial growth. For the paper disc diffusion method, microbial cells were spread onto agar medium, over which a series of discs containing 0-1.0% (w/v) poly(METAC) were placed. Cells were Polymers 2018, 10, 947 4 of 9 cultivated for 24 h at 37 • C and growth of microorganisms around the discs were observed. MIC was defined as the minimum poly(METAC) concentration in discs leading to growth inhibition zones.

Evaluation of Bacterial Aggregation/Precipitation by Poly(METAC) and Poly(METAC)-gel
B. subtilis cells grown in LB liquid medium until the mid-exponential phase were exposed to poly(METAC) or poly(METAC)-gel-1.28 at different concentrations (1-5 mg/1.5 mL) for 24 h. The appropriate amounts of poly(METAC) or poly(METAC)-gel-1.28) were dissolved/dispersed in 500 µL milliQ and mixed with B. subtilis suspension (1 mL). After 24 h, the transmittance of the supernatants was measured using UV-Vis spectroscopy at 500 nm.

Evaluation of the Bactericidal Effect of Poly(METAC) and Poly(METAC)-gel
E. coli cells grown in LB liquid medium until the mid-exponential phase were exposed to poly(METAC) or MilliQ water (as control) and immediately subjected for the next procedures. To evaluate the bactericidal activity of the polymer, cells treated with poly(METAC) were stained with SYTO9 and propidium iodide (PI) using the LIVE/DEAD BacLight Bacterial Viability Kit (Life Technologies, Carlsbad, CA, USA), by following the manufacturer's instructions, and observed under a fluorescence microscope. To evaluate bacterial survival in the presence of poly(METAC), E. coli cells exposed to different concentrations of poly(METAC) were washed once with phosphate buffered saline [21], spread on LB agar medium, and incubated at 37 • C for 24 h, after which the colonies were counted. Adsorbed bacteria on poly(METAC)-gel were observed by confocal microscopy. The poly(METAC)-gels were immersed into an E. coli suspension for 10 min and washed 2 times with large amounts of pH 7.4 phosphate-buffered saline (PBS). Bacterial cells on the poly(METAC)-gels were stained using the LIVE/DEAD BacLight Bacterial Viability Kit.

Characterizations
Number-average molecular weight (M n ) and molecular weight distribution (M w /M n , M w : weight-average molecular weight) of the synthesized poly(METAC) were determined by gel permeation chromatography (GPC) (Shimadzu, Kyoto, Japan) at room temperature, with SB-802.5 HQ and SB-804 HQ columns (Shodex, Tokyo, Japan) connected to a RID-20A refractive index detector (Shimadzu, Kyoto, Japan). Elution was performed with 0.5 M sodium acetate/0.5 M acetic acid buffer at a flow rate of 1.0 mL/min. Transmittance of mixture suspensions of bacteria and polymeric materials at 500 nm was recorded at 25 • C on a UV-Vis spectrometer V-650 (JASCO International Co., Ltd., Tokyo, Japan). A microplate reader Infinite M1000-SSY (TECAN, Kanagawa, Japan), a fluorescence microscope MX (MEIJI Techno, Saitama, Japan) with an excitation light source FL-PWJ (MEIJI Techno, Saitama, Japan), and a confocal microscope LSM 700 (Carl Zeiss, Oberkochen, Germany) were used to evaluate the bactericidal effect of poly(METAC) copolymers.

Results
Molecular weight (M n ) and molecular weight distribution (M w /M n ) of poly(METAC) were determined to be 87,400 g/mol and 2.83, respectively (measured by GPC). The synthesized poly(METAC) inhibited the growth of all the tested microorganisms: Gram-positive bacteria (S. aureus and B. subtilis), Gram-negative bacteria (P. aeruginosa and E. coli), and yeasts (C. albicans and Ss. cerevisiae). The determined minimum inhibitory concentrations (MICs) for these microorganisms are shown in Table 1. The MICs determined by broth dilution method (in liquid media) were found to be 123 µg/mL (MSSA), 123 µg/mL (MRSA), 123 µg/mL (P. aeruginosa), 370 µg/mL (E. coli), 123 µg/mL (B. subtilis), 370 µg/mL (C. albicans), and 370 µg/mL (Sa. cerevisiae). The MICs determined by paper disc diffusion method (on solid media) were found to be 370 µg/mL (MSSA), 370 µg/mL (MRSA), 370 µg/mL (P. aeruginosa), 3300 µg/mL (E. coli), 370 µg/mL (B. subtilis), 1100 µg/mL (C. albicans), and >10,000 µg/mL (Sa. cerevisiae). The MICs of broth dilution method were lower than that of paper disc diffusion method, for all the microorganisms. These results suggested that , when compared with S. epidermidis. The resistance was explained as resulting from the low negative charge and hydrophobicity of the S. aureus surface [22]. In fact, the MICs of every S. aureus strain (clinical isolate species, two MSSA and six MRSA) were all over 3200 µg /mL. On the other hand, the MICs of eleven types of S. epidermidis were in the range 25-100 µg/mL. Moreover, the adhesion of poly(DMAEMA) to S. aureus was lower than that of S. epidermidis, as measured by flow cytometry. The polymeric structures also affect the antimicrobial and adsorptive properties on the target bacteria. Shirbin et al. prepared a macroporous cryogel using cationic polypeptide of poly(lysine)-b-poly(valine), and the cryogel showed a "trap and kill" effect against E. coli [23]. The cationic four-arm star glycopolymer-peptides showed different MICs depending on the glycol-types such as glucose, galactose, or mannose (E. coli: 10-256 µg/mL, P. aeruginosa: 32-512 µg/mL, and S. aureus 16-128 µg/mL) [24]. Next, poly(METAC)-gels were tested on their abilities to induce bacterial aggregation, precipitation, and cell-death. The poly(METAC)-gels-1.28 were prepared via redox polymerization using N,N -methylenebis(acrylamide) as a cross-linker [25]. Figure 2 shows the transmittance changes of B. subtilis suspensions treated by poly(METAC) or poly(METAC)-gel-1.28 after 24 h. The control showed that less than 5% of the observed transmittances were due to the suspended B. subtilis. Similarly, less than 5% of the observed transmittances of the B. subtilis suspensions were due to the adding of the poly(METAC) solution, which were similar to control. On the other hand, the transmittance tended to increase with the amount of poly(METAC)-gel-1.28, and reached 90% with 2 mg of the gel. The weak bacterial aggregation/precipitation in the presence of poly(METAC) is caused by the electrostatic repulsion of the adsorbing poly(METAC) on the bacterial surface. The bacteria trapped on the poly(METAC)-gel-1.28 cannot be re-suspended via the electrostatic interaction between the anionic bacterial surface and cationic gel surface, resulting in the high transmittance values. In the nonionic poly(2-hydroxyethyl methacrylate (HEMA))-gel, the transmittance was lower than 5% ( Figure S1). The interaction of nonionic poly(HEMA)-gels with bacteria was smaller than that of the cationic polymers. Similar results were reported by Berlutti's group [26]. Williams et al. prepared poly(METAC)-based cationic temperature-responsive gels, which show bacteriostatic and bactericidal properties against S. aureus when compared with that of nonionic gels [27]. Next, the biocidal ability of the poly(METAC) and poly(METAC)-gels was investigated. Figure  3 shows the LIVE/DEAD test of E. coli in the presence of MilliQ ( Figure 3A) or 1 wt % of poly(METAC) ( Figure 3B). The E. coli were stained with SYTO9/PI, which leads to the exhibition of red fluorescence by the death cells due to the treatment with poly(METAC). In another experiment, E. coli were exposed to poly(METAC) at different concentrations (10 mg/mL-41 μg/mL), spread on the nutrient agar, and the colony forming units (CFUs) were measured after 24 h. Figure 3C shows the CFU at each poly(METAC) concentration. CFUs decreased with increasing poly(METAC) concentration, and reached a plateau (about 100 CFU/mL) at over 1.1 mg/mL. The prevention of the E. coli growth by poly(METAC) was about 0.4 million-fold higher than that of the control without poly(METAC). Adsorbed bacteria on poly(METAC)-gels were observed by the confocal microscopy. The poly(METAC)-gels were immersed in the E. coli suspension for 10 min, and were washed 2 times with large amounts of pH 7.4 PBS. The bacteria on the poly(METAC)-gels were stained with the live/dead agent. Figure 4A-C shows confocal microscopy images of the poly(METAC)-gels with different cross-linker amounts, 0.65, 1.86, and 3.10 mol %. Almost all bacteria on the poly(METAC)gels were dead as shown by the red fluorescence. The adsorbed bacteria were also observed by scanning electron microscopy (SEM) measurement ( Figure 4D). He et al. prepared a cationic hydrogel film via a light-triggered cross-linking [28]. The antimicrobial activity against E. coli was shown to depend on the amount of the ethylene glycol-based cross-linkers. At 20% of cross-linking degree, approximately 100% of antibacterial activity was reached. Yang et al. prepared a hydrogel consisting of METAC and nonionic monomers bound by disulfide bonds, and the gels successfully prevented the adsorption of proteins and E. coli on the surface [29]. The physical destruction of the adsorbing E. coli on the gel surface was observed via SEM measurement. Other nonionic polymers such as ethylene glycol-based copolymers also can prevent the adsorption of bacteria [30,31]. The METAC is a methacrylate type of monomer and the polymeric structure can be easily controlled via a combination of living radical polymerizations and click chemistry. The antibacterial properties will be customized by the structural formulation of poly(METAC). Next, the biocidal ability of the poly(METAC) and poly(METAC)-gels was investigated. Figure 3 shows the LIVE/DEAD test of E. coli in the presence of MilliQ ( Figure 3A) or 1 wt % of poly(METAC) ( Figure 3B). The E. coli were stained with SYTO9/PI, which leads to the exhibition of red fluorescence by the death cells due to the treatment with poly(METAC). In another experiment, E. coli were exposed to poly(METAC) at different concentrations (10 mg/mL-41 µg/mL), spread on the nutrient agar, and the colony forming units (CFUs) were measured after 24 h. Figure 3C shows the CFU at each poly(METAC) concentration. CFUs decreased with increasing poly(METAC) concentration, and reached a plateau (about 100 CFU/mL) at over 1.1 mg/mL. The prevention of the E. coli growth by poly(METAC) was about 0.4 million-fold higher than that of the control without poly(METAC). Adsorbed bacteria on poly(METAC)-gels were observed by the confocal microscopy. The poly(METAC)-gels were immersed in the E. coli suspension for 10 min, and were washed 2 times with large amounts of pH 7.4 PBS. The bacteria on the poly(METAC)-gels were stained with the live/dead agent. Figure 4A-C shows confocal microscopy images of the poly(METAC)-gels with different cross-linker amounts, 0.65, 1.86, and 3.10 mol %. Almost all bacteria on the poly(METAC)-gels were dead as shown by the red fluorescence. The adsorbed bacteria were also observed by scanning electron microscopy (SEM) measurement ( Figure 4D). He et al. prepared a cationic hydrogel film via a light-triggered cross-linking [28]. The antimicrobial activity against E. coli was shown to depend on the amount of the ethylene glycol-based cross-linkers. At 20% of cross-linking degree, approximately 100% of antibacterial activity was reached. Yang et al. prepared a hydrogel consisting of METAC and nonionic monomers bound by disulfide bonds, and the gels successfully prevented the adsorption of proteins and E. coli on the surface [29]. The physical destruction of the adsorbing E. coli on the gel surface was observed via SEM measurement. Other nonionic polymers such as ethylene glycol-based copolymers also can prevent the adsorption of bacteria [30,31]. The METAC is a methacrylate type of monomer and the polymeric structure can be easily controlled via a combination of living radical polymerizations and click chemistry. The antibacterial properties will be customized by the structural formulation of poly(METAC).

Conclusions
In conclusion, poly(
Thus, these poly(METAC)-based copolymers can potentially be used as antibacterial materials.