Antibacterial activity and mechanism of sanguinarine against Providencia rettgeri in vitro

Background Sanguinarine (SAG), a benzophenanthridine alkaloid, occurs in Papaveraceas, Berberidaceae and Ranunculaceae families. Studies have found that SAG has antioxidant, anti-inflammatory, and antiproliferative activities in several malignancies and that it exhibits robust antibacterial activities. However, information reported on the action of SAG against Providencia rettgeri is limited in the literature. Therefore, the present study aimed to evaluate the antimicrobial and antibiofilm activities of SAG against P. rettgeri in vitro. Methods The agar dilution method was used to determine the minimum inhibitory concentration (MIC) of SAG against P. rettgeri. The intracellular ATP concentration, intracellular pH (pHin), and cell membrane integrity and potential were measured. Confocal laser scanning microscopy (CLSM), field emission scanning electron microscopy (FESEM), and crystal violet staining were used to measure the antibiofilm formation of SAG. Results The MIC of SAG against P. rettgeri was 7.8 μg/mL. SAG inhibited the growth of P. rettgeri and destroyed the integrity of P. rettgeri cell membrane, as reflected mainly through the decreases in the intracellular ATP concentration, pHin and cell membrane potential and significant changes in cellular morphology. The findings of CLSM, FESEM and crystal violet staining indicated that SAG exhibited strong inhibitory effects on the biofilm formation of P. rettgeri and led to the inactivity of biofilm-related P. rettgeri cells.


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127 Intracellular pH (pH in ) test 128 The pHin was evaluated according to the method presented by . 300 L 129 overnight cultures of P. rettgeri were transferred to 30 mL TSB and then incubated at 37C for 8 130 h. After incubation, the cells were centrifuged (8000 ×g, 10 min), rinsed twice with 50 mM HEPES 131 and 5 mM EDTA (mixed solution at pH 8.0), and resuspended in 20 mL mixed solution. 132 Subsequently, 3 M carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Beyotime 133 Bioengineering Institute) was added to the sample. The sample was incubated at 37C for 20 min, 134 then washed once with the mixture solution (pH 7.0; 50 mM PBS and 10 mM MgCl2), and 135 resuspended in the buffer. To eliminate nonconjugated CFDA-SE, glucose (10 mM final 136 concentration) was added and incubated at 37C for another 30 min. Cells were then washed twice, 137 resuspended with 50 mM potassium phosphate buffer (pH 7.0), and stored in ice. 138 Fluorescently stained cell suspensions were treated with SAG (0, 1, and 2 MIC) and 139 transferred into a black opaque 96-well flat-bottomed plate (Nunc, Copenhagen, Denmark). After 140 treatment for 20 min, fluorescence intensity was assessed at excitation wavelengths of 440 and 141 490 nm and emission wavelength of 520 nm, where the excitation slit width was 9 nm and the 142 emission were 20 nm. pHin was defined as the ratio of the fluorescence signal at the pH-sensitive 143 wavelength of 490 nm to the fluorescence signal at the pH-insensitive wavelength of 440 nm. The 144 measurements were performed by a fluorescence microplate reader (Thermo Fisher Scientific, 145 Finland). During the experiment, the system was maintained at 25C, and the fluorescence of the 146 control group was measured and subtracted from the value of the treated suspension.

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The calibration curves were established for CFDA-SE-loaded cells with mixed solutions of 148 different pH values. The mixture including glycine (50 mM), citric acid (50 mM), Na 2 HPO 4 2H 2 O 149 (50 mM), and KCl (50 mM). The pH was adjusted to various values (2-10) with NaOH or HCl, 150 and valinomycin (10 M) and nigrosine (10 M) were added to adjust the pH in and pH out . Finally, 151 fluorescence intensity was evaluated at 25C. 152 153 Determination of membrane potential 154 The membrane potential was determined according to the method by Wang et al. (2018), with 155 minor modifications. The fluorescent probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol 156 [DiBAC 4 (3)] was used to determine the changes in bacterial membrane potential. Bacterial 157 suspensions (1 × 10 5 CFU/mL) were centrifuged at 4000 ×g for 10 min and washed twice with 10 158 mM PBS (pH 7.0). The cells were treated with different concentrations of SAG (0, 1, and 2 MIC) 159 and incubated at 37C for 2 h. Then, the resulting samples were incubated with DiBAC 4 (3) at 25C 160 for 10 min in the dark and washed once with PBS. The fluorescence microplate reader was utilized 161 to determine the florescence intensity at excitation/emission wavelengths of 492/515 nm. The 162 excitation and emission slit widths were 3 and 5 nm, respectively. 163 164 Evaluation of bacterial membrane integrity 165 The modified method of Liu et al. (2018) was applied to assess the cell membrane integrity of 166 P. rettgeri cells using confocal laser scanning microscopy (CLSM; Zeiss LSM 880 with Airyscan, 167 Bonn, Germany). Overnight cultures were diluted in TSB medium a concentration of 1 × 10 5 168 CFU/mL and treated with SAG (0, 1, and 2 MIC) for 2 h. Subsequently, the cells were harvested 169 by centrifugation at 8000 ×g for 5 min, and then resuspended with 10 mM PBS of equivalent 170 volume. Then, the samples were mixed with SYTO 9 and propidium iodide (PI) at 25C for 15 171 min to visualize the cells. Cells were collected and washed with 10 mM PBS to remove excess 172 dyes, and finally measured with a CLSM.
173 Transmission electron microscopy (TEM) 174 TEM (G2 F20 S-Twin; FEI, Hillsboro, OR, USA) was used to analyze cell morphology as 175 described by Joung et al. (2016) with some modification. Overnight cultures of P. rettgeri (1 × 10 5 176 CFU/mL) were diluted and exposed to three concentrations of SAG (0, 1, and 2 MIC) for 4 h at 177 37C. Thereafter, the cultures were centrifuged (10,000 ×g, 8 min), and washed twice with 0.85% 178 NaCl. The suspension was removed, and cell pellets were fixed with 2.5% glutaraldehyde for 12 179 h at 37C. Then, cells were dehydrated through graded alcohols (20%, 50%, 70%, 80%, 90%, and 180 100%) for 15 min; the resulting cell pellets were embedded in resin. Ultrathin samples were incised 181 by ultramicrotome, and uranyl acetate stain was used for TEM. P.rettgeri cells grown on the slides for 12-h were exposed to SAG (0, 1/16, 1/8, and 1/4 MIC) 211 and cultured for 24 h at 37℃, and then washed twice with 0.85% NaCl. The samples of P. rettgeri 212 biofilm were stained with CFDA-SE and then incubated for 15 min at 25C for direct visual 213 observation of the biofilm formation using a CLSM. Fluorescence intensity of the cells was 214 measured at excitation/emission wavelengths of 488/542 nm for CFDA-SE. The changes in major biofilm matrix levels within 24-h-old biofilms of mono or dual species 218 in the presence of SAG were detected by CLSM of the biofilm matrix combined with different 219 dyes. Briefly, biofilms were prepared using the procedure described in the crystal violet biofilm 220 assay section. The resulting samples were washed thrice with 1 mM sterile PBS (pH 7.4) and 221 labeled with three fluorescent dyes: (1) 25% (v/v) SYPRO Ruby, which stains most classes of 222 proteins, and 2 μM SYTO9 dye; (2) 5 μg/mL WGA dye, which labels polysaccharides, and 5 223 μg/mL water-soluble FM® dyes; and (3) 2 μM PI dye and 2 μM SYTO9 dye. Subsequently, the 224 biofilm was washed twice with 1 mM PBS to remove all planktonic bacteria, where the 225 excitation/emission wavelengths were 450/610 nm for SYPRO Ruby (red), 498/517 nm for 226 SYTO9 (green), 488/617 nm for PI (red), 495/519 nm for WGA (green), and 479/565 nm for 227 water-soluble FM® dyes (red). Color confocal images were observed with CLSM. To evaluate the diffusion of antibiotics into biofilms, the diffusion of gatifloxacin into biofilms 231 was identified based on the intrinsic fluorescence of CLSM. The biofilms described above were 232 placed on glass coverslips inside the 24-well plate for 48 h at 37°C in the presence of SAG at 0, 233 1/16, 1/8, and 1/4 MIC, withdrawn, and gently washed thrice with 10 mM PBS, added with 234 gatifloxacin at a final concentration of 0.4 mg/mL, and further incubated at 37°C for 4 h. Then, 3 235 μM SYTO 9 was added and incubated for 15 min to observe gatifloxacin diffusion within biofilms. 236 The samples were washed with 10 mM PBS thrice and observed with CLSM. The emission peak 237 of gatifloxacin at 495 nm was recorded upon excitation at 291 nm. At least three random fields 238 were visualized for each biofilm, and representative images were displayed. All experiments in this study were conducted thrice independently. Each biological replicate 242 was needed to conduct two technical replicates. Data are expressed as mean ± standard deviation 243 (SD). Results were analyzed using SPSS 8.0 software and Origin 8.5 statistics. Analysis of 244 variance (ANOVA) was performed to determine any significant differences (P ≤ 0.01). According to the MIC results presented in Table 1, P. rettgeri manifested antibiotic resistance, 250 as evidenced by the MIC of ampicillin (32 μg/mL), cefepime (16-64 μg/mL), ertapenem (8 251 μg/mL), imipenem (16 μg/mL), tobramycin (8-16 μg/mL), amikacin (2-64 μg/mL), 252 piperacillin/tazobactam (64-128 μg/mL), and nitrofurantoin (32-512 μg/mL). SAG exhibited 253 excellent antibacterial activity against P. rettgeri 1-7, with the MIC values of 7.8, 7.8, 7.8, 7.8, 7.8, 254 3.9, and 3.9 μg/mL, respectively. The biomass of P. rettgeri was decreased when treated with SAG at MIC in media ( Figure 1); 258 however, cells treated with SAG at 2 MIC exhibited a significant decline in the number of viable 259 cells, which was lower than the detection limit after 24 h. However, the growth curves exhibited 260 weaker increases and lower growth rates at concentrations of 1/8 and 1/4 MIC than those at other 261 MIC. Higher concentrations of SAG resulted in a significant reduction in the relative number of 262 viable cells, while the opposite has been found in lower SAG concentrations. 263 264 SAG treatment decreased intracellular ATP concentrations, pHin, and membrane 265 potential of P. rettgeri 266 When P. rettgeri cells were treated with SAG at 1 and 2 MIC, the intracellular ATP 267 concentration was significantly decreased (P < 0.01) in the SAG-treated group compared with the 268 untreated control group ( Figure 2A); however, there were no significant differences between the 269 cells treated with SAG at 1 and 2 MIC, but the decrease in intracellular ATP concentration was 270 positively followed by the increase in SAG concentration. Simultaneously, the extracellular ATP 271 concentrations of P. rettgeri cells increased in a concentration-dependent manner, and its action 272 was in a time-dependent manner ( Figure 2B).

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The pH in of P. rettgeri after SAG treatment changed significantly, as presented in Figure 2C. 274 In this study, the original pH in of P. rettgeri was 5.97 ± 0.25, and treatment with SAG at 1 and 2 275 MIC decreased this pH in significantly to 4.53 ± 0.25 (P < 0.01) and 3.47 ± 0.25 (P < 0.01), 276 respectively.

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Moreover, the fluorescence intensities of P. rettgeri treated with SAG at 1 MIC for 2 h were 278 less than those of the untreated group ( Figure 2D). Consistently, with an increasing concentration 279 of SAG from 1 to 2 MIC, the membrane potential decreased significantly (P < 0.01). 280 281 SAG treatment increased the cell membrane permeability of P. rettgeri 282 The cells with intact membranes stained with CFDA-SE exhibited bright green fluorescence, 283 but membrane-damaged cells loaded with PI exhibited red fluorescence. In Figure 3, P. rettgeri 284 cells of the untreated control group exhibited bright green fluorescence, thereby revealing the 285 physical integrity of the cell membrane. However, the results demonstrated significantly reduced 286 green fluorescence and increased red fluorescence when treated with 1 MIC of SAG. As the SAG 287 concentrations increased, green fluorescence declined, and red fluorescence gradually increased.
288 Treatment with SAG led to changes in the cell morphology of P. rettgeri 289 In this study, TEM was used to assess the level of cell wall damage and intracellular 290 modification in SAG-treated P. rettgeri. The P. rettgeri cells without treatment exhibited visible 291 outline and the peptidoglycan layer ( Figure 4A), while P. rettgeri cells treated with SAG at 1 MIC 292 were malformed, damaged, and out of proportion ( Figure 4B). However, P. rettgeri cells treated 293 with SAG at 2 MIC showed a wavy contour of the cytoplasmic membrane and dense, 294 undifferentiable cellular content, indicating an obvious shrinkage of the cytoplasm ( Figure 4C). 295 Consequently, SAG-treated P. rettgeri could damage the cell membrane and cell wall outline.

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The cell morphology after treatment with SAG presented significant changes using FESEM. 297 Compared with the normal smooth cell surface of the untreated group ( Figure 5A), cells treated 298 with SAG at 1 MIC exhibited significant enlargement, uneven size, and rough surface ( Figure 5B). 299 In addition, cells treated with SAG at 2 MIC exhibited significant surface collapse and expansion 300 on the cell membrane as a result of the destruction of the bacterial cell wall (Figure 5C), which 301 indicated a positive correlation between the increase in the concentrations of SAG and the damage 302 degree of cell membrane. 303 304 Inhibitory effect of SAG on the biofilm formation of P. rettgeri 305 The crystal violet assay, FESEM and CLSM were used to analyze the inhibitory effects of 306 SAG on the biofilm formation of P. rettgeri. As presented in Figure 6, SAG exhibited a significant 307 inhibitory effect on the biofilm formation of P. rettgeri at different concentrations (P < 0.01). The 308 biofilm formation index of the untreated control group was approximately 3.6. Biofilm formation 309 was inhibited by 68% in the presence of 1/16 MIC SAG; furthermore, when the SAG concentration 310 was higher than 1/4 MIC, the biofilm formation in P. rettgeri was suppressed by more than 95%. 311 The results of FESEM and CLSM further showed that biofilm formation was significantly 312 inhibited at 1/16, 1/8, and 1/4 MIC in the SAG-treated group compared with the untreated group 313 (Figure 7). The inactivation effect of SAG against 36-h-old biofilm-associated P. rettgeri cells is 318 presented in Figure 8. The untreated group was almost entirely green, as observed with CLSM 319 (Figure 8A and E), indicating that most of the cell membranes of P. rettgeri cells embedded in 320 biofilms were integrated and viable. However, when P. rettgeri cells within biofilms were exposed 321 to 16 MIC SAG, the CLSM observation displayed abundant red florescence, revealing that most 322 of the cell membranes of P. rettgeri within biofilms were impaired, as presented in Figure 8D and 323 H. Furthermore, with an increasing concentration of SAG from 4 to 8 MIC, the green signal on the 324 membrane disappeared gradually and the red signal increased gradually ( Figure 8B, C, F, and G), 325 indicating that intact and viable biofilms changed with the increasing concentration of SAG. These 326 results suggested that SAG presented the killing effect on the P. rettgeri cells within biofilms. The biofilm matrix commonly comprises proteins, nucleic acid [environmental DNA 331 (eDNA)], and carbohydrates, which provide structural rigidity and protection from the external 332 environment to control gene regulation and nutrient adsorption (Hobley et al., 2015). Thus, the 333 changes in the biofilm matrix composition could affect biofilm formation. Therefore, the effects 334 of SAG on the proteins, nucleic acid (eDNA), and carbohydrates of P. rettgeri were investigated. 335 The changes in major biofilm matrix levels within 24-h-old biofilms of mono or dual species in 336 the presence of SAG were detected by CLSM in Figure 9. Different reagents were used to mark 337 the eDNA and proteins red and the carbohydrates green. The untreated groups of nucleic acids and 338 proteins exhibited almost red florescence, as observed with CLSM; with the increasing 339 concentration of SAG, the red signal was gradually reduced ( Figure 9A and C). The untreated 340 group of carbohydrates exhibited green florescence, as observed with CLSM; with an increasing 341 concentration of SAG, the green signal gradually disappeared ( Figure 9B), indicating that nucleic 342 acid, protein, and carbohydrate contents decrease with an increasing concentration of SAG.
343 344 Effects of SAG on biofilm diffusion 345 Gatifloxacin was used to verify the effects of SAG on biofilm diffusion, which was monitored 346 using fluorescence CLSM. Providencia rettgeri biofilms were grown with different SAG 347 concentrations for 48 h and then added with gatifloxacin for 4 h. Formed biofilms were stained 348 with SYTO9 to allow the visualization of nucleic acid (green fluorescence).

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Gatifloxacin diffused significantly at 0 MIC ( Figure 10A). However, in mixed biofilms with 350 1/4 MIC of SAG, the gatifloxacin signal was minimally detected ( Figure 10D). With an increasing 351 concentration of SAG, the signal diffusion became weaker, thus proving that SAG inhibits biofilm 352 formation. To the best of our knowledge, the present study is the first to evaluate the antibacterial 357 activity and mechanism of SAG against P. rettgeri. The findings of TEM, CLSM, and FESEM 358 revealed significant effects of SAG against P. rettgeri biofilms, including its inhibitory effect on 359 biofilm substance expression and formation as well as on biofilm inactivation. We found that 360 SAG could decrease intracellular ATP concentration and changes in pH in and reduce the 361 membrane potential.

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Here SAG demonstrated strong activity against P. rettgeri biofilms. SAG not only inhibited 363 biofilm formation but also destroyed the intact and viable biofilm. At 1/16 MIC, SAG inhibited 364 biofilm formation by approximately 68%, whereas at 1/4 MIC, more than 95% of the biofilm was 365 inhibited, thus an outstanding antibacterial effect of SAG was observed on P. rettgeri. 366 ATP depletion is a common biological change associated with cell damage, suggesting that 367 ATP is used as a potential indicator of the effects of antimicrobial agents on the intact and viable 368 cell membrane. These results presented that SAG-treated P. rettgeri exhibited a remarkable    Effects of SAG on the growth kinetics of P. rettgeri cells