Bacterial Isolates and Chemicals
7 different isolates of MDR Pseudomonas aeruginosa, including 5 polymyxin B susceptible and 2 resistant isolates, were specially selected from a strain repository at the Department of Microbiology, Shiyan Renmin Hospital. All of those isolates were obtained from different inpatients and specimens and confirmed with the MALDI-TOF mass spectrometry (MS) method. Pseudomonas aeruginosa ATCC 27853 was used as the control strain. Polymyxin B MICs had been interpreted accordingly to the EUCAST clinical breakpoints, version 12.0. Before each experiment, every isolate was cultured onto M-H agar plate and incubated at 37℃ for 24h, and then one colony was selected for further study. Both resveratrol and polymyxin B used in this study were purchased from Solarbio (Beijing, China) and dissolved with sterile water to prepare stock solution with the original concentration of 20mg/ml for polymyxin B and 100mg/ml for resveratrol. Tiny amount of dimethyl sulfoxide (DMSO) was used to assist the dissolution of resveratrol and well-designed DMSO control groups were always included for each experiment.
Susceptibility Testing and Genetic Analysis
MICs of resveratrol and polymyxin B were determined with the Broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guideline. Briefly, bacterial cell suspension prepared with cation-regulated Mueller-Hinton broth (CAMHB) was dispensed into wells of 96-well microtiter plate and made the final bacterial concentration of 5 × 105 CFU/ml for each well. Polymyxin B and resveratrol were separately added into those wells to get desired concentration, ranging from 32μg/ml to 0.125μg/ml with two-fold dilution for polymyxin B, and from 512μg/ml to 32μg/ml with two-fold dilution for resveratrol. After incubating at 37℃ for 24h, the result was then read, and the MIC was defined as the lowest concentration of drug required to visibly inhibit the growth of bacteria. PCR was performed on two polymyxin B resistant isolates with specially designed primers to detect five polymyxin B-resistance related genes including PmrA, PmrB, PhoP, PhoQ, and mcr-1, and the final products were sequenced and analyzed by Biomed (Beijing, China).
Basing on the susceptibility testing result, checkerboard assay was further conducted to determine the interaction between polymyxin B and resveratrol on antibacterial activity. In this experiment, polymyxin B and resveratrol were prepared by two-fold serial dilutions and mixed to produce different concentration combinations. The result was read after incubated at 37℃ for 24 hours and interpreted as follows: FICI ≤0.5, synergistic effects; 0.5<FICI ≤ 1.0, additive effects; 1.0<FICI ≤ 2.0, no interaction; and FICI > 2.0, antagonistic effects. The result was confirmed with at least three independent experiments.
Time-kill studies were performed as described previously with slight modification with all isolates. Resveratrol used in this experiment was maintained at the concentration of 64μg/ml, while the concentration of Polymyxin B for each isolate was determined as the 1/4 MIC of polymyxin B. Bacteria suspension of 5 × 105 CFU/ml was freshly prepared and incubated with polymyxin B and resveratrol alone or in combination at 37℃. Cell counts were determined at the time points of 0, 1, 2, 4, 6, 8, 24 hours respectively, by plating 100μl culture abstracted at different time points on M-H agar plates with appropriate dilution. Synergistic activity was defined as ≥2log10 decrease in CFU/mL by two-drug combination group compared with every single drug group.
Bacterial cell suspension of approximately 5 × 105 CFU/ml was freshly prepared with LB broth and dispensed into wells of 96-well microtiter plate. After statically incubated at 37°C for 24h, the liquid phase was discarded and the wells were gently washed with PBS for five times. Biofilm was fixed by adding 99% methanol to each well for 30 minutes. The fixative was then removed and the plate was allowed to air dry before the addition of crystal violet for 15 minutes to stain the adherent biofilm. The well was washed five times with PBS to remove free cells and 100μl of 7% acetic acid was then added to solubilize the dye combined by biofilm. Absorbance, which reflected the established biofilm, was measured at 595nm on a reader. All of those isolates were ranked according to their OD value from large to small, and the top three isolates were selected for further study.
Biofilm formation inhibition analysis
Three isolates with high biofilm formation capability were selected and biofilm formation analysis was performed as described above. Bacterial cell suspension of approximately 5 × 105 CFU/ml was dispensed into wells of 96-well microtiter plate and mixed with polymyxin B and resveratrol alone or in combination. To ensure that the inhibitory function is mainly on biofilm formation rather than on bacteria growth, the subinhibitory concentration of drug that has less than 10% influence on bacteria growth was employed in this experiment. The influence on bacteria growth was measured by incubating the bacteria suspension of 5 × 105 CFU/ml with polymyxin B and resveratrol alone or in combination at 37°C for 24h and calculated as the decreased percentage of cell number compared with the control group. Resveratrol concentration of 8μg/ml was employed in this experiment, which has less than 10% influence on bacteria growth. While the concentration of polymyxin B for each isolate was determined, by incubating the bacterial suspension of 5 × 105 CFU/ml with a series of diluted polymyxin B in combination with 8μg/ml resveratrol, as the highest polymyxin B concentration of those combinations which has less than 10% influence on bacteria growth. With each newly prepared bacterial suspension, two experiments were performed simultaneously, one was for inhibitory function on biofilm formation analysis, the other for influence on bacteria growth analysis. After incubating at 37°C for 24h, OD value and cell count were measured respectively, and the result was confirmed with at last three independent experiments.
Bactericidal activity analysis with established biofilm
The isolate with the highest biofilm formation capability was selected for this experiment. Biofilm was cultivated as described above and the cells protected by established biofilm were obtained by discarding the content and gently washing the wells with sterilized PBS solution for five times. Change in bacteria sensitivity of this isolate to polymyxin B was confirmed by comparing the influence of polymyxin B on the growth of cells with or without established biofilm. Briefly, 24 wells with established biofilm were divided into two groups. One group was for calculating the average quantity of cells attached to the bottom of well. 100ul sterilized PBS solution was added into each well, and a sterile pipette tip was used to scrape and blow the bottom of the well to release the adherent cells and mix the suspension. The cell number was counted by plating the suspension onto M-H agar plates with appropriate dilution. The other group was for analyzing the sensitivity of those cells protected by established biofilm. 100ul cation-regulated Mueller-Hinton broth with 8μg/mL polymyxin B was added into each well and incubated at 37°C for 24h. Then total cell number in each well, including both the adherent and planktonic cells, was counted by plating onto M-H ager plates with appropriate dilution. While when the mean number of cells attached to the bottom of well with established biofilm had been obtained with the former group, 100ul freshly prepared bacterial suspension, which could provide approximately similar（equal or small above but no more than 10%）amount of cells as the calculated result, was mixed with 8μg/mL polymyxin B and added into wells of 96-well microtiter plate. After incubated at 37°C for 24h, the total cell number was counted and compared with that of the group with established biofilm.
As for bactericidal activity analysis on cells protected by biofilm, every 12 wells with established biofilm from the same batch were divided into a group. 100ul cation-regulated Mueller-Hinton broth, mixed with polymyxin B and resveratrol alone or in combination, was added into each well. Given the decreased sensitivity of cells with established biofilm, relatively high concentration of drugs, 32μg/mL for polymyxin B and 128μg/ml for resveratrol, were employed in this experiment. After incubating at 37°C for 24h, the total cell number was counted and statistical analysis was performed. The result of this experiment was confirmed with three independent experiments.
Every performance was done in triplicate at least. Statistical analysis was carried out with Student’s t-test and P < 0.01 was considered as significant.