Bacterial infection, especially MDR bacterial infection, has always been a tricky problem in clinical practice and also a big threat to people's health. Every year, people all over the world suffer a lot from bacterial infection and huge economic losses are associated with preventing and treating bacterial infection[32–34]. Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that contributes a lot to clinical bacterial infection. And due to the multiple antibiotic resistance mechanisms of Pseudomonas aeruginosa, it is easy for this bacterium to acquired antibiotic resistance[35–36]. In recent few years, with the high prevalence of MDR Pseudomonas aeruginosa, and the limited number of antibacterial agents that can be available for MDR bacterial infection, the treatment of Pseudomonas aeruginosa infection is challenging.
Polymyxin B is one of the very few antibiotic agents that can be used for the treatment of MDR Gram-negative bacterial infection. However, in recent few years, polymyxin B resistance has been frequently reported in various kinds of bacteria including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, as well as Pseudomonas aeruginosa[28,38−39]. Though the polymyxin B resistance mechanism of Pseudomonas aeruginosa is still not completely understand, a common appreciated one is the specific modification of the lipid A component of the outer membrane lipopolysaccharide (LPS) which has negative charge and is the initial target of polymyxin B. It has been reported that some specific mutations in PhoP/PhoQ and PmrA/PmrB two-component system (TCS) can lead to the overexpression of LPS modifying genes, which in turn result in polymyxin B resistance in Pseudomonas aeruginosa[41–43]. Another well-known mechanism is associated with the presence of gene mcr-1. Gene mcr-1 encodes a lipid A phosphoethanolamine transferase that can catalyze the modification of lipid A moiety on bacterial lipopolysaccharide (LPS), so the plasmid carrying mcr-1 gene can also cause polymyxin resistance in Pseudomonas aeruginosa. Mutations of PmrB, PmrA, and PhoQ can be detected in all polymyxin B resistant isolates in this study, suggesting that polymyxin B resistance of those isolates may be associated with the mutations of those genes, and the conclusion of this study is likely to be suitable for those types of Pseudomonas aeruginosa. However, gene mcr-1 could not be found in any of those isolates, that was one limitation of this study.
Even though having been regarded as the last resort agent for the treatment of MDR bacteria infection, the poor clinical efficacy and the high risk of nephrotoxicity of polymyxin have seriously restricted its clinical application[17, 45]. Undoubtedly, any drug that can improve the sensitivity of MDR Pseudomonas aeruginosa to this special antibiotic can greatly benefit patients. Given this consideration, resveratrol is selected as a candidate, since it can be well tolerated by human body and has been reported to have synergistic effect with different antibiotics against various kinds of bacteria species[46–48]. In this study, both the checkerboard assay and time-kill assay have demonstrated that the combination of resveratrol and polymyxin B has synergistic effect against MDR Pseudomonas aeruginosa, which indicates that, with the assistance of resveratrol, a lower concentration of polymyxin B is required for the treatment of MDR Pseudomonas aeruginosa infection. While even though in clinical practice, polymyxin B was recommended to be given with the maximum doses that could be tolerated by patients, increased sensitivity indicates that better bactericidal activity and shorter treatment course can be expected with this defined concentration of polymyxin B . Given being well tolerated by human body, as well as showing synergistic effect with polymyxin B against Pseudomonas aeruginosa, resveratrol is considered to be an ideal partner of polymyxin B for the combination therapy of MDR Pseudomonas aeruginosa infection. It can be expected to improve the clinical efficacy of polymyxin B by increasing the bactericidal activity and reducing the risk of nephrotoxicity by shortening the treatment course.
For long-term consideration, the combination therapy of polymyxin B and resveratrol is likely to slow the evolution of polymyxin B resistance in Pseudomonas aeruginosa. According to the mutant selection window hypothesis, the development of antibiotic resistance in clinical practice is actually due to the accumulation of naturally occurring resistant mutants, and the combination strategy that can intervene in this enrichment step can be favorable for preserving the usefulness of antibiotics [50–51]. Though need to be confirmed with future long-term clinic data, the hypothesis that combination therapy of polymyxin B and resveratrol can slow the development of polymyxin B resistance in Pseudomonas aeruginosa has been partly proved in this study, with the fact that some polymyxin B resistant isolates can still be effectively eradicated by polymyxin B with the concentration of 2ug/ml or even lower when in combination with 64ug/ml resveratrol, which indicate that those resistant mutants are less likely to be accumulated during antibiotic combination therapy compared with the polymyxin B monotherapy.
Biofilm is a typical character for many Pseudomonas aeruginosa isolates, and it can protect bacteria from surrounding environmental pressure, confer bacterial cells with high antibiotic resistance, and even impede phagocytosis[30, 52]. So inhibiting biofilm formation will be an advantage for the treatment of Pseudomonas aeruginosa infection. Antibiofilm has been regarded as an important index for evaluating the antibacterial effect of antibiotics against those cells with high biofilm formation capability [53–54]. In this study, we have demonstrated that polymyxin B and resveratrol have combination effect on inhibiting the biofilm formation of Pseudomonas aeruginosa. This result indicates that, when used with resveratrol, a lower concentration of polymyxin B is required for inhibiting biofilm formation, and a better antibiofilm effect can be excepted. One concern of the combination antibiofilm strategy is for respiratory Pseudomonas aeruginosa infection where the cells are more likely to colonize and form biofilm and relative lower antibiotic concentration can be achieved with systemic administration. If the biofilm formation process can not be effectively curbed, the persistent respiratory Pseudomonas aeruginosa infection may result.
Established biofilm is frequently observed with chronic wound Pseudomonas aeruginosa infection and can result in poor wound healing[55–56]. According to previous reports, established biofilm can mediate high concentration antibiotic resistance, which means that those bacterial cells designated as susceptible to polymyxin B by traditional antibiotic susceptibility interpretation can not be effectively eliminated by polymyxin B with conventional recommended does strategy. Even though the loading does of polymyxin B for systemic administration has been greatly limited for the high risk of nephrotoxicity, there is no such restriction for topical use, so high concentration of polymyxin B can still be used topically for the treatment of bacteria infection [58–59]. In this study, polymyxin B of 8ug/ml was employed for analyzing the sensitivity of Pseudomonas aeruginosa with established biofilm, and the increased resistance of Pseudomonas aeruginosa to polymyxin B was confirmed by comparing with those cells without established biofilm. To investigate if resveratrol can re-sensitize those cells protected by established biofilm to polymyxin B, polymyxin B and resveratrol were used together and the bacterial activity was monitored by cell counting. And the result has demonstrated that polymyxin B in combination with resveratrol has better bacterial activity against those cells with established biofilm compared with the polymyxin B monotherapy. This result suggests that if polymyxin B is used in combination with resveratrol in the treatment of Pseudomonas aeruginosa infection with established biofilm, a better clinical efficacy can be expected.