体外和体内抗多药耐药铜绿假单胞菌合成抗菌肽 WLBU2 的生物膜活性。
In vitro and in vivo antibiofilm activity of the synthetic antimicrobial peptide WLBU2 against multiple drug resistant Pseudomonas aeruginosa strains.
机构信息
Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
出版信息
BMC Microbiol. 2023 May 15;23(1):131. doi: 10.1186/s12866-023-02886-x.
BACKGROUND
The global crisis of antibiotic resistance increases the demand for the novel promising alternative drugs such as antimicrobial peptides (AMPs). Here, the antibiofilm activity of the WLBU2 peptide against Pseudomonas aeruginosa (P. aeruginosa) isolates was investigated in this study.
METHODS
Two clinical MDR and carbapenem resistant P. aeruginosa (CRPA) isolates, and standard P. aeruginosa ATCC 27,853 were investigated. The MIC and MBC of WLBU2 were determined. The MBIC was determined to evaluate inhibitory activity of WLBU2 on biofilm formation and MBEC to dispersal activity on preformed biofilm. The relative expression levels of biofilm-associated genes including rhlI, rhlR, lasI and lasR were analyzed using RT-qPCR. In vivo evaluation of inhibitory effect of WLBU2 on biofilm formation was performed in the murine models of P. aeruginosa biofilm-associated subcutaneous catheter infection.
RESULTS
MIC and MBC of WLBU2 for both MDR and ATCC 27,853 P. aeruginosa strains were 8 and 16 µg/mL, respectively, while both the MIC and MBC against the CR strain were 4 µg/mL. MBIC was estimated to be 64 µg/ml for all strains. MBEC against MDR and ATCC 27,853- P. aeruginosa strains was 128 µg/ml and against CRPA was 64 µg/ml. The bacterial adhesion to a static abiotic solid surface (the surface in the polypropylene microtiter wells) was significantly inhibited at 1/4× MIC in all P. aeruginosa strains and at 1/8× MIC in CRPA strain (P < 0.05). Following treatment with WLBU2 at 1/8× MIC, significant inhibition in biofilm formation was observed in all isolates (P < 0.05). Results of the colorimetric assay showed that WLBU2 at 4× MIC was able to disperse 69.7% and 81.3% of pre-formed biofilms on abiotic surface produced by MDR and standard (ATCC 27,853) P. aeruginosa, respectively (P < 0.03), while a 92.2% reduction in the CRPA biofilm was observed after treatment with 4× MIC WLBU2 (P < 0.03). The expression levels of all genes in isolates treated with 1/2 MIC of WLBU2 were down-regulated by more than four-fold compared to the untreated isolates (P < 0.05). WLBU2 significantly inhibited biofilm formation in murine catheter-associated CRPA infection model at 1/4×MIC, 1/2×MIC, and 1×MIC by 33%, 52%, and 67%, respectively.
CONCLUSION
Considering relatively strong inhibitory and eradication potency of WLBU2 on the P. aeruginosa biofilms in in vitro and in vivo conditions, the peptide can be considered as a promising candidate for designing an antibiofilm drug.
背景
抗生素耐药性的全球危机增加了对新型有前途的替代药物（如抗菌肽）的需求。本研究旨在研究 WLBU2 肽对铜绿假单胞菌（P. aeruginosa）分离株的抗生物膜活性。
方法
研究了 2 株临床多药耐药和碳青霉烯耐药铜绿假单胞菌（CRPA）分离株和标准铜绿假单胞菌 ATCC 27,853。测定了 WLBU2 的 MIC 和 MBC。测定 MBIC 以评估 WLBU2 对生物膜形成的抑制活性，测定 MBEC 以评估其对预形成生物膜的分散活性。使用 RT-qPCR 分析生物膜相关基因 rhlI、rhlR、lasI 和 lasR 的相对表达水平。在铜绿假单胞菌生物膜相关皮下导管感染的小鼠模型中进行了 WLBU2 对生物膜形成抑制作用的体内评价。
结果
WLBU2 对 MDR 和 ATCC 27,853 铜绿假单胞菌株的 MIC 和 MBC 分别为 8 和 16 µg/mL，而对 CR 株的 MIC 和 MBC 均为 4 µg/mL。所有菌株的 MBIC 估计为 64 µg/ml。对 MDR 和 ATCC 27,853-铜绿假单胞菌株的 MBEC 为 128 µg/ml，对 CRPA 的 MBEC 为 64 µg/ml。在所有铜绿假单胞菌株中，在 1/4×MIC 时，细菌对静态非生物固体表面（聚丙烯微量滴定孔中的表面）的粘附显著受到抑制，在 CRPA 株中，在 1/8×MIC 时，细菌对静态非生物固体表面的粘附显著受到抑制（P<0.05）。用 1/8×MIC 的 WLBU2 处理后，所有分离株的生物膜形成均受到显著抑制（P<0.05）。比色法结果表明，在 4×MIC 时，WLBU2 能够分别分散 MDR 和标准（ATCC 27,853）铜绿假单胞菌在非生物表面形成的生物膜的 69.7%和 81.3%（P<0.03），而在 4×MIC WLBU2 处理后，CRPA 生物膜减少 92.2%（P<0.03）。与未经处理的分离株相比，用 1/2 MIC 的 WLBU2 处理的所有基因的表达水平均下调了 4 倍以上（P<0.05）。在 1/4×MIC、1/2×MIC 和 1×MIC 时，WLBU2 分别抑制了 33%、52%和 67%的小鼠导管相关 CRPA 感染模型中的生物膜形成。
结论
考虑到 WLBU2 对铜绿假单胞菌生物膜的体外和体内抑制和清除作用较强，该肽可被视为设计抗生物膜药物的有前途的候选物。
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