肽抗生素hPAB-β抗金黄色葡萄球菌L型活性分析

Antibacterial activity of peptide antibiotic hPAB-β against the L-form of Staphylococcus aureus

目的观察毕赤酵母高密度发酵表达的肽抗生素hPAB-β对L型细菌的抗菌活性。方法采用新青Ⅱ纸片扩散法诱导金黄色葡萄球菌标准株ATCC25923L型,通过形态学观察、滤过实验、回复实验以及电镜观察鉴定L型菌;采用酵母高密度发酵制备肽抗生素hPAB-β,通过稀释法检测hPAB-β对L型细菌的抗菌活性并测定其最小抑菌浓度(MIC)。结果用新青Ⅱ可成功诱导金黄色葡萄球菌L型;所得L型菌能通过0.45μm的滤膜;撤去抗生素后,L型能回复为野生菌;电镜下见多数金葡菌L型的细胞壁部分缺失,少数完全缺失;稀释法检测显示hPAB-β对金葡菌L型具有良好的抗菌活性,其MIC值为32μmol/L。结论肽抗生素hPAB-β对金葡菌L型有良好的抗菌活性。

Objective To determine the antibacterial activity of hPAB-β, a peptide antibiotic derived from human beta defensin 2, against the L-form of Staphylococcus aureus （S. aureus）. Methods The typical L-form of S. aureus strain ATCC25923 was induced by oxacillin paper disk diffusion method, and their morphological characters, colony formation, filtering features, ultra structure and reversion phenomenon were investigated. The peptide antibiotic hPAB-β was prepared by step-wise purification from high cell-density fermentative supernatant of the recombinant Pichia pastoris, and the antibacterial activity of hPAB-β against L-form of S. aureus was detected by serial doubling dilution method in a 96-well plate. Three other antibiotics （oxacillin, vancomycin and chloramphenicol） were chosen as controls in present study. The minimum inhibitory concentrations （MIC） of these antibiotics against the wild type and L-form of S. aureus were also evaluated. Results The L-form of S. aureus was successfully induced and could permeate through a 0.45μm filter. After the inducer （oxacillin） was removed, the L-form of S. aureus could reverse to the wild type. The cell wall of most of induced L-forms was partially absent, and a few of them completely lost their cell wallas observed under the electronic microscope. The MIC of the hPAB-β against L-form of S. aureus was 32μmol/L, which was 4 times higher than that of hPAB-β for the wild type of S. aureus. Conclusion The antibacterial activity of hPAB-β against the L-form of S. aureus has been successfully detected. The results of present study will provide the direct evidence for peptide antibiotic hPAB-β as a potential therapeutic agent for the infection of S. aureus L-form.