蓝莓提取物对产超广谱β-内酰胺酶肺炎克雷伯菌的抑菌活性

Inhibitory activity of blueberry extract against extended spectrum β-lactamase-producing Klebsiella pneumoniae

摘要:

【背景】随着细菌耐药性的增强，产超广谱β-内酰胺酶肺炎克雷伯菌的出现严重危害食品安全和人体健康。蓝莓中含有丰富的多酚和花青素，是天然抗菌材料的优选。【目的】分析蓝莓提取物对3株产超广谱β-内酰胺酶肺炎克雷伯菌的抑制活性及作用机制，并以牛乳为样品检测食品基质对蓝莓提取物抑菌活性的影响。【方法】利用甲醇制备巴尔德温和黑珍珠蓝莓提取物并检测提取物中总酚和花青素含量，利用纸片扩散法检测肺炎克雷伯菌的耐药性，利用最小抑菌浓度(minimum inhibitory concentration，MIC)、最小杀菌浓度(minimum bactericidal concentration，MBC)和生长曲线研究蓝莓提取物的抗菌活性，通过共聚焦激光扫描显微镜观察蓝莓提取物对细胞膜完整性的影响，并检测其在牛乳中的抑菌活性。【结果】巴尔德温和黑珍珠蓝莓提取物中的总酚含量分别为2.3 mg/g和3.5 mg/g，花青素含量分别为67.5 mg/100 g和92.5 mg/100 g。两种蓝莓提取物对肺炎克雷伯菌KP106、KP305和KP408的MIC均为25 mg/mL，MBC均为50 mg/mL。生长曲线表明，2xMIC的2种蓝莓提取物能够在6-8 h内将肺炎克雷伯菌杀死。共聚焦激光扫描显微镜分析表明，蓝莓提取物能够破坏肺炎克雷伯菌细胞膜的完整性。模拟牛乳样品试验表明，100 mg/mL的黑珍珠蓝莓提取物能够完全抑制牛乳中肺炎克雷伯菌的生长。【结论】蓝莓提取物可通过破坏肺炎克雷伯菌的细胞膜而达到抑菌效果，在牛乳中也具有抑制肺炎克雷伯菌的潜力。该研究为蓝莓应用于牛乳中耐药肺炎克雷伯菌的防控提供了理论参考。

Abstract:

[Background] With the enhancement of drug resistance, the extended spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) seriously threatens food safety and human health. Blueberry, rich in polyphenols and anthocyanins, is a potential natural antibacterial candidate. [Objective] To analyze the inhibitory activity and mechanism of blueberry extracts against three ESBL-KP strains and test the effect of food matrix (cow milk) on the antibacterial activity of the extracts. [Methods] Fruit extracts of Vaccinium corymbosum L. and V. ashei were prepared with methanol, and the content of total phenols and anthocyanins in the extracts was determined. The antibiotic resistance of K. pneumoniae was detected by disk diffusion test. The antibacterial activity of blueberry extracts was evaluated based on minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and growth curves of strains. The effect of blueberry extracts on cell membrane integrity was detected by confocal laser scanning microscopy, and its antibacterial activity in milk was detected. [Results] The total phenol content in fruit extracts of V. ashei and V. corymbosum L. was 2.3 mg/g and 3.5 mg/g, respectively, and the anthocyanin content was 67.5 mg/100 g and 92.5 mg/100 g respectively. The MIC and MBC of two blueberry extracts against K. pneumoniae KP106, KP305, and KP408 were 25 mg/mL and 50 mg/mL, respectively. The growth curve showed that the two extracts at 2xMIC could kill K. pneumoniae within 6–8 h. Confocal laser scanning microscopy demonstrated that blueberry extracts could destroy the integrity of cell membrane. The simulated cow milk sample test showed that 100 mg/mL fruit extract of V. corymbosum L. could completely inhibit the growth of ESBL-KP in milk. [Conclusion] Blueberry extract exerts inhibitory effect on K. pneumoniae by destroying the cell membrane of the bacteria, and has the potential to prevent and control K. pneumoniae in milk. This study provides a theoretical reference for the application of blueberry in the prevention and control of drug-resistant K. pneumoniae in milk.