Exploring the activity and mechanism of Bidens pilosa L. extract against MRSA
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摘要:
目的 以耐甲氧西林金黄色葡萄球菌(MRSA)标准菌株和临床菌株为研究对象，探究鬼针草水提物对MRSA的抗菌效果及作用机制。 方法 采用牛津杯法、微量肉汤稀释法评价鬼针草水提物对金黄色葡萄球菌ATCC25923、MRSA标准菌株ATCC43300以及50株MRSA临床分离株的抑菌作用，并确定其最小抑菌浓度(MIC)；测定鬼针草水提物处理后菌液的电导率变化，以及核酸物质、可溶性蛋白等大分子的渗漏情况；利用qRT-PCR技术检测MRSA相关耐药基因的表达变化。 结果 鬼针草水提物针对ATCC25923、ATCC43300及MRSA临床分离株的MIC分别为125 mg/mL、250 mg/mL和15.625~250 mg/mL。鬼针草水提取物作用于MRSA能使其细胞膜通透性改变，进而导致核酸、可溶性蛋白等大分子物质外泄至细胞外。鬼针草水提物能抑制icaA基因的表达。 结论 鬼针草水提物能显著抑制MRSA的活性，其机制可能与影响MRSA细胞膜通透性以及生物膜耐药基因icaA相关。 -
关键词:
- 鬼针草 /
- 耐甲氧西林金黄色葡萄球菌 /
- 抗菌活性 /
- 抑菌机制
Abstract:Objective To evaluate the antibacterial activity of Bidens Pilosa L. extract against Methicillin-resistant Staphylococcus aureus (MRSA) and to elucidate its potential mechanism of action. Methods Oxford cup method and microbroth dilution method were used to evaluate the antibacterial effect of B. pilosa L. extract on S. aureus strain ATCC25923, MRSA standard strain ATCC43300, and fifty clinical MRSA isolates, and to determine the minimum inhibitory concentration (MIC). Changes in bacterial suspension conductivity and leakage of nucleic acids and soluble proteins were determined after the action of B. pilosa L. extract on MRSA. In addition, qRT-PCR technology was used to detect the expression changes of resistance-related genes. Results The MIC values of B. pilosa L. extract against ATCC25923, ATCC43300, and clinical MRSA strains were 125 mg/mL, 250 mg/mL, and 15.625-250 mg/mL, respectively. The extract of B. pilosa L. increased MRSA cell membrane permeability, leading to leakage of large molecules such as nucleic acids and soluble proteins. The extract of B. pilosa L. inhibited the expression of the biofilm-associated gene icaA. Conclusion The extract of B. pilosa L. can significantly inhibit the activity of MRSA. Its mechanism may be related to the impact on MRSA cell membrane permeability and suppression of biofilm-related gene expression. -
图 1 牛津杯法抑菌试验结果
注：A~H分别为鬼针草对M22、M24、M29、M33、M37、M39、M43、M46的牛津杯抑菌结果；其中编号1、4、7、10、13、16、19、22为鬼针草提取液原液，2、5、8、11、14、17、20、23为浓度1/2的鬼针草提取液，3、6、9、12、15、18、21、24为浓度1/4的鬼针草提取液，C为阳性对照(盐酸左氧氟沙星氯化钠注射液，浓度为125 μg/mL)，NC为阴性对照(无菌水)。
Figure 1. Antibacterial results evaluated by the Oxford cup method
图 2 鬼针草水提物抗MRSA的动态抑菌曲线
注：与对照组比较，aP＜0.05，bP＜0.01。
Figure 2. Dynamic antibacterial activity curve of Bidens pilosa L. extract against MRSA
图 3 鬼针草水提物作用MRSA后上清液电导率变化
注：与对照组比较，aP＜0.05。
Figure 3. Changes in the conductivity of Bidens pilosa L. extract on MRSA
图 4 鬼针草水提物作用MRSA后上清液260 nm吸光度变化
注：与对照组比较，aP＜0.01。
Figure 4. Changes in absorbance at 260 nm in MRSA following treatment with Bidens pilosa L. extract
表 1 引物序列
Table 1. Primer sequences
引物名称 序列(5’→3’) 16S-F CGTGCTACAATGGACAATACAAA 16S-R ATCTACGATTACTAGCGATTCCA icaA-F GACGTTGGCTACTGGGATACTGA icaA-R ACACATGGCAAGCGGTTCATAC 
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表 2 鬼针草水提物抗MRSA抑菌试验结果
Table 2. Statistical analysis of the antibacterial activity of Bidens pilosa L. extract against MRSA
试验菌 抑菌圈 试验菌 抑菌圈 试验菌 抑菌圈 试验菌 抑菌圈 试验菌 抑菌圈 M1 - M11 + M21 + M31 + M41 + M2 + M12 + M22 + M32 + M42 + M3 + M13 + M23 + M33 - M43 + M4 + M14 - M24 + M34 + M44 - M5 - M15 + M25 + M35 + M45 + M6 - M16 + M26 + M36 + M46 + M7 + M17 + M27 + M37 + M47 + M8 + M18 + M28 - M38 + M48 + M9 - M19 + M29 + M39 + M49 + M10 - M20 + M30 + M40 + M50 + 注：“+”表示有抑菌圈；“-”表示无抑菌圈。
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表 3 鬼针草水提物抗MRSA的MIC测定结果
Table 3. The MIC result of Bidens pilosa L. extract against MRSA
试验菌 M2 M3 M4 M7 M8 M11 M12 M13 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 M25 M26 M27 M29 M30 MIC(mg/mL) 125 15.625 125 125 15.625 250 125 125 125 125 125 125 250 125 250 125 250 125 125 125 250 125 125 试验菌 M31 M32 M34 M35 M36 M37 M38 M39 M40 M41 M42 M43 M45 M46 M47 M48 M49 M50 ATCC25923 ATCC43300 MIC(mg/mL) 250 125 125 250 125 250 250 125 125 250 250 62.5 250 125 250 250 250 250 125 250
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表 4 鬼针草水提物作用MRSA后上清液可溶性蛋白含量比较(x±s)
Table 4. Determination of soluble protein content in the supernatant following treatment of MRSA with Bidens pilosa L. extract(x±s)
时间 对照组 1/2 MIC 1 MIC 0 min 33.32±11.09 41.02±4.79 251.89±32.95 60 min 34.51±13.66 1 351.29±397.76 3 071.59±670.51 120 min 49.87±19.21 2 141.28±658.27 5 015.69±924.81
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