三叶青提取物TH-w3对金黄色葡萄球菌生物膜的抑制和清除作用

doi:10.3877/cma.j.issn.2095-5820.2022.04.007

目的

探讨三叶青提取物TH-w3对金黄色葡萄球菌生物膜形成的抑制和清除作用。

方法

TH-w3是用80%的乙醇提取后的三叶青干燥块根水煮后减压回收蒸干产品；实验菌株为本实验室临床分离培养并保存的耐甲氧西林金黄色葡萄球菌（MRSA）和甲氧西林敏感金黄色葡萄球菌（MSSA）各30株；菌落计数法绘制金黄色葡萄球菌生物膜形成曲线；二倍稀释法肉眼观察+酶标仪比浊法测定最小抑菌浓度（MIC）；结晶紫法检测TH-w3对金黄色葡萄球菌生物膜的抑制及清除作用。

结果

对各30株MRSA和MSSA的成膜能力进行比较，MRSA菌株成膜阳性22株（73.3%），MSSA成膜阳性菌株3株（10.0%），成膜阳性率MRSA菌株与MSSA菌株比较差异有统计学意义（χ²=43.35，P＜0.01）；TH-w3对“MRSA（+）组”和“MSSA（+）组”菌株的MIC均为8.00 μg/ml；TH-w3对“MRSA（+）组”和“MSSA（+）组”菌株生物膜的抑制和清除作用均具有浓度依赖性，在1个MIC浓度下抑制率和清除率最高，可达68%以上。相同MIC的TH-w3对金黄色葡萄球菌生物膜的抑制和清除作用随培养时间延长明显升高，至48 h后提升渐缓。在1/4 MIC浓度下，诱导24 h后MSSA（+）的抑制率高于MRSA（+）（P＜0.05）。在1/4和1/2 MIC浓度下，诱导24 h后 MSSA（+）的清除率高于MRSA（+）（P＜0.01）。

结论

三叶青提取物TH-w3对金黄色葡萄球菌生物膜有明显抑制和清除作用，具有临床转化价值。

关键词: 三叶青, 金黄色葡萄球菌, 生物膜, 抑制作用, 清除作用

Objective

To explore the inhibition and scavenging effects of TH-w3 extracted from Tetrastigma hemsleyanum on the formation of Staphylococcus aureus biofilm.

Methods

TH-w3 was prepared from dried root of Tetrastigma hemsleyanum by reducing pressure and boiling dry after extraction with 80% ethanol. 30 strains of Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive staphylococcus aureus (MSSA) were isolated, cultured and preserved respectively. Colony-counting method was used to draw the biofilm formation curve of Staphylococcus aureus. The minimum inhibitory concentration (MIC) of TH-w3 were determined by visual inspection and absorbance detection via double ratio dilution method. The inhibition and scavenging effects of TH-w3 on the formation of Staphylococcus aureus biofilm were observed by Crystal violet staining.

Results

22 strains of MRSA (73.3%) and 3 strains of MSSA (10.0%) were observed positive membrane formation. The biofilm formation rates between MRSA and MSSA were statistically significant (χ²=43.35, P＜0.01). The MIC of TH-w3 for MRSA and MSSA in positive biofilm-forming groups were both 8.00 μg/ml. The inhibition and scavenging effects of TH-w3 on MRSA and MSSA showed concentration-dependent, and the inhibition and scavenging rates reached the highest (more than 68%) at the concentration of 1 MIC. The inhibitory and clearing effects of TH-w3 with the same MIC on the biofilm of Staphylococcus aureus expressed a smooth increase after 48 hrs. The inhibition and scavenging rates for MSSA showed higher than MRSA in 1/2 and 1/4 MIC group after induction for 24 hours　(P＜0.05).

Conclusions

The trifoliate extract TH-w3 showed significant inhibition and scavenging effects on Staphylococcus aureus biofilm, which promised a potential clinical translational value.

Key words: Tetrastigma hemsleyanum, Staphylococcus aureus, Microbial film, Inhibition, Scavenging effects

图1 三叶青干燥块茎各成份提取路线图

表1 MRSA和MSSA成膜能力比较

分组	菌株数	阳性率［%（n/N）］	阴性率［%（n/N）］
MRSA	30	73.3（22/30）	26.7（8/30）
MSSA	30	10.0（3/30）^a	90.0（27/30）
合计	60	41.7（25/60）	58.3（35/60）

表1 MRSA和MSSA成膜能力比较

分组	菌株数	阳性率［%（n/N）］	阴性率［%（n/N）］
MRSA	30	73.3（22/30）	26.7（8/30）
MSSA	30	10.0（3/30）^a	90.0（27/30）
合计	60	41.7（25/60）	58.3（35/60）

表2 肉眼观察MIC的测定

肉眼观察结果	TH-w3浓度（μg/ml）
肉眼观察结果	0	0.25	0.50	1.00	2.00	4.00	8.00	16.00
MIC	+++	+++	++	++	++	+	-	-

表2 肉眼观察MIC的测定

肉眼观察结果	TH-w3浓度（μg/ml）
肉眼观察结果	0	0.25	0.50	1.00	2.00	4.00	8.00	16.00
MIC	+++	+++	++	++	++	+	-	-

图2 TH-w3 对“MRSA(+)组”和“MSSA(+)组”菌株的MIC注：2A为MRSA（+）组；2B为MSSA（+）组；两组比较，^aP＜0.01

图3 TH-w3对MRSA（+）和MSSA（+）生物膜的抑制作用注：3A为MRSA（+）组；3B为MSSA（+）组

图4 TH-w3对MRSA（+）和MSSA（+）生物膜的清除作用注：4A为MRSA（+）组；4B为MSSA（+）组

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Inhibitory and scavenging effects of cloverleaf extract TH-w3 on Staphylococcus aureus biofilm

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Inhibitory and scavenging effects of cloverleaf extract TH-w3 on Staphylococcus aureus biofilm