鱼腥草素钠对铜绿假单胞菌生物被膜的清除作用

目的研究鱼腥草素钠对铜绿假单胞菌早期生物被膜及成熟期生物被膜的清除作用。方法用96孔板建立体外铜绿假单胞菌生物被膜,给药24 h后MTT法检测生物被膜最低清除浓度(sessile minimal eradicating concentration,SMEC),扫描电镜下观察药物对生物膜形态影响。结果鱼腥草素钠对铜绿假单胞菌生物被膜的SMEC50,早期是15.6 mg/L,成熟期是250 mg/L,在早期生物被膜向成熟生物被膜发展过程中SMEC50均在250 mg/L,镜下观察250mg/L药物质量浓度能清除大部分的生物被膜,同时可使铜绿假单胞菌发生形态变异。结论鱼腥草素钠对早期及成熟期的生物被膜有一定的清除作用,对早期生物被膜的清除作用更强于成熟期生物被膜。     

鱼腥草素钠增强亚胺培南抗铜绿假单胞菌生物被膜的作用

目的：研究鱼腥草素钠联合亚胺培南增强抗铜绿假单胞菌生物被膜的作用。方法：2倍稀释法测定被试药物最小抑菌浓度（MIC）,结晶紫染色法检测1/2MIC,1MIC,2MIC 的鱼腥草素钠（SH）、亚胺培南（IMP）单药组和1/2MIC的SH与IMP各药物浓度联合组对黏附菌的清除量、生物被膜生长量、藻酸盐产生量的影响。FDA-PI荧光素双染法荧光显微镜下观察各药物组膜分散后死活细菌、生物被膜形态。结果：鱼腥草素钠能增强亚胺培南抗绿假单胞菌生物被膜的作用,尤以2MIC药物浓度联合组作用最强（与阴性对照组比较P<0.001）。荧光显微镜下观察死活细菌、生物被膜形态支持了上述结果。结论：鱼腥草素钠联合亚胺培南能增强抗生物被膜作用,预期两药联合会提高相关感染的临床疗效。     

鱼腥草水提液对铜绿假单胞菌生物被膜的影响及与阿奇霉素的抗菌协同作用

目的研究鱼腥草水提液体外对铜绿假单胞菌生物被膜的影响及与阿奇霉素的抗菌协同作用。方法应用微量倍比稀释法测定鱼腥草水提液、阿奇霉素对铜绿假单胞菌的最小抑菌浓度(MIC),棋盘稀释法测定二者协同抗菌作用,MTT法测定鱼腥草水提液对铜绿假单胞菌生物被膜内细菌代谢的影响,镜下观察该药对铜绿假单胞菌生物被膜的形态影响。结果实验结果提示,鱼腥草水提液对铜绿假单胞菌的MIC为250 g.L-1,协同实验表明与阿奇霉素有抗菌相加作用。鱼腥草水提液对铜绿假单胞菌生物被膜内细菌代谢的SMIC501,3,7d分别是31.25,62.5,62.5 g.L-1,SMIC80分别是250,250,250 g.L-1。SMIC80药物浓度显示对铜绿假单胞菌生物膜形态形成有干扰作用。结论鱼腥草水提液与阿奇霉素抗菌相加作用明显,并能抑制铜绿假单胞菌生物膜的形成,为鱼腥草治疗由生物膜引起的慢性感染提供了依据。     

传统药物对铜绿假单胞菌生物被膜作用研究进展

铜绿假单胞菌是目前院内感染致病菌之一,引起多系统感染,其耐药机制复杂,生物被膜是其耐药的一个独特机制。研究提示一些中药或其他传统药物对铜绿假单胞菌生物被膜具有抑制作用,本文以近3年有关这方面的研究做一个综述。     

鱼腥草素钠联合EDTA-Na2对生物被膜菌的影响

目的 探索鱼腥草素钠(SH)联合乙二胺四乙酸二钠(EDTA-Na₂)对生物被膜菌的影响,寻找潜在的抗生素替代品.方法 通过微量稀释法以及棋盘法,测定SH和EDTA-Na₂对铜绿假单胞菌(Pseudomonas aeruginosa,PA)、金黄色葡萄球菌(Staphylococcus aureus,SA)、白色念珠菌(Candida albicans,CA)的最小抑菌浓度(MIC)和最小膜清除浓度(MBEC)以及两药协同点;通过平板稀释涂布法测定3种菌在协同点的生存曲线;扫描电子显微镜法(SEM)观察PA、SA和CA生物被膜的形态.结果 SH对PA、SA和CA的MIC分别为2.048、0.064、0.064 mg/mL,MBEC分别为2.048、0.256、0.512 mg/mL;EDTA-Na₂对PA、SA和CA的MIC分别为3.75、0.938、0.117 mg/mL,MBEC分别为15、3.75、30 mg/mL;SH联合EDTA-Na₂(SH/EDTA-Na₂)对PA、SA和CA的MIC(mg/mL)分别为0.256/0.938、0.008/0.233、0.008/0.029,MBEC(mg/mL)分别为0.512/3.722、0.032/0.469、0.064/0.938;SEM观察到两药联合对细菌的生长抑制和生物被膜的清除有明显作用,载体上的细菌显著减少,未见或仅有少量的生物被膜结构,与空白对照组比较差异明显.结论 SH与EDTA-Na₂具有协同作用,二者联合能够显著增强SH对PA、SA和CA的生长抑制和生物被膜的清除作用.     

麻杏石甘汤对铜绿假单胞菌生物被膜的影响

目的研究麻杏石甘汤对铜绿假单胞菌生物被膜的影响。方法微量倍比稀释法测定麻杏石甘汤、阿奇霉素对铜绿假单胞菌的最小抑菌浓度(MIC),棋盘稀释法测定二者协同抗菌作用,MTT法评价二者对铜绿假单胞菌生物被膜SMIC(SMIC50、SMIC80)的影响,镀银染色法观察麻杏石甘汤对铜绿假单胞菌生物被膜形态的影响。结果麻杏石甘汤SMIC50和SMIC80分别为250,1 000 mg.ml-1;500 mg.ml-1的麻杏石甘汤对铜绿假单胞菌生物被膜的形态有显著影响。结论麻杏石甘汤对铜绿假单胞菌生物被膜有较强的抑制作用。     

鱼腥草与左氧氟沙星联合应用对生物被膜细菌的清除作用

目的探讨鱼腥草对细菌生物被膜的影响,观察鱼腥草与左氧氟沙星联合应用对铜绿假单胞菌(Pseudomonas aeruginosa,PA)生物被膜的作用.方法用试管二倍稀释法测定鱼腥草和左氧氟沙星的最低抑菌浓度;用平板培养法建立生物被膜细菌体外模型;用MTT活菌计数法测定存活细菌数;用扫描电镜观察药物对生物被膜的破坏及清除作用.结果铜绿假单胞菌培养7 d后可形成稳定的生物被膜.鱼腥草或左氧氟沙星单独作用,对生物被膜的形态及存活细菌数无明显改变.二者联合应用,能破坏生物被膜的结构,显著降低膜内存活细菌数.结论鱼腥草与左氧氟沙星联合运用对生物被膜细菌具有协同杀灭作用.     

穿心莲内酯对铜绿假单胞菌PAO1生物被膜形成的影响

目的:采用激光共聚焦显微镜(CLSM)技术考察穿心莲内酯对铜绿假单胞菌PAO1生物被膜形成的影响,进一步揭示穿心莲内酯的抗菌机制。方法:使用玻璃爬片生物被膜培养法,在不同浓度穿心莲内酯(10μg/ml、1μg/ml、0.1μg/ml和0.01μg/ml)条件下孵育PAO1,并分别于24h、48h及72h取出玻璃爬片,用硫氰酸荧光素标记的刀豆蛋白和碘化吡啶双重免疫荧光染色,采用激光共聚焦显微镜技术观察穿心莲内酯对铜绿假单胞菌生物被膜形成的影响,并测量生物被膜的厚度。结果:断层图像显示和空白对照组比较,用药后PAO1生物被膜生长不完全,出现多种形态,其中在0.1μg/ml剂量下,生物被膜呈破碎絮状;与空白对照组比较,穿心莲内酯在0.1μg/ml、0.01μg/ml剂量下,可明显影响PAO1生物被膜的生长厚度。结论:穿心莲内酯可能具有干扰铜绿假单胞菌PAO1生物被膜形成的作用。     

鱼腥草素钠对铜绿假单胞菌群感效应系统影响的研究

目的研究鱼腥草素钠(SH)对铜绿假单胞菌(Pseudomonas aeruginosa,PA)群感效应系统的影响,以期探明SH抗PA的作用机制。方法微量稀释法测定SH和阿奇霉素(AZM)对PA的最小抑菌浓度(MIC);紫外分光光度法测定SH对PA LasA蛋白酶活性和绿脓菌素生成的影响;平板法观察PA的形态;半定量PCR法和实时荧光定量PCR(q RT-PCR)法检测群感效应相关的基因lasI、rhlI、lasR、rhlR、phzM、pqsA、pslA、lasA、las B、toxA表达量的变化。结果 SH和AZM对PA的MIC分别为512μg/m L和64μg/m L;紫外分光光度法结果显示SH能够抑制LasA蛋白酶的活性和绿脓菌素的合成;SH能够显著影响PA的形态;半定量PCR和q RT-PCR结果显示在SH作用下lasI、rhlI、lasR、lasA、pslA、phz M基因表达显著发生变化。结论 SH具有抗PA群感效应属性,通过抑制PA的群感效应系统发挥抗菌作用。     

苦参提取物与头孢他啶联用对铜绿假单胞菌生物被膜清除作用影响的体外研究

目的:通过建立生物被膜(Bacterial Biofilm,BF)体外模型,探讨苦参提取物在体外对铜绿假单胞菌BF的破坏清除作用及其与头孢他啶(CAZ)联用的协同杀菌作用.方法:用试管二倍稀释法检测最低抑菌浓度;BF快速银染法鉴定生物被膜;连续稀释法进行细菌茵落计数.结果:1/4、1/16 MIC苦参与1/4、1/2头孢他啶合用对铜绿假单胞茵有抑菌作用,2药单独使用均无抑茵作用.结论:2药联用对铜绿假单胞茵生物被膜有明显的清除作用,值得临床推广.     

亚抑菌浓度鱼腥草素钠及与红霉素联合对表皮葡萄球菌生物被膜的作用

目的:观察亚抑菌浓度(亚-MIC)的鱼腥草素钠及与红霉素联用对表皮葡萄球菌生物被膜的影响.方法:连续稀释法测定鱼腥草素钠和红霉素对表皮葡萄球菌的MIC;棋盘格法测定鱼腥草素钠和红霉素联用对表皮葡萄球菌悬浮菌的作用;体外构建表皮葡萄球菌生物被膜,XTT递减法评价亚抑菌浓度的鱼腥草素钠及与红霉素联用对表皮葡萄球菌黏附及对生物被膜内细菌代谢的影响;扫描电镜观察亚抑菌浓度下2药单独及联合用药后对表皮葡萄球菌生物被膜形态的影响情况.结果:鱼腥草素钠、红霉素对表皮葡萄球菌的MIC分别为62.5,7.812 5 mg·L-1;1/8MIC鱼腥草素钠、1/2MIC红霉素联用对表皮葡萄球菌悬浮菌的作用为协同,亚抑菌浓度的鱼腥草素钠、红霉素以及二者联用对表皮葡萄球菌生物被膜菌黏附和代谢均有抑制作用,对表皮葡萄球菌牛物被膜的形态有影响.结论:亚抑菌浓度的鱼腥草素钠、红霉素对表皮葡萄球菌牛物被膜有抑制作用;鱼腥草素钠和红霉素联合用药对表皮葡萄球菌悬浮菌和生物被膜的抑制具有协同作用,在对生物被膜菌代谢的抑制和对牛物被膜形态的影响方面效果尤为显著.     

鱼腥草素钠抑制铜绿假单胞菌致病相关运动能力的研究

鱼腥草素钠(sodium houttuyfonate,SH)是一种有效抑制致病菌感染的传统中药鱼腥草的活性成分的衍生物,但其抑菌机制尚没有被阐明.本研究通过铜绿假单胞菌运动能力试验发现,SH可在有效抑制与细菌致病性相关的浮泳运动、蹭行运动和集群运动能力.平板分析法实验结果表明24 h内SH对浮泳运动、蹭行运动和48 h内SH对集群运动的抑制趋势呈现显著的浓度依赖性特点;并且在1倍SH最小抑菌浓度(minimum inhibitory concentration,MIC) (512 mg·L-1)条件下,细菌的运动能力和阳性对照阿奇霉素(1倍MIC 16mg·L-1)一样基本丧失.进一步,基因表达分析发现SH显著抑制铜绿假单胞菌的鞭毛和菌毛结构基因flgB和pilG的表达,提示SH抑制细菌运动能力的可能机制是通过抑制细菌运动相关的特殊结构鞭毛和菌毛的合成而产生.因此,该研究结果首次证明SH可有效抑制细菌的运动能力,并探讨了抑制机制,为该药物在临床上治疗条件性致病菌铜绿假单胞菌提供理论依据.     

苦参煎剂对大鼠慢性铜绿假单胞菌生物膜肺部感染模型的影响

目的:探讨苦参对大鼠慢性铜绿假单胞菌(PA)生物膜肺炎模型的影响.方法:由支气管内直接注入PA(菌种为PAO579)藻酸盐微粒(1×10~9CFU·mL~(-1)),建立慢性PA生物膜感染模型,并于术后第2天开始给予3种不同剂量的苦参煎剂(3,6,12 g·kg~(-1))或灭菌生理盐水(NS)灌胃治疗,每天1次,连续治疗14 d,2周后评估各组的肺组织病理学、血清PA特异性抗体IgG水平、肺部细胞因子IFN-γ反应的变化及KB纸片琼脂扩散试验.结果:PA感染2周后,3种剂量的苦参组均可明显减轻PA感染大鼠肺部大体观病变和降低肺脓肿发生率(P<0.001);镜下观察发现苦参各治疗组主要表现为慢性炎症(以单个核细胞浸润为主),苦参中、高剂量组有急性炎症(以多形核白细胞浸润为主)的动物数明显低于模型组(P<0.05);免疫学指标检测发现苦参中、高剂量组能够显著下调血清PA特异性IgG抗体(P<0.05或JP<0.001),且血清IgG水平与苦参剂量呈显著负相关(r=-0.95,P<0.01);苦参各治疗组的肺部IFN-γ水平显著高于模型组(P<0.01),尤其是苦参高剂量组较模型组高10倍之多(P<0.001).同时,苦参各治疗组IFN-γ水平与苦参剂量呈正相关关系(r=0.9,P<0.02).另外,KB纸片琼脂扩散法检测发现苦参具有较弱的抗PA活性,而生理盐水无抗PA活性.结论:苦参可能通过调控感染机体的免疫反应,诱导Th1型反应,从而对PA肺感染动物有多方面的治疗作用,而12 g·kg~(-1)的苦参是本研究的最佳剂量.     

黄连解毒汤抗铜绿假单胞菌生物被膜 及与阿奇霉素协同抗菌作用

目的:研究黄连解毒汤抗铜绿假单胞菌牛物被膜及与阿奇霉素协同抗菌作用.方法:微量倍比稀释法测定黄连解毒汤对铜绿假单胞菌的最小抑菌浓度(MIC),棋盘稀释法测定黄连解毒汤和阿奇霉素协同抗菌作用,MTT法测定黄连解毒汤对铜绿假单胞菌生物被膜的最小抑膜浓度(SMIC),显微镜下观察药物对生物膜形态的影响.结果:黄连解毒汤对铜绿假单胞菌的MIC 100 g·L-1,阿奇霉素200 mg·L-1,两药联合后MIC分别为25 g·L-1和25 mg·L-1抑菌分级指数(FIC)0.1875,提示两药协同作用明显.黄连解毒汤对铜绿假单胞菌生物被膜的SMIC5.1,3d均为15.6 g·L-1,7 d31.25 g·L-1;SMIC801,3,7d均为250 g·L-1,形态观察提示黄连解毒汤SMIC80浓度对铜绿假单胞菌生物被膜的抑制作用明显.结论:黄连解毒汤具有抗铜绿假单胞菌生物被膜作用,与阿奇霉素有协同抗菌作用.     

Uterine relaxant effects of Curcuma aeruginosa Roxb. rhizome extracts

The effects and plausible mechanism of action of Curcuma aeruginosa Roxb. (Zingiberaceae) rhizome chloroform and methanol extracts on the uterine contraction were investigated using isolated uterus strips from estrogen primed rats. The contractile responses were recorded isometrically with a Grass FT03 force transducer connected to a MacLab system. The experiments were carried out on both nonstimulated, agonist- and KCl-stimulated uteri. In the nonstimulated uterus, the two extracts (10–400 μg/ml) had no significant effect. In contrast, in the stimulated uterus, the chloroform and methanol extracts exerted concentration-dependent inhibition of the contractions induced by oxytocin (1 mU/ml), prostaglandin F_2α (PGF_2α, 0.5 μg/ml), ACh (3 × 10⁻⁶ M) and KCl (40 mM) with the IC₅₀ (inhibition of force) of 31.4, 58.59, 56.21 and 29.28 μg/ml; and 57.79, 69.3, 223.8 and 69.19 μg/ml, respectively. Verapamil, the reference L-type calcium channel blocker, exhibited a similar pattern of inhibition with the IC₅₀ of 0.03, 0.25, 0.35 and 0.04 μg/ml. The IC₅₀ of diclofenac against a PGF_2α-induced contraction was 31.36 μg/ml. It is known that the contraction induced by agonists and KCl is mainly due to calcium influx through the voltage-gated L-type calcium channels opened indirectly or directly by agonist–receptor activation and KCl. Thus, it is speculated that the two plant extracts might inhibit uterine contraction by interrupting the influx of Ca²⁺ probably through voltage-gated L-type calcium channels. This possibility was further substantiated by the ability of the extracts to shift the CaCl₂–contraction curves to the right. As the methanol extract also reduced the contraction of oxytocin in Ca²⁺-free EDTA solution; thus, it is suggested that part of its action may be involved with an intracellular mechanism. The effect of the two extracts did not involve the activation of β₂-adrenoceptors since their effects were unaffected by propranolol. Based on the inhibitory effect of the extracts on the oxytocin-induced contraction, it is concluded that the extracts might be useful as tocolytic agents for the prevention of preterm labor. Their effects on the inhibition of PGF_2α-induced contractions also seem useful for the treatment of dysmenorrhea. There are reports by others that the plant rhizome contains β-pinene and sesquiterpenes. In addition, there is evidence that these compounds possess spasmolytic effects in the rat intestine and uterus. Therefore, the uterine relaxant effect of the plant extracts could be due to β-pinene and some sesquiterpene lactones contents. The methanol extract is less potent than the chloroform extract, and this might be due to the lower amount of terpene compounds or different compounds may involve in this action.     

黄芩苷对非白念珠菌生物膜抑制作用的研究

目的:研究黄芩苷对光滑念珠菌、近平滑念珠菌、克柔念珠菌、季也蒙念珠菌等非白念珠菌生物膜的影响.方法:96孔微量培养板上构建4种非白念珠菌生物膜;微量稀释法检测黄芩苷对4种非白念珠菌最低抑菌浓度(MIC);XTT减低法评价黄芩苷对4种非白念珠菌生物膜SMIC及菌细胞黏附性的影响.结果:4种非白念珠菌在96孔板中可形成成熟的生物膜;黄芩苷对4种非白念珠菌MIC分别是125,250,125,62.5 mg·L~(-1);黄芩苷对4种非白念珠菌生物膜的SMIC50分别是＞1 000,500,125,250 mg·L~(-1);SMIC80均大于或等于1 000 mg·L~(-1).黄芩苷对4种非白念珠菌的黏附有一定的抑制效应.结论:黄芩苷对近平滑念珠菌、克柔念珠菌、季也蒙念珠菌生物膜有较强的抑制作用.     

Antimicrobial effect of sodium houttuyfonate on Staphylococcus epidermidis and Candida albicans biofilms

Objective

To study antimicrobial effect of Sodium houttuyfonate (SH) on Staphylococcus epidermidis (SE) and Candida albicans (CA).

Methods

The prepared strain broths (OD₆₀₀=0.05) containing SE and CA were firstly used to test the minimal inhibitory concentrations (MICs) of SH, azithromycin (AZM) and fluconazole (FLU) by micro-dilution method. Then the biofilms of SE and CA were matured in 96-well plates, and co-cultured with SH, AZM and FLU for 1, 2 and 3 days to assess the antibiofilm efficacies of the agents with different concentrations by crystal violet staining method. At last, the treated biofilms of SE and CA by 2×MIC agents were observed by scanning electronic microscope.

Results

The MICs of SE and CA were 256 and 1024 µg/mL, respectively. After the 1st, 2nd and 3rd day of medications, the suppressions of biofilm were about 60% (P<0.01), 76% (P=0.000) and 75% (P=0.000) by 2×MIC SH, the suppressions of biofilm were about 90% (P=0.000), 88% (P=0.000) and 90% (P=0.000) by 2×MIC SH, which could be testified by scanning electron microscope results. However, the inhibitions of biofilm attachment had no significant difference for SE by SH and azithromycin and CA by SH and fluconazole.

Conclusion

SH had widely anti-pathogenic effect on pathogenic biofilm formation of either bacteria or fungus, had more influence on enclosed cells of SE and CA than the traditional antibiotics, revealing its target might be the extracellular polymeric substances, and was more active to inhibit the growth of CA than SE.