Biofilm dispersion is the last step in the development of biofilms, and allows bacteria to spawn novel biofilms in new locales. In the previous studies, we found that sodium houttuyfonate (SH) is effective at inhibiting biofilm formation and motility of Pseudomonas aeruginosa. Here, we investigated the effect of SH against the biofilm dispersion of P. aeruginosa by an in vitro model. The results show that the plant derivative, SH, could effectively inhibit both biofilm dispersion of P. aeruginosa, and gene and protein expression of the key biofilm regulator BdlA in a dose-dependent manner. Furthermore, our presented results suggest that SH can penetrate into the biofilm of P. aeruginosa to repress the biofilm life cycle. Therefore, these results indicate that the antimicrobial activity of SH may be partially due to its ability to disrupt biofilm dispersion in P. aeruginosa.

目的 利用指示菌紫色杆菌CV026菌株,建立和优化稳定的细菌菌群传感效应信号分子高丝氨酸内酯平板分析方法.方法 优化受测铜绿假单胞菌高丝氨酸内酯的提取方法,分析铜绿假单胞菌培养时间对信号分子检测的影响.另一方面,优化指示菌株CV026的培养基成分,并优化紫色菌素的提取方法,以完善信号分子的检测反应.结果 恒温水浴挥发法可有效提取高丝氨酸内酯,并且发现当铜绿假单胞菌的培养时间为3d时,高丝氨酸内酯最易被检出.进一步,试验结果显示添加L-Try可提高指示菌株紫色菌素的产量,并利于高丝氨酸内酯的检测.此外,比较发现采用乙醇溶解紫色菌素的方法可更高效的测定紫色菌素的量.结论 本课题建立的检测方法将更有效的检测细菌菌群传感系统信号分子高丝氨酸内酯,将有利于细菌菌群传感机制研究和抑制细菌菌群传感药物的开发工作.

OBJECTIVE: To investigate the in vivo inhibitory effects of sodium houttuyfonate(SH) on symptom pattern of Qi-deficiency in rats induced by infection of bacterial biofilm on rat respiratory tract.METHODS: Symptom pattern is a term used in Traditional Chinese Medicine(TCM) to define a cluster of symptoms in a medical condition. Based on the pattern, TCM therapies are administered. The symptom pattern used in this study was lung-Qi deficiency pattern identified in rats, which was induced by nasal intubation drip of Pseudomonas aeruginosa(P. aeruginosa)(two strains) to form bacterial biofilm on airway combined with stimulation of cold and fatigue. We measured the variations of the symptoms of the pattern, weight, spleen and thymus index, blood gas, lung bronchial tissue pathology and cytokine of rat in different treatments and control groups.RESULTS: The rats of SH-treatment groups had not showed typical symptoms comparing with model group in the early stage of infection. The weight,spleen and thymus index of the SH-treatment groups were significantly higher comparing with untreated model group. The SH-treatment groups also showed higher O2 partial pressure and lower CO2 partial pressure than model group. Furthermore, we found that the bronchopulmonary section of SH-treatment groups not showed typical pathogenic variation in model group. The comparison of cytokine concentration in different groups indicated that SH could prevent the over-production of cytokine to reduce the inflammation occurrence.CONCLUSION: In the early stage of airway infection by biofilm of P. aeruginosa, application of SH can prevent the occurrence of lung-Qi deficiency pattern.

Objective To investigate the mechanism of sodium houttuyfonate (SH) against Pseudomonas aeruginosa(PA) targeting quorum sensing system.Methods The microdilution method was adopted to determine the minimal inhibitory concentration (MIC) of SH and azithromycin (AZM) for PA;The effects of SH on LasA proteases activity and pyocyanin production were detected by UV spectrophotometry;The semi-quantitative PCR and qRT-PCR were used to detect the expression changes of quorum sensing regulatory genes (lasI,rhll,lasR,rhlR,phzM,pqsA,pslA,lasA,lasB,and toxA).Results The MIC ofP.aeruginosa for SH and AZM was 512 μg/mL and 64 μg/mL,respectively;SH could significantly inhibit the activity of LasA proteases,and pyocyanin of bacteria;The morphology ofP.aeruginosa was observed by agar plates;Furthermore,semi-quantitative PCR and qRT-PCR results indicated that the expression oflasI,rhlI,lasR,lasA,pslA,and phzM was significantly impact by SH.Conclusion The results of present study suggest that SH possesses anti-quorum sensing properties and plays a role against infection by anti-quorum sensing activity.

Sodium houttuyfonate (SH) has been found to be mildly anti-pathogenetic against the planktonic and biofilm phenotypes of Pseudomonas aeruginosa. Here, we have attempted to investigate further the anti-pathogenicity of SH and EDTA-Na2 in combination and in vitro and in vivo against planktonic and biofilm phenotypes of Pseudomonas aeruginosa (16 strains), Staphylococcus aureus (13 strains) and Candida albicans (13 strains). The antimicrobial activity of SH against all three pathogens increased dramatically when it was combined with EDTA-Na2, in vitro. Toxic reactions to the drugs when administered orally were insignificant in mice; no abnormalities were observed in the internal organs, such as the lungs and kidneys. Finally, the results of in vivo studies indicate that SH could extend the lifespan of infected animals when administered in combination with EDTA-Na2. Therefore, the results of the present study lead us to suggest that SH could be a promising antimicrobial agent and that SH combined with EDTA-Na2 has the potential to be an excellent choice of drug for combating bacterial infections clinically.

The increasing multidrug resistance of Pseudomonas aeruginosa has become a serious public-health problem. In the present study, the inhibitory activities of sodium houttuyfonate (SH) against biofilm formation and alginate production in a clinical strain of P.aeruginosa (AH16) were investigated in vitro using crystal violet dying and standard curve methods, respectively. The cellular morphology of P. aeruginosa treated with SH was observed using a scanning electron microscope. Furthermore, reverse transcription-quantitative polymerase chain reaction was used to identify differences in the expression levels of genes associated with alginate biosynthesis as a result of the SH treatment. The results indicated that SH significantly inhibited biofilm formation, and decreased the levels of the primary biofilm constituent, alginate, in P. aeruginosa AH16 at various stages of biofilm development. In addition, scanning electron microscopy observations demonstrated that SH markedly altered the cellular morphology and biofilm structure of P. aeruginosa. Furthermore, the results from the reverse transcription-quantitative polymerase chain reaction analysis indicated that SH inhibited biofilm formation by mitigating the expression of the algD and algR genes, which are associated with alginate biosynthesis. Therefore, the present study has provided novel insights into the potent effects and underlying mechanisms of SH-induced inhibition of biofilm formation in a clinical strain of P. aeruginosa.

Sodium houttuyfonate (SH) is a derivative of effective component of a Chinese material medica, Houttuynia cordata, which is applied in anti-infection of microorganism. But, the antimicrobial mechanisms of SH still remain unclear. Here, we firstly discovered that SH effectively inhibits the three types of virulence related motility of.Pseudomonas aeruginosa, i.e., swimming, twitching and swarming. The plate assay results showed that the inhibitory action of SH against swimming and twitching in 24 h and swarming in 48 h is dose-dependent; and bacteria nearly lost all of the motile activities under the concentration of 1 x minimum inhibitory concentration (MIC) (512 mg x L(-1) same as azithromycin positive group (1 x MIC, 16 mg x L(-1)). Furthermore, we found that the expression of structural gene flgB and pilG is down-regulated by SH, which implies that inhibitory mechanism of SH against motility of P. aeruginosa may be due to the inhibition of flagella and pili bioformation of P. aeruginosa by SR Therefore, our presented results firstly demonstrate that SH effectively inhibits the motility activities of P. aeruginosa, and suggest that SH could be a promising antipseudomonas agents in clinic.

Objective To explore the effects of Sodium houttuyfonate initially prevent the happening of the lung Qi deficiency syndrome in rats inducted by pseudomonas aeruginosa biofilm. Methods Healthy SD rats were randomly divided into normal control group,P. aeruginosa standard strains(ATCC27853,Pa) group and clinical strains of P. aeruginosa(AH16) group including the model group,Sodium houttuyfonate(SH) treatment group and azithromycin(AZM) positive control group,respectively. The cultures of standard(Pa) and clinical(AH16) strains were dropped into the noses of the rats combined with stimulations of cold and fatigue,respectively,causing respiratory tract of chronic infection in rats to induce the occurrence of lung Qi deficiency syndrome in rats. After successful infected,the treatment group and positive control group of animals were treated by sodium houttuyfonate and azithromycin,respectively. The syndrome changes in blood gas were observed. The changes of Interleukin-8(IL-8) in alveolar lavage fluid were determined using Enzyme-linked immunosorbent assay. The HE dyeing observation lung and bronchial tissue pathological variations were identified under microscope. Results The rat features in model group accorded with the symptoms of lung Qi deficiency syndrome; the symptom of the drug therapy group was not significant comparing to the lung Qi deficiency symptoms of. Blood gas analysis showed that significant change compared with normal control group in the model group rats; the infections blood arterial partial pressure of oxygen(PO2) were dropped significantly(P 0. 05,or P 0. 01),while partial pressure of carbon dioxide(PCO2) were significantly higher(P 0. 05). Between treatment group compared with normal control group,PO2 and PCO2in rat blood were no significant difference(P 0. 05); Compared with normal control group,IL-8 levels of model group rats lung lavage fluid were significantly elevated(P 0. 05 or P 0. 01); Pathological analysis showed that model group existed apparent lung and bronchial inflammation,to conform to the lung tissue changes in chronic bronchitis,and emphysema. Pathological changes in sodium houttuyfonate treatment group and azithromycin positive control group were not significant in lungs of rats. Conclusion Sodium houttuyfonate can initially prevent the happening of the lung qi deficiency syndrome in rats induced by Pseudomonas aeruginosa biofilm.

Quorum sensing (QS) is a means of cell-to-cell communication that uses diffusible signaling molecules that are sensed by the population to determine population density, thus allowing co-ordinate gene regulation in response to population density. In Pseudomonas aeruginosa, production of the QS signaling molecule, N-acyl homoserine lactone (AHL), co-ordinates expression of key factors of pathogenesis, including biofilm formation and toxin secretion. It is predicted that the inhibition of AHL sensing would provide an effective clinical treatment to reduce the expression of virulence factors and increase the effectiveness of antimicrobial agents. We previously demonstrated that sodium houttuyfonate (SH), commonly used in traditional Chinese medicine to treat infectious diseases, can effectively inhibit QS-regulated processes, including biofilm formation. Here, using a model system, we demonstrate that SH causes the dose-dependent inhibition of AHL production, through down-regulation of the AHL biosynthesis gene, lasI. Addition of SH also resulted in down-regulation of expression of the AHL sensor and transcriptional regulator, LasR, and inhibited the production of the QS-regulated virulence factors, pyocyanin and LasA. These results suggest that the antimicrobial activity of SH may be due to its ability to disrupt QS in P. aeruginosa.

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、0.512mg/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.

目的 探究鱼腥草素钠(Sodium Houttuyfonate:SH)对呼吸道被膜菌铜绿假单胞菌(Pseudomonas aeruginosa:PA)致肺气虚证大鼠肺部β-防御素的影响.方法 采用呼吸道被膜菌铜绿假单胞菌滴鼻法致大鼠呼吸道慢性感染,辅以寒冷疲劳刺激,诱发大鼠肺气虚证模型,用鱼腥草素钠干预,设不给药组为模型组和未感染大鼠作为正常对照组,8周后观察证候表现,镜下观察肺部组织病理改变,酶联免疫吸附法测定支气管灌洗液中β-防御素含量.结果 模型组符合肺气虚证候表现.鱼腥草素钠组、阿奇霉素(azithromycin:AZM)阳性对照组及正常对照组均无明显的肺气虚症候表现.病理学检查显示,模型组的大鼠支气管粘膜有大量的脱落、炎症细胞浸润和肺气肿的发生,而药物治疗组及正常对照组无炎症反应,肺部支气管粘膜完好;模型组肺部灌洗液β-防御素含量明显增高,药物治疗组β-防御素增高不明显,两组相比差异显著(P＜0.01),药物治疗组和正常对照组相比无显著差异(P＞0.05).结论 β-防御素增高可以加剧肺部损伤,在铜绿假单胞菌感染大鼠过程中,鱼腥草素钠使大鼠气道β-防御素维持在正常水平,是抑制气道病变以及炎症反应从而干预肺气虚证的发生的重要因素之一.

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 2x MIC agents were observed by scanning electronic microscope.RESULTS: The MICs of SE and CA were 256 and 1024 mu g/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 2xMIC SH, the suppressions of biofilm were about 90% (P=0.000), 88% (P=0.000) and 90% (P=0.000) by 2xMIC 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. (C) 2013 JTCM. All rights reserved.