美罗培南对las、rhl基因缺陷型铜绿假单胞菌生物膜的体外作用

目的:研究美罗培南对铜绿假单胞菌(PA)PAO1野生型及其PAO1 lasI rhlI、PAO1 lasR rhlR基因缺陷型菌株生物膜的作用。方法:(1)试管双倍稀释法测定美罗培南对铜绿假单胞菌PAO1野生型及其PAO1 lasI rhlI和PAO1 lasR rhlR基因缺陷型菌株的最低抑菌浓度(MIC)和最低杀菌浓度(MBC)。(2)采用生理盐水-吸痰管系统进行铜绿假单胞菌PAO1野生型、PAO1 lasI rhlI和PAO1 lasR rhlR基因缺陷型生物膜培养7d后,用美罗培南26 mg·L~(-1)作用24h,然后用扫描电子显微镜观察生物膜的变化。结果:(1)美罗培南对PAO1野生型、PAO1 lasl rhlI基因缺陷型和PAO1 lasR rhlR基因缺陷型3种铜绿假单胞菌菌株的MIC均为1mg·L~(-1),MBC均为2 mg·L~(-1)。(2)7 d后PAO1野生型和PAO1 lasR rhlR基因缺陷型铜绿假单胞菌都可形成较厚、有孔状通道的生物膜,PAO1 lasl rhlI基因缺陷型形成明显稀薄的早期生物膜结构;经过美罗培南作用后,PAO1野生型铜绿假单胞菌的生物膜结构明显稀薄,而PAO1 lasI rhlI、PAO1 IasR rhlR基因缺陷型铜绿假单胞菌的生物膜结构基本被破坏。结论:铜绿假单胞菌的las、rhl基因可影响其生物膜的形成及生物膜对美罗培南的敏感性。     

氨溴索对铜绿假单胞菌生物膜群体感应系统的影响

目的探讨氨溴索对铜绿假单胞菌生物膜群体感应(QS)系统的影响。方法平板法培养成熟的铜绿假单胞菌PAO1野生型菌株和QS系统缺陷株△lasR△rhlR基因缺陷型、△lasI△rhlI基因缺陷型菌株生物膜,分别予生理盐水和氨溴索1.875 g·L~(-1)、3.75 g·L~(-1)作用8 h。通过荧光探针SYTO9/PI标记,激光共聚焦显微镜(CLSM)观察不同浓度氧溴索作用前后不同菌株生物膜的结构变化,利用生物膜图象结构分析软件(ISA)对氨溴索作用前后的生物膜结构参数进行定量分析。结果 PAO1野生型菌株可见大片状生物膜,细菌密集。2个基因缺陷型菌株的生物膜菌落较稀疏,氨溴索作用后不同菌株生物膜厚度、平均扩散距离和结构熵均显著减少,区域孔率增加,有剂量相关性(P<0.05),PAO1野生型菌株生物膜的变化幅度比QS系统缺陷株更大(P<0.05)。结论氨溴索破坏铜绿假单胞菌生物膜的结构,具有拮抗QS系统的特性,高浓度氨溴索(3.75 g·L~(-1))效果更显著。     

铜绿假单胞菌lasR/rhlR基因缺陷对小鼠腹腔生物被膜感染的影响

目的 探讨铜绿假单胞菌(PA)群体感应(QS)系统的lasR/rhlR基因缺陷对小鼠腹腔PA生物被膜(BF)感染的影响.方法 体外利用无菌引流管切片建立铜绿假单胞菌PAO1野生型和PAO1 lasR/rhlR基因缺陷型菌株生物被膜感染的载体,将两种感染载体和无菌载体分别移植至正常小鼠腹腔构建生物被膜感染模型,术后3d检...     

RNA干扰铜绿假单胞菌群体感应系统对生物膜影响的研究

目的 观察小干扰RNA(small interfering RNA,siRNA)分子干扰铜绿假单胞菌群体感应系统后,对铜绿假单胞菌生物膜的影响.方法 首先针对lasR、rhlR和rhlI基因设计4条siRNA分子,分别是si-asr1、si-hlr1、si-hlr2和si-hli1,体外转录法制备上述siRNA分子.将50nmol/L siRNA分子与4μl脂质体混合,然后转染铜绿假单胞菌成熟期生物膜,应用平板菌落计数法检测siRNA转染后96h内生物膜活细菌数目;siRNA转染生物膜48h后,应用1%复红染色法光学显微镜观察生物膜形态改变,同时应用RT-PCR法检测生物膜弹性蛋白酶B基因lasB表达量.结果 si-hlr1、si-hlr2和si-hli1分别转染铜绿假单胞菌生物膜48h,生物膜出现解体,生物膜内活细菌数目无明显改变.si-asr1转染铜绿假单胞菌生物膜48h后,生物膜形态及生物膜内活细菌数目无明显改变.si-asr1、si-hlr2和si-hli1分别转染铜绿假单胞菌生物膜48h,生物膜lasB基因表达量分别下调了49%、21%和18%.si-hlr1转染生物膜48h后,lasB基因表达量无明显改变.结论 应用siRNA分子干扰铜绿假单胞菌群体感应系统的lasR、rhlR和rhlI三个关键性基因,可以促进生物膜解体,同时能部分抑制生物膜lasB基因表达,但是不能减少生物膜内活细菌数.     

左氧氟沙星对铜绿假单胞菌密度感应系统及毒力因子的调控

目的：评价铜绿假单胞菌在亚抑制浓度（ SIC）的左氧氟沙星作用下毒力因子表型及基因表达的变化并探索其可能的调控途径。方法用临床标准菌株PAO1及其密度感应系统（ QS）的基因突变株,测定不同浓度下菌株生长曲线,以不影响细菌生长的最高抗菌素浓度为亚抑制浓度。分别测定各菌株在SIC的抗菌素作用下生物膜形成、绿脓菌素和鼠李糖脂的变化。用实时定量聚合酶链式反应（ RT－PCR）测定菌株毒力蛋白编码基因以及QS基因在SIC的左氧氟沙星作用下表达的变化。用生物发光法测定QS信号分子在SIC的抗菌素作用下的变化。结果在SIC的左氧氟沙星作用下,PAO1的毒力因子编码基因和QS调控基因（lasR、rhlR）表达均显著增加,PAO1野生株的毒力因子也增加,但lasR、rhlR突变株无此变化,同时QS信号分子水平也无显著变化。结论亚抑制浓度的左氧氟沙星促进铜绿假单胞菌毒力因子的产生,这一效应是通过lasR和rhlR调节实现的,但这一调控作用可能并不是直接通过AHL信号分子完成。     

靶向rhl I基因siRNA分子抑制豚鼠铜绿假单胞菌感染

摘要：目的 探讨靶向rhlI基因的siRNA分子能否抑制豚鼠体内铜绿假单胞菌生物膜感染。方法 应用气管注入法制备铜绿假单胞菌生物膜感染豚鼠模型,18只豚鼠模型随机分成3组,分别经尾静脉注射siRNA分子（50μg.mL-1）与脂质体混合物（siRNA组）、乳酸环丙沙星（环丙沙星组）或生理盐水（对照组）。检查肺和支气管组织病理改变。取左肺组织匀浆和支气管肺泡灌洗液(BALF)进行细菌学检查。结果 siRNA组豚鼠肺和支气管组织结构未见明显异常,环丙沙星组和对照组豚鼠肺和支气管组织结构异常,表现为肺组织实质变性伴炎性细胞浸润,支气管黏膜脱落,管壁变薄。siRNA组豚鼠左肺和BALF的细菌总数均显著低于环丙沙星组（p＜0.001）,siRNA组豚鼠体内分离的铜绿假单胞生物膜形成能力均显著低于环丙沙星组（p＜0.001）。结论 靶向rhlI基因的siRNA分子能够破坏豚鼠体内的铜绿假单胞生物膜,减少感染组织内细菌总数,使病变组织结构恢复正常,有望成为治疗细菌生物膜感染的新一代药物。     

铜绿假单胞菌群体感应系统分子受体基因lasR与Ⅰ类整合酶intI1基因表达的相关性分析

目的通过研究铜绿假单胞菌Ⅰ类整合子阳性株群体感应系统基因lasR与Ⅰ类整合子整合酶基因intI1表达水平的相关性,初步探讨群体感应系统对Ⅰ类整合子的调控机制。方法通过荧光定量RT-PCR方法检测铜绿假单胞菌群体QS信号分子受体基因lasR和Ⅰ类整合子整合酶基因intI1在液相菌与生物被膜菌中mRNA的表达水平,分析两基因表达的相关性。结果铜绿假单胞菌在生物被膜中群体感应系统lasR基因及Ⅰ类整合子intI1基因的mRNA表达水平高于液相菌,两者表达水平呈现正相关性(r=0.695,P<0.05)。结论铜绿假单胞菌群lasR和intI1基因表达呈正相关,提示intI1基因表达可能与群体感应系统的调控作用有关。     

金银花水煎液抗绿脓杆菌生物膜作用及其与庆大霉素的协同作用

目的观察金银花水煎液体外抗铜绿假单胞菌生物膜的作用及与庆大霉素的协同作用。方法通过试管稀释法测定金银花水煎液和庆大霉素对铜绿假单胞菌的最小抑菌浓度(MIC),用MTT法建立体外铜绿假单胞菌生物膜并检定金银花水煎液和庆大霉素不同MIC浓度的抗铜绿假单胞菌生物膜的作用。结果金银花水煎液对绿脓杆菌的MIC为125g.L-1,SMIC也为125 g.L-1;庆大霉素对绿脓杆菌的MIC为1.56 mg.L-1,SMIC为25 mg.L-1,与金银花的协同作用对绿脓杆菌的SMIC为1.56 mg.L-1。结论金银花水煎液在体外能抑制铜绿假单胞菌对固体表面的黏附能力及生物膜形成能力,并能破坏铜绿假单胞菌已形成的生物膜,增强庆大霉素对生物膜内铜绿假单胞菌的清除作用。     

D-环丝氨酸抑制铜绿假单胞菌群体感应活性研究

摘要：目的 以铜绿假单胞菌模式菌株PAO1为研究对象,研究抗结核药物D-环丝氨酸通过靶向抑制病原菌群体感应 (quorum-sensing,QS)系统实现抑制病原菌毒力发挥的新应用潜力。方法 用不同浓度的D-环丝氨酸处理铜绿假单胞菌,通过 系列表型实验结合荧光定量PCR以评估D-环丝氨酸对群体感应所调节的毒力因子和相应基因表达的影响,并利用秀丽隐杆线虫 感染模型评估D-环丝氨酸对铜绿假单胞菌毒力抑制的体内活性。结果 D-环丝氨酸表现出良好的铜绿假单胞菌生物膜、绿脓菌 素以及蛋白水解酶抑制活性,显著抑制了QS系统调控基因和下游功能基因的表达,并且可以显著提高秀丽隐杆线虫在铜绿假单 胞菌感染过程的存活率。结论 D-环丝氨酸可以有效抑制铜绿假单胞菌群体感应系统,有望开发成功的抗生素替代药物。     

亚抑菌浓度莫匹罗星对烧伤创面分离铜绿假单胞菌生物膜的影响

目的:研究烧伤患者伤口分离铜绿假单胞菌的耐药性,并探讨亚抑菌浓度莫匹罗星对铜绿假单胞菌生物膜的作用。方法:采用琼脂平板倍比稀释法检测30株临床分离铜绿假单胞菌的最低抑菌浓度(MIC),菌落计数法分析亚抑菌浓度(1/4 MIC)莫匹罗星对铜绿假单胞菌黏附性的影响,96孔板结晶紫染色法检测亚抑菌浓度莫匹罗星对铜绿假单胞菌生物膜形成的影响。结果:我院临床烧伤患者分离的铜绿假单胞菌对莫匹罗星、哌拉西林、亚胺培南、美罗培南、左氧氟沙星、庆大霉素、奈替米星具有较高的耐药性,而对头孢他啶和氨曲南的敏感性较高。亚抑菌浓度莫匹罗星对临床分离铜绿假单胞菌的黏附性和生物膜形成均有抑制作用。结论:莫匹罗星可抑制铜绿假单胞菌生物膜形成,但其抑制程度具有菌株差异性。     

磷霉素与氨基糖苷类药物联用对体外铜绿假单胞菌生物被膜形态学的影响

目的:通过研究磷霉素与氨基糖苷类药物(阿米卡星、异帕米星)联合应用对铜绿假单胞菌生物被膜的体外联合效应,探讨磷霉素对氨基糖苷类药物在抑制铜绿假单胞菌生物被膜形成的作用。方法:采用改良的组织培养平板法建立铜绿假单胞菌PAO1生物被膜模型,应用银染法快速鉴定法观察经不同剂量的两类药物单用与联合作用处理后的PAO1生物被膜结构,对其形态学进行描述,并采用计算机图像处理系统进行图像分析。结果:银染法快速鉴定的形态学结果显示,PAO1生物被膜上分布着浓密黑染交织物质,其间隙中聚集着短杆状细菌体;同一剂量下,联药组的黑染交织物较单药组少;磷霉素分别与阿米卡星和异帕米星联用时的抑制被膜形成作用基本相似。计算机图像处理系统的分析显示,平均光密度值随着药物的剂量增大而逐渐减小,联药组的平均光密度值显著低于单药组的平均光密度值(P<0.05)。结论:研究发现,磷霉素与氨基糖苷类药物联用对铜绿假单胞菌生物被膜形成的抑制具有显著增效作用。     

Effects of iron depletion on antimicrobial activities against planktonic and biofilm Pseudomonas aeruginosa

Objectives Iron plays an important role in the development of Pseudomonas aeruginosa biofilm. Here we evaluated effects of iron depletion on the antimicrobial activity of ceftazidime, tobramycin and ciprofloxacin against planktonic and biofilm Pseudomonas aeruginosa. Methods We tested the sensitivities of wild‐type PAO1, type‐IV pilus mutant PAO‐ΔpilHIJK and the quorum‐sensing mutant PAO‐JP2 P. aeruginosa planktonic cultures and biofilms to antibiotics under iron‐depleted conditions. Key findings In planktonic bacteria, the minimum concentration that inhibited visible growth (MIC) of ciprofloxacin was increased slightly in an iron‐depleted environment in all three strains, whereas the MIC of tobramycin was similar in iron‐depleted and control environments. The MIC of ceftazidime increased in the PAO‐JP2 strain when iron was depleted. Tobramycin achieved the best bactericidal effect in biofilms. Viable counts were reduced by one log under iron‐depleted conditions in all three strains when tobramycin reached 4 MIC and when ceftazidime and ciprofloxacin reached 8 MIC. Conclusions This study suggests that once the biofilm is formed, iron depletion may only slightly promote the bactericidal effect of antibiotics on PAO1, PAO‐ΔpilHIJK and PAO‐JP2. Although these changes were relatively small, iron as one of the environmental factors should not be ignored when evaluating bactericidal effect of antibiotics. The combination of an iron chelator and antibiotics may have therapeutic value under certain bacterial growth conditions.     

磷霉素与氨基糖苷类药物联用对体外铜绿假单胞菌生物被膜中活菌数的测定

目的通过研究磷霉素与氨基糖苷类药物（阿米卡星、异帕米星）联合应用对铜绿假单胞菌生物被膜的体外联合效应,探讨磷霉素对氨基糖苷类药物在抑制铜绿假单胞菌生物被膜形成方面的作用。方法采用改良的组织培养平板法建立铜绿假单胞菌生物被膜模型,应用活菌计数法观察经不同剂量的两类药物单用与联合作用处理后的铜绿假单胞菌生物被膜的活菌数量,对其进行比较与分析。结果联药组处理的生物被膜内活菌数较单药组显著下降,表明磷霉素与阿米卡星/异帕米星二者联合应用后,生物被膜的形成被抑制,存活细菌数量显著减少。结论研究发现磷霉素与氨基糖苷类药物联用对铜绿假单胞菌生物被膜形成的抑制具有显著增效作用。     

Potent Irreversible Inhibitors of LasR Quorum Sensing in Pseudomonas aeruginosa

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磷霉素与氨基糖苷类药物联用对铜绿假单胞菌生物被膜的影响

目的通过研究磷霉素与氨基糖苷类药物(阿米片星、异帕米星)联合应用对铜绿假单胞菌生物被膜的体外联合效应,探讨磷霉素对氨基糖苷类药物在抑制铜绿假单胞菌生物被膜形成的作用。方法采用改良的组织培养平板法建立铜绿假单胞菌(PAO1)生物被膜模型,应用扫描电镜观察经不同剂量的两类药物单用与联合作用处理后的PAO1生物被膜结构,对其形态学进行分析比较。结果扫描电镜的形态学结果显示,PAO1细菌呈杆状,为浓厚的粘液样物质紧密包绕,聚集成团,菌体之间相互粘连,生物被膜形态完整。经不同浓度的磷霉素、阿米卡星、异帕米星、磷霉素+阿米卡星、磷霉素+异帕米星处理后,随着药物剂量的增加,细菌数减少,聚集程度降低,细菌间粘液状物显著减少。磷霉素+阿米卡星和磷霉素+异帕米星联用的抑制被膜形成作用基本相似,磷霉素的抑制被膜形成作用较阿米卡星和异帕米星差,联药组的抑制被膜形成作用较单药组显著。结论磷霉素与氨基糖苷类药物联用对铜绿假单胞菌生物被膜形成的抑制具有显著增效作用。     

Siphonocholin isolated from red sea sponge Siphonochalina siphonella attenuates quorum sensing controlled virulence and biofilm formation

Increasing incidence of multi-drug resistant bacterial pathogens, especially in clinical settings, has been developed into a grave health situation. The drug resistance problem demands the necessity for alternative unique therapeutic policies. One such tactic is targeting the quorum sensing (QS) controlled virulence and biofilm production. In this study, we evaluated a marine steroid Siphonocholin (Syph-1) isolated from Siphonochalina siphonella against Chromobacterium violaceum (CV) 12472, Pseudomonas aeruginosa (PAO1), Methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii (BAA) for biofilm and pellicle formation inhibition, and anti-QS property. MIC of Syph-1 against MRSA, CV, PAO1 was found as 64 µg/mL and 256 µg/mL against BAA. At selected sub-MICs, Syph-1 significantly (P ≤ 0.05) decreased the production of QS regulated virulence functions of CV12472 (violacein) and PAO1 [elastase, total protease, pyocyanin, chitinase, exopolysaccharides, and swarming motility]. The Syph-1 significantly decreased (p = 0.005) biofilm formation ability of tested bacterial pathogens, at sub-MIC level (PAO1 > MRSA > CV > BAA) and pellicle formation in A. baumannii (at 128 µg/mL). Molecular docking and simulation results indicated that Siph-1 was bound at the active site of BfmR N-terminal domain with high affinity. This study highlights the anti-QS and anti-biofilm activity of Syph-1 against bacterial pathogens reflecting its broad spectrum anti-infective potential.     

Therapeutic possibilities for diffuse panbronchiolitis

Diffuse panbronchiolitis can be thought of as one of the biofilm diseases of which cystic fibrosis is another example. The patient often has persistent infection with Pseudomonas aeruginosa and, in 1982, the survival rate to 5 years in these patients was only 37%. Recently, the prognosis of these patients has remarkably improved by using long-term treatment with 14-membered macrolides. However, the reason for the effect of macrolides in these patients is still obscure. To try to understand this pneumonia, some experiments were performed to assess the effect of macrolides on P. aeruginosa biofilm. An in vitro biofilm of P. aeruginosa was prepared on Teflon after 6 days' incubation and the effects of antibiotics on the biofilm were assessed. Floating, but not biofilm bacteria were killed by meropenem, ciprofloxacin, tosufloxacin and cefoperaxone. Ciprofloxacin with either azithromycin or clarithromycin killed bacteria within the biofilm, whereas ciprofloxacin with clindamycin, josamycin or other 16-membered macrolides was ineffective. Clarithromycin destroyed the biofilm, releasing the bacteria into the medium. The ability of P. aeruginosa to adhere to murine tracheal cells was also reduced by clarithromycin. These kinds of macrolides may have some sort of permeability effect on the biofilm surface and this may bear some relation to the good clinical performance of macrolides in diffuse panbronchiolitis.     

Formation of Pseudomonas aeruginosa inhibition zone during tobramycin disk diffusion is due to transition from planktonic to biofilm mode of growth

Pseudomonas aeruginosa PAO1 (tobramycin MIC?=?0.064 µg/mL) was used to perform agar diffusion tests employing tobramycin-containing tablets. Bacterial growth and formation of inhibition zones were studied by stereomicroscopy and by blotting with microscope slides and staining with methylene blue, Alcian blue and a fluorescent lectin for the P. aeruginosa PSL, which was studied by confocal laser scanning microscopy. Diffusion of tobramycin from the deposit was modelled using a 3D geometric version of Fick's second law of diffusion. The time-dependent gradual increase in the minimum biofilm eradication concentration (MBEC) was studied using a Calgary Biofilm Device. The early inhibition zone was visible after 5 h of incubation. The corresponding calculated tobramycin concentration at the border was 1.9 µg/mL, which increased to 3.2 µg/mL and 6.3 µg/mL after 7 h and 24 h, respectively. The inhibition zone increased to the stable final zone after 7 h of incubation. Bacterial growth and small aggregate formation (young biofilms) took place inside the inhibition zone until the small aggregates contained less than ca. 64 cells and production of polysaccharide matrix including PSL had begun; thereafter, the small bacterial aggregates were killed by tobramycin. Bacteria at the border of the stable inhibition zone and beyond continued to grow to a mature biofilm and produced large amount of polysaccharide-containing matrix. Formation of the inhibition zone during agar diffusion antimicrobial susceptibility testing is due to a switch from a planktonic to biofilm mode of growth and gives clinically important information about the increased antimicrobial tolerance of biofilms.