大黄素对白假丝酵母菌抑菌作用的体外实验研究

目的研究大黄素对白假丝酵母菌的活性及其产生的生物膜的抑菌作用。方法将滴有不同浓度的大黄素药液的滤纸片放在长有白假丝酵母菌的固体培养基上,观察抑菌效果的区别。在体外建立白假丝酵母菌生物膜模型,用浓度为2.500、1.250、0.625、0.312和0.165mg/mL的大黄素溶液分别作用于已建立好的白假丝酵母菌生物膜模型,观察5个浓度的大黄素对白假丝酵母菌的抑菌效果。利用激光共聚焦显微镜(CLSM)观察各个浓度下的生物膜。结果随着大黄素浓度的增加,抑菌环直径增大,抑菌率升高。CLSM观察:大黄素作用于白假丝酵母菌生物膜,使生物膜活性降低。结论大黄素对白假丝酵母菌及其生物膜有抑制作用。     

伊犁黑蜂蜂胶对口腔主要致龋细菌及生物膜生长的实验研究

目的研究伊犁黑蜂蜂胶对口腔常见致龋细菌及其生物膜生长的影响,观察其防龋效果。方法 (1)通过液体稀释法测定伊犁黑蜂蜂胶对口腔常见致龋菌的最小抑菌浓度(MIC)及最小杀菌浓度(MBC);(2)生物膜形成抑制试验测定伊犁黑蜂蜂胶对口腔常见致龋菌的最小生物膜形成抑制浓度(MBIC50);(3)通过结晶紫染色法测定伊犁黑蜂蜂胶对口腔常见致龋菌的最小生物膜清除浓度(MBEC);结果(1)伊犁黑蜂蜂胶对变形链球菌、远缘链球菌、嗜酸乳杆菌、血链球菌、粘性放线菌和内氏放线菌的最低抑菌浓度分别是0.78、0.39、1.56、0.39、0.20和0.20 mg/mL,最低杀菌浓度分别为1.56、0.78、3.125、0.78、0.39和0.39mg/mL;(2)伊犁黑蜂蜂胶对变形链球菌、远缘链球菌、血链球菌、粘性放线菌和内氏放线菌的MBIC50分别是0.39、0.39、0.39、0.05和0.10mg/mL;(3)伊犁黑蜂蜂胶对变形链球菌、远缘链球菌、血链球菌、粘性放线菌和内氏放线菌的MBEC分别是6.25、1.56、3.1256、0.78和0.78mg/mL。结论伊犁黑蜂蜂胶对口腔主要致龋细菌的生长具有抑制作用,能够抑制主要致龋细菌单菌生物膜的形成,并具有一定清除作用,是具有一定防龋效果的天然药物。     

薰衣草精油对白念珠菌体外抗菌活性及生物膜的影响

目的研究薰衣草精油对白念珠菌的体外抗菌活性和生物膜的影响,为临床真菌感染的治疗提供实验依据。方法采用纸片扩散法、连续稀释法测定薰衣草精油对白念珠菌的敏感性、最低抑菌浓度(MIC)和最低杀菌浓度(MBC);采用XTT法检测其抑制白念珠菌生物膜50%细胞活性的浓度(SMIC50),镜下观察对生物膜形态的影响。结果白念珠菌对薰衣草精油高度敏感,薰衣草精油对白念珠菌的MIC为12.5μL/mL,MBC为25μL/mL;对生物膜的SMIC50是100μL/mL,对白念珠菌的菌丝生长有抑制作用。结论薰衣草精油在体外对白念珠菌有抑菌作用。     

溶菌酶对微小小单胞菌生物膜的影响

目的测定溶菌酶对微小小单胞菌及其生物膜的抑菌作用,并测出最小抑菌浓度(MIC)、最小杀菌浓度(MBC)和抑菌率。方法采用对倍稀释的方法,测定溶菌酶对微小小单胞菌的MIC、MBC;在96孔板中体外建立微小小单胞菌生物膜模型,采用MTT法检测溶菌酶对微小小单胞菌生物膜的影响;在六孔板中建立生物膜模型,使用激光共聚焦显微镜(CLSM)观察不同浓度溶菌酶对微小小单胞菌生物膜作用后的变化。结果溶菌酶对微小小单胞菌的MIC为0.0195 mg/mL,MBC为0.3125 mg/mL;CLSM观察结果显示,溶菌酶对微小小单胞菌生物膜的抑制作用随着浓度的增加而增强。结论溶菌酶对微小小单胞菌及其生物膜的生长和活性均具有抑制作用。     

伊犁黑蜂蜂胶对不同状态下变形链球菌乳酸脱氢酶及其相关基因影响的实验研究

目的目的通过新疆伊犁黑蜂蜂胶乙醇提取物(Ethanol Extract of Propolis,EEP)对不同状态下变形链球菌乳酸脱氢酶活性及其相关基因表达影响的作用,研究伊犁黑蜂蜂胶抑制变形链球菌产酸的原因并探讨其可能的防龋机制。方法 (1)分别培养浮游状态与生物膜状态下生长的变形链球菌,根据实验分组用含梯度浓度EEP的BHI培养基、50 mg/L氟化钠的BHI培养基作用18 h,通过还原性辅酶I氧化法测定乳酸脱氢酶活性。(2)分别培养浮游状态与生物膜状态下生长变形链球菌,根据实验分组用含梯度浓度EEP的BHI培养基、含50 mg/L氟化钠的BHI培养基作用18 h,反转录-实时荧光定量PCR(RTq PCR)法测定各组乳酸脱氢酶编码基因ldh表达情况。结果 (1)在浮游状态与生物膜状态下,EEP组和Na F组乳酸脱氢酶活性均有降低,差异具有统计学意义(P0.05)。(2)浮游状态时,实验组组和阳性对照组ldh表达明显受到抑制(P0.05);生物膜状态下,实验组在1 MBEC、1/2 MBEC、1/4 MBEC浓度时ldh表达受到抑制(P0.05),Na F组ldh表达差异没有统计学意义(P0.05)。结论伊犁黑蜂蜂胶能够抑制浮游状态与生物膜状态下变形链球菌乳酸脱氢酶活性及其编码基因ldh表达,来抑制细菌产酸,伊犁黑蜂蜂胶可能是通过此途径抑制变形链球菌产酸,从而达到防龋的效果。     

土槿乙酸对白假丝酵母菌生物膜的抑制作用

目的探讨土槿乙酸(pseudolaric acid B,PAB)对体外白假丝酵母菌生物膜的影响。方法甲基四氮盐(XTT)法检测不同浓度PAB和AMB(两性霉素B)对白假丝酵母菌生物膜的抑制作用。血清芽管试验检测不同浓度PAB对芽管生成的影响。结果 PAB对白假丝酵母菌生物膜的SMIC50(抑制生物膜50%的药物浓度)为256~512μg/m L;1024和512μg/m L PAB对早期2 h生物膜的抑制率分别为(99.5±0.28)%和(97.1±0.38)%;512μg/m L PAB对早期(2 h)、中期(8 h)及成熟期(24 h)生物膜的抑制率分别为(97.1±0.38)%、(90.4±0.32)%和(80.1±0.67)%;不同浓度PAB的血清芽管试验显示,64μg/m L PAB可以完全抑制白假丝酵母菌的出芽生长,16μg/m L PAB可以抑制83.5%的白假丝酵母菌出芽生长。结论 PAB对体外白假丝酵母菌生物膜有抑制作用,对白假丝酵母菌的出芽生长过程抑制作用显著。     

胶体纳米银对白假丝酵母菌的抑制作用

目的探讨胶体纳米银对白假丝酵母菌的抑制作用。方法以白假丝酵母菌为研究对象,采用倍比稀释及涂布法,检测胶体纳米银对白假丝酵母菌的抑制作用及影响因素。结果化学纳米银及物理纳米银对白假丝酵母菌的最小抑菌浓度及最小杀菌浓度相同,分别为3.13μg/mL和6.25μg/mL。12.50μg/mL的化学纳米银及物理纳米银可在5 min内完全杀死白假丝酵母菌。胶体纳米银的抑菌作用受到杀菌环境的影响,特别氯离子对胶体纳米银的杀菌作用有明显的影响。结论胶体纳米银对白假丝酵母菌具有明显的抑菌作用,其抑制作用易受到氯离子及蛋白质的的影响。     

没食子鞣质对变形链球菌生长抑制的实验研究

目的研究没食子鞣质对浮游状态和生物膜状态下变形链球菌生长抑制的作用,探讨药物的防龋功效。方法采用纸片扩散法测定不同浓度没食子鞣质溶液对变形链球菌的抑菌圈大小,采用试管稀释法测定没食子鞣质对变形链球菌生长的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)值大小,用半定量可见光测定A值的方法测定生物膜最低抑菌浓度(MBEC)值。结果没食子鞣质对变形链球菌的生长具有明显的抑制作用,经液体稀释法实验测定,没食子对变形链球菌的MIC为4 mg/m L,MBC为16 mg/m L,经半定量可见光测定A值的方法测定没食子鞣质对变形链球菌单菌生物膜生长的MBEC值为8 mg/m L。结论没食子鞣质对变形链球菌有抑制作用。     

三颗针对白假丝酵母的抑制作用

目的研究三颗针对白假丝酵母的抑制作用。方法采用溴化噻唑蓝四唑法(MTT)和琼脂平板法测定三颗针对白假丝酵母的最低抑菌浓度(MIC)和最低杀菌浓度(MFC);利用倒置荧光显微镜观察三颗针对白假丝酵母菌丝形成的影响;采用MTT法测定三颗针对白假丝酵母菌丝萌发不同时期的抑制率。结果三颗针对白假丝酵母具有较强的抑制作用。其中,采用琼脂平板法测得三颗针抑制该菌的MIC为2mg/mL,MFC为4mg/mL。三颗针能减缓该菌的生长速度或使其停止生长,且浓度越高作用效果越明显。其中4mg/mL的三颗针作用白假丝酵母6h后,能够完全抑制菌丝形成。对于24h后已经萌发为菌丝态的白假丝酵母,三颗针可抑制菌丝的继续生长,与对照组相比,4mg/mL的三颗针对于萌发2h后的白假丝酵母的菌丝抑制率为76.7%(P0.01)。结论三颗针能抑制白假丝酵母菌丝的生长和萌发。     

牡丹皮水煎剂对体外白念珠菌生物膜的抑制作用

研究牡丹皮水煎剂对体外白念珠菌生物膜的影响。体外构建白念珠菌生物膜并采用XTT减低法评价牡丹皮水煎剂对白念珠菌成熟生物膜影响以及包被生物材料对白念珠菌生物膜形成的影响。结果显示,牡丹皮水煎荆对白念珠菌悬浮菌MIC为1．56mg／mL;对白念珠菌生物膜SMIC50与SMIC50分别是3．12和6．25mg／mL;药物包被96孔板对白念珠菌生物膜形成有一定的抑制效应。实验表明牡丹皮水煎剂对体外白念珠菌生物膜有较强抑制作用。     

The assessment of the antibacterial and antifungal activities of aspirin, EDTA and aspirin–EDTA combination and their effectiveness as antibiofilm agents

Aims:  To evaluate the antimicrobial activities of aspirin, EDTA and an aspirin-EDTA (A-EDTA) combination against Pseudomonas aeruginosa , Escherichia coli and Candida albicans in planktonic and biofilm cultures.
Methods and Results:  Minimal inhibitory concentrations (MIC) and minimal biocidal concentrations (MBC) were determined using twofold broth microdilution and viable counting methods, respectively. Aspirin's recorded MIC values ranged from 1·2 to 2·7 mg ml⁻¹. Checkerboard assay demonstrated a synergism in antimicrobial activity upon combination. Aspirin's minimal biofilm eradication concentration values (MBEC) against the established biofilms ranged between 1·35 and 3·83 mg ml⁻¹. A complete eradication of bacterial biofilms was achieved after a 4-h treatment with the A-EDTA combination.
Conclusion:  Both aspirin and EDTA possess broad-spectrum antimicrobial activity for both planktonic and biofilm cultures. Aspirin used at the MBEC for 24 h was successful in eradicating P. aeruginosa , E. coli and C. albicans biofilms established on abiotic surfaces. Moreover, the exposure to the A-EDTA combination (4 h) effected complete bacterial biofilm eradication.
Significance and Impact of the Study:  There is a continuous need for the discovery of new antimicrobial agents. Aspirin and EDTA are 'nonantibiotic drugs', the combination of which can be used successfully to treat and eradicate biofilms established on abiotic surfaces.     

Biofilm susceptibility to metal toxicity

This study compared bacterial biofilm and planktonic cell susceptibility to metal toxicity by evaluating the minimum inhibitory concentration (MIC), the planktonic minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) using the MBEC device. In total, 17 metal cations and oxyanions, chosen to represent groups VIB to VIA of the periodic table, were each tested on biofilm and planktonic cultures of Escherichia coli JM109, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853. In contrast to control antibiotic assays, where biofilm cultures were 2 to 64 times less susceptible to killing than logarithmically growing planktonic bacteria, metal compounds killed planktonic and biofilm cultures at the same concentration in the vast majority of combinations. Our data indicate that, under the conditions reported, growth in a biofilm does not provide resistance to bacteria against killing by metal cations or oxyanions.     

壳聚糖-纳米羟基磷灰石复合制剂对粪肠球菌抗菌作用的体外研究

目的观察壳聚糖一纳米羟基磷灰石（CS／nHA）复合制剂对粪肠球菌生物膜的体外抗菌作用,为研究该药物对根管内粪肠球菌生物膜抗菌的作用打下基础,探索激光共聚焦显微镜（CLSM）技术在研究生物膜领域中的优势。方法采用液体二倍稀释法,测定壳聚糖／纳米羟基磷灰石复合制剂对游离状态下粪肠球菌的最小抑菌浓度和最小杀菌浓度;将粪肠球菌在体外培养成生物膜后,利用CLSM观察研究该生物膜中细菌活性。结果CS／nHA制剂在0．467mg／mL时对游离状态下的粪肠球菌具有抑菌和杀菌作用,与对照组相比差异有统计学意义。使用CS／nHA制剂前后粪肠球菌生物膜活菌百分比显著下降,与对照组相比差异有统计学意义（P〈0．05）。结论CS／nHA制剂对粪肠球菌有较强的抗菌作用,将其作为根管封闭剂中的抗菌成分具有一定的应用前景。     

丹皮酚对变形链球菌生物膜影响的体外研究

目的测定丹皮酚对变形链球菌的抑菌作用;共聚焦显微镜观察丹皮酚对变形链球菌生物膜结构和活性的影响。方法梯度法测定丹皮酚对变形链球菌的MIC(最小抑菌浓度)、MBC(最小杀菌浓度);体外构建变形链球菌生物膜模型,共聚焦显微镜观察不同浓度丹皮酚对变形链球菌生物膜作用后形态结构的影响并进行红绿荧光定量分析其活性变化。结果丹皮酚对变形链球菌的MIC为6.25mg/mL,MBC为25mg/mL;激光共聚焦显微镜观察丹皮酚对变形链球菌生物膜作用后其生物膜结构变稀疏,细菌链变短,生物膜活性也随丹皮酚浓度的提高而逐渐降低。结论丹皮酚对变形链球菌和变形链球菌生物膜结构及其活性均具抑制作用。     

Persister cells, the biofilm matrix and tolerance to metal cations in biofilm and planktonic Pseudomonas aeruginosa

In this study, we examined Pseudomonas aeruginosa ATCC 27853 biofilm and planktonic cell susceptibility to metal cations. The minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) required to eradicate 100% of the planktonic population (MBC 100), and the minimum biofilm eradication concentration (MBEC) were determined using the MBEC trade mark-high throughput assay. Six metals - Co(2+), Ni(2+), Cu(2+), Zn(2+), Al(3+) and Pb(2+)- were each tested at 2, 4, 6, 8, 10 and 27 h of exposure to biofilm and planktonic cultures grown in rich or minimal media. With 2 or 4 h of exposure, biofilms were approximately 2-25 times more tolerant to killing by metal cations than the corresponding planktonic cultures. However, by 27 h of exposure, biofilm and planktonic bacteria were eradicated at approximately the same concentration in every instance. Viable cell counts evaluated at 2 and 27 h of exposure revealed that at high concentrations, most of the metals assayed had killed greater than 99.9% of biofilm and planktonic cell populations. The surviving cells were propogated in vitro and gave rise to biofilm and planktonic cultures with normal sensitivity to metals. Further, retention of copper by the biofilm matrix was investigated using the chelator sodium diethlydithiocarbamate. Formation of visible brown metal-chelates in biofilms treated with Cu(2+) suggests that the biofilm matrix may coordinate and sequester metal cations from the aqueous surroundings. Overall, our data suggest that both metal sequestration in the biofilm matrix and the presence of a small population of 'persister' cells may be contributing factors in the time-dependent tolerance of both planktonic cells and biofilms to high concentrations of metal cations.     

The specific effect of gallic acid on Escherichia coli biofilm formation by regulating pgaABCD genes expression

Escherichia coli (E. coli) is associated with an array of health-threatening contaminations, some of which are related to biofilm states. The pgaABCD-encoded poly-beta-1,6-N-acetyl-D-glucosamine (PGA) polymer plays an important role in biofilm formation. This study was conducted to determine the inhibitory effect of gallic acid (GA) against E. coli biofilm formation. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of GA against planktonic E. coli were 0.5 and 4 mg/mL, and minimal biofilm inhibitory concentration and minimal biofilm eradication concentration values of GA against E. coli in biofilms were 2 and 8 mg/mL, respectively. Quantitative crystal violet staining of biofilms and ESEM images clearly indicate that GA effectively, dose-dependently inhibited biofilm formation. CFU counting and confocal laser scanning microscopy measurements showed that GA significantly reduced viable bacteria in the biofilm. The contents of polysaccharide slime, protein, and DNA in the E. coli biofilm also decreased. qRT-PCR data showed that at the sub-MIC level of GA (0.25 mg/mL) and expression of pgaABC genes was downregulated, while pgaD gene expression was upregulated. The sub-MBC level of GA (2 mg/mL) significantly suppressed the pgaABCD genes. Our results altogether demonstrate that GA inhibited viable bacteria and E. coli biofilm formation, marking a novel approach to the prevention and treatment of biofilm-related infections in the food industry.