构树叶提取物对金黄色葡萄球菌的抑制作用及机理研究

为了研究构树叶提取物的抑菌作用及其机理,以金黄色葡萄球菌为供试菌,采用牛津杯法测定构树叶提取物的抑菌效果,通过测定供试菌生长曲线、细胞膜渗透性和蛋白质合成来研究构树叶提取物的抑菌机理。结果表明,构树叶水提取物和75%乙醇提取物抑菌圈直径分别为17.5和18.0 mm,最小抑菌浓度均为6.8 mg/mL,最小杀菌浓度均为12.50 mg/mL。构树提取物能显著抑制金黄色葡萄球菌蛋白质合成,从而抑制其生长,但对其细胞膜的通透性影响不大。因此,构树叶提取物通过抑制金黄色葡萄球菌蛋白质合成发挥其抑菌效果。     

四种兽用抗生素的体外抑菌效果评价

本实验用试管药物稀释了四种兽用抗生素对猪常见病原菌和正常菌的最小抑菌浓度 （minimal inhibitory concentration, MIC） 和最小杀菌浓度 （minimal bactericidal concentration, MBC）。通过实验发现：各种菌对四种药物的敏感性存在很大的差异,大多数细菌对药物C和D较敏感而对药物A和B存在较严重的耐药。     

四黄止痢复方醇提物(CSME)抗猪链球菌机制研究北大核心

为了研究四黄止痢复方醇提物(CSME)对猪链球菌的抑菌机制,采用微孔-平板法测定其对猪链球菌的最小抑菌浓度(MIC);用吸光光度法检测其对猪链球菌生长曲线、细胞膜和细胞壁通透性、能量代谢活力的影响。结果表明,CSME对猪链球菌的MIC为75 g/L,能显著抑制猪链球菌生长,破坏其细胞膜、细胞壁结构,使内容物外泄,并抑制了琥珀酸脱氢酶(SDH)和苹果酸脱氢酶(MDH)的活性。CSME对猪链球菌有抑制作用,其抑菌作用是通过破坏细胞膜和细胞壁、导致细菌新陈代谢紊乱的机制来实现的。     

地榆提取物对多重耐药大肠埃希氏菌的抑菌活性及机制研究

为探讨地榆不同部位提取物对多重耐药大肠埃希氏菌(MDR E.coli)体外抑菌效果及抑菌机制,通过系统溶剂提取法萃取制备地榆石油醚、乙酸乙酯、正丁醇和水部位提取物,用微量肉汤稀释法检测各部位提取物对MDR E.coli的抑菌活性,筛选最佳抑菌活性部位提取物,并通过对MDR E.coli的细胞壁、细胞膜、菌体总蛋白合成的影响探讨抑菌机制。结果表明,地榆正丁醇部位提取物对MDR E.coli的抑制作用最强,其最小抑菌浓度(MIC)为3.125 mg/mL,在浓度为MIC和0.5×MIC时作用菌体12 h, MDR E.coli菌量分别为(2.37±0.59)×10~(6 )CFU/mL和(2.05±0.57)×10~8 CFU/mL;培养基中碱性磷酸酶(AKP)活性分别为(1.498±0.023)U/L和(0.768±0.088)U/L;在260 nm处培养基上清液OD值分别为0.363±0.003和0.168±0.009,均极显著高于对照组(P0.001);菌体总蛋白条带出现明显缺失。地榆正丁醇部位提取物对MDR E.coli的抑菌作用最强,其作用机制是通过破坏菌体细胞壁、增大菌体细胞膜通透性,干扰细菌总蛋白合成抑制细菌的生长。     

救必应中药血清与抗菌药联合对产ESBLs细菌抑菌效果的研究

本试验主要探讨救必应中药血清与抗菌药联合诱导细菌传代的体外抑菌活性。制备救必应中药血清,采用二倍微量稀释法体外检测抗菌药的最小抑菌浓度(MIC),然后救必应中药血清与抗菌药联合诱导产超广谱β-内酰胺酶(extended spectrumβ-lactamases,ESBLs)细菌传代。救必应中药血清与抗菌药联合诱导细菌传代对耐药大肠杆菌有不同程度的抑菌活性。救必应中药血清可明显地增强β-内酰胺类抗菌药、氨基糖苷类抗菌药、喹诺酮类抗菌药和卡巴氧类抗菌药对耐药菌的抑制作用。     

正己酸对大肠杆菌与金黄色葡萄球菌的抑制效果研究

对大肠杆菌和金黄色葡萄球菌进行正己酸的抗性试验,以研究正己酸对病原菌的抑制作用。试验采用等浓度梯度稀释、光密度值测定、平板涂布方法、牛津杯法,分别测定大肠杆菌及金黄色葡萄球菌的的最小抑菌浓度(minimum inhibitory concentration,MIC)以及最小杀菌浓度(minimum bactericidal concentration,MBC),绘制1/2MIC以及MIC处理过的大肠杆菌以及金黄色葡萄球菌的生长曲线,抑菌圈直径,结合扫描电镜图片分析最小杀菌浓度处理后的细胞形态结构的变化。结果表明:正己酸对大肠杆菌的最小抑菌浓度为700μg/mL、对金黄色葡萄球菌的最小抑菌浓度为1 000μg/mL;对大肠杆菌的最小杀菌浓度为1 000μg/mL、对金黄色葡萄球菌的最小杀菌浓度为1 300μg/mL;当正己酸浓度为1 600μg/mL时,电镜下,大肠杆菌数量较少,金黄色葡萄球菌数量少且呈现出细胞破裂萎缩现象。综上,正己酸对大肠杆菌和金黄葡萄球菌有抑制作用,破坏了金黄葡萄球菌的细胞膜结构。     

杆菌肽锌在畜禽养殖中的应用

肝菌肽锌 (ＺｉｎｃＢａｃｉｔｒａｃｉｎ)对畜禽能够起到显著的抑菌促生长作用 ,主要源于其独特的三重抑菌机理 :①杆菌肽锌是类脂质焦磷酸的特异性抑制剂 ,能抑制细菌细胞壁合成的脱磷酸化过程 ,影响磷脂载体的运转及向细胞壁支架输送粘肽 ,从而抑制细胞壁的合成 ;②与敏感菌的细胞膜相结合 ,损伤细胞膜 ,使细胞膜通透性增加 ,导致膜内物质外流 ;③干扰敏感菌细胞内原浆蛋白的合成。因此 ,杆菌肽锌是一种强杀菌剂 ,能强烈抑制革兰氏阳性菌 ,特别是梭状芽孢杆菌、葡萄球菌、链球菌、棒状杆菌等病原菌对之极为敏感 ,对耐青霉素的金黄色葡萄…     

白术、酸枣仁提取物对金黄色葡萄球菌的抑菌效果研究

试验旨在研究菊科植物白术干燥根茎切片提取物(白术提取物)、鼠李科植物酸枣的种子提取物(酸枣仁提取物)单独和联合作用对金黄色葡萄球菌的抑菌效果。通过微量肉汤法测定抑菌浓度和抑制率;用酶标仪测定相应OD值以检测核酸和蛋白质含量、乳酸脱氢酶(LDH)活力及碱性磷酸酶(AKP)活力,以确定对细菌细胞膜和细胞壁的影响,用十二烷基硫酸钠-聚丙烯酰胺(SDSPAGE)凝胶电泳检测金黄色葡萄球菌菌体总蛋白含量变化;用结晶紫染色法检测对细菌生物膜的抑制作用。结果表明：0.4 mg/mL白术、酸枣仁提取物单独及0.1 mg/mL二者联合作用对金黄色葡萄球菌均具有显著抑菌效果;与对照组相比,白术、酸枣仁提取物单独和联合作用均使菌液上清液OD₂₆₀、OD₂₈₀数值和AKP活力显著增加,菌体LDH活力和总蛋白含量降低,抑制了生物膜的形成。说明白术、酸枣仁提取物单独和联合作用均可显著抑制金黄色葡萄球菌,且联合作用抑菌效果显著强于各药物单独作用。     

安吉白茶对柱状黄杆菌抑菌机理的研究

为探讨安吉白茶对柱状黄杆菌的抑菌机理,本试验通过测定安吉白茶提取液与细菌作用前后培养液电导率和紫外吸收物的变化,以及菌体磷代谢和可溶糖的变化,初步阐明了安吉白茶对柱状黄杆菌的抑菌机理。研究结果显示,经安吉白茶提取液处理后,细菌培养液的电导率和可溶糖浓度均增大,菌悬液中的紫外吸收物也随作用时间的延长而增加,表明安吉白茶提取液可破坏细胞膜的结构、导致细胞通透性增加,进而使细胞内容物外泄。此外,经安吉白茶处理后的柱状黄杆菌对磷的消耗量降低,以致严重影响了核酸、磷脂等细胞重要成分的合成及能量代谢,导致细菌正常生理功能的丧失。结果表明,安吉白茶可通过破坏菌体细胞膜及干扰磷代谢等途径抑制柱状黄杆菌的生长。     

石榴皮水提物对猪源多重耐药大肠杆菌的抑菌作用研究

【目的】筛选腹泻仔猪中携毒力基因且多重耐药的大肠杆菌菌株,评估中药水提物对该菌株的抑菌活性,并探索中药抑菌机制。【方法】通过PCR方法与Kirby-Bauer(K-B)药敏纸片法评估临床大肠杆菌菌株的肠毒素基因携带情况和耐药性;通过微量肉汤稀释法评估中药水提物对多重耐药大肠杆菌菌株的抑菌活性,确定最小抑菌浓度(minimal inhibitory concentration, MIC)和最小杀菌浓度(minimal bactericidal concentration, MBC);通过电导率仪以及相关试剂盒检测评估石榴皮水提物对多重耐药菌株作用不同时间后菌液电导率、碱性磷酸酶(alkaline phosphatase, AKP)含量及菌体内ATP含量的变化;通过SDS-PAGE和蛋白含量检测评估菌体内可溶性蛋白的含量变化;通过扫描电镜观察大肠杆菌形态变化。【结果】PCR检测14株临床大肠杆菌中肠毒素基因携带率达到78.57%,抗生素药敏试验结果显示,所有菌株至少对2种抗生素耐药,均属于多重耐药菌株。中药药敏试验结果显示,黄连、黄芩、石榴皮水提物对多重耐药菌株抑菌活性较好,其中石榴皮水提...     

Study on the Bacteriostatic Mechanism of the Water Extracts of Holly Bark on ESBLs-producing Bacterial

The study was undertaken to study the inhibitory effect and mechanism of the water extracts of Holly Bark.Minimal inhibitory concentration (MIC) was tested through twice micro-dilution method and minimal bactericidal concentration (MBC) was tested by agar plate dilution counting method (SDA)of the water extracts of Holly Bark on producing extended spectrum β-lactamases (ESBLs) bacteria,then the bacteriostatic mechanism of the water extracts of Holly Bark through bacteria curve was tested,the cell ultrastrueture was observed,permeabilty changes of cell wall and membrane,nucleic acid synthesis inhibitory experiment were researched.The results showed that the water extracts of Holly Bark could affect bacteria curve compared with the blank control,destroy the cells seriously,permeability of both cell membrane and wall increased,extravasation of cytoplasm appeared,the content of AKP and soluble protein in the bacterial liquid increased,E.coli DNA fluorescence intensity weakened obviously.Research showed that the bacteriostatic mechanism of the water extracts of Holly Bark was based on its roles about changes on the cell wall and membrance permeability,inhibitting nucleic synthesis of bacterial.     

大黄酸对伪中间葡萄球菌的抗菌活性及作用机制

伪中间葡萄球菌（Staphylococcus pseudintermedius）是犬脓皮病主要致病菌,具有多重耐药性,严重威胁犬及人类健康。本研究从脓皮病患犬中分离20株伪中间葡萄球菌,并揭示大黄酸对伪中间葡萄球菌的抗菌活性及抗菌机理。通过测定最小抑菌浓度、最低杀菌浓度、抑菌曲线和黏附抑制试验分析了大黄酸对伪中间葡萄球菌的抑菌活性;检测大黄酸对伪中间葡萄球菌细胞膜完整性、通透性,细胞活性氧（ROS）水平等,结合电镜观察细菌细胞形态和超微结构,从而探究大黄酸的抑菌机理。结果表明,大黄酸对伪中间葡萄球菌的最小抑菌浓度为12.5 μg·mL^-1,亚抑菌浓度能够显著抑制伪中间葡萄球菌生长和黏附;经大黄酸处理后,伪中间葡萄球菌细胞膜通透性改变,胞外β-半乳糖苷酶活性增加（P<0.05）;ROS测定结果显示,大黄酸处理后细菌细胞ROS水平增高3.9倍（1×MIC）和6.4倍（2×MIC）;大黄酸处理后,电镜观察伪中间葡萄球菌表面存在大量分泌物、皱缩,胞壁破裂、电子密度降低、内容物泄漏等。综上,大黄酸可通过改变细菌细胞膜通透性、破坏膜完整性,诱导细菌细胞产生大量ROS等作用机制达到抗菌目的。     

板蓝根微粉水提物抗大肠杆菌活性及其机制的探究

为了探究板蓝根微粉水提物的抗菌活性及其抗菌机制，试验通过扫描电镜、透射电镜检测板蓝根微粉水提物对大肠杆菌形态和结构影响；酶标仪测定板蓝根微粉对大肠杆菌电导率、胞内物质总漏出率影响；测定大肠杆菌培养液中碱性磷酸酶含量以及大肠杆菌菌体内、外蛋白质含量；通过DAPI染色DNA、RNA，检测板蓝根微粉水提物对大肠杆菌核酸的影响；检测板蓝根微粉水提物对大肠杆菌菌体内、外谷丙转氨酶（ALT）、谷草转氨酶（AST）、丙酮酸以及三磷酸腺苷（ATP）等菌体内代谢的影响。结果显示，板蓝根微粉水提物作用大肠杆菌10 h后，扫描电镜检测可见菌体出现溢缩，菌体长度明显变短，断裂形成许多残体，有的中间凹陷，发生变形；透射电镜下可观察大肠杆菌的胞壁界限模糊不清，壁膜呈现锯齿状，弯弯曲曲，变形，有的菌体破碎。总漏出率、电导率以及碱性磷酸酶含量测定结果显示，板蓝根微粉水提物各组D_{600 nm}均高于空白对照组，且呈剂量依赖性。菌体外蛋白质含量从8 h开始与空白对照组差异极显著（P<0.01）；菌体内蛋白质含量从4 h开始与空白对照组差异极显著（P<0.01）。DNA含量在12 h前与空白对照组无显著差异（P>0.05），从16 h开始与空白对照组差异极显著（P<0.01）；RNA含量在8 h开始降低，在12 h时与空白对照组差异显著（P<0.05），从16 h开始差异极显著（P<0.01）。ALT和AST浓度测定无显著性差异（P>0.05）。培养液和菌体内的丙酮酸含量均高于空白对照组，且从4 h开始与空白对照组差异极显著（P<0.01）。培养液中的ATP含量与空白对照组差异极显著（P<0.01）；菌体内ATP含量从4 h开始与空白对照组差异极显著（P<0.01）。综上，板蓝根微粉水提物可以通过破坏细胞壁、细胞膜的完整性，抑制细菌遗传物质合成和代谢，影响丙酮酸和ATP含量从而实现抗大肠杆菌作用。     

Study on the Effect of the Rosemarinic Acid Combined with Antibacterial Agents on the Bacterial with Gene fosA3

This research was aimed to investigate the effect of rosemarinic acid combined with antibacterial agents against bacterial carrying gene fosA3 in vitro.Resistance gene types were determined by identification of bacteria isolated in clinics.Antibacterial activity and fungicidal activity of rosemarinic acid were tested by oxford cup method and spread-plate method.The minimal inhibiton concertration (MIC) of rosemarinic acid and antibacterial agents were tested through twice micro-dilution method,fractional inhibitory concentration index (FICI) of rosemarinic acid combined with antibacterial agents were determined by microdilution checkerboard techniques.Escheriohia coli carrying multidrug resistance gene fosA3 was isolated.The MIC of rosemarinic acid was 640 μg/mL,when application of rosemarinic acid combined with ciprofloxacin,levofloxacin,coly-mycin,gatifloxacin,amikacin,ceftazidime,ceftiofur sodium,FICI≤0.5,showed an additive effect;With ceftriaxone sodium,norfloxacin,mequindox,0.5fosA3 Escheriohia coli.Antibacterial activities of antibacterial agents against the fosfomycin resistance gene fosA3 Escherichia coli were enhanced significantly by rosemarinic acid.     

迷迭香酸与抗菌药联合对含fosA3基因细菌抑菌效果的研究

试验旨在探讨迷迭香酸与抗菌药联合体外抗含fosA3耐药基因大肠杆菌的效果。本试验对临床分离的细菌进行鉴定,确定细菌所含耐药基因种类,用牛津杯法测定迷迭香酸的抑菌活性,平板涂布法测定迷迭香酸的杀菌活性,采用96孔反应板二倍微量稀释法分别检测迷迭香酸与抗菌药的最小抑菌浓度(MIC),再用微量棋盘稀释法测定迷迭香酸与抗菌药联合作用后的分级抑菌浓度指数(FICI)。结果显示,分离到含fosA3耐药基因大肠杆菌;迷迭香酸的MIC为640 μg/mL,迷迭香酸与环丙沙星、左旋氧氟沙星、黏杆菌素、加替沙星、阿米卡星、头孢他啶、头孢噻呋钠联合应用后其FICI≤0.5,有协同作用;与头孢曲松钠、诺氟沙星、痢菌净联合应用后0.5fosA3基因大肠杆菌有一定的抑菌活性和杀菌活性,其与部分抗菌药联合应用后,抗菌药体外抗菌活性显著增强。     

Effect of Baicalin on Cell Permeability of Escherichia coli

The objective of this study was to explore the antibacterial mechanism of baicalin on Escherichia coli. Based on the determination of baicalin on Escherichia coli minimum inhibitory concentration (MIC) and growth curve, the influence of baicalin on Escherichia coli cells permeability was researched by measuring the conductivity and alkaline phosphatase (AKP) content of bacterial solution. The experimental results showed that baicalin had significantly inhibitory effect on Escherichia coli, and its MIC was 6.25 mg/mL.Baicalin could significantly increase the conductivity and AKP content of Escherichia coli bacteria solution.The results suggested that baicalin on antibacterial activity was mainly due to the changes of the cell membrane permeability and the damage of the cell wall.     

黄芩苷对大肠杆菌细胞通透性的影响

本试验旨在探索黄芩苷对大肠杆菌的抑菌机制。在测定黄芩苷对大肠杆菌的最小抑菌浓度和生长曲线的基础上,通过测定菌液电导率和碱性磷酸酶含量,研究黄芩苷对大肠杆菌细胞通透性的影响。试验结果表明,黄芩苷对大肠杆菌具有明显的抑菌效果,其最小抑菌浓度为6.25 mg/mL;黄芩苷可引起大肠杆菌菌液的电导率和碱性磷酸酶含量明显升高。证实了黄芩苷通过增加大肠杆菌细胞膜与细胞壁通透性,从而发挥抑菌作用。     

Study on the Anti-Escherichia coli Activity and Mechanism of Water Extract of Radix isatidis Powder

In order to explore the antibacterial activity and mechanism of the Radix isatidis powder water extract,the effects of the Radix isatidis powder water extract on the morphology and structure of Escherichia coli (E.coli) were tested by scanning electron microscopy and transmission electron microscopy.The effects of the Radix isatidis powder water extract on the conductivity and total leakage rate of E.coli and the content of alkaline phosphatase in the culture medium of the content of protein,DNA and RNA were stained by DAPI to detect the effect of the Radix isatidis powder water extract on the nucleic acid of E.coli,and the effect of the Radix isatidis powder water extract on the metabolism of E.coli in vivo,in vitro,ALT,AST,pyruvic acid and ATP.The results showed that after the Radix isatidis powder water extract acted on E.coli for 10 h,it was observed by SEM that the bacteria appeared to overflow and shrink,the length of the bacteria became shorter obviously,many residues were formed due to breakage,some of them were sunken in the middle and deformed.Under TEM,it was observed that the boundary of the cell wall of E.coli was unclear,the wall membrane was zigzag,deformed and some of the bacteria were broken.The protein content outside the cell was significantly different from that in the blank control group from 8 h (P<0.01), and that in the cell from 4 h (P<0.01). DNA content had no significant difference with blank control group before 12 h (P>0.05), but had significant difference with blank control group from 16 h (P<0.01); RNA content began to decrease at 8 h, and was significantly different from blank control group at 12 h (P<0.05), and was extremely significant from 16 h (P<0.01). There was no significant difference between ALT and AST (P>0.05). The pyruvate content in culture medium and bacteria was higher than that in blank control group, and the difference was very significant from 4 h (P<0.01). The ATP content in the culture medium was significantly different from that in the blank control group (P<0.01), and the ATP content in the cell was significantly different from that in the blank control group from 4 h (P<0.01).In conclusion,the Radix isatidis powder water extract could inhibit the synthesis and metabolism of bacterial genetic material and the content of pyruvate and ATP by destroying the integrity of cell wall and cell membrane.     

新型黏虫抗菌肽AAP-1对大肠杆菌的抗菌作用

为评价一种新型黏虫抗菌肽（armyworm antimicrobial peptide 1，AAP-1）的抗菌活性，本试验首先采用固相化学合成法合成AAP-1，并通过高效液相色谱纯化和质谱检验，用抑菌圈法测定AAP-1对大肠杆菌的抗菌效果，倍比稀释法检测最小抑菌浓度（minimum inhibitory concentration，MIC），细菌平板计数法测定时间抗菌曲线；最后，通过超微量分光光度计评价AAP-1对大肠杆菌核酸泄露的影响，核酸凝胶电泳评价AAP-1对大肠杆菌胞内DNA的影响，透射电子显微镜观察评价AAP-1对大肠杆菌的整体作用效果。结果表明，合成的AAP-1纯度高于98%，分子质量为4 262.17 u；AAP-1对大肠杆菌具有良好的抗菌活性，MIC为7.8 μg/mL；对大肠杆菌的抗菌作用具有浓度依赖性，且在60 min内抗菌效果逐渐增强；AAP-1能够导致大肠杆菌核酸泄露，致使胞内DNA总量减少，且呈现浓度依赖性；AAP-1也会导致大肠杆菌细胞膜破坏、胞内电子密度明显降低、胞质溶解等现象。综上，本研究化学合成了一种新型抗菌肽AAP-1，且纯度高达98%，其对大肠杆菌具有良好的抗菌效果。本研究可为深入研究AAP-1对大肠杆菌的抗菌机制提供前期数据，可为APP-1的临床应用奠定理论基础。     

Antibacterial Effect of Novel Armyworm Antimicrobial Peptide (AAP-1) Against Escherichia coli

In order to evaluate the bactericidal activity of a novel armyworm antimicrobial peptide (AAP-1),the bactericidal effect of AAP-1 on Escherichia coli (E.coli) was investigated.Firstly,AAP-1 was synthesized by solid-phase chemically synthesis,and purified by high-performance liquid chromatography and tested by mass spectrometry.Then,the bactericidal effect of AAP-1 against E.coli was determined by the bacteriostatic circle method.The minimum inhibitory concentration (MIC) was detected by the double dilution method.The time kill curve was measured by bacterial plate counting method.Finally,the effect of AAP-1 on the nucleic acid leakage of E.coli was evaluation by ultra-micro spectrophotometer.The effect of AAP-1 on the intracellular DNA of E.coli was tested by nucleic acid gel electrophoresis.The overall effect of AAP-1 on E.coli was observed through transmission electron microscope.The results showed that the purity of the synthesized AAP-1 was more than 98%,and its molecular weight was 4 262.17 u.AAP-1 showed good antibacterial activity against E.coli,with a MIC of 7.8 μg/mL.AAP-1 had a concentration-dependent bactericidal effect on E.coli,and within 60 min the bactericidal effect gradually increased.AAP-1 could cause the nucleic acid leakage of E.coli,and resulted in a reduction in the total amount of intracellular DNA of E.coli with concentration-dependent.AAP-1 could also cause cell membrane destruction,significantly reduce intracellular electron density,resulted in cytoplasmic dissolution of E.coli,and so on.In summary,this study suggested that a novel antimicrobial peptide AAP-1 could be chemically synthesized with a purity of up to 98% and showed its good bactericidal effect on E.coli.This study could provide preliminary data for the in-depth study of the bactericidal mechanism of AAP-1 against E.coli and laid a theoretical foundation for the clinical application of AAP-1.