替米考星对胞内金黄色葡萄球菌的抗菌活性

为探究大环内酯类药物替米考星对胞内金黄色葡萄球菌的抗菌活性,本试验以金黄色葡萄球菌ATCC 29213为靶细菌,以小鼠单核巨噬细胞(RAW264.7)为载体细胞,构建金黄色葡萄球菌胞内感染体系,绘制胞内金黄色葡萄球菌的生长曲线,测定替米考星对胞内金黄色葡萄球菌的最低抑菌浓度、最低杀菌浓度、防突变浓度、耐药突变选择窗、抗菌后效应和杀菌曲线。结果显示,胞内金黄色葡萄球菌在0～4、4～18、18～24和24～48 h分别处于迟缓期、对数期、稳定期和衰亡期。最低抑菌浓度、最低杀菌浓度、防突变浓度和耐药突变选择窗分别为4、8、12.8和4～12.8μg/mL。抗菌后效应和杀菌曲线结果显示,替米考星对胞内金黄色葡萄球菌的杀菌特点表现出明显的时间依赖性。结果表明,替米考星对胞内金黄色葡萄球菌表现出较强的抗菌活性,可为治疗由胞内金黄色葡萄球菌引起的奶牛乳腺炎制定合理给药方案提供参考依据。     

纳米抗菌药物抗金黄色葡萄球菌感染研究进展

金黄色葡萄球菌Staphylococcus aureus耐药性问题日益严重,由其引起的奶牛乳腺炎的临床治疗面临着严峻的挑战。通过新型纳米载体系统所制备的纳米抗菌药物治疗金葡菌感染尤为重要。通过总结金葡菌感染机制、常规药物及纳米抗菌药物在治疗金葡菌感染中的应用,讨论并分析纳米抗菌药物在兽医临床上治疗金葡菌感染所面临的挑战,对纳米抗菌药物治疗金葡菌感染的应用前景进行展望。     

固体脂质纳米(SLNs)在抗寄生虫药物中应用的研究进展

基于抗寄生虫药物应用面临的问题以及在预防和治疗寄生虫病中的重要作用,固体脂质纳米(SLNs)因其独特的优势成为当前研究的热点。从SLNs可以提高抗寄生虫药物的口服吸收、增强胞内感染治疗疗效、提高药物安全性和渗透性能以及靶向性传递等四个方面阐述了目前SLNs在抗寄生虫药物传递中的研究现状,为后续进一步研究提供参考。     

抗胞内菌感染纳米药物研究进展

纳米技术所制备的纳米抗菌药物可有效改善药物胞内转运能力,为提高胞内菌感染的治疗提供新的策略。本文通过总结常见兼性或专性胞内菌的感染以及纳米抗菌药物治疗胞内菌感染的现状,讨论并分析纳米抗菌药物在兽医临床上治疗胞内菌感染所面临的挑战,并展望纳米抗菌药物在治疗胞内感染性疾病的应用前景。     

金黄色葡萄球菌胞外分泌蛋白的核酸酶活性研究

为研究金黄色葡萄球菌胞外分泌蛋白的核酸酶活性,本研究复苏培养金黄色葡萄球菌后取培养上清液,采用透析得到金黄色葡萄球菌胞外分泌蛋白,结果显示获得的该蛋白浓度为48.5μg/mL。采用琼脂糖凝胶电泳法、琼脂扩散法和琼脂培养法检测金黄色葡萄球菌胞外分泌蛋白的核酸酶活性,利用琼脂糖凝胶电泳法探究温度、pH、金属离子对核酸酶活性的影响。结果显示,金黄色葡萄球菌胞外分泌蛋白表现出降解λDNA的核酸酶活性,且最适温度和pH值分别为50℃和9.0,在低温和酸性条件下核酸酶的活性较弱,但胞外核酸酶对70℃以上的耐受性较差。不同浓度的Ba^2+、Mg^2+和Zn^2+对胞外分泌蛋白的核酸酶活性无影响;低浓度(0.01 mmol/L^1 mmol/L)的Ca^2+、Ni^2+、Cu^2+和Mn^4+可以促进胞外核酸酶切割λDNA的活性;高浓度的Na^+、K^+和Fe^3+可以提高胞外核酸酶切割λDNA的活性;添加Co^2+(0.01 mmol/L^10 mmol/L)可以促进胞外分泌蛋白的核酸酶活性。本研究证实了金黄色葡萄球菌胞外分泌蛋白的核酸酶活性,为进一步研究胞外分泌蛋白在金黄色葡萄球菌和宿主互作中的确切作用奠定了基础。     

固体脂质纳米载体促进药物吸收的研究进展

因受到溶解速率、酶和酸碱环境等因素的影响,一些具有良好药理活性的不溶性药物的使用和吸收会受到一定的限制。药物的吸收效果不仅取决于药物的理化性质,更与其载体系统有关,合适的载体能够促进药物吸收,提高药物的生物利用度,使药物在局部控制释放,以使治疗效果最大化。固体脂质纳米载体可以提高多种药物特别是亲脂化合物的胃肠道吸收和生物利用度,在促进药物的转运和吸收方面具有重要意义。对于固体脂质纳米载体的研究进展以及仍面临的问题,将从作用、影响其促吸收的因素和促吸收的机制等方面进行综述,以指导和推动固体脂质纳米载体在促进药物吸收方面的应用。     

夏枯草等14味中药对金黄色葡萄球菌的体外抑菌活性

采用索氏提取,超声波和水煎煮方法提取夏枯草等14味中药的有效部位,应用二倍试管稀释法分别测定其提取物对金黄色葡萄球菌的最小抑菌浓度（MIC）和最小杀菌浓度（MBC）。结果显示,夏枯草、虎杖、何首乌、连翘、柴胡、穿心莲、地榆7味中药有效部位对金黄色葡萄球菌的抗菌活性最强,其MIC为1．953～3lI250g／L,MBC为3．90～31．25g／L;佛手、黄柏2味中药有效部位具有较强的体外抗菌活性,其MIC值为62．5g／L,MBC值为125．0g／L;大青叶、板蓝根、金银花、杜仲、白头翁5味中药有效部位的抗菌活性较差,其MIC值为125．0g／L,MBC值为250．0,≥250．0g／L。不同中药有效部位对金黄色葡萄球菌的抗菌活性有所差异,其中夏枯草、虎杖、何首乌、地榆和佛手有效部位对金黄色葡萄球菌具有较好的抗菌活性。     

纳米技术提高抗菌药物治疗胞内菌感染的研究进展

对胞内菌感染性疾病常规治疗所面临的难题、纳米技术治疗胞内菌感染的优势以及纳米药物对不同胞内菌感染性疾病的治疗研究进展进行总结,分析了纳米技术提高抗菌药物进入细胞能力的机制和影响因素,针对纳米抗菌药物在治疗胞内菌感染疾病面临的挑战提出了纳米抗菌药物在胞内感染性疾病治疗领域的发展策略。     

7种抗菌药物对金黄色葡萄球菌及大肠杆菌体外抗生素后效应的研究

采用稀释法去除抗生素，用菌落计数测定细菌生长曲线的方法测定环丙沙星、洛美沙星、诺氟沙星、恩诺沙星、庆大霉素、青霉素及氨苄西林对金葡球菌及大肠杆菌的抗生素后效应(PAE)。结果：几种药物在各浓度组对金黄色葡萄球菌均呈现明显的PAE，氟喹诺酮类药物及庆大霉素对大肠杆菌产生较长的PAE，而β-内酰胺类药物作用后的PAE则很短。PAE的存在提示：在兽医临床设计给药方案时，适当延长给药间隔时间，减少给药次数，仍能维持抗菌效果。     

金银花提取物对金黄色葡萄球菌的抗菌作用研究

目的:研究金银花对金黄色葡萄球菌的抗菌作用。方法:将金银花用50%乙醇提取,石油醚萃取后取水相,采用试管二倍稀释法测定其对金黄色葡萄球菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),建立小鼠尾静脉注射感染金黄色葡萄球菌模型检测其对小鼠体内的保护率,并采用比色法研究其对细菌体内琥珀酸脱氢酶(SDH)比活性的影响。结果:金银花对金黄色葡萄球菌的MIC和MBC分别为15.6 mg/mL和31.2 mg/mL,能降低小鼠人工感染金黄色葡萄球菌死亡率,但差异不显著(P>0.05);金银花能极显著降低细菌体内SDH比活性(P<0.01)。结论:金银花对金黄色葡萄球菌具有抑菌作用,其抑菌作用是通过降低SDH比活性来实现的。     

Antibacterial activity of florfenicol composite nanogels against Staphylococcus aureus small colony variants

BackgroundFlorfenicol might be ineffective for treating Staphylococcus aureus small colony variants (SCVs) mastitis.ObjectivesIn this study, florfenicol-loaded chitosan (CS)-sodium tripolyphosphate (TPP) composite nanogels were prepared to allow targeted delivery to SCV infected sites.MethodsThe formulation screening, the characteristics, in vitro release, antibacterial activity, therapeutic efficacy, and biosafety of the florfenicol composite nanogels were studied.ResultsThe optimized formulation was obtained when the CS and TPP were 10 and 5 mg/mL, respectively. The encapsulation efficiency, loading capacity, size, polydispersity index, and zeta potential of the optimized florfenicol composite nanogels were 87.3% ± 2.7%, 5.8% ± 1.4%, 280.3 ± 1.5 nm, 0.15 ± 0.03, and 36.3 ± 1.4 mv, respectively. Optical and scanning electron microscopy showed that spherical particles with a relatively uniform distribution and drugs might be incorporated in cross-linked polymeric networks. The in vitro release study showed that the florfenicol composite nanogels exhibited a biphasic pattern with the sustained release of 72.2% ± 1.8% at 48 h in pH 5.5 phosphate-buffered saline. The minimal inhibitory concentrations of commercial florfenicol solution and florfenicol composite nanogels against SCVs were 1 and 0.25 µg/mL, respectively. The time-killing curves and live–dead bacterial staining showed that the florfenicol composite nanogels were concentration-dependent. Furthermore, the florfenicol composite nanogels displayed good therapeutic efficacy against SCVs mastitis. Biological safety studies showed that the florfenicol composite nanogels might be a biocompatible preparation because of their non-toxic effects on the renal tissue and liver.ConclusionsFlorfenicol composite nanogels might improve the treatment of SCV infections.     

Antibacterial activity of enrofloxacin loaded gelatin-sodium alginate composite nanogels against intracellular Staphylococcus aureus small colony variants

BackgroundThe poor intracellular concentration of enrofloxacin might lead to treatment failure of cow mastitis caused by Staphylococcus aureus small colony variants (SASCVs).ObjectivesIn this study, enrofloxacin composite nanogels were developed to increase the intracellular therapeutic drug concentrations and enhance the efficacy of enrofloxacin against cow mastitis caused by intracellular SASCVs.MethodsEnrofloxacin composite nanogels were formulated by an electrostatic interaction between gelatin (positive charge) and sodium alginate (SA; negative charge) with the help of CaCl₂ (ionic crosslinkers) and optimized by a single factor test using the particle diameter, zeta potential (ZP), polydispersity index (PDI), loading capacity (LC), and encapsulation efficiency (EE) as indexes. The formation mechanism, structural characteristics, bioadhesion ability, cellular uptake, and the antibacterial activity of the enrofloxacin composite nanogels against intracellular SASCVs strain were studied systematically.ResultsThe optimized formulation was comprised of 10 mg/mL (gelatin), 5 mg/mL (SA), and 0.25 mg/mL (CaCl₂). The size, LC, EE, PDI, and ZP of the optimized enrofloxacin composite nanogels were 323.2 ± 4.3 nm, 15.4% ± 0.2%, 69.6% ± 1.3%, 0.11 ± 0.02, and −34.4 ± 0.8 mV, respectively. Transmission electron microscopy showed that the enrofloxacin composite nanogels were spherical with a smooth surface and good particle size distributions. In addition, the enrofloxacin composite nanogels could enhance the bioadhesion capacity of enrofloxacin for the SASCVs strain by adhesive studies. The minimum inhibitory concentration, minimum bactericidal concentration, minimum biofilm inhibitory concentration, and minimum biofilm eradication concentration were 2, 4, 4, and 8 μg/mL, respectively. The killing rate curve had a concentration-dependent bactericidal effect as increasing drug concentrations induced swifter and more radical killing effects.ConclusionsThis study provides a good tendency for developing enrofloxacin composite nanogels for treating cow mastitis caused by intracellular SASCVs and other intracellular bacterial infections.     

Enhancement of antibacterial activity of tilmicosin against Staphylococcus aureus by solid lipid nanoparticles in vitro and in vivo

This study aimed to enhance the antibacterial activity of tilmicosin by solid lipid nanoparticles (SLN). Tilmicosin-loaded hydrogenated castor oil (HCO)-SLN was prepared using a hot homogenisation and ultrasonication method. The physicochemical characteristics of SLN were investigated by scanning electron microscopy (SEM) and photon correlation spectroscopy (PCS). The antibacterial activity of tilmicosin-SLN against Staphylococcus aureus was evaluated by growth inhibition and colony-counting method. A therapeutic study of tilmicosin-SLN was conducted by subcutaneous injection in a mouse mastitis model infected with S. aureus by teat canal infusion. Therapeutic efficacy was assessed by physical appearance of the mammary gland and measurement of colony-forming units (CFU) per gland. The results showed that the diameter, polydispersivity index, zeta potential, encapsulation efficiency and loading capacity of the nanoparticles were 343±26 nm, 0.33±0.08, -7.9±0.4 mV, 60.4±3.3% and 11.2±0.47%, respectively. Tilmicosin-SLN showed a sustained-release effect and sustained and enhanced antibacterial activity in vitro. SLN significantly enhanced the therapeutic efficacy of tilmicosin determined by lower CFU counts and a decreased degree of inflammation. These results demonstrated that the HCO-SLN is an effective carrier to enhance the antibacterial activity of tilmicosin.     

槲皮素-替米考星混合物纳米粒的制备及对金黄色葡萄球菌小菌落变异株抗菌活性研究

【目的】 制备一种适用于治疗由金黄色葡萄球菌小菌落变异株(Staphylococcus aureus small colony variants,SASCVs)引起奶牛乳腺炎感染的槲皮素-替米考星混合物纳米粒,评价其对SASCVs菌株的抗菌活性。【方法】 通过对槲皮素(0.05、0.10、0.15和0.20 g)和替米考星原料药用量(0.2、0.3、0.4和0.5 g)及转速(500、1 000、1 500 r/min)、反应时间(0.5、1.0、2.0 h)、反应温度(25、50、75 ℃)、溶剂浓度(5.0%、7.5%、10.0%)进行考察,以外观性状为指标,筛选槲皮素-替米考星混合物纳米粒最优配方和制备条件;以微观形态、沉降体积比、再分散性、粒径和Zeta电位为指标,对制备的槲皮素-替米考星混合物纳米粒表征;通过琼脂扩散法、微量肉汤稀释法和体外杀菌曲线,对比槲皮素-替米考星混合物纳米粒和市售替米考星溶液对SASCVs菌株的抗菌活性,评价槲皮素-替米考星混合物纳米粒的优势。【结果】 当替米考星为0.4 g、槲皮素为0.1 g、溶剂为20 mL 7.5%的聚乙二醇溶液,在转速为1 000 r/min、反应时间为1 h、反应温度为50 ℃时,所制备的槲皮素-替米考星混合物纳米粒最优,其溶液外观性状显示颜色清亮,无沉淀和絮状物;微观形态显示该混合物纳米粒分布均匀,颗粒大小一致;沉降体积比为1,再分散性良好,平均粒径为741.9 nm,Zeta电位为-7.69 mV。替米考星标准品、槲皮素-替米考星混合物纳米粒和市售替米考星溶液对SASCVs菌株的抑菌圈直径分别为2.54、1.51和1.38 cm,对SASCVs菌株的最低抑菌浓度分别为1、1和2 μg/mL;体外杀菌曲线表明,槲皮素-替米考星混合物纳米粒对SASCVs菌株表现出明显的浓度依赖性。【结论】 本研究所制备的槲皮素-替米考星混合物纳米粒对SASCVs菌株具有较强杀菌能力,可为治疗由SASCVs菌株引起的奶牛乳腺炎提供基础研究资料。     

Tilmicosin‐ and florfenicol‐loaded hydrogenated castor oil‐solid lipid nanoparticles to pigs: Combined antibacterial activities and pharmacokinetics

The combined antibacterial effects of tilmicosin (TIL) and florfenicol (FF) against Actinobacillus pleuropneumoniae (APP) (n = 2), Streptococcus suis (S. suis) (n = 2), and Haemophilus parasuis (HPS) (n = 2) were evaluated by chekerboard test and time‐kill assays. The pharmacokinetics (PKs) of TIL‐ and FF‐loaded hydrogenated castor oil (HCO)‐solid lipid nanoparticles (SLN) were performed in healthy pigs. The results indicated that TIL and FF showed synergistic or additive antibacterial activities against APP, S. suis and HPS with the fractional inhibitory concentration (FIC) ranging from 0.375 to 0.75. The time‐kill assays showed that 1/2 minimum inhibitory concentration (MIC) TIL combined with 1/2 MIC FF had a stronger ability to inhibit the growth of APP, S. suis, and HPS than 1 MIC TIL or 1 MIC FF, respectively. After oral administration, plasma TIL and FF concentrations could maintain about 0.1 μg/ml for 192 and 176 hr. The SLN prolonged the last time point with detectable concentrations (T_last), area under the concentration–time curve (AUC_0‐t), elimination half‐life (T_½ke), and mean residence time (MRT) by 3.1, 5.6, 12.7, 3.4‐fold of the active pharmaceutical ingredient (API) of TIL and 11.8, 16.5, 18.1, 12.1‐fold of the API of FF, respectively. This study suggests that the TIL‐FF‐SLN could be a useful oral formulation for the treatment of APP, S. suis, and HPS infection in pigs.     

重组鸡Cathelicidins类抗菌肽的融合表达及其对金黄色葡萄球菌抗菌活性的研究

Cathelicidins抗菌肽在家禽天然免疫系统中发挥重要作用,本研究将已构建的含Cathelicidins抗菌肽成熟肽的阳性克隆菌株Rosetta(DE3)/pET30-CathL1S、pET30-CathL2S、pET30-CathL3S分别在37℃、1.0 mmol/L IPTG的条件下进行诱导表达。SDS-PAGE和Western blotting分析结果表明,Cathelicidins成熟肽片段均在大肠杆菌中以融合形式成功表达,表达产物的表观分子质量分别为7、8、7.5 ku。琼脂糖孔穴扩散法检测结果显示,表达产物对多种革兰氏阴性菌和阳性菌均具有很好的抑制活性,其对金黄色葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis)、藤黄微球菌(Micrococcus luteus)、大肠杆菌(Escherichia coli)、鸡白痢沙门氏菌C79-13 (Salmonella pullorum)、巴氏杆菌、鸭疫里默氏杆菌及绿脓杆菌等供试菌株均有较强的抑制作用,尤其对抗生素耐药菌株有明显的抑制作用。以雏鸡为实验模型研究重组抗菌肽对金黄色葡萄球菌的体内抗菌活性,结果表明,注射中、高剂量CathL-1抗菌肽试验组(5、10 mg/kg),在试验结束后取肝脏组织匀浆液涂布于TMP平板,未分离到葡萄球菌。各抗菌肽试验组平均增重均高于感染对照组,其中低剂量抗菌肽试验组(2.5 mg/kg)体内抑菌效果虽与普通抗生素相当,但其增重效果尤为明显。     

利血平增敏法检测恩诺沙星药物残留的研究

目的了解恩诺沙星对金黄色葡萄球菌(金葡菌)的抗菌活性及利血平对其抗菌活性的影响。方法采用对倍稀释浓度琼脂平板法测定抗菌药物的最低抑菌浓度(MIC)值,同法测定利血平对恩诺沙星对金葡菌MIC值的影响。结果利血平可有效增强恩诺沙星的抗菌活性,增大了恩诺沙星抑制金葡菌的抑菌圈直径,提高了恩诺沙星在组织中检测的灵敏度。研究发现利血平加入后中心浓度由0.5ul/ml变为0.0625ul/ml,最低检测限由0.125μg/ml降低到0.015625μg/ml。最低检测限减低了8倍。     

抑制奶牛乳房炎源金黄色葡萄球菌的乳酸菌的筛选

为获得抑制奶牛乳房炎源金黄色葡萄球菌(S. aureus)的乳酸菌(LAB),本研究从新疆巴音布鲁克牧区鲜牛乳和哈萨克族乳制品奶疙瘩样品中分离培养LAB,通过传统的分离鉴定与16S rDNA基因序列测序相结合的方法鉴定LAB种类,同时以临床奶牛乳房炎源金黄色葡萄球菌S. aureus N2为指示菌,采用双层琼脂扩散法检测分离株的抑菌能力。通过测定生长曲线确定分离株的生长稳定期,进而利用硫酸铵沉淀法透析提取稳定期内分离株的细菌素,并检测其细菌素抑菌效价。结果显示:从样品中筛选获得5株能够抑制指示菌生长的LAB,分别为希氏乳杆菌、干酪乳杆菌、粪肠球菌、戊糖片球菌和乳酸乳球菌亚种。生长规律曲线表明20 h^30 h为5株LAB的稳定期,此期培养液pH值维持在3.8~4.5。从培养20 h的5株LAB上清液中提取到了细菌素,经检测其具有抑菌活性,抑菌效价分别为457 IU/mL、1 023 IU/mL、676 IU/mL、1 862 IU/mL和1 023 IU/mL。本研究结果表明5株LAB通过在生长稳定期内维持较低酸性环境(pH<4.5),代谢产生细菌素对乳房炎源S. aureus发挥抑制生长作用。本研究为S. aureus性奶牛乳房炎的生物防治提供了实验依据。     

利血平增敏法检测环丙沙星药物残留的研究

为了解环丙沙星对金黄色葡萄球菌（金葡菌）的抗菌活性及利血平对其抗菌活性的影响,采用对倍稀释浓度琼脂平板法测定抗菌药物的最低抑菌浓度(MIC)值,同法测定利血平对环丙沙星对金葡菌MIC值的影响。结果表明利血平增强了环丙沙星的抗菌活性,增大了环丙沙星抑制金葡菌的抑菌圈直径,提高了环丙沙星在组织中检测的灵敏度。我们研究发现利血平加入后中心浓度由0.5 μl/ml变为0.0625 μl/ml,最低检测限由0.125 μg/ml降低到0.015625 μg/ml。