抗菌肽LL-37的抑菌作用及其稳定性研究

采用微量2倍稀释法测定抗菌肽LL-37对大肠杆菌、沙门菌和金黄色葡萄球菌的最小抑菌浓度(MIC),并进一步研究温度、pH和保存时间对LL-37稳定性的影响。结果显示:LL-37对大肠杆菌、沙门菌和金黄色葡萄球菌的最小抑菌浓度分别为3.12、1.56和0.78μg/mL。热稳定性试验显示:重组抗菌肽121℃加热21 min、100℃加热3 h仍有较好的活性。酸碱稳定性试验结果显示:LL-37在pH 2.0～12.0时均具有一定的活性,pH 5.0～6.0时活性最好。此外,-20℃为此抗菌肽长期保存的最佳条件。     

抗菌肽BSN-37的抑菌活性及其稳定性分析

试验旨在研究抗菌肽BSN-37的抑菌活性和稳定性。采用微量倍比稀释法测定抗菌肽BSN-37的最小抑菌浓度(MIC),菌落计数法分析抗菌肽BSN-37对大肠杆菌CVCC1568的杀菌动力学特征,扩散法测定抗菌肽BSN-37的盐离子稳定性、酸碱稳定性、血清稳定性、胰酶稳定性、热稳定性、反复冻融稳定性、室温保持稳定性、药效保持时间稳定性。结果显示,抗菌肽BSN-37对革兰氏阴性菌(大肠杆菌和沙门氏菌)的MIC为1.5625～25μg/mL,对革兰氏阳性菌(枯草芽孢杆菌和短小芽孢杆菌)的MIC为1.5625～12.5μg/mL,对真菌(白色念珠菌)没有活性;抗菌肽BSN-37可在90min内完全杀灭大肠杆菌CVCC1568;除了Fe2+和胰酶会影响抗菌肽BSN-37的抑菌活性外,在75～100℃的高温、150～250mmol/L高浓度盐离子(Na+、K+、Ca2+和Mg2+)、20%～25%饱和浓度的Cu2+、pH 4～10、20%～25%的血清、10～12次的反复冻融、10～12d室温保存等条件下仍能保持较好的抑菌活性,药效保持时间可达7.5d。本试验结果表明,抗菌肽BSN-37具有替代抗生素成为新抗菌药物的潜力,为抗菌肽BSN-37的临床应用提供了理论依据。     

抗菌肽LL-37在毕赤酵母SMD1168中的高效表达及活性鉴定

为获得高效表达的抗菌肽LL-37,根据人源抗菌肽LL-37的氨基酸序列,选择毕赤酵母(Pichia pastoris)密码子的偏嗜性,通过SOEing法改造合成LL-37基因片段,克隆到pGAPZαA质粒中,获得分泌型重组酵母表达载体pGAPZαA-LL-37。pGAPZαA-LL-37通过限制性内切酶AvrII酶切线性化后,经电穿孔法转入毕赤酵母细胞SMD1168。经Zeocin抗性筛选,得到高拷贝转化子,PCR检测表明LL-37基因与毕赤酵母染色体稳定结合。在GAP启动子调控下,LL-37蛋白获得分泌表达,其上清表达量约为237mg/L。表达产物耐热性强,在100℃条件下30min内仍保持一定的抗菌活性。表达产物对大肠杆菌DH5α、大肠杆菌D31和猪链球菌的最小抑菌浓度(MIC)分别为1.56μg/mL、3.12μg/mL和6.25μg/mL。     

抗菌肽对猪源大肠杆菌和猪链球菌体外抑菌活性研究

<正>1材料和方法1.1材料(1)试验菌株。8株猪大肠杆菌(E.coil)分离自大肠菌感染的猪体,8株猪链球菌株分离自感染的病猪,均来源于河南省内猪场,由河南农业大学牧医工程学院传染病实验室自行分离保存。(2)试剂和药物。抗菌肽和牛肉膏(生化试剂)购自北京奥星生物技术有限公司,批号20070720;蛋白胨(生化试剂)购自北京奥星生物技术有限公司,批号20081102;氯化钠(分析纯)购自天津北方天医化学试剂厂,批号20080924;磷酸氢二钾(分析纯)购自上海恒信化学试剂有限公司,批号20080602。     

猪源抗菌肽PR-39对畜禽常见病原菌的抑菌活性及与抗生素协同杀菌效应研究

实验旨在测定猪源抗菌肽PR-39对常见病原菌的抑菌活性,以及其与常用抗生素的协同杀菌效应。实验结果表明:PR-39对常见畜禽病原菌均有一定的抑菌活性,但对革兰氏阳性菌的敏感性较差;将PR-39与阿莫西林、土霉素、硫酸链霉素联合使用效果良好。研究结果为PR-39的实际应用提供理论数据,同时体现出PR-39替代抗生素开发成新型饲料添加剂的巨大潜力。今后可重点研究PR-39的杀菌机制以及联合不同抗生素协同杀菌的相关机理,为抗菌肽在新饲料配方中的使用奠定实验基础。     

抗菌肽CJH对常见畜禽病原菌抑菌活性及与抗生素协同杀菌效应研究北大核心

抗菌肽CJH是从金环蛇毒液中提取出的一种抗菌活性极强的免疫活性多肽物质。试验测定了抗菌肽CJH对不同革兰氏阴性菌和阳性菌的抑菌活性及杀菌速率,并与常用抗生素的测定结果进行了对比;通过微量肉汤稀释棋盘实验法测定了抗菌肽CJH联用不同抗生素的杀菌效果。结果表明:相比几种常用抗生素,抗菌肽CJH具有广谱的抗菌活性,且杀菌能力较强;抗菌肽CJH与阿莫西林、土霉素联用有协同效应。试验为抗菌肽CJH的实际应用及为了探明其减少甚至替代抗生素成为新型饲料添加剂的潜力提供参考。     

国产黄霉素对畜禽常见病原菌体外抑菌活性及小白鼠急性毒性试验研究

采用杯碟法、试管2倍稀释法及简化寇氏法分别对国产黄霉素进行了畜禽常见病原菌体外抑菌活性和小白鼠急性毒性试验研究。体外抑菌活性试验结果表明，黄霉素对4株不同动物源性的金黄色葡萄球菌均有非常敏感的抑菌作用，抑菌活性明显比喹乙醇强；对猪丹毒杆菌、李氏杆菌、无乳链球菌、停乳链球菌的抑菌活性与喹乙醇相当或各有千秋；但对部分革兰氏阴性菌的抑菌活性明显比喹乙醇弱。小白鼠急性毒性试验结果表明，黄霉素对小白鼠的口服LD50为9310mg／kg，Lq50的95%可信限为8953～9678mg／kg，毒性很小，作为抗生素饲料添加剂使用是安全的。     

抗菌肽对抗生素耐药菌株的抑菌活性研究

[目的]研究抗菌肽对抗生素耐药菌株的抑菌活性。[方法]利用抗性平板划线法从腹泻病牛血便中筛选分离出1株耐药菌,通过16S rDNA序列进行鉴定,采用琼脂孔穴扩散法通过梯度盐酸壮观霉素（spectinomycin,Spe⁺）、氨苄青霉素（ampicillin,Amp⁺）、硫酸卡那霉素（kanamycin,Kan⁺）和氯霉素（chloramphenicol,Cm⁺）试验确定该菌药敏特性,并利用1种抗菌肽制剂对该菌株进行药敏试验。[结果]经BLAST比对分析该菌16S rDNA序列,鉴定该耐药菌为科氏葡萄球菌（Staphylococcus cohnii）,此菌对Amp⁺敏感,但对试验中其他抗生素均有耐药性,各梯度抗菌肽对该耐药菌均具有明显的抑菌活性。[结论]抗菌肽能有效抑制耐药科氏葡萄球菌的生长,有望在畜牧生产中代替抗生素使用。     

复方中药制剂对9种畜禽肠道病原菌的体外抑菌试验

畜禽泻痢性疾病是畜禽常发病,严重制约着养殖业的可持续发展。本试验采用牛津杯法分别测定了复方中药制剂水提液、药典四黄止痢颗粒水提液和西药对照药物复方阿莫西林溶液对9种畜禽肠道病原菌（猪大肠杆菌K88、K99、987P,猪沙门氏菌SM2和鸡大肠杆菌O18、O37、O39,鸡沙门氏菌SM1、SM503）的抑菌试验。结果表明,所有药物对9种供试菌均有不同的抑菌效果,对9种畜禽肠道病原菌抑菌直径大小顺序分别是复方阿莫西林溶液、复方中药制剂和药典四黄止痢颗粒水提液。表明复方中药制剂对畜禽肠道致病菌有良好的抑菌作用,完全可以替代抗生素治疗该类病原菌引起的下痢性疾病。     

Antibacterial activity of cefquinome against equine bacterial pathogens

Cefquinome is known for its use as an antibacterial drug in cattle and pigs. The objective of this study was to evaluate the antibacterial activity of cefquinome against equine pathogenic bacteria. The minimum inhibitory concentration (MIC) of cefquinome was determined for a total of 205 strains, which had recently been isolated in Europe from diseased horses (respiratory infection, foal septicaemia). The bactericidal activity was tested against 19 strains using the time killing method. The post-antibiotic effect (PAE) and post-antibiotic sub-MIC effect (PA SME) were determined against 12 strains. Cefquinome showed high activity against Actinobacillus equuli and streptococci (MIC(90) of 0.016 and 0.032microg/mL), Enterobacteriaceae (MIC(90)=0.125microg/mL) and staphylococci (MIC(90)=0.5microg/mL). The activity was limited against Rhodococcus spp. and Pseudomonas spp. Cefquinome was shown to be a time dependent bactericidal antibiotic against the target pathogens, killing occurring at a concentration close to the MIC. A PAE of 0.5-10h was calculated against streptococci whereas no PAE was observed for Escherichia coli. A longer PA SME was determined for streptococci (3.3 to >24h with a killing effect) and E. coli (0.5-13.9h). Cefquinome was shown to have a broad spectrum of activity which covers many equine pathogens.     

头孢噻呋钠对21种畜禽常见病原菌的体外抑菌作用

目的:探讨头孢噻呋钠对21种畜禽常见病原菌的抑制作用,为人工感染试验和临床用药提供依据.方法:采用试管二倍稀释法体外抑菌试验.结果:国产头孢噻呋钠对猪链球菌、猪放线杆菌、猪鸡巴氏杆菌、猪鸡大肠杆菌、猪鸡沙门氏菌、鸡金葡菌、产气荚膜梭菌、肺炎克雷伯、停乳链球菌、无乳链球菌的MIC 0.5～0.06 μg/ml,对猪丹毒、奶牛乳房炎、链球菌、猪金葡菌、牛金葡菌的MIC 32～2 μg/ml,优于青霉素.对绿脓杆菌、、猪胃肠炎的MIC 64～125 μg/ml,高于青霉素.     

In vitro antimicrobial activity of sulfonamides against some porcine pathogens

The minimal inhibitory concentrations (MIC) of sulfonamides were determined against Bordetella bronchiseptica (n = 10), Pasteurella multocida (n = 10), Haemophilus pleuropneumoniae (n = 20), and Streptococcus suis (n = 10) strains isolated from pigs with atrophic rhinitis, pneumonia, or meningitis. Sulfonamides tested in an agar dilution method were sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamethazine, sulfadoxine, sulfisoxazole, sulfamerazine, sulfamethoxazole, sulfamethoxypyridazine, sulfanilamide, sulfatroxazole, and sulfisomidine. Results indicated that monotherapy of S suis infections with sulfonamides should not be encouraged because the MIC50 of all sulfonamides investigated was greater than 32 micrograms/ml. The MIC50 of the sulfonamides against B bronchiseptica ranged from 0.5 to 8 micrograms/ml, against P multocida from 2 to 32 micrograms/ml, and against H pleuropneumoniae from 8 to 64 micrograms/ml. The MIC50 of sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamerazine, and sulfamethoxazole for the gram-negative bacteria did not exceed 16 micrograms/ml. Among these compounds, sulfamethoxazole had the highest activity. The frequently prescribed sulfamethazine had an overall low antimicrobial activity.     

Antibacterial effect of bovine lactoferrin against udder pathogens

The antibacterial effect of lactoferrin (Lf) was tested on isolates of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and coagulase-negative staphylococci (CNS) as well as on Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae), originally isolated from bovine mastitis. Concentrations of Lf used were 0.67 mg/ml, 1.67 mg/ml, and 2.67 mg/ml. Growth of udder pathogens was monitored by turbidometry either in broth culture or in whey prepared from normal milk. We focused on 3 different growth variables: lag time, slope, and maximum absorbance of bacterial growth curves. Growth inhibition was seen in the broth but hardly at all in whey. The isolates of E. coli and CNS did not grow sufficiently well in whey to draw any conclusions. The most effective inhibitory activity of Lf was seen against E. coli and P. aeruginosa. All 5 E. coil isolates had similar growth patterns. Inhibition of growth by Lf was concentration-dependent. The concentration of 0.67 mg/ml in broth and whey was generally too low for a significant inhibitory effect.     

In vitro evaluation of various quinolone antibacterial agents against veterinary mycoplasmas and porcine respiratory bacterial pathogens

The in vitro activities of 12 quinolones and four antibiotics were determined against 15 veterinary mycoplasmal species and four species of bacteria commonly involved in respiratory infections in pigs. The newer quinolones were markedly more active in vitro against a wide range of mycoplasmas than nalidixic acid and the earlier quinolones. Against Mycoplasma hyopneumoniae ciprofloxacin was the most active quinolone with a geometric mean minimal inhibitory concentration (MIC) against 16 strains of 0.01 microgram ml-1 compared with 0.04 microgram ml-1 for tiamulin, 0.06 microgram ml-1 for tylosin, 0.17 microgram ml-1 for oxytetracycline and 0.23 microgram ml-1 for gentamicin. M hyosynoviae was less sensitive to the quinolones with mean MICs of 0.6 microgram ml-1 for ofloxacin and 0.7 microgram ml-1 for ciprofloxacin compared with 0.034 microgram ml-1, or less, for tiamulin. Norfloxacin and its 6-chloro analogue were both mycoplasmacidal in vitro at five or 10 times their MICs against M hyopneumoniae UCD4. Tiamulin was mycoplasmastatic. The quinolones were also active against porcine Bordetella bronchiseptica and Pasteurella multocida strains and Haemophilus species. Ciprofloxacin was the most active quinolone with mean MICs of 0.58 microgram ml-1 against B bronchiseptica (nine strains), 0.026 microgram ml-1 against P multocida (five strains) and 0.01 microgram ml-1, or less, against Haemophilus pleuropneumoniae (nine strains) and H parasuis (two strains) compared with mean MICs of from 0.5 microgram ml-1 to 64 micrograms ml-1, or more, for the antibiotics. This combination of excellent mycoplasmacidal activity against M hyopneumoniae and good antibacterial activity, suggests that the quinolones have great potential for treating respiratory infections in pigs, including enzootic pneumonia.