鸡肠道抗菌肽的提取及活性研究

取新鲜鸡肠黏膜超声波破碎,乙酸浸提,经Sephadex G-50层析后,收集具有抑菌活性的组分进行超滤离心,抑菌实验表明滤过物具有抑菌活性.滤过物经SDS-PAGE分析为一个条带,分子量约为6.078KD,得到电泳纯的鸡肠道抗微生物肽.该肽具有一定的耐热性,但经90℃以上热处理后活性下降较大;pH值对其活性影响较大,在pH6～8时活性最强.     

抗菌肽CRAMP的安全性、稳定性及其在清除铜绿假单胞菌生物被膜中的作用

旨在考察鼠源抗菌肽CRAMP的安全性、稳定性及其在清除铜绿假单胞菌生物被膜中的作用。采用兔血红细胞悬液的溶血性和小鼠腹腔巨噬细胞(RAW264.7)的细胞毒性考察CRAMP的安全性;采用不同温度、蛋白酶、金属离子和酸碱梯度对CRAMP抗菌活性的影响,考察其稳定性;通过在6孔细胞培养板中构建铜绿假单胞菌(PAO1株)成熟生物被膜,以人源抗菌肽LL-37和3种抗菌药为对照,采用结晶紫染色法检测生物被膜量,平板菌落计数法对生物被膜活菌数进行计数,苯酚-硫酸法结合Folin-酚试剂法检测生物被膜胞外基质含量,激光扫描共聚焦显微镜(CLSM)观察CRAMP干预PAO1生物被膜形态学的变化。结果显示,CRAMP在所测试浓度下对兔血红细胞溶血率均<2%;在62.5~125 mg·L^-1时对 RAW264.7没有细胞毒性。温度(25~100 ℃)、两种盐离子(Na⁺、K⁺)以及pH为5~10时对CRAMP的抗菌活性没有影响;胃蛋白酶、木瓜蛋白酶、胰蛋白酶均不同程度影响了CRAMP的抗菌活性。6孔细胞培养板中,4倍MIC 的CRAMP极显著降低了PAO1生物被膜量(减少率为76.74%,P<0.01),极显著减少了生物被膜中的活菌数量(减少了1.2个CFU·mL^-1,P<0.01),效果优于LL-37和3种抗菌药。CRAMP组的胞外基质显著低于空白对照组和LL-37组(P<0.05)。CLSM结果显示：与对照组相比较,4MIC浓度下,CRAMP组的细菌总荧光强度(PI+SYTO)显著低于空白对照组和LL-37组(P<0.05);与空白对照组相比,CRAMP组和LL-37组的死菌荧光强度/活菌荧光强度比值 (PI/SYTO)均极显著增大(P<0.01)。综上表明,CRAMP具有低溶血性、低细胞毒性,除蛋白酶作用不稳定外,具有较好的稳定性,对PAO1成熟生物被膜具有明显的清除作用,且效果优于LL-37,具有良好的药物开发前景。     

饲料中添加天蚕素抗菌肽对南美白对虾免疫功能、抗病力的影响

本研究以南美白对虾为研究对象,探究饲料中天蚕素抗菌肽的合适添加量。在饲料中依次添加不同浓度水平（0%、1%、2%、3%、4%和5%）的天蚕素抗菌肽,配制为6种配合饲料,用以投喂南美白对虾,饲养周期8周。结果显示：与对照组相比,饲料中添加天蚕抗菌肽对南美白对虾增重率、蛋白质沉积率和特定生长率有显著影响（P＜0.05）,高水平天蚕素抗菌肽会抑制南美白对虾的增重率和特定生长率,南美白对虾的饵料系数显著下降,但对南美白对虾存活率和摄食率无显著影响（P＞0.05）|随着饲料中天蚕素抗菌肽的添加水平不断增加,3%组南美白对虾的ACP、POD和SOD含量达到最大水平,与对照组有明显差异（P＜0.05）|随着饲料中天蚕素抗菌肽的添加水平的不断上升,3%组南美白对虾的淀粉酶、脂肪酶和胃蛋白酶含量最高,与对照组存在明显差异（P＜0.05）。结果表明,饲料中添加天蚕素抗菌肽显著提高南美白对虾的增重率和特定生长率,提高南美白对虾的生长发育速度,从而增强机体免疫力,提高南美白对虾的抗病能力。 [关键词]天蚕抗菌肽|南美白对虾|抗病力|免疫功能     

A framework for assessing the efficacy of antimicrobials in the control of necrotic enteritis in broiler chickens

This review presents a framework for assessing the efficacy of antimicrobials used to control necrotic enteritis (NE) caused byClostridium perfringens (CP) in the context of susceptibility testing and clinical efficacy, and their potential interactions with the intestinal microbiota of poultry. Practitioners have traditionally based their choice of antimicrobial agent on antimicrobial susceptibility testing, but there appears to be a lack of correlation with clinical efficacy for in-feed antimicrobials (particularly bacitracin and virginiamycin). Resistance patterns of CP and antimicrobials have been monitored using epidemiological cutoffs for minimal inhibitory concentration (MIC), which are not intended to guide therapy. Also, most data have been determined using CP isolates from healthy birds (i.e., potentially from commensal strains not known to be clinically relevant). It is believed that NE is caused by specific virulent CP strains (and potentially other bacteria) that proliferate and displace these commensals. The presence of resistant commensals is not necessarily detrimental (and may even be beneficial) if they inhibit the single CP strain dominance effect observed in acute NE. The choice of antimicrobial therapy in a clinical setting should thus be based on a variety of factors, including an accurate diagnosis, results of efficacy studies, prior experience at the premises in question, and interpretation of MIC data, recognizing that it is not necessarily well correlated with clinical efficacy.     

Isolation and antimicrobial susceptibility of Plesiomonas shigelloides from great cormorants (Phalacrocorax carbo hanedae) in Gifu and Shiga Prefectures,Japan

Plesiomonas shigelloides is a causal agent of gastroenteritis, sepsis and meningitis in humans. We examined the prevalence of P. shigelloides among great cormorants (Phalacrocorax carbo hanedae) in Japan and the antimicrobial susceptibility of isolates. P. shigelloides was isolated from 33 (47.8%) of 69 fecal samples from great cormorants in 2014. All 33 isolates were subjected to antimicrobial susceptibility testing using broth microdilution methods, which showed resistance to ampicillin (31 isolates, 93.9%), tetracycline (two isolates, 6.1%) and trimethoprim (one isolate, 3.0%). The high prevalence of P. shigelloides in the great cormorants implicates the possible microbiological risk to public health.     

抗菌肽对发酵床饲养仔猪抗氧化指标、免疫指标及肠道主要菌群数量的影响

为研究发酵床饲养模式下饲粮中添加抗菌肽对仔猪生长性能、抗氧化指标、免疫指标及盲肠主要菌群数量的影响,选用108头35日龄,体重13kg左右的健康苏钟猪,随机分为3组,每组3重复,每重复12头猪,分别饲喂基础饲粮(对照组)、基础饲粮+40mg/kg杆菌肽锌+20mg/kg硫酸抗敌素(抗生素组)和基础饲粮+300 mg/kg抗菌肽(抗菌肽组),试验期49d。结果表明:(1)与对照组相比,饲粮中添加抗菌肽能够显著提高仔猪血清和肝脏中谷胱甘肽过氧化物酶活性(P0.05),极显著降低肝脏丙二醛含量(P0.01);抗菌肽组肝脏谷胱甘肽过氧化物酶活性比抗生素组极显著提高(P0.01)。(2)与对照组相比,饲粮中添加抗菌肽能够极显著提高仔猪十二指肠上皮内淋巴细胞数量(P0.01);抗菌肽组仔猪血清IgA含量分别比对照组和抗生素组提高4.03%、2.27%,抗菌肽组空肠上皮内淋巴细胞数量比对照组提高10.70%,但差异均不显著(P0.05)。(3)与对照组相比,饲粮中添加抗菌肽能够显著降低仔猪盲肠中大肠杆菌数量(P0.05);细菌总数降低5.98%,但差异不显著(P0.05)。综上所述,本试验条件下,饲粮中添加300mg/kg的抗菌肽能够提高仔猪抗氧化和免疫功能;减少仔猪盲肠中大肠杆菌数量,而对盲肠有益菌群无不良影响。     

Comparative Phenotypic and Genotypic Analyses of Salmonella Rissen that Originated from Food Animals in Thailand and United States

Salmonella enterica serovar Rissen has been recognized as one of the most common serovar among humans and pork production systems in different parts of the world, especially Asia. In the United States, this serovar caused outbreaks but its epidemiologic significance remains unknown. The objectives of this study were to compare the phenotypic (antimicrobial susceptibility) and genotypic attributes of Salmonella Rissen isolated in Thailand (Thai) and the United States (US). All the Thai isolates (n = 30) were recovered from swine faecal samples. The US isolates (n = 35) were recovered from swine faecal samples (n = 29), cattle (n = 2), chicken (n = 2), dog (n = 1) and a ready‐to‐eat product (n = 1). The antimicrobial susceptibility of isolates was determined using the Kirby‐Bauer disk diffusion method with a panel of 12 antimicrobials. Pulse‐field gel electrophoresis (PFGE) was used to determine the genotypic diversity of isolates. All Thai isolates showed multidrug resistance (MDR) with the most frequent antibiotic resistance shown against ampicillin (100%), sulfisoxazole (96.7%), tetracycline (93.3%), streptomycin (90%) and chloramphenicol (30%). About half of the isolates of USA origin were pan‐susceptible and roughly 30% were resistant to only tetracycline (R‐type: Te). Salmonella Rissen isolated from Thailand and the USA in this study were found to be clonally unrelated. Genotypic analyses indicated that isolates were clustered primarily based on the geographic origin implying the limited clonality among the strains. Clonal relatedness among different host species within the same geography (USA) was found. We found genotypic similarity in Thai and US isolates in few instances but with no epidemiological link. Further studies to assess propensity for increased inter‐regional transmission and dissemination is warranted.     

分泌抗金黄色葡萄球菌细菌素乳酸菌的分离鉴定

为分离和筛选产抗金黄色葡萄球菌乳酸菌素的优势乳酸菌,利用乳酸菌分离培养基MRS从收集的各种腌制菜汁中分离培养乳酸菌,通过细菌培养特性、革兰氏染色特点、生理生化特性初步鉴定,同时根据Genbank中乳酸菌的16SrDNA序列设计特异性引物,采用PCR方法进一步鉴定,并以金黄色葡萄球菌为指示菌对乳酸菌的发酵上清液进行抑菌特性研究。结果表明,从腌渍菜汁中分离获得90株产酸菌,通过形态学、生理生化特性和PCR鉴定,结果73株产酸菌为乳酸杆菌;分泌产物抑菌试验表明,有10株菌具有抑制金黄色葡萄球菌活性,经酸排除和过氧化氢排除试验,仍然有5株乳酸菌的分泌产物具有抑制金黄色葡萄球菌活性。可见,从腌渍菜汁分离到的乳酸菌具有抑制金黄色葡萄球菌活性的特性,主要是通过分泌乳酸菌素来发挥作用。