东海海洋有抗菌活性细菌的筛选及系统发育分析

为了筛选开发新的抗生素,从海水、海泥以及刺鲳、鳗鱼、黄鱼、安康鱼、鲽鱼、马头鱼的鳃和胃肠道中分离得到195株海洋细菌,以金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、黑曲霉和啤酒酵母作为指示菌,用滤纸片法定性测定它们的抗菌活性,筛选到抗菌活性物质产生菌22株.对其中6个抗菌活性较强的菌株进行16S rDNA序列分析,结果显示,它们分别是克氏菌属的Kocuria rhizophila、葡萄球菌属的Staphylococcus saprophyticus、芽孢杆菌属的Bacillus sphaericus和Bacillus thuringiensis、冷杆菌属的Psychrobacter faecalis和变形菌属的Proteus penneri.系统发育分析表明这6个菌株归于3个类群:1个为高(G C)%的革兰阳性菌;3个为低(G C)%的革兰阳性菌;另2个为γ-变形菌.     

鸭不同肠段内菌群结构分析与拟杆菌分布研究

旨在分析健康鸭十二指肠、空肠、回肠和盲肠内容物菌群组成、多样性特征以及拟杆菌分布。本研究选择健康高邮鸭20只,公母各半,70日龄时无菌采集十二指肠、空肠、回肠和盲肠内容物,提取肠道内容物细菌基因组,利用IonS5TMXL平台进行高通量测序,分析肠道内容物菌群结构与丰度特征以及拟杆菌的分布。结果表明,十二指肠、空肠内容物菌群丰度显著高于回肠和盲肠内容物（P<0.05）,回肠内容物菌群多样性最低;十二指肠和空肠内菌群群落结构较为相似,与回肠,特别是盲肠的相似度较小。健康鸭肠内优势菌门为拟杆菌门（Bacteroidetes）、厚壁菌门（Firmicutes）、变形菌门（Proteobacteria）和梭杆菌门（Fusobacteria）,上述菌门在各肠段内容物中相对丰度不同;不同肠段内容物中定植了不同差异微生物物种,十二指肠、空肠、回肠和盲肠内容物中差异菌门分别是变形菌门、放线菌门（Actinobacteria）、厚壁菌门和拟杆菌门。鸭肠道中共分析到28种拟杆菌种,其中B.acidifaciens、B.barnesiae、B.caccae、B.caecicola、B.coprocola、B.sp和B.luti在盲肠内容物中显著聚类,且B.caecigallinarum、B.plebeius和B.barnesiae在鸭盲肠中优势定植。结果显示,鸭肠段空间显著影响了其内容物中菌群丰度与多样性,不同肠段内定植了差异的优势微生物物种,这可能与肠段小环境以及功能一致,在盲肠内容物中优势定植拟杆菌,推测可能与鸭盲肠生理生化功能相关。     

Microbiota characterization of sheep milk and its association with somatic cell count using 16s rRNA gene sequencing

This work aimed to use 16S ribosomal RNA sequencing with the Illumina MiSeq platform to describe the milk microbiota from 50 healthy Assaf ewes. The global observed microbial community for clinically healthy milk samples analysed was complex and showed a vast diversity. The core microbiota of the sheep milk includes five genera: Staphylococcus, Lactobacillus, Corynebacterium, Streptococcus and Escherichia/Shigella. Although there are some differences, some of these genera are common with the microbiota core pattern of milk from other species, especially with dairy cows. The microbial composition of the studied samples, based on the definition of amplicon sequence variants, was analysed through a correlation network. A preliminary analysis by grouping the milk samples based on their somatic cell count (SCC), which is considered an indicator of subclinical mastitis (SM), showed certain differences for the core of the samples identified as SM. The differences in the microbiota diversity pattern among samples might also suggest that subclinical mastitis would be associated with the significant increase in some genera that are inhabitants of the mammary gland and a remarkable concomitant reduction in the microbial diversity. Additionally, we have also presented here a preliminary analysis to assess the impact of the sheep milk microbiome on SCC, as an indicator of subclinical mastitis. The results here reported provide a first characterization of the sheep milk microbiota and settle the basis for future studies in this field.     

瑶山亚种树鼩源嗜水性气单胞菌的分离鉴定及生物学特性研究

为确诊广西中医药大学瑶山亚种树鼩人工驯化养殖中心的瑶山亚种树鼩发病死亡原因,并调查分离菌株的致病性与耐药情况,试验通过平板划线分离获得病原菌,进一步对分离菌进行形态观察、培养特性、生化试验及16S rRNA序列分析,纯化获得一株致病性嗜水性气单胞菌;利用昆明小鼠的致病性试验测定了该分离菌株的半数致死量（LD₅₀）,利用药敏纸片法分析该分离株对常用药物的敏感性,并应用PCR方法进行菌株耐药基因的检测。结果显示,从病死瑶山亚种树鼩分离到一株嗜水性气单胞菌,镜检显示为革兰氏阴性菌,形态呈短杆状。该菌16S rRNA序列与日本淡水湖源嗜水性气单胞菌SWCY-3.27株同源性高达100%,遗传进化关系最近。该分离菌对成年昆明小鼠的LD₅₀为1×10⁷ CFU,毒力高于模式菌株ATCC 7966,近似于中国流行株NJ-35和J-1等,致病性试验鉴定该菌为强毒力菌株。生化鉴定七叶苷、阿拉伯糖、氧化酶呈阳性,各种生化鉴定符合嗜水气单胞菌生化特性。药敏检测显示,该菌株对米诺环素、链霉素、妥布霉素等9种药物敏感,对四环素、红霉素、青霉素等10种药物耐药。以PCR扩增基因方法对分离菌进行6类12种耐药基因的检测,发现分离菌存在氨基糖苷类（Aph-（3）-lia）和β-内酰胺类（mecA）两类抗生素的耐药基因,与药敏试验结果基本吻合。本研究结果表明,引起此次树鼩死亡的病原菌为嗜水性气单胞菌,可用高敏药物进行针对性治疗,为防治嗜水性气单胞菌感染导致的树鼩疾病提供参考依据。     

腹泻犊牛与健康犊牛粪便菌群结构组成与功能研究

本研究旨在探究腹泻与健康犊牛粪便微生物的结构组成与分布特征,筛选出一些可作为评价犊牛腹泻参考指标的特异性菌群。试验采集10份腹泻和10份健康夏南牛犊牛的粪便样本并提取粪便总DNA,运用16S rRNA高通量测序技术,比较腹泻组和健康组在微生物多样性方面（组成及结构）存在的差异,获得两组间具有统计学差异的Biomarker,并对差异功能基因进行注释。结果表明,腹泻犊牛粪便微生物的多样性小于健康犊牛;在门水平上,与健康组相比,腹泻组犊牛粪便中微生物相对丰度显著升高的是变形菌门和梭杆菌门,螺旋菌门显著降低（P<0.05）;在属水平上,与健康组相比,腹泻组犊牛粪便微生物中埃希氏-志贺菌属、梭杆菌属、乳杆菌属和梭状芽孢杆菌属相对丰度明显升高,不可培养拟杆菌目S24-7群、拟普雷沃菌属和普雷沃氏菌科的菌属显著降低（P<0.05）。通过对COG功能预测分析,腹泻组在翻译、核糖体结构和起源类群明显降低。通过KEGG代谢途径分析,腹泻组粪便中显著升高的代谢通路是碳水化合物代谢和外源性物质降解和代谢,显著降低的代谢通路分别是细胞增殖和死亡、复制和修复、翻译和转录（P<0.05）。本研究分析了腹泻组和健康组之间的优势菌群,筛选出可以作为犊牛腹泻参考指标的10个Biomarker,并对功能差异基因进行注释,为进一步研究犊牛腹泻的治疗及预防奠定了基础。     

腹泻仔猪与健康仔猪肠道微生物多样性比较

目的通过研究对比哺乳期腹泻仔猪与健康仔猪粪便菌群,探讨腹泻对哺乳期仔猪肠道微生物的影响。方法通过高通量16S rDNA测序技术对腹泻组仔猪（n=6）和健康组仔猪（n=3）粪便样本进行测序,比较两组仔猪肠道微生物群落的组成和结构。结果腹泻组仔猪和健康组仔猪粪便菌群差异显著（P<0.05）,腹泻组仔猪的肠道菌群多样性显著（P<0.05）低于健康组仔猪。与健康组仔猪相比,在门水平上,腹泻组仔猪的梭杆菌门和变形菌门的相对丰度显著（P<0.05）增加,而厚壁菌门的相对丰度显著（P<0.05）下降;在属水平上,腹泻组仔猪的梭杆菌属（Fusobacterium）、普雷沃菌属（Prevotella）、埃希菌属（Escherichia）、乳杆菌属（Lactobacillus）、厌氧弧菌属（Anaerovibrio）、拟普雷沃菌属（Alloprevotella）和Fusobacteriaceae_unclassified的相对丰度显著（P<0.05）增加,而大部分厚壁菌门菌属的相对丰度显著（P<0.05）降低。结论通过分析对比腹泻仔猪与健康仔猪肠道微生物多样性,为预防和治疗哺乳期仔猪腹泻提供了理论依据。     

Evaluation of feline oral squamous cell carcinomas for p16 protein immunoreactivity and the presence of papillomaviral DNA

Oral squamous cell carcinomas (OSCCs) develop commonly in cats. While the cause of the feline neoplasms is unknown, a quarter of human OSCCs are caused by papillomavirus (PV) infection. As PV DNA has been previously detected in a feline OSCC, it was hypothesised that PV infection could be a significant cause of feline OSCCs. Human OSCCs that are caused by PVs contain increased p16^CDKN2A protein (p16), which can be detected using immunohistochemistry. In cats, increased p16 immunoreactivity has been reported within PV-associated skin lesions. This study evaluated p16 immunoreactivity within 30 feline OSCCs. Additionally, PCR was used to amplify PV DNA from the OSCCs. Increased p16 immunoreactivity was present within 2 OSCCs. However, as PV DNA was not amplified from any OSCC in this study, it cannot be confirmed that the increased p16 was caused by PV infection. Therefore, these results do not support the hypothesis that PVs are a significant cause of OSCCs in cats. Loss of p16 expression is considered an important process in the development of human non-PV-induced OSCCs. In contrast, loss of p16 immunoreactivity was only present in 2 feline OSCCs. This suggests that human and feline OSCCs develop due to different molecular mechanisms.