Root-Associated Endophytic Bacterial Community Composition of Asparagus officinalis of Three Different Varieties

Asparagus (Asparagus officinalis L) is an economically important crop, rich in nutrients, and is also conducive to solving ecological and environmental problems. Plants may acquire benefits from root-associated endophytic bacteria. However, the composition of the endophytic bacterial community associated with the roots of asparagus is poorly elucidated. In this study, the nine root samples of asparagus from three different varieties including Asparagus officinalis var. Grande (GLD), A. officinalis var. Jinglvlu3 (JL3) and A. officinalis var. Jingzilu2 (JZL) were investigated by high-throughput sequencing technology of the 16S rDNA V5-V7 hypervariable region of endophytic bacteria. A total of 16 phyla, 29 classes, 90 orders, 171 families, and 312 genera were identified. Endophytic bacteria diversity and bacteria structure was different among the three varieties and was influenced by rhizosphere soil properties and varieties. In the GLD variety, the main phyla were Proteobacteria, Actinobacteria, and Firmicutes. The main phylum in JL3 and JZL varieties was Proteobacteria. The observations showed that GLD had the highest diversity of endophytes as indicated by the Shannon index (GLD > JZL > JL3). The order of the endophytes richness was GLD > JL3 > JZL. The PCA and PCoA analysis revealed the microbial communities were different between three different asparagus varieties, and the microbial composition of GLD and JZL was more similar. This report provides an important reference for the study of endophytic microorganisms of asparagus. Supplementary informationThe online version contains supplementary material available at (10.1007/s12088-021-00926-6) contains supplementary material, which is available to authorized users.     

一株猪源化脓隐秘杆菌的鉴定、生物学特性研究与基因组分析

[背景] 化脓隐秘杆菌作为一种条件致病菌,常与蓝耳病病毒、猪圆环病病毒、巴氏杆菌等混合感染猪,引起各种非特异性化脓性感染,部分临床症状与副猪嗜血杆菌相似。本试验从江西某猪场一起因呼吸道症状急性死亡病猪的肺脏分离到一株具有β溶血特性、疑似化脓隐秘杆菌的细菌病原。[目的] 鉴定该病原的菌属种类、生物学特性及基因组特征,为该菌疾病的基础研究与临床防控奠定理论基础。[方法] 采用透射电镜、扫描电镜结合PCR鉴定等方法对菌属种类进行鉴定;通过动物实验、药敏试验,分析菌株的生物学特性;借助全基因组测序、生物信息学等技术发掘该菌的基因组特征。[结果] 形态观察、16S rRNA基因鉴定及系统发育分析证实,该分离菌株是化脓隐秘杆菌,命名为JX18;药敏试验表明,该菌株对克林霉素和林可霉素不敏感,对其余所选药物均敏感;动物实验结果显示,对小鼠腹腔攻毒后,小鼠腹部出现明显脓肿病灶,最高剂量组的小鼠全部死亡;全基因组测序结果表明,该菌株携带plo、nanH、nanP、fimA、fimC、fimE等重要毒力基因,并且与其他化脓隐秘杆菌菌株毒力基因的相似性较高;根据同源基因数据库（Cluster of Orthologous Groups,COG）预测,菌株JX18中参与碳水化合物代谢的基因占比最高;通过毒力基因数据库（virulence factor database,VFDB）、UniProtKB/Swiss-Prot等数据库预测,JX18菌株基因组中携带有多个组氨酸激酶、反应调节因子等与细菌双组分调控系统相关基因,以及多种与粘附、侵入及分泌系统相关的毒力基因。[结论] 在江西省分离鉴定出一株猪源强致病性化脓隐秘杆菌,丰富了猪源化脓隐秘杆菌病原信息,为进一步开展猪源化脓隐秘杆菌相关研究与防控奠定了理论依据。     

布鲁氏菌毒力因子研究进展

布鲁氏菌是一种革兰氏阴性、兼性胞内寄生菌,可引起人畜共患病布鲁氏菌病。布鲁氏菌致病机制复杂,可通过表达多种毒力因子等方式躲避或抑制宿主免疫系统的攻击并发挥其对机体的致病效应,实现其在宿主体内的长期存活。因此,布鲁氏菌病易转化为慢性感染。本文对目前已发现的多种布鲁氏菌毒力因子相关研究进展进行综述,以期进一步认识布鲁氏菌病的致病机理,为布鲁氏菌病防治提供参考。     

溶藻弧菌群体基因组学研究

溶藻弧菌(Vibrio alginolyticus)是一种能够对人类以及鱼、虾、贝类等水产品致病的弧菌,给人类健康带来威胁,也给水产养殖业造成巨大的经济损失。目前该物种基于全基因组的遗传多样性和重要遗传元件研究报道较少。本研究对采集自全国4个省份的68株溶藻弧菌进行高通量测序,获得全基因组序列,并结合113株公开发表的全球序列数据,利用fineSTRUCTURE软件、VFDB毒力因子库和CARD、ResFinder耐药数据库,对溶藻弧菌的种群结构和毒力、耐药因子分别进行解析。结果表明:溶藻弧菌可分为谱系1和谱系2。两个谱系在美洲和亚洲均有分布,但欧洲仅分离到谱系1菌株;共鉴定发现12个克隆群,其中一个克隆群内菌株存在跨洋传播现象。该物种携带tlh、OmpU、IlpA等多种不同功能的毒力因子;毒力因子在两个谱系间的分布无特异性,但存在地域间差异:其中欧洲菌株携带VP1611、vcrD、vopD和fleR/flrC的比率低于其他地区,而基因IlpA的携带率则明显高于其他地区,我国广西菌株中fleR/flrC基因携带率低于其他省份,且不携带IlpA。多个基因组携带blaCARB-42、tet(34)、tet(35)、parE、CRP、rsmA、TxR和fos等与多种抗生素耐受相关的基因,其中TxR和fos基因在谱系2中的出现频率远高于谱系1;此外,TxR基因在亚洲菌株中的携带率高于美洲和欧洲地区,而在我国四川菌株中的携带率则低于其他省份。在5个基因组中(VA24、VA28、2014V-1011、ZJ-T和Vb1833)观察到质粒或ICE等携带多种耐药基因的大片段。本研究通过群体基因组学的研究方法,揭示了溶藻弧菌的种群结构组成和毒力、耐药相关元件的分布,为进一步了解溶藻弧菌的遗传特征和致病机制提供必要基础,为该病原的监测、预防和控制工作提供科学支撑。     

CRISPR在食源性致病菌进化分析、检测分型及毒力耐药调控中的应用进展

规律成簇间隔的短回文重复序列 (Clustered regularly interspaced short palindromic repeats,CRISPR) 与其相关蛋白基因系统可通过限制基因的水平转移而有效防御噬菌体等外源基因元件的入侵,不同细菌之间的CRISPR结构有所差异。基于CRISPR系统的差异性,文中对近几年CRISPR在食源性致病菌进化分析、检测与分型、毒力与耐药中的应用研究进行概述,并对基于CRISPR序列特点开发的细菌检测分型方法以及CRISPR与食源性致病菌的毒力、耐药性之间的相关性进行重点总结分析。此外,文中探讨了CRISPR在进化分析、检测与分型、毒力与耐药应用中的不足,提出将CRISPR分型方法标准化、完善与扩充致病菌CRISPR数据库、进一步探究噬菌体与细菌之间的共进化关系等建议,为进一步探索CRISPR功能提供参考。     

海南地区与环渤海湾美人鱼发光杆菌美人鱼亚种水产动物分离株的表型与遗传特征分析

美人鱼发光杆菌美人鱼亚种（Photobacterium damselae subsp.damselae,PDD）是一种广泛分布于海洋环境内的重要病原菌,可导致多种海洋生物患病死亡,近年来在我国不同养殖区域内均有发现和报道。[目的] 通过生理代谢表型、毒力基因分布和分子遗传分析,系统比较我国海南地区和环渤海湾不同株系PDD的表型与遗传多样性特征。[方法] 使用选择性培养基测定PDD的蔗糖发酵能力、运动性、溶血性和磷脂酶活性;通过PCR检测质粒pPHDD1复制起始点基因pPHDD1 replication origin和毒力基因dly,hlyA_pl,hlyA_ch,plpV;基于toxR基因序列构建系统发育树,分析16株PDD的遗传关系。[结果] 4株PDD蔗糖发酵测试呈阳性,12株PDD蔗糖发酵测试呈阴性。不同株系PDD运动能力存在显著差异,其中环渤海湾分离株Pdd1608、Pdd2009和Pdd1704的运动能力较强。仅有环渤海湾分离株Pdd1608表现为强溶血性、强磷脂酶活性;海南地区分离株Pdd1612和Pdd0912表现为弱溶血性、无磷脂酶活性;其余菌株表现为弱溶血性、弱磷脂酶活性。菌株Pdd1608的毒力基因谱为pPHDD1 replication origin-dly-hlyA_p-hlyA_ch-plpV,其余菌株的毒力基因谱为hlyA_ch-plpV。系统发育分析揭示不同株系PDD具有高度的遗传多样性。[结论] 流行于海南地区和环渤海湾的PDD可能都由多克隆种群组成,它们具有复杂的表型特征、相似的毒力基因分布和高度的遗传多样性。     

Crystal Structure and Pathophysiological Role of the Pneumococcal Nucleoside-binding Protein PnrA

Nucleotides are important for RNA and DNA synthesis and, despite a de novo synthesis by bacteria, uptake systems are crucial. Streptococcus pneumoniae, a facultative human pathogen, produces a surface-exposed nucleoside-binding protein, PnrA, as part of an ABC transporter system. Here we demonstrate the binding affinity of PnrA to nucleosides adenosine, guanosine, cytidine, thymidine and uridine by microscale thermophoresis and indicate the consumption of adenosine and guanosine by ¹H NMR spectroscopy. In a series of five crystal structures we revealed the PnrA structure and provide insights into how PnrA can bind purine and pyrimidine ribonucleosides but with preference for purine ribonucleosides. Crystal structures of PnrA:nucleoside complexes unveil a clear pattern of interactions in which both the N- and C- domains of PnrA contribute. The ribose moiety is strongly recognized through a conserved network of H-bond interactions, while plasticity in loop 27–36 is essential to bind purine- or pyrimidine-based nucleosides.Further, we deciphered the role of PnrA in pneumococcal fitness in infection experiments. Phagocytosis experiments did not show a clear difference in phagocytosis between PnrA-deficient and wild-type pneumococci. In the acute pneumonia infection model the deficiency of PnrA attenuated moderately virulence of the mutant, which is indicated by a delay in the development of severe lung infections. Importantly, we confirmed the loss of fitness in co-infections, where the wild-type out-competed the pnrA-mutant. In conclusion, we present the PnrA structure in complex with individual nucleosides and show that the consumption of adenosine and guanosine under infection conditions is required for virulence.     

African Swine Fever Virus MGF-110-9L-deficient Mutant Has Attenuated Virulence in Pigs

African swine fever virus(ASFV) is the etiological agent of African swine fever(ASF), an often lethal disease in domestic and wild pigs. ASF represents a major threat to the swine industry worldwide. Currently, no commercial vaccine is available because of the complexity of ASFV or biosecurity concerns. Live attenuated viruses that are naturally isolated or genetically manipulated have demonstrated reliable protection against homologous ASFV strain challenge. In the present study, a mutant ASFV strain with the deletion of ASFV MGF-110-9 L(ASFV-D9 L) was generated from a highly virulent ASFV CN/GS/2018 parental strain, a genotype II ASFV. Relative to the parental ASFV isolate, deletion of the MGF-110-9 L gene significantly decreased the ability of ASFV-D9 L to replicate in vitro in primary swine macrophage cell cultures. The majority of animals inoculated intramuscularly with a low dose of ASFV-D9 L(10 HAD50) remained clinically normal during the 21-day observational period. Three of five ASFV-D9 L-infected animals displayed low viremia titers and low virus shedding and developed a strong virus-specific antibody response, indicating partial attenuation of the ASFV-D9 L strain in pigs. The findings imply the potential usefulness of the ASFV-D9 L strain for further development of ASF control measures.     

利用细菌人工染色体技术构建整合F基因的重组MDV疫苗株*

目的:预防马立克氏病病毒(MDV)和新城疫病毒(NDV)混合感染鸡引起的疾病,构建表达NDV F蛋白的MDV疫苗株CVI988 BAC重组载体,并包装成重组病毒,为疫苗免疫提供更多的重组疫苗选择。方法:首先利用PCR扩增带有卡那霉素(Kanamycin,Kana)抗性基因片段的F基因,采用同源重组的方法将其整合到CVI988 BAC上,进一步诱导I-SceI表达敲除Kana基因而获得重组质粒CVI988 BAC-F。通过磷酸钙法转染鸡胚成纤维细胞获得重组病毒。结果:Western blot和间接免疫荧光实验证实重组病毒能够表达F蛋白。病毒生长曲线和蚀斑大小测定结果表明,F基因的插入不影响病毒的体外增殖。结论:利用BAC技术成功构建了整合F基因的重组MDV病毒CVI988 BAC-F,为MDV重组疫苗研发,防控NDV与MDV共感染奠定了基础。     

优化两步输注美罗培南联合参麦注射液对重症感染患者血清感染指标、细菌清除率和T淋巴细胞亚群的影响

目的:探讨优化两步输注美罗培南联合参麦注射液对重症感染患者血清感染指标、细菌清除率和T淋巴细胞亚群的影响。方法:选取2017年1月-2018年10月在我院重症医学科(ICU)住院的153例重症感染患者,按随机数字表法将患者分为传统输注美罗培南组(A组,54例)、优化两步输注美罗培南组(B组,49例)、优化两步输注美罗培南联合参麦注射液组(C组,50例)。比较治疗后三组患者的总体疗效、血清感染指标和T淋巴细胞亚群水平及不良反应发生情况。结果:B组、C组的临床有效率、细菌清除率、28天生存率高于A组(P<0.05);B组、C组机械通气时间、ICU住院时间、总住院时间少于A组,且C组少于B组(P<0.05);治疗后B组、C组CD3⁺、CD4⁺、CD4⁺/CD8⁺高于A组,且C组高于B组(P<0.05);B组、C组CD8⁺、白细胞计数(WBC)、降钙素原(PCT)、C-反应蛋白(CRP)水平低于A组,且C组低于B组(P<0.05)。三组患者不良反应发生率比较无差异(P>0.05)。结论:优化两步输注美罗培南联合参麦注射液治疗重症感染患者疗效确切,可提高细菌清除率,改善患者免疫状态,促进患者康复。