Detection of Laribacter hongkongensis using species-specific duplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (ISR)

Aims: For the rapid detection of Laribacter hongkongensis, which is associated with human community‐acquired gastroenteritis and traveller’s diarrhoea, we developed a duplex species‐specific PCR assay. Methods and Results: Full‐length of the 16S–23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR‐based sequencing. Two species‐specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species‐specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2·1 × 10^?2 ng μl^?1 genomic DNA which corresponds to 5000 CFU ml^?1. Conclusions: The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. Significance and Impact of the Study: This species‐specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.     

Identification and characterization of bacterial isolates from the Mir space station

Twenty bacterial isolates (supplied by NASA) from the Mir space station water system were identified using Vitek GNI+ test card, API 20NE, and 16S rRNA gene sequencing. The identification of only one isolate agreed among the three techniques. The utility of the API 20NE and Vitek GNI+ test card approaches for identifying these isolates was Limited. Although 16S rRNA gene sequencing effectively identified many of the bacteria to the genus level, 74% of the isolates could not be identified to the species level. Isolates were also characterized based on motility and hydrophobicity. About 40% of the isolates were motile and four isolates were hydrophobic, suggesting that many of the bacteria have the potential to colonize surfaces and form biofilms. These findings demonstrate the difficulties in identifying bacteria from some environments to the species level and have implications for determining the risks of contamination in water systems of space shuttles and stations.     

硇洲岛潮汐带牡蛎相关可培养细菌多样性

[目的]了解南海硇洲岛潮汐带香港巨牡蛎(Crassostrea hongkongensis)相关可培养细菌的多样性.[方法]应用纯培养法分离样品中的细菌(含放线菌),采用基于16S rRNA基因序列的系统发育分析方法研究这些分离菌株的生物多样性.[结果]用补充0-25％(W/V) NaC1的MA、MH和NA培养基从样品中分离到102株细菌,在形态观察和部分生理生化实验结果的基础上去冗余,选取74个代表性菌株进行基于16S rRNA基因序列的系统发育分析.结果表明,这些菌株归为38个物种,属于4个大的系统发育类群(Gamma-Proteobacteria,Alpha-Proteobacteria,Bacteroidetes,Firmicutes)的16个科、18个属.大多数菌株属于Gamma-Proteobacteria亚门(45株,60.8％),其余依次是Firmicutes门(12株,16.2％)、Bacteroidetes门(11株,14.9％)和Alpha-Proteobacteria亚门(6株,8.1％).大多数菌株与其系统发育关系最密切的有效发表种典型菌株之间(16S rRNA基因序列相似性为94.3％-99.8％)存在一定的遗传差异,其中JSM 111069可能代表Carnobacteriaceae科的潜在新属;菌株JSM 111039和JSM 111085可能分别代表Bacillus属的两个新物种,菌株JSM 111020、JSM 111072和JSM 111090可能分别代表Pseudoalteromonas、Proteus和Idiomarina属的新物种.[结论]南海硇洲岛潮汐带牡蛎中存在较为丰富的原核生物多样性,并潜藏着一定数量的微生物新类群(物种).     

仙山湖国家湿地公园湿地与旱地土壤细菌群落结构特征比较

【背景】湿地土壤微生物群落结构会因环境的变化自我调整,因而不同湿地环境会形成不同土壤菌群结构和分布特征。【目的】研究旱地与湿地土壤不同利用方式对土壤细菌的影响,以仙山湖国家湿地公园湿地和旱地两种土地利用方式(0−20 cm)作为研究对象。【方法】利用高通量测序技术对土壤细菌群落的物种组成、α多样性和丰度等进行分析,并结合土壤理化因子分析影响菌群结构的环境因子。【结果】仙山湖湿地系统土壤中存在的细菌隶属于49个门147个纲。在仙山湖湿地土壤的优势菌门(OTU>1%)中,旱地的优势菌门为酸杆菌门(Acidobacteria)、变形菌门(Proteobacteria)、绿弯菌门(Chloroflexi)、泉古菌门(Crenarchaeota)、放线菌门(Actinobacteria)、蓝藻门(Cyanobacteria)、芽单胞菌门(Gemmatimonadetes)、浮霉菌门(Planctomycetes)和拟杆菌门(Bacteroidetes);湿地的优势菌门为变形菌门(Proteobacteria)、蓝藻门(Cyanobacteria)、拟杆菌门(Bacteroidetes)、绿弯菌门(Chloroflexi)、酸杆菌门(Acidobacteria)、厚壁菌门(Firmicutes)和硝化螺旋菌门(Nitrospirae)。在优势菌属(OTU>0.3%)中,旱地的优势菌属为Candidatus Solibacter、柯里氏念珠菌属(Candidatus Koribacter)、红游动菌属(Rhodoplanes)、伯克霍尔德菌属(Burkholderia)和分枝杆菌属(Mycobacterium),湿地的优势菌属为地杆菌属(Geobacter)、拟杆菌属(Bacteroides)、慢生型根瘤菌属(Bradyrhizobium)、Dok59、神奇甲基菌属(Candidatus Methylomirabilis)和黄杆菌属(Flavobacterium)。湿地土壤与旱地土壤优势OTU均与有机质含量呈正相关,与含水量、pH呈负相关。此外,线性判别分析(linear discriminant analysis effect size, LEfSe)显示了旱地与湿地的土壤生物标记物与土壤理化性质的关系。【结论】土地利用方式变化能显著改变仙山湖国家湿地公园湿地土壤的细菌菌群结构,可为湿地土壤的保护提供理论依据。     

PCR反应条件对16S rRNA基因扩增子测序准确性的影响

【背景】16S rRNA基因扩增子测序技术是一种不依赖培养而获得样本中细菌种群结构、相对丰度等信息的方法。高通量测序技术实验步骤较多,每一步骤细微的差别都可能在最终的测序结果中放大,并造成测序结果与实际情况的偏差。【目的】基于MiSeq测序平台,探讨PCR反应体系中扩增引物序列、退火温度、模板起始量、扩增循环数和变性时间等5个因素对16S rRNA基因测序结果的影响。【方法】对mock DNA的16S rRNA基因扩增子进行测序,分别分析不同的扩增引物、退火温度、模板起始量、循环数和变性时间对数据准确性的影响。【结果】不同的扩增引物对检测结果有较大的影响,采用的4组引物中,引物B (V3–V4,341F/806R)的准确性最好,引物A(V3–V4,341F/805R)次之。比较不同退火温度(52、55和60℃)对检测准确性的影响,退火温度60℃的结果最接近理论值。模板起始量(2、10和50 ng)的检测结果显示,mock DNA起始量为2ng的结果准确性最高。相较于其他3组(15+18、25+8和30+8),循环数为(20+8)的检测结果最接近mock DNA的理论值。不同变性时间(3...     

绝经综合征患者肠道菌群特征

研究绝经综合征患者的肠道菌群特征,为该类患者的治疗提供参考。

选取77例绝经综合征患者为病例组（P组）,24例绝经前后健康女性为健康对照组（H组）,采用16S rRNA基因测序技术检测两组对象肠道菌群结构,计算临床参数与肠道菌群的Spearman相关系数并采用PICRUSt2进行菌群的功能预测。

两组对象肠道菌群的alpha和beta多样性差异均无统计学意义（均P＞0.05）。LEfSe分析显示两组对象肠道菌群存在显著差异的物种（种水平）有14个（LDA分数＞2.0）。Spearman相关分析发现,雌二醇与聚集杆菌、动物双歧杆菌动物亚种和吉氏不动杆菌均呈正相关（均P＜0.05）,这3种细菌在绝经前后健康女性中数量较高,而卵泡刺激素、黄体生成素水平与上述3个物种均呈负相关（均P＜0.05）。功能预测显示,与心血管疾病和碳水化合物代谢相关的KEGG L3代谢途径在P组患者肠道菌群中富集（均P＜0.05）。

绝经综合征患者存在肠道菌群结构紊乱,表现为与性激素水平相关的聚集杆菌、动物双歧杆菌动物亚种和吉氏不动杆菌丰度降低,同时在绝经综合征患者中存在独特的代谢途径。

     

采用高通量测序技术研究抗生素对小鼠肠道菌群的影响

目的 探讨不同浓度抗生素对小鼠肠道菌群多样性和结构的影响,并预测相关功能变化。方法 15只SPF级ICR小鼠随机分为正常组、低浓度抗生素组和高浓度抗生素组,连续灌胃5 d后,采集小鼠新鲜粪便样本。利用Illumina MiSeq测序平台,对细菌的16S rRNA V3‒V4区进行高通量测序,并对测序结果进行生物信息学分析。结果 高、低浓度抗生素组小鼠肠道菌群组成与正常组存在明显差异。与正常组相比,高剂量组小鼠肠道肠球菌属、志贺埃希菌属相对丰度显著升高（t=‒2.71,P=0.026;t=‒2.30,P<0.05）;分节丝状菌属、拟普雷沃菌属相对丰度显著降低（t=2.88,P=0.020;t=2.49,P=0.037）,理研菌属极显著降低（t=3.79,P=0.005）。低剂量组小鼠肠道菌群变形菌纲成为优势菌,芽胞杆菌属、粪球菌_2、苏黎世杆菌属、普雷沃菌属_2、普雷沃菌属_7、志贺埃希菌属、沙雷菌属和放线菌属等相对丰度显著升高（均P<0.05）;梭杆菌属、泛菌属极显著升高（t=‒3.19,P=0.013;t=‒3.50,P=0.008）;分节丝状菌属、理研菌属相对丰度显著降低（t=2.69,P=0.028;t=2.33,P=0.048）。PICRUSt功能预测分析显示,抗生素组显著增加人类疾病、细胞过程和环境信息处理功能层的基因拷贝数,显著降低有机系统、遗传信息处理和代谢功能层的基因拷贝数。结论 广谱抗生素能破坏小鼠肠道的微生态平衡,有必要深入研究抗生素对心血管、免疫性、感染性及神经退行性疾病发展的潜在作用。     

宁夏地区春季奶牛场牛舍土壤细菌群落特征

【背景】现代规模化生产模式下,牛舍环境管理是影响奶牛高效健康生产的重要因素。【目的】探讨牛场不同牛舍土壤细菌群落特征,为奶牛健康生产提供理论依据。【方法】采集宁夏某规模化奶牛场的哺乳犊牛岛、断奶犊牛舍、育成牛舍、低产泌乳牛舍、高产头胎泌乳牛舍、高产经产泌乳牛舍、干奶牛舍和病牛舍这8个不同牛舍的土样,每个牛舍6个重复,共48份土样。利用16S rRNA基因扩增子测序分析细菌群落结构与多样性,并对细菌群落的功能进行预测。【结果】不同牛舍土样细菌群落组成存在差异,并且8个牛舍中高产头胎泌乳牛舍土样的细菌群落多样性最高。哺乳犊牛岛土壤与其他牛舍土壤细菌群落在门水平上差异较大;泌乳期牛舍土样之间的细菌群落结构相似度较高。在门的水平上,拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和厚壁菌门(Firmicutes)是这8个牛舍土样共有的优势菌门。在属的水平上,嗜盐碱的盐单胞菌属(Halomonas)、具有潜在降解特性的Fermentimonas和栖海面菌属(Aequorivita)及致病菌的鸟杆菌属(Ornithobacterium)是犊牛期牛舍土样的优势菌属;嗜盐碱的Truepera是育成牛舍土样的优势菌属;致病菌的不动杆菌属(Acinetobacter)和Parapedobacter、耐药菌的Pedobacter是泌乳期牛舍土样的优势菌属。【结论】致病菌和参与硝酸盐呼吸的细菌主要分布在哺乳犊牛岛,嗜盐碱菌主要分布在断奶犊牛舍和育成牛舍,产甲烷的细菌主要分布在高产头胎泌乳牛舍。本研究分析了不同牛舍土壤细菌群落多样性,为奶牛健康生产提供理论依据。     

中央戈壁石下生物土壤结皮中细菌群落结构和多样性

【背景】戈壁石下生物土壤结皮(Biological soil crusts,BSCs)中的微生物在干旱、半干旱地区生态系统物质循环中起重要作用,其细菌群落结构和多样性因气候和地理环境的不同而存在较大差异。中央戈壁面积大,其石下BSCs中细菌群落结构和多样性却知之甚少。【目的】比较中央戈壁石下BSCs和周围裸土中细菌群落结构和多样性的异同,揭示环境因子对它们的影响以及细菌之间的相互作用关系。【方法】利用Illumina MiSeq对16S rRNA基因进行高通量测序,使用生物信息学分析方法分析细菌群落结构和多样性及其影响因素;基于CoNet软件绘制物种共现性网络图,利用Cytoscape 3.5.1软件将网络图可视化。【结果】石下BSCs的速效磷(Available phosphorus,AP)、速效氮(Available nitrogen,AN)和叶绿素a (Chlorophyll a,Chl a)的含量均明显高于周围裸土(最高高出471%),而pH则略低于周围裸土;石下BSCs中细菌的最优势菌门为Cyanobacteria(45.85%-53.77%),优势属(9.25%-18.42%)有Trichocoleus、Chroococcidiopsis和属于Cyanobacteria纲的1个未知属;临近裸土中细菌的最优势菌门为Actinobacteria (38.82%-44.69%),优势属(5%)有Arthrobacter、Rubrobacter和其它3个属于放线菌门或酸杆菌门的未知属;在土壤理化因子中,AP对石下细菌群落组成的影响最大;除Actinophytocola属和Cyanobacteria纲中的未知属外,其他微生物之间均存在较强的互作关系,其中以共存的互作关系为主(约占60%),点度中心性、接近中心性和中介中心性均较高的节点都属于Cyanobacteria门和α-Proteobacteria纲。【结论】中央戈壁石下BSCs中的细菌群落结构和多样性明显不同于临近裸土,Cyanobacteria门和α-Proteobacteria纲的细菌是该地区石下早期BSCs形成和发育最初及最重要的驱动力。     

Genetic differences among three species of the genus Trichiurus (Perciformes: Trichiuridae) based on mitochondrial DNA analysis

The mitochondrial DNA segment encoding the 16S ribosomal RNA (16S rRNA) gene sequence (ca. 600 bp) was compared among Trichiurus sp. 2 (sensu Nakabo, 2002) (obtained from various areas of Japan), T. japonicus Temminck and Schlegel (collected from various localities within Japan), and true T. lepturus Linnaeus (caught off the Atlantic coast of the United States and Brazil) of the family Trichiuridae using 10, 10, and 15 specimens, respectively. Based on phylogenetic analysis using a neighbor-joining (NJ) algorithm, the haplotypes of Trichiurus sp. 2, T. japonicus, and T. lepturus indicated three distinct monophyletic lineages, being supported by 100% bootstrap values with no haplotypes overlapping or sharing among the lineages. Trichiurus sp. 2, T. japonicus, and T. lepturus are genetically different from each other, suggesting that they are three distinct species.     

2020微生物组测序与分析专题序言

微生物是人体、动植物、土壤、沉积物、水体、空气等生境中最重要的生命体。对这些生境中微生物的分析已经成为一项基础的研究技术。微生物组测序与分析作为近年来快速发展的技术,已经在人类健康、环境污染治理、食品工业以及农牧业等领域得到了广泛应用。为了梳理和总结微生物组测序与分析技术的现状、发展状况和应用前景,本专题收录了16篇本领域的论文,分别从样本保存和处理、单菌基因组测序与分析、特殊生境中的微生物组特征分析、微生物组相关数据库和算法以及微生物组测序与分析专家共识等方面,详细介绍了微生物组测序与分析领域的发展态势,为推动我国微生物组测序与分析产业和科研的快速发展、促进微生物组相关产业的良性发展提供必要的参考。     

Actinobacterial diversity from marine sediments collected in Mexico

Seventeen different media known to support the growth and isolation of members of the class Actinobacteria were evaluated as selective isolation media for the recovery of this microbial group from marine sediments samples collected in the Gulf of California and the Gulf of Mexico. A general selective isolation procedure was employed for six sediments and nearly 300 actinomycetes were recovered from the selective isolation plates. Full 16S rRNA gene sequencing revealed that the isolates belonged to several actinobacterial taxa, notably to the genera Actinomadura, Dietzia, Gordonia, Micromonospora, Nonomuraea, Rhodococcus, Saccharomonospora, Saccharopolyspora, Salinispora, Streptomyces, “Solwaraspora” and Verrucosispora. Previous works on marine sediments have been restricted to the isolation of members of the genera Micromonospora, Rhodococcus and Streptomyces. This study provides further evidence that Actinobacteria present in marine habitats are not restricted to the Micromonospora-Rhodococcus-Streptomyces grouping. Indeed, this first systematic study shows the extent of actinobacterial diversity that can be found in marine sediments collected in Mexico and probably, worldwide. The 16S rRNA gene sequences of marine isolates A1, AA2, AA6, AB1, AB2, AG1, AI2, AK1, AL2, AO1, AO3, AR1, AW1, B1, BB1, BC1, C5, R1, R2, R3, AV1, AE1, AI1, AN1 and AP1 determined in this study have been deposited under GenBank accession numbers EU714241–EU714258 and FJ462359–FJ462365, respectively.     

Stability and microbial community structure of a partial nitrifying fixed-film bioreactor in long run

A partial nitrification system was investigated for 471 days under DO varying concentrations for assessing its stability and population dynamics. Within 130 days of operation at feed DO concentration of 1.0 ± 0.1 mg/L, more than 85% of nitrite was accumulated. Efficiency deteriorated when the feed DO concentration was increased to 4.2 ± 0.3 mg/L. Nitrite accumulation could not be re-established on decreasing feed DO to 1.0 ± 0.1 mg/L. Even at DO concentration of <0.05 mg/L, nitrate production was observed; a condition termed as anoxic nitrification. NOB was detected in the biomass even under this condition by Fluorescence in-situ hybridization (FISH) analysis. Through 16S rRNA gene sequencing a major fraction of unknown bacterial sequences closely resembling haloalkalophilic bacteria of marine origin were detected. The study indicated that these bacterial species might play a role in anoxic nitrification and that NOB could survive extreme low DO condition.     

早产儿高胆红素血症与肠道菌群的相关性

目的探讨高胆红素血症早产儿与非高胆红素血症早产儿肠道菌群的差异,为该病的治疗提供思路和依据。方法选择2017年1月至6月出生后即在本院新生儿科治疗的早产儿(胎龄35+1～36+6周)为研究对象,其中高胆红素血症早产儿(黄疸组)124例,非高胆红素血症早产儿(对照组)118例。采集两组对象出生后24 h内的首次胎便,采用高通量测序技术对粪便样本中细菌16S rRNA基因进行检测,分析肠道菌群组成情况。结果黄疸组早产儿肠道微生物群落组成与对照组在门水平上相似度较高。在属水平上,两组对象肠道菌群均以Ralstonia、Rhodococcus、Enterococcus为主。对照组患儿肠道Lactobacillus的相对丰度显著高于黄疸组(t=-2.073 0,P=0.039 1)。黄疸组患儿肠道Ralstonia的相对丰度与总胆红素水平呈负相关(r=-0.314 8,P=0.000 3)。结论高胆红素血症早产儿与非高胆红素血症早产儿肠道菌群的种类和丰度存在差异。Lactobacillus可能是高胆红素血症的潜在生物标记物。