Volume: 20, Issue: 2

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The Regulation of Gonadal Somatic Cell Differentiation in Humans

Min Chen, Fei Gao

Page 219-222

Original Research

Dissecting Human Gonadal Cell Lineage Specification and Sex Determination Using A Single-cell RNA-seq Approach

Rui Wang, Xixi Liu, Li Li, Ming Yang, Jun Yong, Fan Zhai, Lu Wen, Liying Yan, Jie Qiao, Fuchou Tang

Gonadal somatic cells are the main players in gonad development and are important for sex determination and germ cell development. Here, using a time-series single-cell RNA sequencing (scRNA-seq) strategy, we analyzed fetal germ cells (FGCs) and gonadal somatic cells in human embryos and fetuses. Clustering analysis of testes and ovaries revealed several novel cell subsets, including POU5F1+SPARC+ FGCs and KRT19+ somatic cells. Furthermore, our data indicated that the bone morphogenetic protein (BMP) signaling pathway plays cell type-specific and developmental stage-specific roles in testis development and promotes the gonocyte-to-spermatogonium transition (GST) in late-stage testicular mitotic arrest FGCs. Intriguingly, testosterone synthesis function transitioned from fetal Sertoli cells to adult Leydig cells in a stepwise manner. In our study, potential interactions between gonadal somatic cells were systematically explored and we identified cell type-specific developmental defects in both FGCs and gonadal somatic cells in a Turner syndrome embryo (45, XO). Our work provides a blueprint of the complex yet highly ordered development of and the interactions among human FGCs and gonadal somatic cells.
研究问题： 人类性腺体细胞对性腺的发育、性腺的性别决定以及生殖细胞的发育发挥着重要的作用。基于小鼠模型，我们已经对哺乳动物性腺的发育、性别分化以及性发育障碍具有较多了解。但是由于物种的差异以及人类样品的相对稀缺性等原因，目前对人类性腺的发育，尤其对于人类性腺体细胞的发育的研究还不够深入。 研究方法： 为了进一步探索人类性腺的性别决定和谱系分化的机制，该研究利用单细胞转录组测序技术10X 平台对6-23周的人类16个胎儿性腺的55,404个性腺细胞进行了系统、深入研究。并通过对睾丸组织细胞共培养探究了BMP信号通路对生殖细胞和睾丸体细胞的作用和调控机制。 主要结果： 1. 鉴定发现了新的性腺生殖细胞和体细胞亚型 2. 特纳综合征胎儿（45， XO）中的胎儿生殖细胞（FVGC）和性腺体细胞显示出细胞类型特异性缺陷。 3. 功能实验证明BMP信号通路在人类胚胎的睾丸发育中发挥细胞类型特异性和发育阶段特异性的关键作用。 4. 睾丸的Leydig细胞和Sertoli细胞之间通过互相调控促进对方的发育进程。 5. 对雌性颗粒细胞和雌雄性类固醇谱系潜在的发育起源进行了推断。

Page 223-245

Original Research

Over 50,000 Metagenomically Assembled Draft Genomes for the Human Oral Microbiome Reveal New Taxa

Jie Zhu, Liu Tian, Peishan Chen, Mo Han, Liju Song, Xin Tong, Xiaohuan Sun, Fangming Yang, Zhipeng Lin, Xing Liu, Chuan Liu, Xiaohan Wang, Yuxiang Lin, Kaiye Cai, Yong Hou, Xun Xu, Huanming Yang, Jian Wang, Karsten Kristiansen, Liang Xiao, Tao Zhang, Huijue Jia, Zhuye Jie

The oral cavity of each person is home to hundreds of bacterial species. While taxa for oral diseases have been studied using culture-based characterization as well as amplicon sequencing, metagenomic and genomic information remains scarce compared to the fecal microbiome. Here, using metagenomic shotgun data for 3346 oral metagenomic samples together with 808 published samples, we obtain 56,213 metagenome-assembled genomes (MAGs), and more than 64% of the 3589 species-level genome bins (SGBs) contain no publicly available genomes. The resulting genome collection is representative of samples around the world and contains many genomes from candidate phyla radiation (CPR) that lack monoculture. Also, it enables the discovery of new taxa such as a genus Candidatus Bgiplasma within the family Acholeplasmataceae. Large-scale metagenomic data from massive samples also allow the assembly of strains from important oral taxa such as Porphyromonas and Neisseria. The oral microbes encode genes that could potentially metabolize drugs. Apart from these findings, a strongly male-enriched Campylobacter species was identified. Oral samples would be more user-friendly collected than fecal samples and have the potential for disease diagnosis. Thus, these data lay down a genomic framework for future inquiries of the human oral microbiome.

Page 246-259

Original Research

Metagenomic Analysis Reveals A Possible Association Between Respiratory Infection and Periodontitis

Zhenwei Liu, Tao Zhang, Keke Wu, Zhongshan Li, Xiaomin Chen, Shan Jiang, Lifeng Du, Saisai Lu, Chongxiang Lin, Jinyu Wu, Xiaobing Wang

Periodontitis is an inflammatory disease that is characterized by progressive destruction of the periodontium and causes tooth loss in adults. Periodontitis is known to be associated with dysbiosis of the oral microflora, which is often linked to various diseases. However, the complexity of plaque microbial communities of periodontitis, antibiotic resistance, and enhanced virulence make this disease difficult to treat. In this study, using metagenomic shotgun sequencing, we investigated the etiology, antibiotic resistance genes (ARGs), and virulence genes (VirGs) of periodontitis. We revealed a significant shift in the composition of oral microbiota as well as several functional pathways that were represented significantly more abundantly in periodontitis patients than in controls. In addition, we observed several positively selected ARGs and VirGs with the Ka/Ks ratio > 1 by analyzing our data and a previous periodontitis dataset, indicating that ARGs and VirGs in oral microbiota may be subjected to positive selection. Moreover, 5 of 12 positively selected ARGs and VirGs in periodontitis patients were found in the genomes of respiratory tract pathogens. Of note, 91.8% of the background VirGs with at least one non-synonymous single-nucleotide polymorphism for natural selection were also from respiratory tract pathogens. These observations suggest a potential association between periodontitis and respiratory infection at the gene level. Our study enriches the knowledge of pathogens and functional pathways as well as the positive selection of antibiotic resistance and pathogen virulence in periodontitis patients, and provides evidence at the gene level for an association between periodontitis and respiratory infection.

Page 260-273

Original Research

Effects of Resistant Starch on Symptoms, Fecal Markers, and Gut Microbiota in Parkinson’s Disease — The RESISTA-PD Trial

Anouck Becker, Georges Pierre Schmartz, Laura Gröger, Nadja Grammes, Valentina Galata, Hannah Philippeit, Jacqueline Weiland, Nicole Ludwig, Eckart Meese, Sascha Tierling, Jörn Walter, Andreas Schwiertz, Jörg Spiegel, Gudrun Wagenpfeil, Klaus Faßbender, Andreas Keller, Marcus M. Unger

The composition of the gut microbiota is linked to multiple diseases, including Parkinson’s disease (PD). Abundance of bacteria producing short-chain fatty acids (SCFAs) and fecal SCFA concentrations are reduced in PD. SCFAs exert various beneficial functions in humans. In the interventional, monocentric, open-label clinical trial “Effects of Resistant Starch on Bowel Habits, Short Chain Fatty Acids and Gut Microbiota in Parkinson’s Disease” (RESISTA-PD; ID: NCT02784145), we aimed at altering fecal SCFAs by an 8-week prebiotic intervention with resistant starch (RS). We enrolled 87 subjects in three study-arms: 32 PD patients received RS (PD + RS), 30 control subjects received RS, and 25 PD patients received solely dietary instructions. We performed paired-end 100 bp length metagenomic sequencing of fecal samples using the BGISEQ platform at an average of 9.9 GB. RS was well-tolerated. In the PD + RS group, fecal butyrate concentrations increased significantly, and fecal calprotectin concentrations dropped significantly after 8 weeks of RS intervention. Clinically, we observed a reduction in non-motor symptom load in the PD + RS group. The reference-based analysis of metagenomes highlighted stable alpha-diversity and beta-diversity across the three groups, including bacteria producing SCFAs. Reference-free analysis suggested punctual, yet pronounced differences in the metagenomic signature in the PD + RS group. RESISTA-PD highlights that a prebiotic treatment with RS is safe and well-tolerated in PD. The stable alpha-diversity and beta-diversity alongside altered fecal butyrate and calprotectin concentrations call for long-term studies, also investigating whether RS is able to modify the clinical course of PD.

Page 274-287

Original Research

Beneficial Effects of Celastrol on Immune Balance by Modulating Gut Microbiota in Experimental Ulcerative Colitis Mice

Mingyue Li, Weina Guo, Yalan Dong, Wenzhu Wang, Chunxia Tian, Zili Zhang, Ting Yu, Haifeng Zhou, Yang Gui, Kaming Xue, Junyi Li, Feng Jiang, Alexey Sarapultsev, Huafang Wang, Ge Zhang, Shanshan Luo, Heng Fan, Desheng Hu

Ulcerative colitis (UC) is a chronic inflammatory bowel disease caused by many factors including colonic inflammation and microbiota dysbiosis. Previous studies have indicated that celastrol (CSR) has strong anti-inflammatory and immune-inhibitory effects. Here, we investigated the effects of CSR on colonic inflammation and mucosal immunity in an experimental colitis model, and addressed the mechanism by which CSR exerts the protective effects. We characterized the therapeutic effects and the potential mechanism of CSR on treating UC using histological staining, intestinal permeability assay, cytokine assay, flow cytometry, fecal microbiota transplantation (FMT), 16S rRNA sequencing, untargeted metabolomics, and cell differentiation. CSR administration significantly ameliorated the dextran sodium sulfate (DSS)-induced colitis in mice, which was evidenced by the recovered body weight and colon length as well as the decreased disease activity index (DAI) score and intestinal permeability. Meanwhile, CSR down-regulated the production of pro-inflammatory cytokines and up-regulated the amount of anti-inflammatory mediators at both mRNA and protein levels, and improved the balances of Treg/Th1 and Treg/Th17 to maintain the colonic immune homeostasis. Notably, all the therapeutic effects were exerted in a gut microbiota-dependent manner. Furthermore, CSR treatment increased the gut microbiota diversity and changed the compositions of the gut microbiota and metabolites, which is probably associated with the gut microbiota-mediated protective effects. In conclusion, this study provides the strong evidence that CSR may be a promising therapeutic drug for UC.
研究问题： 溃疡性结肠炎是一种由多种因素引起的慢性炎症性肠病，可导致肠道炎症性改变和肠道微生物群失调。已有研究表明，雷公藤红素具有很强的抗炎和免疫调节作用。雷公藤红素对DSS诱导的溃疡性结肠炎小鼠模型是否具有治疗作用？其可能机制是什么？ 研究方法： 使用DSS诱导结肠炎小鼠模型，通过组织切片染色、肠道通透性检测、细胞因子检测、流式细胞术、粪菌移植、16S rRNA测序、非靶向代谢组学和体外细胞分化等方法，探究雷公藤红素治疗结肠炎小鼠的效果和其发挥作用的潜在机制。 主要结果： 雷公藤红素可缓解DSS诱导的结肠炎小鼠症状，抑制肠道组织促炎细胞因子而上调抑炎因子的表达，并通过调控T细胞的分化重塑肠道Treg/Th1和Treg/Th17免疫平衡，进而抑制肠道过度炎症反应。然而，使用抗生素“清除”肠道菌群后，雷公藤红素的保护作用消失，随后的粪菌移植实验再一次证实了肠道菌群的介导作用。进一步研究显示雷公藤红素治疗增加了肠道菌群组成的多样性，并通过增加丙酮酸和腺苷等代谢产物，抑制Th1和Th17细胞分化，而促进Treg细胞分化。 数据链接： https://bigd.big.ac.cn/.

Page 288-303

Original Research

Life History Recorded in the Vagino-cervical Microbiome Along with Multi-omes

Zhuye Jie, Chen Chen, Lilan Hao, Fei Li, Liju Song, Xiaowei Zhang, Jie Zhu, Liu Tian, Xin Tong, Kaiye Cai, Zhe Zhang, Yanmei Ju, Xinlei Yu, Ying Li, Hongcheng Zhou, Haorong Lu, Xuemei Qiu, Qiang Li, Yunli Liao, Dongsheng Zhou, Heng Lian, Yong Zuo, Xiaomin Chen, Weiqiao Rao, Yan Ren, Yuan Wang, Jin Zi, Rong Wang, Na Liu, Jinghua Wu, Wei Zhang, Xiao Liu, Yang Zong, Weibin Liu, Liang Xiao, Yong Hou, Xun Xu, Huanming Yang, Jian Wang, Karsten Kristiansen, Huijue Jia

The vagina contains at least a billion microbial cells, dominated by lactobacilli. Here we perform metagenomic shotgun sequencing on cervical and fecal samples from a cohort of 516 Chinese women of reproductive age, as well as cervical, fecal, and salivary samples from a second cohort of 632 women. Factors such as pregnancy history, delivery history, cesarean section, and breastfeeding were all more important than menstrual cycle in shaping the microbiome, and such information would be necessary before trying to interpret differences between vagino-cervical microbiome data. Greater proportion of Bifidobacterium breve was seen with older age at sexual debut. The relative abundance of lactobacilli especially Lactobacillus crispatus was negatively associated with pregnancy history. Potential markers for lack of menstrual regularity, heavy flow, dysmenorrhea, and contraceptives were also identified. Lactobacilli were rare during breastfeeding or post-menopause. Other features such as mood fluctuations and facial speckles could potentially be predicted from the vagino-cervical microbiome. Gut and salivary microbiomes, plasma vitamins, metals, amino acids, and hormones showed associations with the vagino-cervical microbiome. Our results offer an unprecedented glimpse into the microbiota of the female reproductive tract and call for international collaborations to better understand its long-term health impact other than in the settings of infection or pre-term birth.
人体共生菌群细胞数在1014数量级，而阴道、口腔部位的菌群密度仅次于大肠，其中女性阴道菌群细胞数至少有1010数量级，阴道菌群对维持女性的生殖健康至关重要。本研究对两个人群共1148名中国女性多部位样本的宏基因组数据进行研究，包括516位育龄女性（人群1）的宫颈分泌物和粪便样本； 632位女性（人群2）的宫颈分泌物、粪便以及唾液样本。本研究发现了阴道-宫颈菌群新的亚型，并发现包括哺乳、剖腹产等在内的孕产史、婚育年龄等因素对女性生殖道菌群的持久影响，这些因素是影响阴道-宫颈菌群最重要的因素，其影响程度甚至高于月经周期对菌群的影响。此外，研究发现，短双歧杆菌（Bifidobacterium breve）在初始性生活年龄较晚的女性中含量较多；而乳杆菌的含量，尤其是卷曲乳杆菌（Lactobacillus crispatus），跟怀孕史呈负相关。研究还发现一些跟月经不规律、月经量大、痛经、避孕等相关的菌群标志物；乳杆菌在哺乳期女性和绝经期女性的阴道中均变少。研究还收集了其他一些组学数据，多组学分析还发现，激素、免疫、氨基酸、重金属，甚至皮肤状况、心理状态等与也都与阴道-宫颈菌群有潜在的关联，提示生殖道菌群像肠道菌群一样是全面反映人体健康的轴心群落。目前国外对生殖道菌群的研究主要还集中在感染、早产等，本研究首次从多组学的角度对女性生殖道菌群进行全面的探索，展示了与广大女性菌群健康管理相关的更加广泛的研究空间，有待更多人群合作。

Page 304-321

Original Research

Variation of the Vaginal Microbiome During and After Pregnancy in Chinese Women

Xiaoai Zhang, Qingzhi Zhai, Jinfeng Wang, Xiuling Ma, Bo Xing, Hang Fan, Zhiying Gao, Fangqing Zhao, Wei Liu

A comprehensive profiling of the vaginal microbial communities and their variability enables an accurate description of the microbiome in women. However, there is a lack of studies available on Chinese women. In the present study, the composition of the vaginal microbiota during pregnancy and the 6-week postpartum period of 454 Chinese women was characterized by sequencing the V3–V4 region of the 16S ribosomal RNA (rRNA) gene. The vaginal microbiome showed variations during pregnancy and the postpartum period based on the abortion history, hypertensive disorders, delivery mode, and maternal age. Co-variation of 22 bacterial taxa, including the Lactobacillus genus and two of its species, may account for the common characteristics of the vaginal microbiome under scenarios of different medical histories and pregnancy outcomes. In contrast, discriminant bacterial species were significantly different between women who had preterm birth (PTB) with and without premature rupture of membranes (PROM), and the community state type (CST) IV-A without any predominant Lactobacillus species in the microbiota was more prevalent during pregnancy in the PROM-PTB cases, suggesting that specific bacterial species could be considered to distinguish between different types of PTB. By providing data on Chinese women, this study will enrich the knowledge of the human microbiome and contribute to a better understanding of the association between the vaginal microbiome and reproductive health.

Page 322-333

Original Research

Panoramic Insights into Microevolution and Macroevolution of A Prevotella copri-containing Lineage in Primate Guts

Hao Li, Jan P. Meier-Kolthoff, Canxin Hu, Zhongjie Wang, Jun Zhu, Wei Zheng, Yun Tian, Feng Guo

Prevotella copri and its related taxa are widely detected in mammalian gut microbiomes and have been linked with an enterotype in humans. However, their microevolution and macroevolution among hosts are poorly characterized. In this study, extensively collected marker genes and genomes were analyzed to trace their evolutionary history, host specificity, and biogeographic distribution. Investigations based on marker genes and genomes suggest that a P. copri-containing lineage (PCL) harbors diverse species in higher primates. Firstly, P. copri in the human gut consisted of multiple groups exhibiting high genomic divergence and conspicuous but non-strict biogeographic patterns. Most African strains with high genomic divergence from other strains were phylogenetically located at the root of the species, indicating the co-evolutionary history of P. copri and Homo sapiens. Secondly, although long-term co-evolution between PCL and higher primates was revealed, sporadic signals of co-speciation and extensive host jumping of PCL members were suggested among higher primates. Metagenomic and phylogenetic analyses indicated that P. copri and other PCL species found in domesticated mammals had been recently transmitted from humans. Thirdly, strong evidence was found on the extensively horizontal transfer of genes (e.g., genes encoding carbohydrate-active enzymes) among sympatric P. copri groups and PCL species in the same primate host. Our study provides panoramic insights into the combined effects of vertical and horizontal transmission, as well as potential niche adaptation, on the microevolutionary and macroevolutionary history for an enterotype-representative lineage.

Page 334-349

Original Research

Characterization of Changes and Driver Microbes in Gut Microbiota During Healthy Aging Using A Captive Monkey Model

Zhi-Yuan Wei, Jun-Hua Rao, Ming-Tian Tang, Guo-An Zhao, Qi-Chun Li, Li-Ming Wu, Shao-Qiang Liu, Bi-Hai Li, Bai-Quan Xiao, Xing-Yin Liu, Jian-Huan Chen

Recent population studies have significantly advanced our understanding of how age shapes the gut microbiota. However, the actual role of age could be inevitably confounded due to the complex and variable environmental factors in human populations. A well-controlled environment is thus necessary to reduce undesirable confounding effects, and recapitulate age-dependent changes in the gut microbiota of healthy primates. Herein we performed 16S rRNA gene sequencing, characterized the age-associated gut microbial profiles from infant to elderly crab-eating macaques reared in captivity, and systemically revealed the lifelong dynamic changes of the primate gut microbiota. While the most significant age-associated taxa were mainly found as commensals such as Faecalibacterium, the abundance of a group of suspicious pathogens such as Helicobacter was exclusively increased in infants, underlining their potential role in host development. Importantly, topology analysis indicated that the network connectivity of gut microbiota was even more age-dependent than taxonomic diversity, and its tremendous decline with age could probably be linked to healthy aging. Moreover, we identified key driver microbes responsible for such age-dependent network changes, which were further linked to altered metabolic functions of lipids, carbohydrates, and amino acids, as well as phenotypes in the microbial community. The current study thus demonstrates the lifelong age-dependent changes and their driver microbes in the primate gut microbiota, and provides new insights into their roles in the development and healthy aging of their hosts.

Page 350-365

Original Research

Genome of the Giant Panda Roundworm Illuminates Its Host Shift and Parasitic Adaptation

Yue Xie, Sen Wang, Shuangyang Wu, Shenghan Gao, Qingshu Meng, Chengdong Wang, Jingchao Lan, Li Luo, Xuan Zhou, Jing Xu, Xiaobin Gu, Ran He, Zijiang Yang, Xuerong Peng, Songnian Hu, Guangyou Yang

Baylisascaris schroederi, a roundworm (ascaridoid) parasite specific to the bamboo-feeding giant panda (Ailuropoda melanoleuca), represents a leading cause of mortality in wild giant panda populations. Here, we present a 293-megabase chromosome-level genome assembly of B. schroederi to infer its biology, including host adaptations. Comparative genomics revealed an evolutionary trajectory accompanied by host-shift events in ascaridoid parasite lineages after host separations, suggesting their potential for transmission and rapid adaptation to new hosts. Genomic and anatomical lines of evidence, including expansion and positive selection of genes related to the cuticle and basal metabolisms, indicate that B. schroederi undergoes specific adaptations to survive in the sharp-edged bamboo-enriched gut of giant pandas by structurally increasing its cuticle thickness and efficiently utilizing host nutrients through gut parasitism. Additionally, we characterized the secretome of B. schroederi and predicted potential drug and vaccine targets for new control strategies. Overall, this genome resource provides new insights into the host adaptation of B. schroederi to the giant panda as well as the host-shift events in ascaridoid parasite lineages. Our findings on the unique biology of B. schroederi will also aid in the development of prevention and treatment measures to protect giant panda populations from roundworm parasitism.
西氏贝蛔虫是大熊猫的一种专性寄生虫（蛔虫），是导致野生大熊猫死亡的主要原因之一。本研究获得西氏贝蛔虫染色体水平基因组（293 Mb），并从中解析了包括其宿主适应在内的多个生物学问题。比较基因组学揭示蛔虫谱系在宿主分化之后发生“宿主转移”事件的进化历程，这表明蛔虫具有传播与快速适应新宿主的能力。基因组学和解剖学证据，结合虫体皮层结构和基础代谢相关基因的扩展与正向选择分析，表明西氏贝蛔虫通过增加皮层厚度和提高宿主肠道内营养的利用效率来适应大熊猫布满锋利竹刺的肠道环境。此外，本研究还鉴定了西氏贝蛔虫分泌组，并筛选出新的潜在药物和疫苗靶点。总之，该基因组数据为解释西氏贝蛔虫适应大熊猫以及蛔虫谱系宿主转移事件提供了新的见解。同时，西氏贝蛔虫独特生物学特性的研究也将有助于开发预防和治疗蛔虫病的新方法，从而保护大熊猫种群免受蛔虫的侵害。

Page 366-381

Original Research

Consistent Alterations of Human Fecal Microbes After Transplantation into Germ-free Mice

Yanze Li, Wenming Cao, Na L Gao, Xing-Ming Zhao, Wei-Hua Chen

Fecal microbiota transplantation (FMT) of human fecal samples into germ-free (GF) mice is useful for establishing causal relationships between the gut microbiota and human phenotypes. However, due to the intrinsic differences between human and mouse intestines and the different diets of the two organisms, it may not be possible to replicate human phenotypes in mice through FMT; similarly, treatments that are effective in mouse models may not be effective in humans. In this study, we aimed to identify human gut microbes that undergo significant and consistent changes (i.e., in relative abundances) after transplantation into GF mice in multiple experimental settings. We collected 16S rDNA-seq data from four published studies and analyzed the gut microbiota profiles from 1713 human–mouse pairs. Strikingly, on average, we found that only 47% of the human gut microbes could be re-established in mice at the species level, among which more than 1/3 underwent significant changes (referred to as “variable taxa”). Most of the human gut microbes that underwent significant changes were consistent across multiple human–mouse pairs and experimental settings. Consequently, about 1/3 of human samples changed their enterotypes, i.e., significant changes in their leading species after FMT. Mice fed with a controlled diet showed a lower enterotype change rate (23.5%) than those fed with a noncontrolled diet (49.0%), suggesting a possible solution for rescue. Most of the variable taxa have been reported to be implicated in human diseases, with some recognized as the causative species. Our results highlight the challenges of using a mouse model to replicate human gut microbiota-associated phenotypes, provide useful information for researchers using mice in gut microbiota studies, and call for additional validations after FMT. An online database named FMT-DB is publicly available at http://fmt2mice.humangut.info/#/.

Page 382-393

Original Research

First Glimpse of Gut Microbiota of Quarantine Insects in China

Yanxue Yu, Qi Wang, Ping Zhou, Na Lv, Wei Li, Fangqing Zhao, Shuifang Zhu, Di Liu

Quarantine insects are economically important pests that frequently invade new habitats. A rapid and accurate monitoring method to trace the geographical sources of invaders is required for their prevention, detection, and eradication. Current methods based on genetics are typically time-consuming. Here, we developed a novel tracing method based on insect gut microbiota. The source location of the insect gut microbiota can be used to rapidly determine the geographical origin of the insect. We analyzed 179 gut microbiota samples from 591 individuals of 22 quarantine insect species collected from 36 regions in China. The gut microbiota of these insects primarily included Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Proteobacteria, and Tenericutes. The diversity of the insect gut microbiota was closely associated with geographical and environmental factors. Different insect species could be distinguished based on the composition of gut microbiota at the phylum level. Populations of individual insect species from different regions could be distinguished based on the composition of gut microbiota at the phylum, class, and order levels. A method for determining the geographical origins of invasive insect species has been established; however, its practical application requires further investigations before implementation.

Page 394-404

Original Research

Systematic Cross-biospecimen Evaluation of DNA Extraction Kits for Long- and Short-read Multi-metagenomic Sequencing Studies

Jacqueline Rehner, Georges Pierre Schmartz, Laura Groeger, Jan Dastbaz, Nicole Ludwig, Matthias Hannig, Stefan Rupf, Berthold Seitz, Elias Flockerzi, Tim Berger, Matthias Christian Reichert, Marcin Krawczyk, Eckart Meese, Christian Herr, Robert Bals, Sören L. Becker, Andreas Keller, Rolf Müller, The IMAGINE Consortium

High-quality DNA extraction is a crucial step in metagenomic studies. Bias by different isolation kits impairs the comparison across datasets. A trending topic is, however, the analysis of multiple metagenomes from the same patients to draw a holistic picture of microbiota associated with diseases. We thus collected bile, stool, saliva, plaque, sputum, and conjunctival swab samples and performed DNA extraction with three commercial kits. For each combination of the specimen type and DNA extraction kit, 20-gigabase (Gb) metagenomic data were generated using short-read sequencing. While profiles of the specimen types showed close proximity to each other, we observed notable differences in the alpha diversity and composition of the microbiota depending on the DNA extraction kits. No kit outperformed all selected kits on every specimen. We reached consistently good results using the Qiagen QiAamp DNA Microbiome Kit. Depending on the specimen, our data indicate that over 10 Gb of sequencing data are required to achieve sufficient resolution, but DNA-based identification is superior to identification by mass spectrometry. Finally, long-read nanopore sequencing confirmed the results (correlation coefficient > 0.98). Our results thus suggest using a strategy with only one kit for studies aiming for a direct comparison of multiple microbiotas from the same patients.

Page 405-417

Original Research

Precision Methylome and In Vivo Methylation Kinetics Characterization of Klebsiella pneumoniae

Jing Fu, Ju Zhang, Li Yang, Nan Ding, Liya Yue, Xiangli Zhang, Dandan Lu, Xinmiao Jia, Cuidan Li, Chongye Guo, Zhe Yin, Xiaoyuan Jiang, Yongliang Zhao, Fei Chen, Dongsheng Zhou

Klebsiella pneumoniae (K. pneumoniae) is an important pathogen that can cause severe hospital- and community-acquired infections. To systematically investigate its methylation features, we determined the whole-genome sequences of 14 K. pneumoniae strains covering varying serotypes, multilocus sequence types, clonal groups, viscosity/virulence, and drug resistance. Their methylomes were further characterized using Pacific Biosciences single-molecule real-time and bisulfite technologies. We identified 15 methylation motifs [13 N6-methyladenine (6mA) and two 5-methylcytosine (5mC) motifs], among which eight were novel. Their corresponding DNA methyltransferases were also validated. Additionally, we analyzed the genomic distribution of GATC and CCWGG methylation motifs shared by all strains, and identified differential distribution patterns of some hemi-/un-methylated GATC motifs, which tend to be located within intergenic regions (IGRs). Specifically, we characterized the in vivo methylation kinetics at single-base resolution on a genome-wide scale by simulating the dynamic processes of replication-mediated passive demethylation and MTase-catalyzed re-methylation. The slow methylation of the GATC motifs in the replication origin (oriC) regions and IGRs implicates the epigenetic regulation of replication initiation and transcription. Our findings illustrate the first comprehensive dynamic methylome map of K. pneumoniae at single-base resolution, and provide a useful reference to better understand epigenetic regulation in this and other bacterial species.
肺炎克雷伯菌是肠杆菌科克雷伯氏菌属中最为重要的一类致病菌，为中国第二大院感菌，其不断增长的趋势严重威胁人民生命健康和公共卫生安全。针对于目前肺炎克雷伯菌DNA甲基化研究空白的现状，我们以有临床代表性的14株不同分型的肺炎克雷伯菌株为研究对象，利用三代单分子实时测序技术结合重亚硫酸盐测序，首次全景式地揭示了肺炎克雷伯菌的基因组甲基化图谱：本研究共鉴定到15个DNA甲基化基序及对应的甲基化酶，包括13个6mA和2个5mC甲基化基序，其中8个甲基化基序及对应的甲基化酶为首次报道。对于最重要的两个甲基化基序GATC和CCWGG（14株菌株共有），我们发现其在基因组上呈现非随机分布，且半/未甲基化倾向分布于基因间区，说明其可能参与细菌的表观修饰调控。重要的是，本研究通过深度解析细菌生长周期中甲基化组的动态变化，构建了相应的数学模型，很好地模拟了GATC和CCWGG在指数生长期的被动去甲基化和再甲基化的动力学过程，首次实现了全基因组水平单基序位点甲基化速率的精准计算。具体而言，我们发现GATC在oriC区和IGR区具有较慢的甲基化速率，提示肺炎克雷伯菌通过核酸表观修饰系统全面参与了细菌的复制起始和转录调控。总之，本研究揭示了肺炎克雷伯菌的单碱基分辨率的动态甲基化图谱，其普适性也为其它细菌的动态甲基化图谱和核酸表观遗传修饰提供了可借鉴的参考；另外，本研究为肺炎克雷伯菌精细表观修饰调控的研究奠定了坚实的理论基础，为更好地了解细菌的核酸表观修饰调控提供了有益的借鉴。

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