Volume: 17, Issue: 1

Preface

The Fast Track for Microbiome Research

Kang Ning, Yigang Tong

Page 1-3

Review Article

Stereotypes About Enterotype: the Old and New Ideas

Mingyue Cheng, Kang Ning

In 2011, the term “enterotype” first appeared to the general public in Nature, which refers to stratification of human gut microbiota. However, with more studies on enterotypes conducted nowadays, doubts about the existence and robustness of enterotypes have also emerged. Here we reviewed current opinions about enterotypes from both conceptual and analytical points of view. We firstly illustrated the definition of the enterotype and various factors influencing enterotypes, such as diet, administration of antibiotics, and age. Then we summarized lines of evidence that pose the concept against the enterotype, and described the current methods for enterotype analysis. Finally, we showed that the concept of enterotype has been extended to other ecological niches. Based on current studies on enterotypes, it has been clear that more studies with larger sample sizes are needed to characterize the enterotypes. Improved computational methods are also required to build sophisticated models, reflecting the dynamics and resilience of enterotypes.
肠型这一名词于2011年的《自然》期刊中首次提出，其意义是对人体肠道微生物样本进行分类分析。然而随着关于肠型研究的日益增多，有关肠型的存在性与鲁棒性的质疑也开始浮现。在这里，我们从概念以及分析角度回顾了目前关于肠型的观点与意见。我们首先阐明了肠型的定义以及肠型的多种影响因素，例如饮食，抗生素治疗和年龄。此外，我们总结了多条质疑肠型概念的证据，并且描述了目前进行肠型分析的计算方法。最终，我们展示了肠型概念已经延伸到人体其他部位的微生物以及其他物种的肠道微生物。基于目前已有的关于肠型的研究，我们发现要想描述清楚肠型的特征仍需要更大的样本量以及更多样化的样本来源个体。我们也需要改进的算法来建立复杂精细的模型，从而表征肠型的动态特征与恢复能力。

Page 4-12

Review Article

Intestinal Microbiota in Early Life and Its Implications on Childhood Health

Lu Zhuang, Haihua Chen, Sheng Zhang, Jiahui Zhuang, Qiuping Li, Zhichun Feng

Trillions of microbes reside in the human body and participate in multiple physiological and pathophysiological processes that affect host health throughout the life cycle. The microbiome is hallmarked by distinctive compositional and functional features across different life periods. Accumulating evidence has shown that microbes residing in the human body may play fundamental roles in infant development and the maturation of the immune system. Gut microbes are thought to be essential for the facilitation of infantile and childhood development and immunity by assisting in breaking down food substances to liberate nutrients, protecting against pathogens, stimulating or modulating the immune system, and exerting control over the hypothalamic–pituitary–adrenal axis. This review aims to summarize the current understanding of the colonization and development of the gut microbiota in early life, highlighting the recent findings regarding the role of intestinal microbes in pediatric diseases. Furthermore, we also discuss the microbiota-mediated therapeutics that can reconfigure bacterial communities to treat dysbiosis.
数以万亿计的微生物栖居于人体，参与人体的生理与病理过程，在人的整个生命进程中都发挥着重要作用。微生物组在不同的生命阶段具有独特的组成和功能特征。越来越多的证据表明，人体的微生物可能在婴儿发育和免疫系统成熟过程中发挥着重要作用。肠道菌群能够协助分解食物、防御病原体、刺激和调节免疫系统，并能够对下丘脑 - 垂体 - 肾上腺轴施加控制，被认为是促进婴儿和儿童发育的必要条件。本文旨在介绍目前人们对肠道菌群在人类生命早期定植和发育的认识，以及肠道菌群在儿童相关疾病进程中扮演的角色。此外，还探讨了通过对肠道菌群的操纵实现对疾病治疗的方法和最新进展。

Page 13-25

Original Research

Correlation of Gut Microbiome Between ASD Children and Mothers and Potential Biomarkers for Risk Assessment

Ning Li, Junjie Yang, Jiaming Zhang, Cheng Liang, Ying Wang, Bin Chen, Changying Zhao, Jingwen Wang, Guangye Zhang, Dongmei Zhao, Yi Liu, Lehai Zhang, Jun Yang, Guimei Li, Zhongtao Gai, Lei Zhang, Guoping Zhao

Variation of maternal gut microbiota may increase the risk of autism spectrum disorders (ASDs) in offspring. Animal studies have indicated that maternal gut microbiota is related to neurodevelopmental abnormalities in mouse offspring, while it is unclear whether there is a correlation between gut microbiota of ASD children and their mothers. We examined the relationships between gut microbiome profiles of ASD children and those of their mothers, and evaluated the clinical discriminatory power of discovered bacterial biomarkers. Gut microbiome was profiled and evaluated by 16S ribosomal RNA gene sequencing in stool samples of 59 mother–child pairs of ASD children and 30 matched mother–child pairs of healthy children. Significant differences were observed in the gut microbiome composition between ASD and healthy children in our Chinese cohort. Several unique bacterial biomarkers, such as Alcaligenaceae and Acinetobacter, were identified. Mothers of ASD children had more Proteobacteria, Alphaproteobacteria, Moraxellaceae, and Acinetobacter than mothers of healthy children. There was a clear correlation between gut microbiome profiles of children and their mothers; however, children with ASD still had unique bacterial biomarkers, such as Alcaligenaceae, Enterobacteriaceae, and Clostridium. Candidate biomarkers discovered in this study had remarkable discriminatory power. The identified patterns of mother–child gut microbiome profiles may be important for assessing risks during the early stage and planning of personalized treatment and prevention of ASD via microbiota modulation.
孤独症谱系障碍(Autism Spectrum Disorder，ASD)简称孤独症，与自闭症同义，是一组以社交沟通障碍、兴趣或活动范围狭窄以及重复刻板行为为主要特征的神经发育性障碍疾病。自闭症的病因尚不清楚，遗传学研究表明，自闭症的发生发展受遗传因素的影响较大，但其患病风险效应具有高度的可变性，仅靠遗传因素并不能解释其发病原因，因此环境因素不容忽视，一般认为遗传、表观遗传和环境因素相互作用，共同在自闭症的发生发展中发挥作用。动物实验研究表明母代的肠道菌群紊乱会导致子代神经发育障碍的发生。母亲肠道菌群的紊乱可能会增加后代患有自闭症的风险。然而，目前尚无研究对自闭症患儿的母子肠道微生物组进行同时分析并评价其相关性。 该研究分析比较了59对自闭症儿童及其母亲与30对健康对照母子的肠道菌群的群落结构。研究结果显示中国自闭症儿童与健康儿童的肠道微生物组成存在显著差异，并找出了潜在的标志菌：如产碱杆菌科（Alcaligenaceae）和不动杆菌属（Acinetobacter）。自闭症儿童的母亲与健康儿童的母亲相比，肠道中变形菌门（Proteobacteria），α变形菌纲（Alphaproteobacteria），莫拉菌科（Moraxellaceae）和不动杆菌属（Acinetobacter）相对丰度显著升高。虽然儿童与其母亲的肠道微生物群谱之间存在明显的相关性，自闭症儿童肠道内仍有其独特的细菌标志物，如产碱杆菌科（Alcaligenaceae），肠杆菌科（Enterobacteriaceae），梭菌属（Clostridium）等。该研究所发现的肠道细菌标志物可以显著地区分自闭症儿童与健康儿童。母子肠道菌群图谱和标志菌的研究将对自闭症早期风险评估、预防以及通过调节菌群制定个性化的治疗方案提供新的思路。该研究主要得出以下结论： 1. 中国自闭症儿童的肠道菌群与健康儿童相比有显著差异，且存在潜在的与致病相关的肠道菌； 2. 自病症儿童的母亲与健康儿童的母亲的肠道菌群结构也存在显著的差异； 3. 虽然儿童与其母亲的肠道菌群之间存在明显的相关性，自闭症儿童肠道内仍有其独特的细菌标志物； 4. 所发现的细菌标志物具有潜在的自闭症风险评估的价值。

Page 26-38

Original Research

Antibiotic Treatment Drives the Diversification of the Human Gut Resistome

Jun Li, Elizabeth A. Rettedal, Eric van der Helm, Mostafa Ellabaan, Gianni Panagiotou, Morten O.A. Sommer

Despite the documented antibiotic-induced disruption of the gut microbiota, the impact of antibiotic intake on strain-level dynamics, evolution of resistance genes, and factors influencing resistance dissemination potential remains poorly understood. To address this gap we analyzed public metagenomic datasets from 24 antibiotic treated subjects and controls, combined with an in-depth prospective functional study with two subjects investigating the bacterial community dynamics based on cultivation-dependent and independent methods. We observed that short-term antibiotic treatment shifted and diversified the resistome composition, increased the average copy number of antibiotic resistance genes, and altered the dominant strain genotypes in an individual-specific manner. More than 30% of the resistance genes underwent strong differentiation at the single nucleotide level during antibiotic treatment. We found that the increased potential for horizontal gene transfer, due to antibiotic administration, was ∼3-fold stronger in the differentiated resistance genes than the non-differentiated ones. This study highlights how antibiotic treatment has individualized impacts on the resistome and strain level composition, and drives the adaptive evolution of the gut microbiota.
过往的研究结果证明了抗生素可以引起肠道菌群的失衡和抗药性的增加，但是抗生素如何影响人类肠道菌群和抗药性基因在微观层面的的进化、菌株群体动力学及可能影响抗药性扩散的因素，我们了解甚少。为了回答上述问题，我们把基于宏基因组公共数据的分析及一个深度功能分析的多组学前瞻研究结合在一起进行深度挖掘。我们发现，短期抗生素干预会增加抗药性基因谱在个体之间的差异，提升抗药基因的基因拷贝数及选择个体特异性的的细菌优势菌株。同时，在抗生素干预阶段，30%的的抗药型基因经历了单核苷酸水平的分化。而相比于无显著核苷酸分化的基因，核苷酸分化的基因的水平转移倾向增加了超过三倍。总的来说，本研究结果揭示了抗生素干预会驱动抗药性基因的适应性进化和提升水平基因转移倾向，并对肠道菌群在菌株级别构成和抗药性基因谱具有个体特异性的影响。

Page 39-51

Original Research

Effects of Proton Pump Inhibitors on the Gastrointestinal Microbiota in Gastroesophageal Reflux Disease

Yi-Chao Shi, Shun-Tian Cai, Ya-Ping Tian, Hui-Jun Zhao, Yan-Bing Zhang, Jing Chen, Rong-Rong Ren, Xi Luo, Li-Hua Peng, Gang Sun, Yun-Sheng Yang

Proton pump inhibitors (PPIs) are commonly used to lessen symptoms in patients with gastroesophageal reflux disease (GERD). However, the effects of PPI therapy on the gastrointestinal microbiota in GERD patients remain unclear. We examined the association between the PPI usage and the microbiota present in gastric mucosal and fecal samples from GERD patients and healthy controls (HCs) using 16S rRNA gene sequencing. GERD patients taking PPIs were further divided into short-term and long-term PPI user groups. We showed that PPI administration lowered the relative bacterial diversity of the gastric microbiota in GERD patients. Compared to the non-PPI-user and HC groups, higher abundances of Planococcaceae, Oxalobacteraceae, and Sphingomonadaceae were found in the gastric microbiota from the PPI-user group. In addition, the Methylophilus genus was more highly abundant in the long-term PPI user group than in the short-term PPI-user group. Despite the absence of differences in alpha diversity, there were significant differences in the fecal bacterial composition of between GERD patients taking PPIs and those not taking PPIs. There was a higher abundance of Streptococcaceae, Veillonellaceae, Acidaminococcaceae, Micrococcaceae, and Flavobacteriaceae present in the fecal microbiota from the PPI-user group than those from the non-PPI-user and HC groups. Additionally, a significantly higher abundance of Ruminococcus was found in GERD patients on long-term PPI medication than that on short-term PPI medication. Our study indicates that PPI administration in patients with GERD has a significant effect on the abundance and structure of the gastric mucosal microbiota but only on the composition of the fecal microbiota.
质子泵抑制剂（PPI）通常用于减轻胃食管反流病（GERD）患者的症状。然而，PPI治疗对GERD患者胃肠道微生物群的影响仍不清楚。我们使用16S rRNA基因测序检测了PPI使用与来自GERD患者和健康对照（HC）的胃粘膜和粪便样品中的微生物之间的关联。服用PPI的GERD患者进一步分为短期和长期PPI使用组。我们发现PPI给药降低了GERD患者胃微生物的细菌多样性。与非PPI使用组和HC组相比，来自PPI使用组的胃微生物群中发现了更高丰度的Planococcaceae，Oxalobacteraceae和Sphingomonadaceae。此外，长期PPI使用组中的Methylophilus属比在短期PPI使用组中更丰富。尽管α多样性没有差异，但服用PPI的GERD患者和未服用PPI的GERD患者的粪便细菌组成存在显着差异。来自PPI使用组的粪便微生物群中存在的链球菌科，Veillonellaceae，Acidaminococcaceae，Micrococcaceae和Flavobacteriaceae的丰度高于非PPI使用和HC组。此外，长期PPI药物治疗的GERD患者的Ruminococcus丰度明显高于短期PPI药物。我们的研究表明，GERD患者的PPI给药对胃粘膜微生物群的丰度和结构有显着影响，但仅对粪便微生物群的组成有影响。

Page 52-63

Original Research

Inulin Can Alleviate Metabolism Disorders in ob/ob Mice by Partially Restoring Leptin-related Pathways Mediated by Gut Microbiota

Xiaofeng Song, Liang Zhong, Na Lyu, Fei Liu, Boxing Li, Yanan Hao, Yong Xue, Jing Li, Yuqing Feng, Yue Ma, Yongfei Hu, Baoli Zhu

Inulin has been used as a prebiotic to alleviate glucose and lipid metabolism disorders in mice and humans by modulating the gut microbiota. However, the mechanism underlying the alleviation of metabolic disorders by inulin through interactions between the gut microbiota and host cells is unclear. We use ob/ob mice as a model to study the effect of inulin on the cecal microbiota by 16S rRNA gene amplicon sequencing and its interaction with host cells by transcriptomics. The inulin-supplemented diet improved glucose and lipid metabolism disorder parameters in ob/ob mice, alleviating fat accumulation and glucose intolerance. The α diversity of gut microbial community of ob/ob mice was reduced after inulin treatment, while the β diversity tended to return to the level of wild type mice. Interestingly, Prevotellaceae UCG 001 (family Prevotellaceae) was obviously enriched after inulin treatment. A comparative analysis of the gene expression profile showed that the cecal transcriptome was changed in leptin gene deficiency mice, whereas the inulin-supplemented diet partially reversed the changes in leptin gene-related signaling pathways, especially AMPK signaling pathway, where the levels of gene expression became comparable to those in wild type mice. Further analysis indicated that Prevotellaceae UCG 001 was positively correlated with the AMPK signaling pathway, which was negatively correlated with markers of glycolipid metabolism disorders. Our results suggest that the inulin-supplemented diet alleviates glucose and lipid metabolism disorders by partially restoring leptin related pathways mediated by gut microbiota.
菊粉作为益生元可以通过调节小鼠和人的肠道菌群来缓解糖脂代谢紊乱，然而，菊粉通过肠道菌群和宿主细胞相互作用关系来改善代谢紊乱的作用机制仍不清楚。我们使用ob/ob小鼠作为模型，利用16S rRNA扩增子测序技术和转录组测序技术来研究菊粉对盲肠菌群的影响以及菌群和宿主细胞的互作关系。添加了菊粉的饮食可以明显改善ob/ob小鼠的糖脂代谢紊乱相关指标，减轻脂肪沉积和葡萄糖不耐受程度，菊粉干预后小鼠盲肠菌群的α多样性降低，而β多样性趋向于恢复到野生型状态。有趣的是，Prevotellaceae UCG 001（属于Prevotellaceae科）这一菌属在菊粉干预后得到显著富集。基因表达谱的分析结果表明leptin基因缺陷型小鼠的盲肠转录组发生改变，而菊粉干预则可以恢复部分信号通路的丰度，尤其是AMPK signaling pathway，基本恢复到野生型水平。Prevotellaceae UCG 001与AMPK signaling pathway之间呈显著正相关，与糖脂代谢紊乱指标呈现显著负相关。我们的研究结果表明菊粉改善糖脂代谢紊乱可能是通过肠道菌群介导的恢复部分leptin基因相关信号通路来实现的。

Page 64-75

Original Research

Agricultural Risk Factors Influence Microbial Ecology in Honghu Lake

Maozhen Han, Melissa Dsouza, Chunyu Zhou, Hongjun Li, Junqian Zhang, Chaoyun Chen, Qi Yao, Chaofang Zhong, Hao Zhou, Jack A Gilbert, Zhi Wang, Kang Ning

Agricultural activities, including stock-farming, planting industry, and fish aquaculture, can affect the physicochemical and biological characters of freshwater lakes. However, the effects of pollution producing by agricultural activities on microbial ecosystem of lakes remain unclear. Hence, in this work, we selected Honghu Lake as a typical lake that is influenced by agriculture activities. We collected water and sediment samples from 18 sites, which span a wide range of areas from impacted and less-impacted areas. We performed a geospatial analysis on the composition of microbial communities associated with physicochemical properties and antibiotic pollution of samples. The co-occurrence networks of water and sediment were also built and analyzed. Our results showed that the microbial communities of impacted and less-impacted samples of water were largely driven by the concentrations of TN, TP, NO3−-N, and NO2−-N, while those of sediment were affected by the concentrations of Sed-OM and Sed-TN. Antibiotics have also played important roles in shaping these microbial communities: the concentrations of oxytetracycline and tetracycline clearly reflected the variance in taxonomic diversity and predicted functional diversity between impacted and less-impacted sites in water and sediment samples, respectively. Furthermore, for samples from both water and sediment, large differences of network topology structures between impacted and less-impacted were also observed. Our results provide compelling evidence that the microbial community can be used as a sentinel of eutrophication and antibiotics pollution risk associated with agricultural activity; and that proper monitoring of this environment is vital to maintain a sustainable environment in Honghu Lake.
农业生产活动，包括畜牧养殖、种植业和鱼类养殖等活动。农业生产活动中产生的污染会影响湖泊的理化性质和生态系统。先前的研究大多集中在农业生产活动对湖泊的宏观生态系统的影响上，而农业活动产生的污染对湖泊微生物生态系统影响研究却比较少，特别是农业生产活动中的抗生素对湖泊微生态的影响还缺少相关的研究。在本文中，我们选择洪湖这一典型的湖泊作为研究农业活动对湖泊微生态系统研究的对象，探究农业生产活动中的污染对湖泊微生态的影响。我们收集了洪湖及其附近的河流和池塘的18个位点的水样和底泥样本，共28个，水样和底泥样本各14个。这些样本依据农业生产活动对其影响的程度可分为受影响程度高（impacted）和受影响程度低（less-impacted）的水样和底泥样本。使用高通量测序技术对这些样本中微生物的16S rRNA V4-V5区进行测序，解析出这些样本的微生物群落组成和功能组成，并将理化因素和抗生素污染与微生物群落组成进行了关联分析，探索了农业生产活动对湖泊微生态影响及其在空间位置上的规律。结果表明，水体中的TN、TP、NO3--N和NO2--N是影响水体微生物群落结构的主要因素，而底泥中的Sed-OM和Sed-TN则是影响底泥微生物群落结构的主要驱动力。我们揭示了抗生素对于湖泊微生态的影响，其中土霉素和四环素是造成水体和底泥样本微生物群落结构的重要因素。我们筛选出可以用来表征农业生产活动对湖泊微生态影响程度的生物标志物。此外，我们也构建了微生物共出现网络，基于共出现网络拓扑结构的分析揭示了农业生产活动对微生物群落中微生物的影响。我们的结果表明微生物群落可以作为农业污染的响应器，而对环境的适当监测对于维持洪湖环境的可持续发展至关重要。

Page 76-90

Original Research

How Microbes Shape Their Communities? A Microbial Community Model Based on Functional Genes

Xiaoqing Jiang, Xin Li, Longshu Yang, Chunhong Liu, Qi Wang, Weilai Chi, Huaiqiu Zhu

Exploring the mechanisms of maintaining microbial community structure is important to understand biofilm development or microbiota dysbiosis. In this paper, we propose a functional gene-based composition prediction (FCP) model to predict the population structure composition within a microbial community. The model predicts the community composition well in both a low-complexity community as acid mine drainage (AMD) microbiota, and a complex community as human gut microbiota. Furthermore, we define community structure shaping (CSS) genes as functional genes crucial for shaping the microbial community. We have identified CSS genes in AMD and human gut microbiota samples with FCP model and find that CSS genes change with the conditions. Compared to essential genes for microbes, CSS genes are significantly enriched in the genes involved in mobile genetic elements, cell motility, and defense mechanisms, indicating that the functions of CSS genes are focused on communication and strategies in response to the environment factors. We further find that it is the minority, rather than the majority, which contributes to maintaining community structure. Compared to health control samples, we find that some functional genes associated with metabolism of amino acids, nucleotides, and lipopolysaccharide are more likely to be CSS genes in the disease group. CSS genes may help us to understand critical cellular processes and be useful in seeking addable gene circuitries to maintain artificial self-sustainable communities. Our study suggests that functional genes are important to the assembly of microbial communities.
长期以来，微生物种群动力学致力于探索微生物群落的多样性和复杂性，是微生物生态学研究关注的一个重要科学问题。近年来，基于对微生物群落进行高通量测序的宏基因组学技术得到迅速的发展，很好地避开了绝大多数微生物难以在现有实验室条件和技术下进行培养这一难题，同时也为微生物种群动力学研究提供了新的途径和方法。从高通量测序数据出发，目前大多数微生物种群动力学模型主要使用16S rRNA数据进行物种水平的分析。本文从宏基因组的全基因组测序数据出发，进行基因及功能注释、物种注释并得到以物种和功能为分类单元的特征，结合环境因子信息，提出了一个基于功能相互作用关系进而量化种群适应度的FCP模型（Functional gene-based Composition Prediction model）。数据检测表明，FCP模型在一个简单的（酸矿水微生物群落）和复杂的（人肠道菌群）微生物系统中均能很好地预测微生物群落在不同环境下的种群结构及其变化。 从FCP模型出发，我们可进一步从分子水平上分析基因缺失对群落结构造成的影响，由此定义了对群落结构稳定至关重要的功能基因——CSS基因（Community Structure Shaping genes）。运用FCP模型，本文设计了一个鉴定CSS基因的方法，并将其应用于酸矿水微生物群落和人肠道菌群。结果显示，CSS基因在生物膜成熟过程中发生了显著变化。在早期样本中，CSS基因富含与环境压力相关的具有防御机制的功能基因；随着生物膜逐渐成熟，膜形成和控制细胞运动的功能类基因显著增加，表明微生物群落在生态演替过程发生了生理变化。与人们熟悉的微生物必需基因（Essential Genes）相比，CSS基因的特点在于显著地富集于移动元件、细胞运动和防御机制等功能，很好地描述了微生物群落对于环境特别是极端环境的适应性。值得关注的是，群落中各物种对于CSS基因的贡献分析结果表明，群落中的低丰度菌，而不是主导菌，对群落结构稳定发挥着重要的作用。此外，我们发现人肠道菌群中CSS基因的数量远高于酸矿水微生物。患病组的CSS基因与健康对照组相比有着明显的不同，而且也得到了相关文献的支持。

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Original Research

GPA: A Microbial Genetic Polymorphisms Assignments Tool in Metagenomic Analysis by Bayesian Estimation

Jiarui Li, Pengcheng Du, Adam Yongxin Ye, Yuanyuan Zhang, Chuan Song, Hui Zeng, Chen Chen

Identifying antimicrobial resistant (AMR) bacteria in metagenomics samples is essential for public health and food safety. Next-generation sequencing (NGS) technology has provided a powerful tool in identifying the genetic variation and constructing the correlations between genotype and phenotype in humans and other species. However, for complex bacterial samples, there lacks a powerful bioinformatic tool to identify genetic polymorphisms or copy number variations (CNVs) for given genes. Here we provide a Bayesian framework for genotype estimation for mixtures of multiple bacteria, named as Genetic Polymorphisms Assignments (GPA). Simulation results showed that GPA has reduced the false discovery rate (FDR) and mean absolute error (MAE) in CNV and single nucleotide variant (SNV) identification. This framework was validated by whole-genome sequencing and Pool-seq data from Klebsiella pneumoniae with multiple bacteria mixture models, and showed the high accuracy in the allele fraction detections of CNVs and SNVs in AMR genes between two populations. The quantitative study on the changes of AMR genes fraction between two samples showed a good consistency with the AMR pattern observed in the individual strains. Also, the framework together with the genome annotation and population comparison tools has been integrated into an application, which could provide a complete solution for AMR gene identification and quantification in unculturable clinical samples. The GPA package is available at https://github.com/IID-DTH/GPA-package.
随着细菌耐药的增加，对于细菌抗生素耐药的监测，在公共卫生和食品安全领域越来越重要。近期，高通量测序技术的重大突破，为人类和其他物种中基因型检测提供了有效的工具，也为建立基因型-表型关联提供了重要手段。但是，由于临床混合样本中，菌群结构复杂，相关的生物信息挖掘工具及软件，仍然缺乏。本研究中，我们建立了一套基于贝叶斯模型的工具，用于复杂细菌菌群中特定基因型检测，称为GPA。在模拟肺炎克雷伯菌基因组数据中，该工具不仅可检测CNV和SNV，而且有效的降低了混合样本中假阳性发现率FDR和平均绝对偏差MAE。进而，我们利用肺炎克雷伯菌的混合测定数据，分析在2009年和2013年两年病人中耐药基因的CNV和SNV突变频率的差异，该结果可成功构建肺炎克雷伯菌基因-耐药表型关联。最后，我们使用了公开的宏基因组数据进行软件评估，表明该方法可用于临床样本中非培养的细菌的鉴定及其耐药基因的定量分析。该工具已经提交至网站 https://github.com/IID-DTH/GPA-package。

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