Escherichia coli (E. coli) is a common microorganism that is widely present in the environment and closely related to human health. The extent of E. coli presence in the human gut has been a subject of ongoing debate. Through whole-genome shotgun metagenomic sequencing, our study revealed that E. coli exists in the human body at a low abundance (average abundance 1.21%), with occasional short-term bursts leading to temporary increases in abundance, with the highest recorded at 50.91%. Further investigations into the factors contributing to these short-term blooms of E. coli showed significant variations in strain types and genomes within fecal samples collected from the same individuals at different time points. Evolutionary tree analysis indicated that samples from different individuals crossed, suggesting a change in the dominant E. coli strains within the human gut. Therefore, it can be inferred that E. coli in the human body are more likely to be transient bacteria rather than permanent residents in the gut. The rapid rate of turnover among months (87.5% within a month) and short-term blooms of E. coli in the human body can establish "latent infections" of nonpathogenic strains in healthy individuals while also posing a potential risk of introducing pathogenic strains, thereby impacting human health. In summary, our study revealed the variation in E. coli abundance and strains within the human gut, influenced by geographic area and temporal factors. These findings contribute to a better understanding of the relationship between E. coli, the gut microbiota, and human health.IMPORTANCE Escherichia coli (E. coli) is a microorganism closely linked to human health, and its presence in the human gut has been a topic of debate. Our study, using whole-genome shotgun metagenomic sequencing, revealed that E. coli exists at a low abundance in the human body, with occasional short-term bursts leading to temporary increases. Strain and genome variations were observed within fecal samples from the same individuals at different time points, suggesting transient rather than permanent residence of E. coli in the gut. The rapid turnover rate and short-term blooms of E. coli can establish latent infections while also posing a risk of introducing pathogenic strains. These findings enhance our understanding of the relationship between E. coli, the gut microbiota, and human health.

The concept of “enterotypes” in microbiome research has attracted substantial interest, particularly focusing on the abundance of Prevotella spp. in the human gut. In this study, the intricate dynamics of Prevotella spp. in the human gut microbiota was investigated, based on the metagenomic method. First, 239 fecal samples from individuals across four regions of China revealed a bimodal distribution, highlighting the abundance and variability in Prevotella spp. within the Chinese population. Second, the longitudinal cohort study included 184 fecal samples from 52 time points collected from seven individuals who demonstrated either the outbreaks or disappearances of Prevotella spp., emphasizing the transient nature of Prevotella abundance levels and suggesting shifts in Prevotella “enterotypes.” Furthermore, a turnover of the dominant Prevotella spp. was observed, indicating the potential presence of diverse subtypes of Prevotella enterotype. Notably, the genomic analysis demonstrated the persistence of specific Prevotella strains within individuals over extended periods, highlighting the enduring presence of Prevotella in the human gut. In conclusion, by integrating the temporal and geographical scales in our research, we gained deeper insights into the dynamics of Prevotella, emphasizing the importance of considering the dynamics at the time and species level in gut microbiota studies and their implications on human health.

Abstract Background: Rapid and accurate pathogen detection is important for effective control of infectious diseases. However, traditional pathogen culture methods have a very long detection time, as well as high rates of false-positive and false-negative results. Third generation sequencing (TGS) technology brings the new possibility of being used as a pathogen detection method. However, the practicability of a pathogen detection report based on TGS is still lacking. There is also a lack of professional and accurate report interpretation. Results: Here, we report on the development of a pathogen detection and analysis tool (NPIP) based on third generation nanopore sequencing technology. We also prove the practicability of nanopore sequencing and NPIPanalysis tools in emergency and clinical pathogen detection by demonstrating its use in a practical case. Conclusions: This platform provides an effective, convenient, and fast analysis tool for clinicians and public health personnel to more successfully apply TGS in pathogen detection.

脱氧核糖核酸(DNA)存储是一种以人工合成的生物大分子DNA作为信息载体的新型存储技术,具有存储密度高、存储量大、存储时间长、损耗率低等优点,有望成为一种新的数据存储介质.随着DNA合成技术(数据写入)和DNA测序技术(数据读取)的快速发展,DNA存储已成为下一代存储技术的热点.然而目前通过二代测序技术实现DNA数据的读取仍然是一个操作复杂、耗时长且成本较高的过程.如何更深入融合和改进DNA合成技术和测序技术,将DNA信息的存储和读取进一步快速高效地实现,是目前需要解决的问题.本研究设计并实践了基于便携式的国产纳米孔测序平台开展DNA存储信息快速读取的可能性,实现了4h内将存储于DNA片段中一首中文诗词进行重新测序和即时解码,验证了国产纳米孔测序技术从存储DNA信息的介质中即时读取信息的能力.

The gut microbiota comprises the collective genomes of microbial symbionts and is composed of bacteria, fungi, viruses, and protists within the gastrointestinal tract of a host. Although the literature associated with gut microbiota is increasing, studies on eukaryotes in the human gut are just beginning to surface. Blastocystis is one of the most common intestinal parasites of humans and animals and is estimated to colonise more than 1 billion people on a global scale. However, the understanding of the genetic characteristics of Blastocystis subtype (ST) at the genome level and its relationship with other members of the gut microbiota is still limited. In this study, by surveying the prevalence and genome characteristics of Blastocystis sp . ST3 in a Chinese population (prevalence % = 6.09%), the association of Blastocystis sp . ST3 with region and time and the structure of the resident gut bacterial population was clarified. We identified novel sequences (50 mitochondrial and 41 genome sequences) and determined their genetic diversity amongst strains within Blastocystis sp . ST3 (4.14 SNPs/kb). Furthermore, we found that colonisation of Blastocystis was strongly associated with increased bacterial richness and higher abundance of several anaerobes. Finally, we performed time series sampling on two Blastocystis- positive individuals and confirmed that Blastocystis could exist continually in the human gut microbiota and persist for a long time, even for 4 years.

In this study, we aimed to understand the characteristics of the gut microbial composition in a healthy Chinese population and to evaluate if they differed across different regions. In addition, we aimed to understand the changes in the gut microbial composition over time. We collected 239 fecal samples from healthy Chinese adults living in four regions and performed a 1-year time cohort study in a small population in Beijing. The Chinese gut microbiota share 34 core bacterial genera and 39 core bacterial species, which exist in all collected samples. Several disease-related microorganisms (DRMs), virulence factors, and antibiotic resistance genes were found in one or more healthy Chinese samples. Differences in gut microbiota were observed in samples from different regions, locations, individuals, and time points. Compared to other factors, time was associated with a lower degree of change in the gut microbiota. Our findings revealed spatial and temporal changes in the gut microbiota of healthy Chinese individuals. Compared to fecal microbiomes of 152 samples in the publicly released the Human Microbiome Project (HMP) project from the United States, samples in this study have higher variability in the fecal microbiome, with higher richness, Shannon diversity indices, and Pielou evenness indexes, at both the genus and species levels. The microbiota data obtained in this study will provide a detailed basis for further understanding the composition of the gut microbiota in the healthy Chinese population. IMPORTANCE China accounts for approximately 1/5th of the world's total population. Differences in environment, ethnicity, and living habits could impart unique features to the structure of the gut microbiota of Chinese individuals. In 2016, we started to investigate healthy Chinese people and their gut microbiomes. Phase I results for 16S rRNA amplicons have been released. However, owing to the limitations of 16S rRNA amplicon sequencing, the gut microbiome of a healthy Chinese population could not be examined thoroughly at the species level, and the detailed changes in the gut microbiota over time need to be investigated. To address these knowledge gaps, we started a phase II study and investigated the basis for variations in the gut microbiome composition in a healthy Chinese population at the species level using shotgun metagenomics technology. In the phase II study, we also conducted a time scale analysis of fecal samples from healthy Chinese subjects, as a pioneered study, which quantitatively clarified the changes in the gut microbiota at both the spatial and temporal levels and elucidated the distribution pattern of DRMs in healthy Chinese individuals.

Studying the dynamics and stability of the human gut microbiota over time is important for exploring their relationship with human health and developing treatment strategies for putative microbiome-related ailments. Here, we collected stool samples from seven healthy Chinese subjects at 1-month intervals between 2016 and 2020. Sequencing and bioinformatics analyses revealed that the bacteria in the collected fecal samples fluctuated over time, and the extent of these changes increased over time. Further, the average shared proportion value obtained using Sourcetracker2 was 63.5% for samples collected from the same individual in the preceding month, and over a 3-year period, this value decreased to 40.7%. Furthermore, the proportion of different bacteria in the gut microbiota of the different subjects fluctuated to varying degrees. Therefore, our results suggested that it is important to consider the effect of time on gut microbiota composition when it is used to evaluate health. Our study opens up a new field of microbiota research, considering not just the instantaneous microbiota, but also the change of the gut microbiota over time.

目的 评估粪便样本的不同保存条件对肠道微生态研究结果的影响.方法 设计相关实验,比较7种不同的保存方法,基于高通量测序技术比对不同时间、不同贮存温度对粪便样本DNA质量、菌群多样性及病原菌检出等结果之间的差异.结果 证实了对于粪便样本采集仍建议采取即刻提取核酸或-20℃保存的方法.同时比较多种保存方法后发现,采用不同样本保存方法受到影响的菌属集中在低丰度菌属(≤0.01％),对应较高丰度的病原菌检测结果的可靠性影响较小,样本中丰度超过千分之一的病原菌检测率超过95％.结论 对于肠道微生态研究建议对粪便样本采取即刻提取核酸或-20℃保存的方法.对于由于其他原因未能妥善保存的粪便样本(如常温保存48 h),仍可对丰度超过千分之一的病原菌进行检测.以上结果对于肠道菌群研究中异地所采集的粪便样本运输和贮存具有一定的指导意义.

In recent years, 16S rRNA amplicon sequencing technology has been widely used to study human gut microbiota and to detect unknown pathogens in clinical samples. However, its resolution to bacterial population can only reach the relative abundance of genus level, and different factors affect the final bacterial profile, such as sample concentrations, PCR cycle numbers and amplification primers. In order to solve these problems, we developed a quantitative 16S rRNA amplicon sequencing method by combining random tag and internal marker method. The new methods improved the accuracy of human gut microbiota, reduced the impact of experimental operation on the results, and improved the comparability between sequencing and other molecular biological methods.

Pathogenic microorganism analysis based on next-generation sequencing technology is an important tool for clinical diagnosis, public health surveillance, and outbreak investigation. However, scientific researchers without the relevant background lack the time, training, or infrastructure to use large data sets or install and use command line tools. Therefore, the bioinformatic team at the Chinese Center for Disease Control and Prevention developed the Microbial Data Analysis Cloud Platform (MDACP) as a safe, professional, and efficient pathogen genetic data analysis platform for rapid microbial data mining, such as for candidate pathogen detection, genome typing, and traceability. MDACP is a web service system based on the Docker platform and can be used for data analysis on various operating systems. The platform focuses on pathogen analysis and continuously develops new analysis processes according to the analysis needs of the users. This platform has a friendly user interface and is easy to operate, allowing users to submit data through data pages or graphical clients, flexibly control parameters according to data conditions, and analyze data in parallel with multiple tasks. Researchers can quickly carry out bioinformatic analyses without coding work, promote follow-up research and information mining of projects, and improve the utilization of big data in the field of disease control. MDACP enables research personnel to conduct data analysis and management and assists clinicians and disease control personnel with mining information, such as pathogen identification, classification, and traceability.

China’s population accounts for about 1/5 th of the world’s total population. Owing to differences in environment, race, living habits, and other factors, the structure of the intestinal flora of Chinese individuals is expected to have unique features; however, this has not been thoroughly examined. Here, we collected faecal samples from healthy adults living in three cities of China and investigated their gut microbiome using metagenomics and bioinformatics technology. We found that 11 core bacterial genera were present in all of the Chinese faecal samples; moreover, several patient characteristics (age, region, body mass index, physical exercise, smoking habits, and alcoholic drink, and yogurt consumption) were found to have different effects on the gut microbiome of healthy Chinese people. We also examined the distribution patterns of disease-related microorganisms (DRMs), revealing which DRMs can potentially be used as markers for assessment of health risk. We also developed a program called “Guthealthy” for evaluating the health status associated with the microbiome and DRM pattern in the faecal samples. The microbiota data obtained in this study will provide a basis for a healthy gut microbiome composition in the Chinese population.

目的 了解四环素类耐药基因在不同国家人体、动物和环境微生态中的分布情况及其多样性.方法 利用生物信息学方法,对2 036份不同来源的微生态样本宏基因组测序数据进行四环素耐药基因的鉴定,研究四环素耐药基因在不同来源、不同国别、不同人体部位的分布及耐药机制.结果 2 036份样本中,四环素耐药基因检出率为44.70％(910份),其中人源样本检出率最高,达78.99％(880/1 114),其次是动物样本,检出率40.98％(25/61).共检出28种四环素耐药基因,248份样本至少检出10种以上四环素耐药基因,在人源性样本中28种基因全部检出,动物源样本中检出6种基因.28种四环素类耐药基因中,包括10种编码核糖体保护蛋白基因,其中5种(tetQ、tet32、tetW、tetO、tetM)在50％以上的人源样本中检出,4种在动物源样本中检出率＞16％.338份人源样本具有国别(中国、丹麦、西班牙、美国和日本)信息,耐药基因分析显示,tetO、tetQ和tetW 3种耐药基因在5个国家均有检出;来源于中国的样本均检出tet32、ta40、tetO、tetQ和taW 5种耐药基因,并匹配上24种四环素耐药基因型.在人体不同部位微生态四环素类耐药基因分布中,肠道中的耐药基因型分布最丰富,共有26种四环素耐药基因匹配到肠道样本中,其中tetQ、tetW、tetO、tet32、tet40 5种耐药基因型检出率＞70％.结论 在人体、动物微生态中存在着大量的、多样的四环素类耐药基因,公众健康和生态环境受到巨大威胁,需要引起高度重视.

Advances in high-throughput sequencing have led to unprecedented growth in the amount of available genome sequencing data, especially for bacterial genomes, which has been accompanied by a challenge for the storage and management of such huge datasets. To facilitate bacterial research and related studies, we have developed the Mypathogen database (MPD), which provides access to users for searching, downloading, storing and sharing bacterial genomics data. The MPD represents the first pathogenic database for microbial genomes and metagenomes, and currently covers pathogenic microbial genomes (6604 genera, 11 071 species, 41 906 strains) and metagenomic data from host, air, water and other sources (28 816 samples). The MPD also functions as a management system for statistical and storage data that can be used by different organizations, thereby facilitating data sharing among different organizations and research groups. A user-friendly local client tool is provided to maintain the steady transmission of big sequencing data. The MPD is a useful tool for analysis and management in genomic research, especially for clinical Centers for Disease Control and epidemiological studies, and is expected to contribute to advancing knowledge on pathogenic bacteria genomes and metagenomes.

BACKGROUND:Pathogen detection in clinical samples based on 16S metagenomic sequencing technology in microbiology laboratories is an important strategy for clinical diagnosis, public health surveillance, and investigations of outbreaks. However, the implementation of the technology is limited by its accuracy and the time required for bioinformatics analysis. Therefore, a simple, standardized, and rapid analysis pipeline from the receipt of clinical samples to the generation of a test report is needed to increase the use of metagenomic analyses in clinical settings.RESULTS:We developed a comprehensive bioinformatics analysis pipeline for the identification of pathogens in clinical samples based on 16S metagenomic sequencing data, named 16SPIP. This pipeline offers two analysis modes (fast and sensitive mode) for the rapid conversion of clinical 16S metagenomic data to test reports for pathogen detection. The pipeline includes tools for data conversion, quality control, merging of paired-end reads, alignment, and pathogen identification. We validated the feasibility and accuracy of the pipeline using a combination of culture and whole-genome shotgun (WGS) metagenomic analyses.CONCLUSIONS:16SPIP may be effective for the analysis of 16S metagenomic sequencing data for real-time, rapid, and unbiased pathogen detection in clinical samples.

BACKGROUND:The rapid spread of Clostridium difficile NAP1/BI/027 (C. difficile 027) has become one of the leading threats of healthcare-associated infections worldwide. However, C. difficile 027 infections have been rarely reported in Asia, particularly in China.RESULTS:In this study, we identified a rare C. difficile bloodstream infection (BSI) from three isolates of a patient during repeated hospital admission. This finding triggered a retrospective epidemiological study to scan all cases and strains emerged from this ward during the past three years. Using medical personnel interviews, medical record reviews and the genomic epidemiology, two outbreaks in 2012 and 2013-2014 were identified. Through using whole genome sequencing, we succeeded to trace the origin of the BSI strain. Surprisingly, we found the genome sequences were similar to C. difficile 027 strain R20291, indicating the occurrence of a rare C. difficile 027 strain in China. Integrated epidemiological investigation and whole genome sequencing of all strains, we constructed a nosocomial transmission map of these two C. difficile 027 outbreaks and traced the origin of the infection.CONCLUSIONS:By genome sequencing, spatio-temporal analysis and field epidemiology investigation, we can estimate their complex transform network and reveal the possible modes of transmission in this ward. Based on their genetic diversity, we can assume that the toilets, bathroom, and janitor's equipment room may be contaminated area, which may be suggested to improve infection control measures in the following health care.

Type IV secretion system (T4SS) can mediate the passage of macromolecules across cellular membranes and is essential for virulent and genetic material exchange among bacterial species. The Type IV Secretion Project 2.0 (T4SP 2.0) database is an improved and extended version of the platform released in 2013 aimed at assisting with the detection of Type IV secretion systems (T4SS) in bacterial genomes. This advanced version provides users with web server tools for detecting the existence and variations of T4SS genes online. The new interface for the genome browser provides a user-friendly access to the most complete and accurate resource of T4SS gene information (e.g., gene number, name, type, position, sequence, related articles, and quick links to other webs). Currently, this online database includes T4SS information of 5239 bacterial strains. Conclusions. T4SS is one of the most versatile secretion systems necessary for the virulence and survival of bacteria and the secretion of protein and/or DNA substrates from a donor to a recipient cell. This database on virB/D genes of the T4SS system will help scientists worldwide to improve their knowledge on secretion systems and also identify potential pathogenic mechanisms of various microbial species.

Objective To investigate the genotype of klebsiella pneumonia strains isolated from eldly inpatients by multiple-locus variable-number tandem-repeat analysis. Methods Totally 184 klebsiella pneumonia strains,isolated from eldly inpatients,were collected,and their genome DNA were extracted. The polymorphism of 7 variable-number tandem-repeat locus in the DNA samples was analyzed by multiple primers polymerase chain reaction and capillary electrophoresis. The clustering analysis of genotyping was carried out with the BioNumerics 5.1 software. Results A total of 139 genotypes were identified in 184 klebsiella pneumonia clinical strains,showing obvious genetic polymorphisms. With clustering analysis of genotypes,all the strains were categorized into three gene clusters (genogroups 1,2,and 3). The genogroup 1 was the biggest cluster,containing 93.06% of the isolated strains. Conclusion There was a predominant cluster in the klebsiella pneumonia strains isolated from eldly inpatients in our center,and the major source of klebsiella pneumonia infection remained the nosocomial infection.

目的：探讨454高通量测序平台应用于临床样本快速检测的可能性。方法应用454高通量测序平台测定血液、呼吸道和消化道临床样本中微生物菌群的16S rDNA 基因的 V3-V4高变异区段序列，采用 BLAST 方法与 RDP 数据库进行比对，基于比对结果，获得不同种属的病原菌的所匹配的 read 数。结果8份临床模拟样本中，7份检测出目标菌属，检出率为87.5%。在血液、肺泡灌洗液和咽拭子这类正常状态下的无菌体液，检测结果较为明确，检出的细菌较为集中，显示了检测结果的可靠性。而对本身含有较多种类细菌的粪便样本，其目标菌比例明显降低。结论实现了80 h 内得到血液、呼吸道和消化道临床样本中含有病原菌情况的检测报告，并探讨了高通量测序技术在今后临床实际应用时进一步提高检测速度和灵敏度的方法。

Clostridium difficile, the etiological agent of Clostridium difficile infection (CDI), is a gram-positive, spore-forming bacillus that is responsible for ∼20% of antibiotic-related cases of diarrhea and nearly all cases of pseudomembranous colitis. Previous data have shown that a substantial proportion (11%) of the C. difficile genome consists of mobile genetic elements, including seven conjugative transposons. However, the mechanism underlying the formation of a mosaic genome in C. difficile is unknown. The type-IV secretion system (T4SS) is the only secretion system known to transfer DNA segments among bacteria. We searched genome databases to identify a candidate T4SS in C. difficile that could transfer DNA among different C. difficile strains. All T4SS gene clusters in C. difficile are located within genomic islands (GIs), which have variable lengths and structures and are all conjugative transposons. During the horizontal-transfer process of T4SS GIs within the C. difficile population, the excision sites were altered, resulting in different short-tandem repeat sequences among the T4SS GIs, as well as different chromosomal insertion sites and additional regions in the GIs.

Objective To investigate the bacterial community composition and diversity in Haemaphysalis longicornis. Methods The metagenomic 16S rDNA V3-V4 profiling of bacteria were sequenced by Illumina Miseq PE300 sequencing platform to describe the bacterial community in H. longicornis ticks, to evaluate bacterial population diversity and to identify the predominant microorganisms and distribution of pathogens. Results A total of 1 070 624 Tags and 145 479 OTUs were obtained for the study. Rarefaction analysis showed a rare factional Shannon?Wiener index reaching a plateau,which indicated that sufficient numbers of sequences had been sequenced for diversity analyses and the richness of bacteria of H. longicornis. The predominated phyla in the H. longicornis were Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes sequentially. Gammaproteobacteria class showed the highest relative abundance. Also we identified a core bacterial community and divided the ticks into two groups. The 21 and 11 unique bacterial communities were detected in individual group, GⅠand GⅡrespectively. A large number of pathogenic bacteria and opportunistic pathogens were found,there were no Spirochaeta and Mycoplasma present in the tick samples. Conclusion The bacterial community structure and distribution of pathogens obtained from H. longicornis ticks showed a high diversity.