The initial state of the human gut microbiome determines its reshaping by antibiotics

Microbiome studies have demonstrated the high inter-individual diversity of the gut microbiota. However, how the initial composition of the microbiome affects the impact of antibiotics on microbial communities is relatively unexplored. To specifically address this question, we administered a second-generation cephalosporin, cefprozil, to healthy volunteers. Stool samples gathered before antibiotic exposure, at the end of the treatment and 3 months later were analysed using shotgun metagenomic sequencing. On average, 15 billion nucleotides were sequenced for each sample. We show that standard antibiotic treatment can alter the gut microbiome in a specific, reproducible and predictable manner. The most consistent effect of the antibiotic was the increase of Lachnoclostridium bolteae in 16 out of the 18 cefprozil-exposed participants. Strikingly, we identified a subgroup of participants who were enriched in the opportunistic pathogen Enterobacter cloacae after exposure to the antibiotic, an effect linked to lower initial microbiome diversity and to a Bacteroides enterotype. Although the resistance gene content of participants'' microbiomes was altered by the antibiotic, the impact of cefprozil remained specific to individual participants. Resistance genes that were not detectable prior to treatment were observed after a 7-day course of antibiotic administration. Specifically, point mutations in beta-lactamase bla_CfxA-6 were enriched after antibiotic treatment in several participants. This suggests that monitoring the initial composition of the microbiome before treatment could assist in the prevention of some of the adverse effects associated with antibiotics or other treatments.     

Comparison of Methods to Identify Pathogens and Associated Virulence Functional Genes in Biosolids from Two Different Wastewater Treatment Facilities in Canada

The use of treated municipal wastewater residues (biosolids) as fertilizers is an attractive, inexpensive option for growers and farmers. Various regulatory bodies typically employ indicator organisms (fecal coliforms, E. coli and Salmonella) to assess the adequacy and efficiency of the wastewater treatment process in reducing pathogen loads in the final product. Molecular detection approaches can offer some advantages over culture-based methods as they can simultaneously detect a wider microbial species range, including non-cultivable microorganisms. However, they cannot directly assess the viability of the pathogens. Here, we used bacterial enumeration methods together with molecular methods including qPCR, 16S rRNA and cpn60 gene amplicon sequencing and shotgun metagenomic sequencing to compare pre- and post-treatment biosolids from two Canadian wastewater treatment plants (WWTPs). Our results show that an anaerobic digestion WWTP was unsuccessful at reducing the live indicator organism load (coliforms, generic E. coli and Salmonella) below acceptable regulatory criteria, while biosolids from a dewatering/pelletization WWTP met these criteria. DNA from other pathogens was detected by the molecular methods, but these species were considered less abundant. Clostridium DNA increased significantly following anaerobic digestion treatments. In addition to pathogen DNA, genes related to virulence and antibiotic resistance were identified in treated biosolids. Shotgun metagenomics revealed the widest range of pathogen DNA and, among the approaches used here, was the only approach that could access functional gene information in treated biosolids. Overall, our results highlight the potential usefulness of amplicon sequencing and shotgun metagenomics as complementary screening methods that could be used in parallel with culture-based methods, although more detailed comparisons across a wider range of sites would be needed.     

Transfer Efficiency of Bacteria and Viruses from Porous and Nonporous Fomites to Fingers under Different Relative Humidity Conditions

Fomites can serve as routes of transmission for both enteric and respiratory pathogens. The present study examined the effect of low and high relative humidity on fomite-to-finger transfer efficiency of five model organisms from several common inanimate surfaces (fomites). Nine fomites representing porous and nonporous surfaces of different compositions were studied. Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis, MS2 coliphage, and poliovirus 1 were placed on fomites in 10-μl drops and allowed to dry for 30 min under low (15% to 32%) or high (40% to 65%) relative humidity. Fomite-to-finger transfers were performed using 1.0 kg/cm² of pressure for 10 s. Transfer efficiencies were greater under high relative humidity for both porous and nonporous surfaces. Most organisms on average had greater transfer efficiencies under high relative humidity than under low relative humidity. Nonporous surfaces had a greater transfer efficiency (up to 57%) than porous surfaces (<6.8%) under low relative humidity, as well as under high relative humidity (nonporous, up to 79.5%; porous, <13.4%). Transfer efficiency also varied with fomite material and organism type. The data generated can be used in quantitative microbial risk assessment models to assess the risk of infection from fomite-transmitted human pathogens and the relative levels of exposure to different types of fomites and microorganisms.     

RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes

16S rRNA amplicon analysis and shotgun metagenome sequencing are two main culture-independent strategies to explore the genetic landscape of various microbial communities. Recently, numerous studies have employed these two approaches together, but downstream data analyses were performed separately, which always generated incongruent or conflict signals on both taxonomic and functional classifications. Here we propose a novel approach, RiboFR-Seq (Ribosomal RNA gene flanking region sequencing), for capturing both ribosomal RNA variable regions and their flanking protein-coding genes simultaneously. Through extensive testing on clonal bacterial strain, salivary microbiome and bacterial epibionts of marine kelp, we demonstrated that RiboFR-Seq could detect the vast majority of bacteria not only in well-studied microbiomes but also in novel communities with limited reference genomes. Combined with classical amplicon sequencing and shotgun metagenome sequencing, RiboFR-Seq can link the annotations of 16S rRNA and metagenomic contigs to make a consensus classification. By recognizing almost all 16S rRNA copies, the RiboFR-seq approach can effectively reduce the taxonomic abundance bias resulted from 16S rRNA copy number variation. We believe that RiboFR-Seq, which provides an integrated view of 16S rRNA profiles and metagenomes, will help us better understand diverse microbial communities.     

Comparative Metagenomics of Anode-Associated Microbiomes Developed in Rice Paddy-Field Microbial Fuel Cells

In sediment-type microbial fuel cells (sMFCs) operating in rice paddy fields, rice-root exudates are converted to electricity by anode-associated rhizosphere microbes. Previous studies have shown that members of the family Geobacteraceae are enriched on the anodes of rhizosphere sMFCs. To deepen our understanding of rhizosphere microbes involved in electricity generation in sMFCs, here, we conducted comparative analyses of anode-associated microbiomes in three MFC systems: a rice paddy-field sMFC, and acetate- and glucose-fed MFCs in which pieces of graphite felt that had functioned as anodes in rice paddy-field sMFC were used as rhizosphere microbe-bearing anodes. After electric outputs became stable, microbiomes associated with the anodes of these MFC systems were analyzed by pyrotag sequencing of 16S rRNA gene amplicons and Illumina shotgun metagenomics. Pyrotag sequencing showed that Geobacteraceae bacteria were associated with the anodes of all three systems, but the dominant Geobacter species in each MFC were different. Specifically, species closely related to G. metallireducens comprised 90% of the anode Geobacteraceae in the acetate-fed MFC, but were only relatively minor components of the rhizosphere sMFC and glucose-fed MFC, whereas species closely related to G. psychrophilus were abundantly detected. This trend was confirmed by the phylogenetic assignments of predicted genes in shotgun metagenome sequences of the anode microbiomes. Our findings suggest that G. psychrophilus and its related species preferentially grow on the anodes of rhizosphere sMFCs and generate electricity through syntrophic interactions with organisms that excrete electron donors.     

Plasmid metagenome reveals high levels of antibiotic resistance genes and mobile genetic elements in activated sludge

The overuse or misuse of antibiotics has accelerated antibiotic resistance, creating a major challenge for the public health in the world. Sewage treatment plants (STPs) are considered as important reservoirs for antibiotic resistance genes (ARGs) and activated sludge characterized with high microbial density and diversity facilitates ARG horizontal gene transfer (HGT) via mobile genetic elements (MGEs). However, little is known regarding the pool of ARGs and MGEs in sludge microbiome. In this study, the transposon aided capture (TRACA) system was employed to isolate novel plasmids from activated sludge of one STP in Hong Kong, China. We also used Illumina Hiseq 2000 high-throughput sequencing and metagenomics analysis to investigate the plasmid metagenome. Two novel plasmids were acquired from the sludge microbiome by using TRACA system and one novel plasmid was identified through metagenomics analysis. Our results revealed high levels of various ARGs as well as MGEs for HGT, including integrons, transposons and plasmids. The application of the TRACA system to isolate novel plasmids from the environmental metagenome, coupled with subsequent high-throughput sequencing and metagenomic analysis, highlighted the prevalence of ARGs and MGEs in microbial community of STPs.     

MEGAN Community Edition - Interactive Exploration and Analysis of Large-Scale Microbiome Sequencing Data

There is increasing interest in employing shotgun sequencing, rather than amplicon sequencing, to analyze microbiome samples. Typical projects may involve hundreds of samples and billions of sequencing reads. The comparison of such samples against a protein reference database generates billions of alignments and the analysis of such data is computationally challenging. To address this, we have substantially rewritten and extended our widely-used microbiome analysis tool MEGAN so as to facilitate the interactive analysis of the taxonomic and functional content of very large microbiome datasets. Other new features include a functional classifier called InterPro2GO, gene-centric read assembly, principal coordinate analysis of taxonomy and function, and support for metadata. The new program is called MEGAN Community Edition (CE) and is open source. By integrating MEGAN CE with our high-throughput DNA-to-protein alignment tool DIAMOND and by providing a new program MeganServer that allows access to metagenome analysis files hosted on a server, we provide a straightforward, yet powerful and complete pipeline for the analysis of metagenome shotgun sequences. We illustrate how to perform a full-scale computational analysis of a metagenomic sequencing project, involving 12 samples and 800 million reads, in less than three days on a single server. All source code is available here: https://github.com/danielhuson/megan-ce     

中国人群肠道微生物鸟枪法宏基因组测序研究进展

肠道微生物与人类健康的相关性研究仍然是当前生命科学研究领域的前沿热点之一。不依赖培养的 16S rRNA基因高通量测序是当前的主要研究手段。但随着测序成本的降低和数据分析方法的日渐成熟,宏基因组鸟枪法测序因具有信息量更大、更全等优势,将逐渐成为今后一段时间内研究肠道微生物组的重要手段。美国在人类微生物组计划的资助下,对30 805份样品进行了肠道微生物宏基因测序分析。通过NCBI PubMed和SRA数据库检索,共发现72项研究收集了约10 000份中国人的肠道样品用于宏基因组测序。但到目前为止,仅56项研究进行了公开发表,其中与代谢性疾病相关的文献16篇,与感染和免疫性疾病相关的文献16篇,与心脑血管疾病相关的文献12篇。由于采样地点以北京、广州、上海等大城市为主,测序平台和测序分析方法均存在较大差异,且大部分研究仍以相关性分析为主,相关研究成果在临床疾病诊疗中所发挥的作用仍非常有限。规范采样方法、标准化测序平台和数据分析流程,开展多中心平行研究将有助于数据整合和比较分析。同时,结合使用转录组、蛋白质组和培养组学等多组学方法开展功能验证和分子作用机制研究,将有利于更好地将肠道微生物研究成果服务于临床疾病诊疗。     

Pro-Inflammatory Flagellin Proteins of Prevalent Motile Commensal Bacteria Are Variably Abundant in the Intestinal Microbiome of Elderly Humans

Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute “cell motility” category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ²⁸. The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13–4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved metagenome assembly from short reads will be required to facilitate in silico analyses of complete complex biochemical pathways for low-abundance target species from shotgun metagenomes.     

Metagenomic microbial community profiling of Unnai hot spring by Ion-Torrent based shotgun sequencing

This is the first report on depicting the pioneering microbiota of Unnai hot spring using shotgun metagenome sequencing approach. Community analysis encompassed a total of 688,059 sequences with the total size 125.31 Mbp and 46% G + C content. Sequencing metagenome reported about 992 species belonged to 40 different phyla dominated by Firmicutes (97.49%), Proteobacteria (1.36%), and Actinobacteria (0.31%). In functional analysis, Non-Supervised Orthologous Groups (NOG) annotation revealed the predominance of poorly characterized reads (82.79%). Moreover, the subsystem classification displayed 19% genes assigned to carbohydrates metabolism, 12% genes allocated to clustering-based subsystems, 10% genes belonged to amino acids and its derivatives. The result suggests the huge bacterial diversity which will be useful for further characterizing the economically important bacteria for biotechnological applications.     

Acquired Genetic Mechanisms of a Multiresistant Bacterium Isolated from a Treatment Plant Receiving Wastewater from Antibiotic Production

The external environment, particularly wastewater treatment plants (WWTPs), where environmental bacteria meet human commensals and pathogens in large numbers, has been highlighted as a potential breeding ground for antibiotic resistance. We have isolated the extensively drug-resistant Ochrobactrum intermedium CCUG 57381 from an Indian WWTP receiving industrial wastewater from pharmaceutical production contaminated with high levels of quinolones. Antibiotic susceptibility testing against 47 antibiotics showed that the strain was 4 to >500 times more resistant to sulfonamides, quinolones, tetracyclines, macrolides, and the aminoglycoside streptomycin than the type strain O. intermedium LMG 3301^T. Whole-genome sequencing identified mutations in the Indian strain causing amino acid substitutions in the target enzymes of quinolones. We also characterized three acquired regions containing resistance genes to sulfonamides (sul1), tetracyclines [tet(G) and tetR], and chloramphenicol/florfenicol (floR). Furthermore, the Indian strain harbored acquired mechanisms for horizontal gene transfer, including a type I mating pair-forming system (MPF_I), a MOB_P relaxase, and insertion sequence transposons. Our results highlight that WWTPs serving antibiotic manufacturing may provide nearly ideal conditions for the recruitment of resistance genes into human commensal and pathogenic bacteria.     

Multidrug-resistant Opportunistic and Pathogenic Bacteria Contaminate Algerian Banknotes Currency

Currency is one of the most exchanged items in human communities as it is used daily in exchange for goods and services. It is handled by persons with different hygiene standards and can transit in different environments. Hence, money can constitute a reservoir for different types of human pathogens. This study aimed to evaluate the potential of Algerian banknotes to shelter opportunistic pathogenic and multiresistant bacteria. To that end, 200 circulating notes of four different denominations were collected from various places and analyzed for their bacterial loads and contents. Besides, predominant strains were identified and characterized by biochemical and molecular methods, and their resistance profiles against 34 antibiotics were determined. Our results indicated that 100% of the studied banknotes were contaminated with bacteria. The total bacterial concentrations were relatively high, and different bacterial groups were grown, showing important diversity. In total, 48 predominant strains were identified as belonging to 17 genera. Staphylococcus and Micrococcus were the most prevalent genera, followed by Bacillus, Pseudomonas, and Acinetobacter. Antibiotic susceptibility testing showed that all the isolates harbored resistance to at least two molecules, and worrying resistance levels were observed. These findings prove that Algerian currency harbors opportunistic multiresistant bacteria and could potentially act as a vehicle for the spread of bacterial diseases and as a reservoir for antibiotic resistance genes among the community. Therefore, no cash payment systems should be developed and generalized to minimize cash handling and subsequent potential health risks.Key words: currency, Algeria, opportunistic bacteria, antibiotic resistance, circulating resistance genes     

Characterization of the rumen microbiome of Indian Kankrej cattle (Bos indicus) adapted to different forage diet

Present study described rumen microbiome of Indian cattle (Kankrej breed) to better understand the microbial diversity and largely unknown functional capacity of the rumen microbiome under different dietary treatments. Kankrej cattle were gradually adapted to a high-forage diet (four animals with dry forage and four with green forage) containing 50 % (K1), 75 % (K2) to 100 % (K3) forage, and remaining concentrate diet, each for 6 weeks followed by analysis of rumen fiber adherent and fiber-free metagenomic community by shotgun sequencing using ion torrent PGM platform and EBI-metagenomics annotation pipeline. Taxonomic analysis indicated that rumen microbiome was dominated by Bacteroidetes followed by Firmicutes, Fibrobacter, Proteobacteria, and Tenericutes. Functional analysis based on gene ontology classified all reads in total 157 categories based on their functional role in biological, molecular, and cellular component with abundance of genes associated with hydrolase activity, membrane, transport, transferase, and different metabolism (such as carbohydrate and protein). Statistical analysis using STAMP revealed significant differences (P?相似文献   

Shallow shotgun sequencing of the microbiome recapitulates 16S amplicon results and provides functional insights

Prevailing 16S rRNA gene-amplicon methods for characterizing the bacterial microbiome of wildlife are economical, but result in coarse taxonomic classifications, are subject to primer and 16S copy number biases, and do not allow for direct estimation of microbiome functional potential. While deep shotgun metagenomic sequencing can overcome many of these limitations, it is prohibitively expensive for large sample sets. Here we evaluated the ability of shallow shotgun metagenomic sequencing to characterize taxonomic and functional patterns in the faecal microbiome of a model population of feral horses (Sable Island, Canada). Since 2007, this unmanaged population has been the subject of an individual-based, long-term ecological study. Using deep shotgun metagenomic sequencing, we determined the sequencing depth required to accurately characterize the horse microbiome. In comparing conventional vs. high-throughput shotgun metagenomic library preparation techniques, we validate the use of more cost-effective laboratory methods. Finally, we characterize similarities between 16S amplicon and shallow shotgun characterization of the microbiome, and demonstrate that the latter recapitulates biological patterns first described in a published amplicon data set. Unlike for amplicon data, we further demonstrate how shallow shotgun metagenomic data provide useful insights regarding microbiome functional potential which support previously hypothesized diet effects in this study system.     

Functional profiling of the gut microbiomes in two different populations of the brown planthopper,Nilaparvata lugens

Previous studies have demonstrated that gut symbionts are involved in the detoxification metabolism of insect hosts, but the relationship between gut symbionts and host detoxification metabolism of the brown planthopper (Nilaparvata lugens, BPH) remains unclear. In the present study, an indoor population (NlIP) and a field population (NlFP) of the BPH were used to characterize the functional profiling of the gut microbiome based on 16S rDNA sequencing. The results show that the NlIP and NlFP strains of N. lugens had different symbiont compositions, and Proteobacteria, Actinobacteria, and Firmicutes were the dominate phyla, accounting for >75% of the total symbiont compositions. Additionally, the NlIP strain had more Pantoea and Stenotrophomonas, while the NlFP strain showed a higher Wolbachia, Actinobacteria, and Herbaspirillum relative abundance. Furthermore, functional content of the metagenome predicted by PICRUSt demonstrated no significant difference in metagenomic function between the NlIP and NlFP strains in the principal component analysis (PCA), and only three types of genes, namely, genes involved with metabolic diseases, poorly characterized genes, and genes involved in circulatory systems, were different between the strains based on KEGG pathway analysis, which also speculated that gut symbionts are not directly involved in the detoxification metabolism for insecticides in the BPH. These results will be helpful for further research into the mechanisms of gut symbionts involved in detoxification metabolism in the BPH.     

Dysbiosis in the Gut Bacterial Microbiome of Patients with Uveitis,an Inflammatory Disease of the Eye

Uveitis (UVT), an inflammatory disease of the eye significantly contributes to vision impairment and blindness. Uveitis is associated with systemic infectious and autoimmune diseases, but in most cases, the aetiology remains unidentified. Dysbiosis in the gut microbiome has been implicated in autoimmune diseases, inflammatory diseases, cancers and mental disorders. In a mice model of autoimmune UVT, it was observed that manipulating the gut microbiome reduces the inflammation and disease severity. Further, alterations in the bacterial gut microbiome and their metabolites were reported in UVT patients from a Chinese cohort. Hence, it is worth comparing the bacterial gut microbiome of UVT patients with that of healthy controls (HC) to ascertain whether dysbiosis of the gut microbiome has implications in UVT. Our analyses showed reduced diversity of several anti-inflammatory organisms including Faecalibacterium, Bacteroides, Lachnospira, Ruminococcus and members of Lachnospiraceae and Ruminococcaceae families, and enrichment of Prevotella (proinflammatory) and Streptococcus (pathogenic) OTUs in UVT microbiomes compared to HC. In addition, decrease in probiotic and antibacterial organisms was observed in UVT compared to HC microbiomes. Heatmap and PCoA plots also indicated significant variations in the microbiomes of UVT versus HC. This is the first study demonstrating dysbiosis in the gut bacterial communities of UVT patients in an Indian cohort and suggests a role of the gut microbiome in the pathophysiology of UVT.     

Distinct composition and metabolic functions of human gut microbiota are associated with cachexia in lung cancer patients

Cachexia is associated with decreased survival in cancer patients and has a prevalence of up to 80%. The etiology of cachexia is poorly understood, and limited treatment options exist. Here, we investigated the role of the human gut microbiome in cachexia by integrating shotgun metagenomics and plasma metabolomics of 31 lung cancer patients. The cachexia group showed significant differences in the gut microbial composition, functional pathways of the metagenome, and the related plasma metabolites compared to non-cachectic patients. Branched-chain amino acids (BCAAs), methylhistamine, and vitamins were significantly depleted in the plasma of cachexia patients, which was also reflected in the depletion of relevant gut microbiota functional pathways. The enrichment of BCAAs and 3-oxocholic acid in non-cachectic patients were positively correlated with gut microbial species Prevotella copri and Lactobacillus gasseri, respectively. Furthermore, the gut microbiota capacity for lipopolysaccharides biosynthesis was significantly enriched in cachectic patients. The involvement of the gut microbiome in cachexia was further observed in a high-performance machine learning model using solely gut microbial features. Our study demonstrates the links between cachectic host metabolism and specific gut microbial species and functions in a clinical setting, suggesting that the gut microbiota could have an influence on cachexia with possible therapeutic applications.Subject terms: Microbiome, Metagenomics, Next-generation sequencing, Metabolomics