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Non-heritable, phenotypic drug resistance toward antibiotics challenges antibiotic therapies. Characteristics of such phenotypic resistance have implications for the evolution of heritable resistance. Diverse forms of phenotypic resistance have been described, but phenotypic resistance characteristics remain less explored than genetic resistance. Here, we add novel combinations of single-cell characteristics of phenotypic resistant E. coli cells and compare those to characteristics of susceptible cells of the parental population by exposure to different levels of recurrent ampicillin antibiotic. Contrasting expectations, we did not find commonly described characteristics of phenotypic resistant cells that arrest growth or near growth. We find that under ampicillin exposure, phenotypic resistant cells reduced their growth rate by about 50% compared to growth rates prior to antibiotic exposure. The growth reduction is a delayed alteration to antibiotic exposure, suggesting an induced response and not a stochastic switch or caused by a predetermined state as frequently described. Phenotypic resistant cells exhibiting constant slowed growth survived best under ampicillin exposure and, contrary to expectations, not only fast-growing cells suffered high mortality triggered by ampicillin but also growth-arrested cells. Our findings support diverse modes of phenotypic resistance, and we revealed resistant cell characteristics that have been associated with enhanced genetically fixed resistance evolution, which supports claims of an underappreciated role of phenotypic resistant cells toward genetic resistance evolution. A better understanding of phenotypic resistance will benefit combatting genetic resistance by developing and engulfing effective anti-phenotypic resistance strategies.

Importance: Antibiotic resistance is a major challenge for modern medicine. Aside from genetic resistance to antibiotics, phenotypic resistance that is not heritable might play a crucial role for the evolution of antibiotic resistance. Using a highly controlled microfluidic system, we characterize single cells under recurrent exposure to antibiotics. Fluctuating antibiotic exposure is likely experienced under common antibiotic therapies. These phenotypic resistant cell characteristics differ from previously described phenotypic resistance, highlighting the diversity of modes of resistance. The phenotypic characteristics of resistant cells we identify also imply that such cells might provide a stepping stone toward genetic resistance, thereby causing treatment failure.

Prevotella copri is the dominant species of the Prevotella genus in the gut, which is genomically heterogeneous and difficult to isolate; hence, scarce research was carried out for this species. This study aimed to investigate the effect of P. copri on hyperglycemia. Thirty-nine strains were isolated from healthy individuals, and three strains (HF2123, HF1478, and HF2130) that had the highest glucose consumption were selected to evaluate the effects of P. copri supplementation on hyperglycemia. Microbiomics and non-target metabolomics were used to uncover the underlying mechanisms. Oral administration of P. copri in diabetic db/db mice increased the expression and secretion of glucagon-like peptide-1 (GLP-1), significantly improved hyperglycemia, insulin resistance, and lipid accumulation, and alleviated the pathological morphology in the pancreas, liver, and colon. P. copri changed the composition of the gut microbiota of diabetic db/db mice, which was characterized by increasing the ratio of Bacteroidetes to Firmicutes and increasing the relative abundance of genera Bacteroides, Akkermansia, and Faecalibacterium. After intervention with P. copri, fecal metabolic profiling showed that fumaric acid and homocysteine contents decreased, and glutamine contents increased. Furthermore, amino acid metabolism and cAMP/PKA signaling pathways were enriched. Our findings indicate that P. copri improved glucose metabolism abnormalities in diabetic db/db mice. Especially, one of the P. copri strains, HF2130, has shown superior performance in improving hyperglycemia, which may have the potential as a probiotic against hyperglycemia.

Importance: As a core member of the human intestinal ecosystem, Prevotelal copri has been associated with glucose metabolic homeostasis in previous studies. However, these results have often been derived from metagenomic studies, and the experimental studies have been based solely on the type of strain DSM 18205^T. Therefore, more experimental evidence from additional isolates is needed to validate the results according to their high genomic heterogeneity. In this study, we isolated different branches of strains and demonstrated that P. copri could improve the metabolic profile of hyperglycemic mice by modulating microbial activity. This finding supports the causal contribution of P. copri in host glucose metabolism.

The gastric microbial community plays a fundamental role in gastric cancer (GC), and the two main anatomical subtypes of GC, non-cardia and cardia GC, are associated with different risk factors (Helicobacter pylori for non-cardia GC). To decipher the different microbial spatial communities of GC, we performed a multicenter retrospective analysis to characterize the gastric microbiota in 223 GC patients, including H. pylori-positive or -negative patients, with tumors and paired adjacent normal tissues, using third-generation sequencing. In the independent validation cohort, both dental plaque and GC tumoral tissue samples were collected and sequenced. The prevalence of H. pylori and oral-associated bacteria was verified using fluorescence in situ hybridization (FISH) assays in GC tumoral tissues and matched nontumoral tissues. We found that the vertical distribution of the gastric microbiota, at the upper, middle, and lower third sites of GC, was likely an important factor causing microbial diversity in GC tumor tissues. The oral-associated microbiota cluster, which included Veillonella parvula, Streptococcus oralis, and Prevotella intermedia, was more abundant in the upper third of the GC. However, H. pylori was more abundant in the lower third of the GC and exhibited a significantly high degree of microbial correlation. The oral-associated microbiota module was co-exclusive with H. pylori in the lower third site of the GC tumoral tissue. Importantly, H. pylori-negative GC patients with oral-associated gastric microbiota showed worse overall survival, while the increase in microbial abundance in H. pylori-positive GC patients showed no difference in overall survival. The prevalence of V. parvula in both the dental plaque and GC tissue samples was concordant in the independent validation phase. We showed that the oral-associated species V. parvula and S. oralis were correlated with overall survival. Our study highlights the roles of the oral-associated microbiota in the upper third of the GC. In addition, oral-associated species may serve as noninvasive screening tools for the management of GC and an independent prognostic factor for H. pylori-negative GCs.

Importance: Our study highlights the roles of the oral-associated microbiota in the upper third of gastric cancer (GC).We showed that the oral-associated species Veillonella parvula and Streptococcus oralis were correlated with overall survival. In addition, oral-associated species may serve as noninvasive screening tools for the management of GC and an independent prognostic factor for Helicobacter pylori-negative GCs.

Hypersaline ecosystems display taxonomically similar assemblages with low diversities and highly dense accompanying viromes. The ecological implications of viral infection on natural microbial populations remain poorly understood, especially at finer scales of diversity. Here, we sought to investigate the influence of changes in environmental physicochemical conditions and viral predation pressure by autochthonous and allochthonous viruses on host dynamics. For this purpose, we transplanted two microbiomes coming from distant hypersaline systems (solar salterns of Es Trenc in Spain and the thalassohaline lake of Aran-Bidgol lake in Iran), by exchanging the cellular fractions with the sterile-filtered accompanying brines with and without the free extracellular virus fraction. The midterm exposure (1 month) of the microbiomes to the new conditions showed that at the supraspecific taxonomic range, the assemblies from the solar saltern brine more strongly resisted the environmental changes and viral predation than that of the lake. The metagenome-assembled genomes (MAGs) analysis revealed an intraspecific transition at the ecotype level, mainly driven by changes in viral predation pressure, by both autochthonous and allochthonous viruses.

Importance: Viruses greatly influence succession and diversification of their hosts, yet the effects of viral infection on the ecological dynamics of natural microbial populations remain poorly understood, especially at finer scales of diversity. By manipulating the viral predation pressure by autochthonous and allochthonous viruses, we uncovered potential phage-host interaction, and their important role in structuring the prokaryote community at an ecotype level.

Bacterial chromosomal type I toxin-antitoxin systems consist of a small protein, typically under 60 amino acids, and a small RNA (sRNA) that represses toxin translation. These gene pairs have gained attention over the last decade for their contribution to antibiotic persistence and phage tolerance in bacteria. However, biological functions for many remain elusive as gene deletions often fail to produce an observable phenotype. For many pairs, it is still unknown when the toxin and/or antitoxin gene are natively expressed within the bacterium. We examined sequence conservation of three type I toxin-antitoxin systems, tisB/istR-1, shoB/ohsC, and zor/orz, in over 2,000 Escherichia coli strains, including pathogenic and commensal isolates. Using our custom database, we found that these gene pairs are widespread across E. coli and have expression potential via BLASTn. We identified an alternative, dominant sequence variant of TisB and confirmed that it is toxic upon overproduction. Additionally, analyses revealed a highly conserved sequence in the zorO mRNA untranslated region that is required for full toxicity. We further noted that over 30% of E. coli genomes contain an orz antitoxin gene only and confirmed its expression in a representative strain: the first confirmed report of a type I antitoxin without its cognate toxin. Our results add to our understanding of these systems, and our methodology is applicable for other type I loci to identify critical regulatory and functional features.IMPORTANCEChromosomal type I toxin-antitoxins are a class of genes that have gained increasing attention over the last decade for their roles in antibiotic persistence which may contribute to therapeutic failures. However, the control of many of these genes and when they function have remained elusive. We demonstrate that a simple genetic conservation-based approach utilizing free, publicly available data yields known and novel insights into the regulation and function of three chromosomal type I toxin-antitoxins in Escherichia coli. This study also provides a framework for how this approach could be applied to other genes of interest.

Gut microbiota of the bumblebee is critical as it modulates the health and fitness of the host. However, the mechanisms underlying the formation and maintenance of the diversity of bumblebee gut bacteria over a long period of evolution have yet to be elucidated. In particular, the gut bacterial diversity and community assembly processes of Bombus pyrosoma across the Chinese border remain unclear. In this study, we systematically carried out unprecedented sampling of 513 workers of the species Bombus pyrosoma across the Chinese landscape and used full-length 16S rRNA gene sequencing to examine their gut microbiota diversity and biogeography. The gut microbiota composition and community structure of Bombus pyrosoma from different geographical locations were diverse. On the whole, the gut bacteria Gilliamella and Snodgrassella are dominant in bumblebees, but opportunistic pathogens Serratia and Pseudomonas are dominant in some sampling sites such as Hb15, Gs1, Gs45, Qhs15, and Ssx35. All or part of environmental factors such as latitude, annual mean temperature, elevation, human footprint, population density, and annual precipitation can affect the alpha diversity and community structure of gut bacteria. Further analysis showed that the assembly and shift of bumblebee gut bacterial communities under geographical variation were mainly driven by the stochastic drift of the neutral process rather than by variable selection of niche differentiation. In conclusion, our unprecedented sampling uncovers bumblebee gut microbiome diversity and shifts over evolutionary time.

Importance: The microbiotas associated with organisms facilitates host health and fitness, and the homeostasis status of gut microbiota also reflects the habitat security faced by the host. In addition, managing gut microbiota is important to improve bumblebee health by understanding the ecological process of the gut microbiome. Thus, we first carried out an runprecedented sampling of 513 workers of the species Bombus pyrosoma across the Chinese landscape and used full-length 16S rRNA gene sequencing to uncover their gut microbiota diversity and biogeography. Our study provides new insights into the understanding of gut microbiome diversity and shifts for Chinese Bumblebee over evolutionary time.

The alarming rise of antibiotic-resistant bacterial infections is driving efforts to develop alternatives to conventional antibiotics. In this context, antimicrobial peptides (AMPs) have emerged as promising candidates for their ability to target a broad range of microorganisms. However, the development of AMPs with optimal potency, selectivity, and/or stability profiles remains a challenge. To address it, computational tools for predicting AMP properties and designing novel peptides have gained increasing attention. PyAMPA is a novel platform for AMP discovery. It consists of five modules, namely AMPScreen, AMPValidate, AMPSolve, AMPMutate, and AMPOptimize, that allow high-throughput proteome inspection, candidate screening, and optimization through point-mutation and genetic algorithms. The platform also offers additional tools for predicting and evaluating AMP properties, including antimicrobial and cytotoxic activity, and peptide half-life. By providing innovative and accessible inroads into AMP motifs in proteomes, PyAMPA will enable advances in AMP development and potential translation into clinically useful molecules. PyAMPA is available at: https://github.com/SysBioUAB/PyAMPA.

Importance: This paper introduces PyAMPA, a new bioinformatics platform designed for the discovery and optimization of antimicrobial peptides (AMPs). It addresses the urgent need for new antimicrobials due to the rise of antibiotic-resistant infections. PyAMPA, with its five predictive modules -AMPScreen, AMPValidate, AMPSolve, AMPMutate and AMPOptimize, enables high-throughput screening of proteomes to identify potential AMP motifs and optimize them for clinical use. Its unique approach, combining prediction, design, and optimization tools, makes PyAMPA a robust solution for developing new AMP-based therapies, offering a significant advance in combatting antibiotic resistance.

Are two adjacent genes in the same operon? What are the order and spacing between several transcription factor binding sites? Genome browsers are software data visualization and exploration tools that enable biologists to answer questions such as these. In this paper, we report on a major update to our browser, Genome Explorer, that provides nearly instantaneous scaling and traversing of a genome, enabling users to quickly and easily zoom into an area of interest. The user can rapidly move between scales that depict the entire genome, individual genes, and the sequence; Genome Explorer presents the most relevant detail and context for each scale. By downloading the data for the entire genome to the user's web browser and dynamically generating visualizations locally, we enable fine control of zoom and pan functions and real-time redrawing of the visualization, resulting in smoother and more intuitive exploration of a genome than is possible with other browsers. Further, genome features are presented together, in-line, using familiar graphical depictions. In contrast, many other browsers depict genome features using data tracks, which have low information density and can visually obscure the relative positions of features. Genome Explorer diagrams have a high information density that provides larger amounts of genome context and sequence information to be presented in a given-sized monitor than for tracks-based browsers. Genome Explorer provides optional data tracks for the analysis of large-scale data sets and a unique comparative mode that aligns genomes at orthologous genes with synchronized zooming.

Importance: Genome browsers provide graphical depictions of genome information to speed the uptake of complex genome data by scientists. They provide search operations to help scientists find information and zoom operations to enable scientists to view genome features at different resolutions. We introduce the Genome Explorer browser, which provides extremely fast zooming and panning of genome visualizations and displays with high information density.

Locusta migratoria is an important phytophagous pest, and its gut microbial communities play an important role in cellulose degradation. In this study, the gut microbial and cellulose digestibility dynamics of Locusta migratoria were jointly analyzed using high-throughput sequencing and anthrone colorimetry. The results showed that the gut microbial diversity and cellulose digestibility across life stages were dynamically changing. The species richness of gut bacteria was significantly higher in eggs than in larvae and imago, the species richness and cellulose digestibility of gut bacteria were significantly higher in early larvae (first and second instars) than in late larvae (third to fifth instars), and the diversity of gut bacteria and cellulose digestibility were significantly higher in imago than in late larvae. There is a correlation between the dynamics of gut bacterial communities and cellulose digestibility. Enterobacter, Lactococcus, and Pseudomonas are the most abundant genera throughout all life stages. Six strains of highly efficient cellulolytic bacteria were screened, which were dominant gut bacteria. Carboxymethyl cellulase activity (CMCA) and filter paper activity (FPA) experiments revealed that Pseudomonas had the highest cellulase enzyme activity. This study provides a new way for the screening of cellulolytic bacteria and lays the foundation for developing insects with significant biomass into cellulose-degrading bioreactors.

Importance: Cellulose is the most abundant and cheapest renewable resource in nature, but its degradation is difficult, so finding efficient cellulose degradation methods is an urgent challenge. Locusta migratoria is a large group of agricultural pests, and the large number of microorganisms that inhabit their intestinal tracts play an important role in cellulose degradation. We analyzed the dynamics of Locusta migratoria gut microbial communities and cellulose digestibility using a combination of high-throughput sequencing technology and anthrone colorimetry. The results revealed that the gut microbial diversity and cellulose digestibility were dynamically changed at different life stages. In addition, we explored the intestinal bacterial community of Locusta migratoria across life stages and its correlation with cellulose digestibility. The dominant bacterial genera at different life stages of Locusta migratoria were uncovered and their carboxymethyl cellulase activity (CMCA) and filter paper activity (FPA) were determined. This study provides a new avenue for screening cellulolytic bacteria and lays the foundation for developing insects with significant biomass into cellulose-degrading bioreactors.

Mixotrophy is an important trophic strategy for bacterial survival in the ocean. However, the global relevance and identity of the major mixotrophic taxa remain largely elusive. Here, we combined phylogenetic, metagenomic, and metatranscriptomic analyses to characterize ubiquitous Arcobacteraceae based on our deep-sea in situ incubations and the global data. The phylogenomic tree of Arcobacteraceae is divided into three large clades, among which members of clades A and B are almost all from terrestrial environments, while those of clade C are widely distributed in various marine habitats in addition to some terrestrial origins. All clades harbor genes putatively involved in chitin degradation, sulfide oxidation, hydrogen oxidation, thiosulfate oxidation, denitrification, dissimilatory nitrate reduction to ammonium, microaerophilic respiration, and metal (iron/manganese) reduction. Additionally, in clade C, more unique pathways were retrieved, including thiosulfate disproportionation, ethanol fermentation, methane oxidation, fatty acid oxidation, cobalamin synthesis, and dissimilatory reductions of sulfate, perchlorate, and arsenate. Within this clade, two mixotrophic Candidatus genera represented by UBA6211 and CAIJNA01 harbor genes putatively involved in the reverse tricarboxylic acid pathway for carbon fixation. Moreover, the metatranscriptomic data in deep-sea in situ incubations indicated that the latter genus is a mixotroph that conducts carbon fixation by coupling sulfur oxidation and denitrification and metabolizing organic matter. Furthermore, global metatranscriptomic data confirmed the ubiquitous distribution and global relevance of Arcobacteraceae in the expression of those corresponding genes across all oceanic regions and depths. Overall, these results highlight the contribution of previously unrecognized Arcobacteraceae to carbon, nitrogen, and sulfur cycling in global oceans.IMPORTANCEMarine microorganisms exert a profound influence on global carbon cycling and ecological relationships. Mixotrophy, characterized by the simultaneous utilization of both autotrophic and heterotrophic nutrition, has a significant impact on the global carbon cycling. This report characterizes a group of uncultivated bacteria Arcobacteraceae that thrived on the "hot time" of bulky particulate organic matter and exhibited mixotrophic strategy during the in situ organic mineralization. Compared with clades A and B, more unique metabolic pathways were retrieved in clade C, including the reverse tricarboxylic acid pathway for carbon fixation, thiosulfate disproportionation, methane oxidation, and fatty acid oxidation. Global metatranscriptomic data from the Tara Oceans expeditions confirmed the ubiquitous distribution and extensive transcriptional activity of Arcobacteraceae with the expression of genes putatively involved in carbon fixation, methane oxidation, multiple sulfur compound oxid