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Microbiome research using amplicon sequencing of microbial marker genes has surged over the past decade, propelled by protocols for highly multiplexed sequencing with barcoded primer constructs. Newer Illumina platforms like the NovaSeq and NextSeq series significantly outperform older sequencers in terms of reads, output, and runtime. However, these platforms are more prone to index-hopping, which limits the application of protocols designed for older platforms such as the Earth Microbiome Project protocols; hence, there is a need to adapt these established protocols. Here, we present an ultra-high-throughput amplicon library preparation and sequencing protocol (HighALPS) incorporating the capabilities of these newer sequencing platforms, designed for both 16S rRNA gene and fungal internal transcribed spacer domain sequencing. Our results demonstrate good run performance across different sequencing platforms and flow cells, with successful sequencing of mock communities, validating the protocol's effectiveness. The HighALPS library preparation method offers a robust, cost-effective, and ultra-high-throughput solution for microbiome research, compatible with the latest sequencing technologies. This protocol allows multiplexing thousands of samples in a single run at a read depth of tens of millions of sequences per sample.IMPORTANCEMarker gene amplicon sequencing on Illumina devices remains the most commonly used technology to profile microbial communities. Yet, most library preparation protocols are not adapted to harness the capabilities and deal with the caveats of the latest Illumina sequencing platforms, which highly outperform older platforms in terms of speed, quality, and output. Here, we present an ultra-high-throughput, cost-effective, and robust library preparation protocol (HighALPS) optimized to fully leverage the capabilities of the latest Illumina sequencing platforms. The combinatorial unique dual index strategy effectively combats miss-assignment of reads due to index-hopping, which is more prevalent in newer platforms. The HighALPS protocol incorporates technological (e.g., novel sequencing chemistry and lab automation platforms) as well as bioinformatics advances (e.g., denoising algorithms which make triplicate amplifications unnecessary) of the last few years to optimize and streamline library preparation for bacterial and fungal communities.

Staphylococcus aureus is a major source of community and nosocomial infections. Due to the extensive application of antibiotics, S. aureus has developed resistance to antibiotics, especially vancomycin, making clinical treatment challenging. Staphylococcal accessory regulator A (SarA) modulates S. aureus virulence by regulating the principal virulence factors. However, its role in vancomycin resistance remains largely unknown. Herein, we found that SarA not only reduces the susceptibility of S. aureus to vancomycin by directly inhibiting the expression of autolysis-related genes, but also enhances resistance to vancomycin by negatively regulating the transcription of an ATP-binding cassette (ABC) transporter, ABC-like, thereby altering the bacterial surface charge and reducing vancomycin's binding efficiency to the cell wall. Moreover, the regulation of antibiotic resistance by SarA is strain-dependent. Our study uncovers the roles of SarA in regulating vancomycin resistance, providing potential targets and ideas for the prevention and control of vancomycin-intermediate S. aureus infections.IMPORTANCEStaphylococcus aureus poses a major threat to public health due to its increasing resistance to vancomycin, a last-line antibiotic. This study reveals that Staphylococcal accessory regulator A regulates vancomycin resistance in S. aureus by suppressing genes related to autolysis and negatively regulating an ATP-binding cassette (ABC) transporter (ABC-like). This regulation of the transporter reduces the bacterial surface charge, impairing the ability of vancomycin to bind to the cell wall. These findings suggest a novel mechanism of antibiotic resistance in S. aureus and identify potential targets for combating vancomycin-intermediate S. aureus infections.

Bifidobacterium adolescentis is one of the most frequently encountered bifidobacterial species present in the adult human gut microbiota, with a prevalence of approximately 60%. Despite its high prevalence, B. adolescentis has not been extensively studied and characterized, and our understanding of its physiological traits, genetic diversity, and potential interactions with other members of the human gut microbiota or with its host is therefore fragmentary. In the current study, a data set comprising 1,682 B. adolescentis genomes was compiled by combining publicly available data and metagenome assemblies from 131 projects to uncover the unique genetic characteristics of this species. A pangenome analysis of B. adolescentis identified 203 clusters of orthologous genes absent from the other five human-associated Bifidobacterium species, six of which were in silico predicted to encode functions unique to this taxon. Furthermore, 2,597 genes were predicted to have been acquired by horizontal gene transfer, including genes encoding extracellular structures involved in interaction with the host and other microorganisms, and phage defense mechanisms against bacteriophages. Detailed phylogenetic analysis revealed seven clusters within the B. adolescentis species, each partially associated with the origin of strain isolation, suggesting phylogenetic differentiation shaped by geographical strain origin. Moreover, a large-scale metagenomic analysis of over 10,000 human gut metagenomes from healthy adults revealed that B. adolescentis co-occurs with 36 putative beneficial commensals and butyrate-producing taxa, highlighting its role as a key bifidobacterial species involved in microbial networking within the adult human gut microbiota.

Importance: To comprehensively explore the biodiversity within a microbial species, the reconstruction of a substantial number of genomes is essential. In this study, we successfully uncovered the genetic diversity of Bifidobacterium adolescentis by retrieving a large number of genomes from human gut metagenomic samples. The complete overview of the B. adolescentis pangenome enabled us to investigate the genetic features that distinguish this gut commensal from other bifidobacterial species residing in the human intestinal microbiota.

Microbes inhabiting soils experience periodic water deprivation. The effects of desiccation on DNA, protein, and membrane integrity are well-described. However, the effects of drying and rehydration on the composition of cellular RNA and metabolites are still poorly understood. Here, we describe how slow drying and rehydration with water vapor influence the composition of RNAs and metabolites in a soil Arthrobacter. While drying reduced cultivability relative to hydrated controls, water vapor rehydration fully restored it. Ribosomal RNA proportions remained constant throughout all treatments, and mRNA profiles showed stable composition during desiccation-changing only during transitions into and out of desiccation-induced dormancy. Six transcriptional modules displayed distinct expression patterns in desiccated-rehydrated samples relative to hydrated controls, including desiccation-rehydration responsive and rehydration-specific profiles. Targeted intracellular metabolomics revealed similarly static profiles during desiccation, with a cluster of ribonucleosides and nucleobases increasing in response to desiccation and returning to baseline levels upon rehydration with water vapor. These findings demonstrate that both mRNA and metabolite profiles remain essentially frozen in desiccated Arthrobacter, with dynamic changes occurring only during state transitions. These results have important implications for environments with frequent drying cycles where stable mRNA in dormant cells combined with intracellular RNA recycling may obscure interpretations of RNA-based environmental analyses that use RNA as a marker of microbial activity. Our results suggest that RNA-based activity assessments in periodically dry environments require careful consideration of dormancy-associated molecular preservation.IMPORTANCEMetabolic activity quickly ceases in drying bacteria as they enter desiccation-induced dormancy. We show that mRNA and metabolite profiles were variable during drying and rewetting but did not change while desiccated. Additionally, water vapor stimulated the shift from the static to active state when exiting desiccation-induced dormancy. These shifts coincided with increased cultivability, indicating water vapor resuscitated dry cells. Because RNAs are transient, labile molecules that are turned over rapidly in growing bacteria, the presence of RNA in the environment is used as a marker for microbial activity. Our research shows this assumption may not hold for desiccated cells, indicating reliance on RNA as a marker of activity in environments that experience drying may obscure estimates of in situ microbial activity.

While numerous deep-branching lineages of Archaea have been found over the last two decades, most of them still remain enigmatic and uncultivated. In their article, Prokofeva et al. report the isolation of two species from a thermoacidophilic lineage previously reported as "Candidatus Marsarchaeota" and propose a renaming to Tardisphaeria (M. I. Prokofeva, A. I. Karaseva, A. S. Tulenkov, A. A. Klyukina, et al., mSystems 10:e00710-25, 2025, https://doi.org/10.1128/msystems.00710-25). Cultivation coupled with genome analysis revealed a strong enrichment and co-occurrence of polysaccharide and sugar metabolisms in these archaea relative to other thermoacidophiles. These polyextreme archaea were also found to make up large abundances (up to 40% relative abundance) in acidic hot springs, indicating they are important for carbon cycling in these environments. These organisms may also host biotechnologically relevant genes for using polysaccharides that are stable at high temperatures and low pH. Overall, these new isolates improve our understanding of archaeal diversity and metabolism and open the door for more studies on these previously inaccessible organisms.

Sinks contaminated with opportunistic pathogens are a source of hospital-acquired infections, responsible for morbidity and mortality in neonatal intensive care units (NICUs). Understanding pathogen behavior in sinks is essential for preventing their spread. Only a few studies have examined how sink environments affect pathogen distribution through changes in drain microbiota. This research uses an integrative approach to study three major bacterial pathogens: Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Serratia marcescens. Sink drains in two NICUs were sampled during 2-month and 5-month periods. The diversity and abundance of opportunistic pathogens were determined at the genotypic level. Their occurrence was analyzed considering microbial communities, water parameters, faucet design, and sink usage. P. aeruginosa, S. marcescens, and S. maltophilia were found in 47%, 39%, and 67% of drain samples, respectively. Low genotype diversity was observed within sinks, with 1-3 genotypes per species/sample. Dominant genotypes persisted throughout the sampling periods, showing the persistence of opportunistic pathogen strains in drains. Quantification of the studied bacterial sequence types ranged from 10³ to 10⁷ DNA copies/mL. The heterogeneous spatial distribution of the three species between individual sink drains was primarily attributed to changes in community composition, chlorine concentrations, and faucet design. We isolated a strain of Delftia tsuruhatensis (Dt1S33), whose presence in the sink environment was negatively correlated with the three opportunistic pathogens. Dt1S33 reduced the capacity of the pathogens to form biofilms in laboratory co-cultures. These findings underscore the key roles of biotic and abiotic factors in the colonization of sink drains by pathogens.IMPORTANCEHospital sinks are critical reservoirs for opportunistic pathogens (OPs), increasing the risk of healthcare-associated infections, especially in vulnerable populations such as neonatal intensive care unit (NICU) patients. Our study found that 39%-67% of sink drains were persistently colonized by Pseudomonas aeruginosa, Serratia marcescens, and Stenotrophomonas maltophilia, with a limited number of genotypes dominating for months. Colonization patterns in drains varied between NICUs, mainly influenced by microbial community composition and sink design. Notably, Delftia tsuruhatensis presence was negatively correlated with OP colonization and inhibited OP biofilm formation in vitro. These results highlight the interplay of abiotic and biotic factors in sink colonization and suggest that antagonistic bacteria could help reduce pathogen persistence. Understanding these dynamics is crucial for developing targeted interventions to mitigate infection risks in high-risk hospital settings.

The human gastrointestinal tract is home to a diverse community of microorganisms from all domains of life, collectively referred to as the gut microbiome. While gut bacteria have been studied extensively in relation to human host health and physiology, other constituents remain underexplored. This includes the gut virome, the collection of bacteriophages, eukaryotic viruses, and other mobile genetic elements present in the intestine. Like gut bacteria, the gut virome has been causatively linked to human health and disease. However, the gut virome is substantially more difficult to characterize, given its high diversity and complexity, as well as multiple challenges related to in vitro cultivation and in silico detection and annotation. In this mini-review, we describe various methodologies for examining the gut virome using both culture-dependent and culture-independent tools. We highlight in vitro and in vivo approaches to cultivate viruses and characterize viral-bacterial host dynamics, as well as high-throughput screens to interrogate these relationships. We also outline a general workflow for identifying and characterizing uncultivated viral genomes from fecal metagenomes, along with several key considerations throughout the process. More broadly, we aim to highlight the opportunities to synergize and streamline wet- and dry-lab techniques to robustly and comprehensively interrogate the human gut virome.

The human gut microbiome is a dynamic ecosystem. Host behaviors (e.g., diet) provide a regular source of environmental variation that induces fluctuations in the abundances of resident microbiota. Despite these displacements, microbial community members remain highly resilient. Population abundances tend to fluctuate around a characteristic steady-state over long timescales in healthy human hosts. These temporary excursions from steady-state abundances, known as sojourn trajectories, have the potential to inform our understanding of the fundamental dynamics of the microbiome. However, to our knowledge, the macroecology of sojourn trajectories has yet to be systematically characterized. In this study, we leverage theoretical tools from the study of random walks to characterize the duration of sojourn trajectories, their shape, and the degree that diverse community members exhibit similar qualitative and quantitative dynamics. We apply the stochastic logistic model as a theoretical lens for interpreting our empirical observations. We find that the typical timescale of a sojourn trajectory does not depend on the mean abundance of a community member (i.e., carrying capacity), although it is strongly related to its coefficient of variation (i.e., environmental noise). This work provides fundamental insight into the dynamics, timescales, and fluctuations exhibited by diverse microbial communities.IMPORTANCEMicroorganisms in the human gut often fluctuate around a characteristic abundance in healthy hosts over extended periods of time. These typical abundances can be viewed as steady states, meaning that fluctuating abundances do not continue towards extinction or dominance but rather return to a specific value over a typical timescale. Here, we empirically characterize the (i) length (i.e., number of days), (ii) relationship between length and height, and (iii) typical deviation of a sojourn trajectory. These three patterns can be explained and unified through an established minimal model of ecological dynamics, the stochastic logistic model of growth.

Listeria monocytogenes, as a significant foodborne pathogen, is not frequently encountered; however, when infections do occur, they can prove highly lethal to specific populations. Antibiotics are still regarded as the primary treatment option for Listeria infections. Nevertheless, under the global antibiotic crisis, there is an urgent demand for innovative and alternative strategies. In our study, we identified taurine, a sulfur-containing free amino acid that can be extracted from a wide variety of foods, as an effective inhibitor of Listeria growth. Furthermore, our findings revealed that taurine administration significantly reduced bacterial burden and concurrently mitigated host-derived inflammation in the mouse model. It was observed that taurine stimulated T-cell proliferation and inhibited pyroptosis via mitogen-activated protein kinase and NLRP3/caspase-1/GSDMD pathways. Our research outcomes position taurine as a promising therapeutic candidate for combating Listeria infections, with an inherent advantage of reduced likelihood for inducing antibiotic resistance compared to conventional antibiotic treatments.

Importance: Listeria monocytogenes infections are lethal to specific groups. With the antibiotic crisis, new treatments are needed. Taurine, a safe dietary compound, was found to inhibit Listeria growth. It targets both L. monocytogenes virulence and host immunopathology, stimulated T-cell proliferation, and inhibited pyroptosis. We establish taurine as the non-antibiotic agent that decouples bacterial cytotoxicity from inflammation-driven tissue damage, offering an immediately translatable strategy for high-risk infections amid the antibiotic resistance crisis.