mSystems最新文献

{"title":"Comparative gut microbiome in diarrheal and non-diarrheal children: an individually matched case-control study.","authors":"Jialiang Xu, Yufeng Fan, Geruo Qu, Jiaqi Li, Yao Peng, Mengyu Wang, Jingyun Zhang, Yuqing Feng, Xiao Liu, Yongfei Hu, Biao Kan, Zhenpeng Li, Mei Zeng, Xin Lu","doi":"10.1128/msystems.00198-26","DOIUrl":"10.1128/msystems.00198-26","url":null,"abstract":"<p><p>Diarrhea, a leading cause of under-five mortality in developing countries, drives therapeutic challenges amid global antibiotic resistance. This study aimed to compare gut microbiota, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and virulence factors (VFs) in diarrheal and non-diarrheal children, explore reasons for asymptomatic pathogen carriage, and develop a microbiome-based model to predict the potential etiology of diarrhea. This individually matched case-control study analyzed 42 paired fecal samples (selected from 716 diarrhea/non-diarrhea cases) to compare gut microbiome profiles, ARGs, MGEs, and VFs using metagenomic sequencing and pathogen-specific PCR. Diarrheal children showed reduced alpha diversity, increased Proteobacteria, and elevated VFs and MGEs. Surprisingly, ARGs were more abundant in non-diarrheal children, suggesting ARG colonization in healthy hosts. <i>Actinomyces</i> was enriched in non-diarrheal pathogen carriers and may be associated with asymptomatic bacterial pathogen carriage. A random forest (RF) model incorporating non-pathogenic bacteria achieved high accuracy in predicting diarrheal status and pathogen carriage. This study reveals distinct microbial ecologies between diarrheal and healthy children. The enrichment of specific bacterial taxa in asymptomatic carriers may be associated with pathogen tolerance. The unexpected ARG abundance in healthy children highlights a hidden antimicrobial resistance reservoir. These findings inform microbiome-based diagnostics and antibiotic stewardship in pediatric diarrhea.IMPORTANCEThis study is significant because it provides robust, individually matched case-control evidence linking childhood diarrhea to gut microbiome dysbiosis and the distribution of pathogenic and resistance-related genetic elements. By integrating metagenomic sequencing with pathogen-specific polymerase chain reaction (PCR), it offers a comprehensive comparison of microbial composition, virulence factors (VFs), mobile genetic elements (MHEs), and antibiotic resistance genes (ARGs) between diarrheal and non-diarrheal children. The findings demonstrate reduced microbial diversity and enrichment of Proteobacteria, virulence factors, and mobile genetic elements in diarrheal cases, highlighting microbiome instability during infection. Importantly, the unexpectedly higher abundance of antibiotic resistance genes in non-diarrheal children underscores the underestimated role of healthy populations as reservoirs of resistance. Furthermore, the establishment of predictive models for diarrhea status and pathogen carriage enhances the translational value of the study. Overall, this work advances the understanding of pediatric diarrheal disease and informs prevention, surveillance, and treatment strategies in the context of global antimicrobial resistance.</p>","PeriodicalId":18819,"journal":{"name":"mSystems","volume":" ","pages":"e0019826"},"PeriodicalIF":5.2,"publicationDate":"2026-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149081162","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rarefaction is better than robust Aitchison PCA and other compositional data analysis methods at controlling for uneven sequencing effort.","authors":"Patrick D Schloss","doi":"10.1128/msystems.00930-26","DOIUrl":"10.1128/msystems.00930-26","url":null,"abstract":"<p><p>Amplicon sequencing typically results in a wide distribution in the number of sequences obtained from each sample. How best to account for this variation has been a persistent problem in the microbial ecology literature. Historically, rarefaction has been used in ecology and this practice was adopted by microbial ecologists. However, rarefaction has been strongly criticized, leading to the development of compositional data analysis and normalization methods. Therefore, I reassessed the benchmarking data that were generated by the developers of one such method, robust Aitchison PCA. I found numerous problems in the Python code that led to the support of robust Aitchison PCA. These problems extended to the creation of simulated data sets, implementation of machine learning methods, and choice of analysis parameters. Furthermore, the analysis of the simulated and case study data sets was done in a manner that was foreign to standard microbiome analyses. I corrected the problems in the original code, added data sets with smaller effect sizes, and expanded the collection of methods that purport to correct for uneven sequencing effort. Contrary to the claims of the original analysis, robust Aitchison PCA was not insensitive to uneven sequencing effort and did not perform as well as rarefaction. In fact, even using the benchmarking framework from the original analysis, rarefaction outperformed robust Aitchison PCA, other compositional data analysis methods, and other normalization methods. Rarefaction remains the preferred method of controlling for uneven sampling effort in amplicon sequence studies.</p><p><strong>Importance: </strong>Efforts to connect the structure of microbial communities with environmental processes and host health have captured widespread interest among scientists and the general public. The methods used to generate the sequencing data that are fundamental to these efforts result in wide variation in the number of sequences per sample. This variation and how to account for it can have detrimental effects on the ability to draw valid conclusions. Compositional data analysis methods, including robust Aitchison principal component analysis (PCA), have grown in popularity for mitigating these effects and have been proposed as alternatives to rarefaction. In this study, I reviewed and fixed the code that was used to benchmark robust Aitchison PCA and expanded the analysis. With this improved and expanded analysis, I found that rarefaction was still superior to robust Aitchison PCA and other methods of controlling for uneven sequencing effort.</p>","PeriodicalId":18819,"journal":{"name":"mSystems","volume":" ","pages":"e0093026"},"PeriodicalIF":5.2,"publicationDate":"2026-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149081307","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proteomic profiling of the <i>Pseudomonas aeruginosa</i> stringent response highlights global adaptive responses during stationary phase.","authors":"Jacquelyn Rich, Samuel J T Wardell, Dorival Martins, Malika Khakhimova, Gowthami Sampathkumar, Tevy Chan, Thi Tuyet Mai Nguyen, Geoffrey McKay, Lorne Taylor, Daniel Pletzer, Dao Nguyen","doi":"10.1128/msystems.01742-25","DOIUrl":"10.1128/msystems.01742-25","url":null,"abstract":"<p><p><i>Pseudomonas aeruginosa</i> (<i>P.a</i>) is a clinically important opportunistic pathogen capable of adapting and surviving a wide range of environments and stress conditions. A central regulator of this adaptability is the stringent response, mediated by the alarmones (p)ppGpp. Activation of the stringent response triggers a global physiological shift away from active metabolism toward stress management and cellular quiescence, a key transition to stationary phase. To date, few studies have investigated the protein-level processes under stringent response control. In this study, we performed shotgun proteomic profiling (LC-MS/MS) of a (p)ppGpp-null <i>relA spoT</i> double knockout mutant (ΔSR) and its isogenic PAO1 parent during late exponential and stationary phase planktonic growth in rich medium. In this first characterization of the stringent response proteome in <i>P.a</i>, we identified 146 differentially abundant proteins (DAP) in late exponential phase ΔSR cells, and 2.3-fold greater DAP (339) in stationary-phase cells. Functional characterization and system-level analysis of the differentially abundant stationary phase proteome highlighted the key role of (p)ppGpp in shutting down a wide range of metabolic processes while inducing adaptive processes such as responses to oxidative stress. Comparative RNA-seq analysis also uncovered incongruent protein and mRNA responses in the ΔSR relative to wild-type cells in several processes, including iron homeostasis and antioxidant defenses. Transcriptomic analysis of the ΔSR also revealed differential expression of numerous sRNAs, including PrrF1/2, raising the possibility that sRNAs may mediate certain stringent-response-dependent post-transcriptional control. Phenotypic validation of the ΔSR proteome confirmed multiple defects in antioxidant defenses and dysregulated pyoverdine biosynthesis.</p><p><strong>Importance: </strong><i>Pseudomonas aeruginosa</i> is an important opportunistic human pathogen that causes difficult-to-treat infections and is highly adaptable to stressful environments. A key orchestrator of this adaptability is the stringent response, mediated by the signaling molecule (p)ppGpp. In this study, we characterized the extensive proteome remodeling in the (p)ppGpp-null <i>relA spoT</i> mutant during late exponential and stationary phase and provided the first protein-level analysis of the stringent response in <i>P. aeruginosa</i>. These data highlight the role of (p)ppGpp in shutting down broad metabolic functions and activating adaptive pathways, including stress-resistance responses, particularly during stationary phase. Using comparative proteomics and RNA-seq, we also find that several processes-such as iron regulation and oxidative stress responses-show mismatched RNA and protein changes, suggesting additional layers of post-transcriptional control, potentially involving small regulatory RNAs.</p>","PeriodicalId":18819,"journal":{"name":"mSystems","volume":" ","pages":"e0174225"},"PeriodicalIF":5.2,"publicationDate":"2026-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148990647","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Prescribed burns drive lasting changes in soil nitrogen cycling and microbial function.","authors":"Alaina O Benot, Gray Waldschmidt, Samuel C Gilvarg, Eva O L Legge, Isaac J Okyere, Chanistha Tiyapun, Sarah K Lucas, Andrew L Vander Yacht, Jennifer L Goff","doi":"10.1128/msystems.00852-26","DOIUrl":"10.1128/msystems.00852-26","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Fire is a major pulse disturbance to soil microbial communities, with broad implications for nutrient cycling; however, regular burning is also a natural and often-essential process maintaining biodiversity in unique and imperiled fire-dependent ecosystems. Prescribed fire is widely used to promote this biodiversity and simultaneously reduce wildfire risk. Although such repeated burning is known to alter surface biodiversity, belowground soil geochemistry, and soil microbial community structure, the functional consequences (i.e&lt;i&gt;.&lt;/i&gt;, the metabolic capabilities that underlie the ecosystem services soil microorganisms provide) remain underexplored. Here, we examined the effects of 30 years of repeated prescribed fire at the Albany Pine Bush-a fire-dependent, inland pitch pine barren ecosystem of the northeastern United States. Compared with the control stands, we observed that this long-term fire management has led to substantial depletion of inorganic soil nitrogen, specifically nitrate. We found no meaningful differences in the higher-level taxonomic composition of soil prokaryotic or fungal communities; however, analysis of metagenome-assembled genomes assembled from these soils revealed several differentially abundant populations. Furthermore, our metagenomic analysis revealed significant changes in the nitrogen-cycling functional potential, specifically decreased dissimilatory nitrate reduction and denitrification potential in repeatedly burned soils. These functional shifts have important implications for both nutrient cycling and emissions of trace nitrogen gases from these soils. Our results suggest that functionally meaningful changes in the soil microbiome can persist between burn events, even when higher-order community membership appears stable. This may imply that repeated fire can deplete reactive nitrogen emissions from soils by lowering the functional capacity of nitrogen-reducing microbes.IMPORTANCEPrescribed fire is widely used by land managers to reduce wildfire risk and promote biodiversity. While the effects of fire on aboveground processes are well understood, much less is known about how repeated burning influences soil biological properties-including the functional role that soil microorganisms play in nutrient cycling and greenhouse gas production. We addressed this gap by studying soils from the Albany Pine Bush, a rare and endangered ecosystem that has experienced regular prescribed fires for 30 years. Long-term fire management significantly altered soil chemistry, specifically lowering the amount of nitrogen in the soil. In addition, we found that fire management decreased the genetic potential of the soil microbial community to produce nitrogen oxides-potent contributors to climate change. Thus, prescribed fire's contribution to greenhouse gas emissions may involve a complex relationship between direct fire-driven emissions, increased fire resilience of promoted vegetation, and-as suggested by our results-the r","PeriodicalId":18819,"journal":{"name":"mSystems","volume":" ","pages":"e0085226"},"PeriodicalIF":5.2,"publicationDate":"2026-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149012735","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Library strategies differentially shape microbial, functional, and host signals in clinical metagenomic sequencing.","authors":"Gengyan Luo, Jin Zhao, Lin-Lin Xie, Yirong Li, Yang-Zhi Rao, Yanni Sun, Xin Lu, John-Sebastian Eden, Liangjun Chen, Mang Shi","doi":"10.1128/msystems.00897-26","DOIUrl":"10.1128/msystems.00897-26","url":null,"abstract":"<p><p>Metagenomic next-generation sequencing (mNGS) is increasingly used in infectious disease diagnostics, yet how library preparation shapes the microbial, functional, and host signals recovered from clinical samples remains poorly defined. Here, we performed a within-sample parallel comparison of three mNGS library preparation strategies-DNA-based libraries (DNAlib), RNA-based libraries (RNAlib), and total nucleic acid-based libraries (TNAlib)-across a diverse range of clinical specimens spanning five sample types. Using a curated clinical infectome as a benchmark, we show that library strategies are not interchangeable but capture distinct biological dimensions of the same specimen. RNAlib provided the most comprehensive standalone recovery of the clinical infectome, with improved detection of RNA viruses and cellular pathogens, enhanced resolution of resistance and virulence signals, and preservation of infection-associated host immune signatures. DNAlib showed stronger baseline recovery of DNA viruses and broader host genome coverage, whereas the TNAlib workflow evaluated here largely behaved as an intermediate strategy rather than a consistent improvement over dedicated DNA- or RNA-based workflows. Together, these results establish that the library preparation protocol is a major determinant of how clinical mNGS data should be interpreted and provide a framework for selecting sequencing strategies according to specific diagnostic and biological questions.IMPORTANCEMetagenomic sequencing is increasingly used in infectious disease research and clinical diagnostics, but different library preparation strategies may recover fundamentally different biological signals from the same sample. These signals include not only pathogens but also background microbes, microbial functional activity, and host immune-response patterns. Here, we systematically compared DNA-, RNA-, and total nucleic acid-based metagenomic sequencing libraries using the same clinical samples processed in parallel. We found that the three strategies did not provide equivalent information. RNA-based sequencing generated the most informative single-library view of infection, particularly for RNA viruses, cellular pathogens, functional microbial signals, and host immune-response patterns. DNA-based sequencing was more effective for DNA virus and host genome recovery, whereas the total nucleic acid sequencing workflow evaluated here generally behaved as an intermediate strategy. These findings show that library preparation can substantially influence the interpretation of metagenomic data.</p>","PeriodicalId":18819,"journal":{"name":"mSystems","volume":" ","pages":"e0089726"},"PeriodicalIF":5.2,"publicationDate":"2026-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148963657","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Non-genetic red blood cell variability can modulate hemoglobin consumption by intracellular <i>Plasmodium falciparum</i>.","authors":"Joseph R Perko, Abhyudai Singh, Secilia I Lopez, Armando Estrada, Shweta Singh, Janice M Moser, Kelly Haupfear, George J Klarmann, Sabrina J Tuson, Vincent B Ho, Shirley Luckhart, Andreas E Vasdekis","doi":"10.1128/msystems.00554-26","DOIUrl":"10.1128/msystems.00554-26","url":null,"abstract":"<p><p>Natural, non-genetic variation among host cells can shape infection outcomes, yet its role in intracellular parasite dynamics remains poorly understood. Here, we show that variability in hemoglobin (Hb) content among individual red blood cells (RBCs) modulates nutrient consumption by <i>Plasmodium falciparum</i>, the most lethal malaria parasite. Using label-free quantitative-phase imaging (QPI), we measured cytosolic Hb in thousands of uninfected and infected RBCs across the 48-h asexual cycle. Hb content varied widely among uninfected cells, and after invasion, infected cells mirrored and amplified this distribution: variability increased from 17% in uninfected RBCs to >100% in schizonts. A minimal predator-prey model accurately reproduced the observed spread in parasite Hb consumption, showing that host variability alone can generate much of the diversity in parasite feeding rates traditionally attributed to parasite factors. These findings provide the first evidence that host phenotypic noise drives variation in Hb consumption among genetically identical parasites. Incorporating host-to-host variability into within-host models could improve predictions of parasite growth, refine the activation kinetics of Hb-dependent antimalarials, and reveal analogous principles in other intracellular infections where host variability and host-parasite crosstalk can shape pathogen outcomes. This systems-level perspective emphasizes the need to account for host heterogeneity in both experimental design and therapeutic strategies.IMPORTANCECell-to-cell phenotypic variability is a fundamental feature of living systems, yet its impact on host-pathogen interactions has been largely overlooked. Here, we show that natural, non-genetic variation in red blood cell hemoglobin strongly shapes how <i>Plasmodium falciparum</i>-the most lethal malaria parasite-consumes Hb inside host cells. Using quantitative imaging and a mathematical model, we demonstrate that host variability alone can explain much of the observed differences in parasite feeding rates. This perspective reframes parasite Hb consumption as an emergent property of host-parasite interactions. More broadly, it highlights how natural variability among host cells can influence the course of intracellular infections, with potential implications for predicting pathogen growth and optimizing drug treatment strategies.</p>","PeriodicalId":18819,"journal":{"name":"mSystems","volume":" ","pages":"e0055426"},"PeriodicalIF":5.2,"publicationDate":"2026-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148891895","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cellular responsiveness as a predictive indicator for population collapse and autonomous control in continuous cultures of <i>Pseudomonas putida</i>.","authors":"Maximilian Sehrt, Hannah Sehrt, Laurie Josselin, Juan Andres Martinez, Frédéric Francis, Frank Delvigne","doi":"10.1128/msystems.00768-26","DOIUrl":"10.1128/msystems.00768-26","url":null,"abstract":"<p><p>How microbial populations respond to repeated environmental transitions determines both their ecological fitness and their utility in biotechnological applications. Using <i>Pseudomonas putida</i> KT2440 equipped with fluorescent biosensors and monitored by automated flow cytometry in the Segregostat platform, we show that exposure to benzoate, a plastic-derived aromatic feedstock, progressively reduces cellular responsiveness, defined as the fraction of cells that successfully activate a gene circuit following an environmental transition. Unlike classical switching costs, which promote phenotypic diversification, benzoate suppresses responsiveness without increasing population entropy, in a concentration-dependent and circuit-independent manner tightly correlated with fitness loss. A resource allocation model incorporating the competing demands of benzoate assimilation, toxicity, and tolerance reveals that this impairment emerges from a three-way competition for limited cellular resources. Above a critical benzoate load, insufficient resources remain available to sustain the adaptive reallocation required for circuit activation. In continuous culture, a non-responsive subpopulation accumulates as a leading indicator of population collapse. Exploiting this signal, we implement a two-stage connected bioreactor system in which benzoate feeding is autonomously regulated based on real-time population structure, enabling complete substrate consumption and stable operation at otherwise destabilizing concentrations. These results establish cellular responsiveness as a quantitative population variable and demonstrate that structure-aware feedback control, acting on population composition rather than bulk physiology, provides a principled route toward autonomous bioprocesses on challenging substrates.</p><p><strong>Importance: </strong>This work contributes at two levels. First, it positions cellular responsiveness as a quantitative and mechanistically grounded population variable. It also shows that cellular responsiveness can be interpreted based on a resource allocation approach, a principle that could be applied to several other biological systems. Second, it demonstrates that monitoring population composition, rather than bulk physiology, provides new engineering principle toward autonomous bioprocess control on challenging substrates.</p>","PeriodicalId":18819,"journal":{"name":"mSystems","volume":" ","pages":"e0076826"},"PeriodicalIF":5.2,"publicationDate":"2026-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149069936","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrated cross-sectoral surveillance of antimicrobial resistance genotypes and phenotypes across disparate reservoirs.","authors":"Thomas D Watts, Thanavit Jirapanjawat, Laura Perlaza-Jiménez, Francesco Ricci, Eve Tudor-Matthew, Eleonora Chiri, Sean K Bay, Rhys Grinter, Rachael Lappan, Trevor Lithgow, Laura C Woods, Chris Greening","doi":"10.1128/msystems.00741-26","DOIUrl":"10.1128/msystems.00741-26","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Antimicrobial-resistant (AMR) bacteria and genes are continually exchanged among humans, animals, and environmental reservoirs. Disparate and siloed surveillance methods present a major challenge for tracing and disrupting AMR transmission, with clinical monitoring focusing on detecting specific pathogens or specific genes of interest, and environmental surveys often relying on inferences drawn from indicator organisms. Here, we demonstrate that, following sample-specific pre-processing, common surveillance approaches can be applied consistently to profile AMR abundance, distribution, and phenotypes across diverse reservoirs in both urban and agricultural settings. Across all sample types, three core methods provided complementary insights: (i) quantitative PCR (qPCR) arrays to measure multiple AMR genes, (ii) gene- and genome-centric metagenomics for comprehensive resistome profiling, and (iii) culture-based genomics with susceptibility testing to link genotypes to phenotypes. We applied this approach to profile 1,032 metagenome-assembled genomes, 66 bacterial isolate genomes, and 78 and 6,642 AMR genes/reference sequences via qPCR and metagenomics, respectively. This integrated framework revealed the prevalence and diversity of resistance mechanisms in both putative pathogens and non-pathogenic bacteria with potentially transmissible genes, with wastewater especially enriched in AMR genes. We detected mismatches between genotype and phenotype predictions and a prevalence of intermediate resistance phenotypes, highlighting how many mechanisms of environmental resistance remain poorly understood. Overall, this study demonstrates that unified, field-leading surveillance methods can be used in diverse environmental and animal samples while highlighting that multiple methods are needed to capture the diverse AMR genotypes and phenotypes in these settings to enable comprehensive monitoring and adaptive solutions to restrict transmission.IMPORTANCEAntimicrobial resistance (AMR) is driven by the exchange of resistant bacteria and genes across interconnected human, animal, and environmental reservoirs, yet fragmented surveillance limits our ability to track transmission and intervene effectively. We demonstrate that widely used surveillance methods can be applied cohesively across diverse sample types, including wastewater, soil, water, agricultural environments, and fecal samples, to generate comparable insights into AMR abundance, diversity, and phenotype. By integrating qPCR, metagenomics, and culture-based genomics, we reveal resistance in both pathogenic and non-pathogenic bacteria, reaffirm wastewater as a major AMR reservoir, and uncover frequent mismatches between genetic predictions and observed susceptibility. The prevalence of intermediate resistance further suggests that many environmental resistance mechanisms remain poorly understood. These findings show that no single method captures AMR complexity and highlight the need for unified","PeriodicalId":18819,"journal":{"name":"mSystems","volume":" ","pages":"e0074126"},"PeriodicalIF":5.2,"publicationDate":"2026-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149069999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Phylogenetic inconsistency of pairwise SNP clustering for inferring tuberculosis transmission in a high-burden, endemic setting: a case study from Thailand.","authors":"Pakorn Aiewsakun, Walter Samuel Hunter, Prasit Palittapongarnpim","doi":"10.1128/msystems.00641-26","DOIUrl":"10.1128/msystems.00641-26","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Whole-genome sequence analysis is now widely used to delineate tuberculosis transmission clusters. A standard practice is to cluster bacterial isolates based on a fixed maximum genome-wide pairwise single nucleotide polymorphism (pwSNP) distance threshold. In this study, we evaluated the phylogenetic consistency of pwSNP-distance clustering with thresholds ranging between 1 and 25 single nucleotide polymorphisms (SNPs) using two contrasting data sets: (i) a data set from the UK (&lt;i&gt;N&lt;/i&gt; = 390) published by T. M. Walker, C. L. C. Ip, R. H. Harrell, J. T. Evans, et al. (Lancet Infect Dis 13:137-146, 2013, https://doi.org/10.1016/S1473-3099(12)70277-3), which was foundational to the establishment of this method, and (ii) a data set from Thailand (&lt;i&gt;N&lt;/i&gt; = 3,341), characterized by persistent transmission and sparse, non-systematic sampling. For the UK data set, the standard pwSNP-distance clustering using thresholds of ≥12 SNPs yielded entirely monophyletic clusters and showed high concordance with a comparative monophyly constrained, tree-based method. In contrast, for the Thai data set, pwSNP-distance clustering often generated non-monophyletic clusters, even by the 25-SNP threshold. The pwSNP-distance and comparative tree-based clustering methods only showed large consistency at thresholds of ≥22 SNPs. This suggests that SNP clusters defined by low distance thresholds (i.e., &lt;12 SNPs for the UK data set, and &lt;22 SNPs for the Thai data set) may lack robustness, and the problem is particularly severe for data sets characterized by persistent transmission, likely due to poorer cluster separation. Moreover, our findings indicate that large cluster sizes, high maximum intra-cluster genetic distances, and broad sample collection time spans may serve as useful indicators of potentially non-monophyletic clusters. We also demonstrate that mixed infections can produce spurious, phylogenetically long-range SNP linkages, underscoring the necessity of strict sequence quality control.IMPORTANCEFixed-threshold pairwise single nucleotide polymorphism (pwSNP)-distance clustering is commonly used to delineate tuberculosis transmission clusters. From an epidemiological perspective, a genuine transmission cluster must be monophyletic, originating from a single source. However, pwSNP-distance clustering is inherently simplistic and can therefore violate this principle, making the assessment of its phylogenetic consistency critical. Our results demonstrate that while this method effectively delineated complete transmission clusters for the data set from the UK, a low-burden and non-persistent transmission setting, it frequently generated non-monophyletic clusters when applied to the Thai data set, characterized by persistent transmission alongside sparse and non-systematic sampling. Furthermore, we found that clusters derived using low distance thresholds could notably vary between the pwSNP-distance and comparative tree-based clustering methods, suggesting li","PeriodicalId":18819,"journal":{"name":"mSystems","volume":" ","pages":"e0064126"},"PeriodicalIF":5.2,"publicationDate":"2026-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148963662","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Landscape of retron diversity across the SPIRE microbial metagenome resource reveals candidate novel type XI-like lineages.","authors":"Nicolás Toro","doi":"10.1128/msystems.00732-26","DOIUrl":"10.1128/msystems.00732-26","url":null,"abstract":"<p><p>Retrons are bacterial genetic elements encoding a specialized reverse transcriptase (RT) that synthesizes multicopy single-stranded DNA and are increasingly recognized as components of bacterial anti-phage defense systems. However, their diversity and ecological distribution across large-scale genomic resources remain poorly characterized. Here, we surveyed retron RTs across the SPIRE representative metagenome collection, a non-redundant, species-level data set spanning diverse microbial habitats. Using a curated panel of type-specific hidden Markov models, we identified retrons representing all canonical types together with additional divergent lineages. Retron distribution showed strong taxonomic and ecological structuring, with some groups restricted to specific bacterial phyla, whereas others were broadly distributed across environmental categories. Systematic novelty assessment identified two candidate type XI-like lineages, TXI_C2like and TXI_noncan_h, characterized by protease-independent architectures and distinct accessory modules associated with WYL- and DnaB_C-containing proteins, respectively. <i>De novo</i> covariance-based analyses further identified candidate msr/msd-like non-coding RNA structures in both lineages, supporting conservation of the canonical RT-ncRNA organizational framework despite extensive sequence divergence. Together, these findings expand the known diversity of retron systems and identify type XI-like retrons as a dynamic and previously underexplored evolutionary group.IMPORTANCERetrons are bacterial genetic elements that are increasingly exploited as programmable tools for genome editing, molecular recording, and biosensing in addition to their natural role in anti-phage defense. Despite this growing biotechnological interest, the true diversity of retrons across the bacterial world has remained largely unmapped. By mining a resource of over 100,000 processed microbial metagenomes, we uncovered thousands of retron sequences spanning known types as well as previously unrecognized lineages and found that their distribution is strongly shaped by both bacterial taxonomy and ecological niche. Among these, we identified two candidate new lineages related to type XI retrons that lack the protease domain typical of this group but instead carry distinct accessory proteins, expanding the known architectural diversity of these systems. These findings broaden the catalog of retron diversity available for functional characterization and biotechnological engineering and provide a framework for prioritizing candidate lineages for future experimental validation.</p>","PeriodicalId":18819,"journal":{"name":"mSystems","volume":" ","pages":"e0073226"},"PeriodicalIF":5.2,"publicationDate":"2026-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148944183","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}