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Halogenated phenazine (HP) compounds have shown promise as antimicrobial agents, particularly against biofilm-associated Gram-positive pathogens. Among these compounds, HP-29 demonstrates potent activity against methicillin-resistant Staphylococcus aureus by inducing rapid iron starvation. As maintenance of trace metals homeostasis is critical for the survival of Streptococcus mutans, this study investigated the antimicrobial efficacy of HP-29 and the impact of metal supplementation on this major oral and occasional systemic pathogen. As anticipated, HP-29 inhibited S. mutans growth in a dose-dependent manner, with iron supplementation alleviating the antimicrobial effect. Cobalt, manganese, or nickel supplementation also mitigated the inhibitory activity of HP-29, but, unexpectedly, the addition of zinc greatly enhanced HP-29 antimicrobial activity. This zinc-driven potentiation of HP-29 extended to other Gram-positive pathogens, including Enterococcus faecalis and S. aureus. Inductively coupled plasma mass spectrometry analysis revealed that intracellular iron content decreased significantly following exposure to HP-29. When combined with zinc, HP-29 triggered a 5-fold increase in intracellular zinc and reduced manganese levels by ~50%. Transcriptome analysis showed that HP-29 treatment, with or without zinc, altered expression of genes linked to iron and manganese uptake as well as zinc efflux, suggesting broad disruption of metal ion regulation. These findings highlight HP-29 as a potent antimicrobial that broadly impairs metal homeostasis. The unexpected synergy of HP-29 with zinc points toward a promising dual-agent therapeutic strategy against Gram-positive pathogens.IMPORTANCEWidespread development of antibiotic resistance has created a constantly moving target when combating infectious microbes. Here, we further explore an antimicrobial halogenated phenazine, HP-29, which is effective against Gram-positive bacteria through disruption of intracellular trace metal equilibrium. We showed that HP-29 inhibits growth of the oral and systemic pathogen Streptococcus mutans and that its antimicrobial effect is greatly potentiated by the addition of zinc. The zinc-mediated enhancement of HP-29's efficacy was also observed in other Gram-positive pathogens, including Enterococcus faecalis and Staphylococcus aureus. Intracellular trace metal quantifications and transcriptome analysis confirmed that HP-29 treatment impairs trace metal homeostasis, an outcome that is exacerbated when S. mutans is treated with both HP-29 and zinc. The observed synergy of HP-29 with zinc supports the development of a dual-agent therapeutic strategy against Gram-positive pathogens.

Emerging viruses remain a threat to human health; however, many aspects of their infection cycle are still poorly understood. Host lipid structures and abundances are observed to be significantly altered during infection, and the mechanisms regulating lipid synthesis and modification remain largely unknown. In this work, we analyzed a large multi-omic data set from three Middle East respiratory syndrome coronavirus (MERS-CoV)-infected primary human lung cell types, all derived from three distinct donors to investigate the changes in lipid species during infection. Analysis of lipidomics data identified perturbations of various lipid classes, and we hypothesized and confirmed that MERS-CoV infection orchestrates an increase in ceramide via sphingomyelinase pathways required for infection. We also identified a minor subset of proteins with lipid-related functions with increased differential expression among a striking majority of lipid-related proteins with decreased differential expression. The most prominent of these is ACSL3, a long-chain acyl-CoA synthetase that is key for the synthesis of triacylglycerides and is associated with lipid droplet formation, an established feature of coronavirus-infected cells. Accordingly, the inhibition of acyl-CoA synthetase activity reduced MERS-CoV replication. These results suggest a model wherein coronaviruses perturb overall cellular metabolism to shift resources to the production of ceramides and triacylglycerides, particularly through acyl-CoA synthetase activity. Our findings suggest a strategy for targeting CoV replication through the inhibition of specific subsets of lipid metabolism.

Importance: Combating emerging viral threats requires an in-depth understanding of how the virus commandeers host resources to facilitate replication. Viral particles are comprised of protein and lipids; hence, the synthesis of both is critical for virus spread. Our studies have demonstrated that the synthesis of two lipid species, ceramides and triacylglycerides, is essential for Middle East respiratory syndrome coronavirus replication and that virus replication is impaired if these synthetic pathways are blocked. These results suggest a model wherein coronaviruses perturb overall cellular metabolism to shift resources to the production of ceramides and triacylglycerides. Our findings suggest a strategy for targeting coronavirus replication through the inhibition of specific subsets of lipid metabolism.

Tomato is a staple crop and an excellent model to study host-microbiota interactions in the plant food chain. In this study, we describe a "lab-in-the-field" approach to investigate the microbiota of field-grown tomato plants. High-throughput amplicon sequencing revealed a three-microhabitat partition, phyllosphere, rhizosphere, and root interior, differentiating host-associated communities from the environmental microbiota. An individual bacterium, classified as Acinetobacter sp., emerged as a dominant member of the microbiota at the plant-soil continuum. To gain insights into the functional significance of this enrichment, we subjected rhizosphere specimens to shotgun metagenomics. Similar to the amplicon sequencing survey, a "microhabitat effect," defined by a set of rhizosphere-enriched functions, was identified. Mobilization of mineral nutrients, as well as adaptation to salinity and polymicrobial communities, including antimicrobial resistance genes (ARGs), emerged as a functional requirement sustaining metagenomic diversification. A metagenome-assembled genome representative of Acinetobacter calcoaceticus was retrieved, and metagenomic reads associated with this species identified a functional specialization for plant-growth promotion traits, such as phosphate solubilization, siderophore production, and reactive oxygen species detoxification, which were similarly represented in a tomato genotype-independent fashion. Our results revealed that the enrichment of a beneficial bacterium capable of alleviating plant abiotic stresses appears decoupled from ARGs facilitating microbiota persistence at the root-soil interface.IMPORTANCETomatoes are at center stage in global food security due to their high nutritional value, widespread cultivation, and versatility. Tomatoes provide essential vitamins and minerals, contribute to diverse diets, and support farmer livelihoods, making them a cornerstone of sustainable food systems. Beyond direct dietary benefits, the intricate relationship between tomatoes, their associated microbiota, and antimicrobial resistance gene (ARG) is increasingly recognized. Tomato plants host diverse microbial communities in association with their organs, which influence plant health and productivity. Crop management impacts the composition and function of these communities, contributing to the prevalence of ARGs in the soil and on the plants themselves. These genes can potentially transfer to human pathogens, posing a food safety and public health risk. Understanding these complex interactions is critical for developing sustainable agricultural practices capable of mitigating the impact of climatic modifications and the global threat of antimicrobial resistance.

The study of the human microbiome (mirroring broader practice across biomedical science) has historically defaulted to the use of simplified, socially constructed "boxes," such as racial and ethnic labels, that fail to accurately capture human variation and fundamentally misdirect the search for mechanisms to explain differences in health outcomes. Five years ago, I proposed a "frameshift," a fundamental conceptual shift away from relying on these categories and toward a more nuanced, careful approach to the complexity of human variation. Moving "out of the box" means tackling the difficult but essential work of analyzing microbial variation through a systems lens, connecting large-scale ecosocial drivers to individual mechanisms and outcomes. In this Full Circle review, I discuss rapid progress in the field toward this new framework and argue that by adopting transdisciplinary methods, we can generate more accurate, actionable, and equitable solutions for human health.

Synergy between influenza A virus (IAV) and Streptococcus pneumoniae is a long-recognized and clinically important problem. Recent work has demonstrated that IAV particles can directly bind to the bacterial surface and that bacterial-viral complexes exhibit enhanced bacterial colonization and invasive disease, increased viral environmental survival leading to increased efficacy of airborne transmission, and enhanced vaccine response to both pathogens over simultaneous co-infection without direct interactions. However, the molecule(s) responsible for mediating the direct interaction are yet to be characterized. In this study, we demonstrate that the broadly conserved Gram-positive bacterial cell wall glycan lipoteichoic acid (LTA) is one of the molecules that can mediate this interaction. This interaction between viral particles and bacterial cell-envelope glycans is also demonstrated in interactions between enteric viruses and enteric bacteria, suggesting a conserved mechanism of trans-kingdom interactions. We show that LTA will compete for binding between IAV and S. pneumoniae, that disruption of genes responsible for LTA presentation at the cell surface will reduce viral binding, and that viral neuraminidase can bind LTA. This work adds to the growing body of literature on direct bacterial-viral interactions between human-associated bacteria and pathogenic viruses and can provide novel insights into the lethal synergy of influenza-pneumococcal co-infections.IMPORTANCECo-infection between influenza A virus (IAV) and Streptococcus pneumoniae leads to severe disease. Recently, it was demonstrated that IAV particles can bind to the surface of bacterial cells and that direct interactions will enhance both bacterial and viral pathogenesis as well as immune responses to each pathogen. However, it is unclear what bacterial and viral components are responsible for the interaction. We demonstrate that a carbohydrate component of the bacterial cell wall can bind to IAV particles. This is similar to direct interactions observed between enteric viruses and cell wall components of enteric bacteria. This work adds to the body of knowledge about trans-kingdom interactions between human-associated bacteria and human pathogenic viruses, as well as providing novel insights into the serious clinical problem of influenza-pneumococcal synergy.

The infant gut microbiome plays a critical role in the early development of the immune system, brain function, metabolism, and defense against pathogens. However, data from underrepresented populations, like Iceland, with its distinct dietary and lifestyle habits, remain limited. This paper presents the initial findings from the Icelandic Diet and the Infant Gut Microbiome Development (IceGut) study. Fecal samples were collected at multiple time points, representing 328 unique study identifiers, with one to five samples per child, from before the introduction of solid foods up to 5 years of age, and postpartum samples from 214 mothers. Microbial composition and predicted functional potential were assessed using 16S rRNA gene sequencing. Children in the cohort followed typical gut microbiome maturation, but at 1 year of age, they showed a notably higher relative abundance of Blautia than reported in comparable cohorts. This time point marked a transition in both taxonomic composition and predicted functional gene counts. By 5 years, the children had higher observed richness than their mothers but lower Shannon and Simpson diversities. At 2 and 5 years, and in the mothers, samples positive for archaea had significantly higher alpha diversity than samples that tested negative for archaea. Mothers with gestational diabetes mellitus (GDM) exhibited a higher relative abundance of Blautia but a lower alpha diversity. The variance in offspring gut microbiome explained by maternal GDM became progressively stronger over time, being significant at the age of 5 and explaining 2.5% of the variance.

Importance: This study provides the first comprehensive analysis of gut microbiome development in Icelandic children, covering the time from before the introduction of solid foods to 5 years of age. Although the overall developmental patterns of the gut microbiome in Icelandic children were similar to what has been seen in other studies, interesting differences were observed, such as a higher abundance of Blautia at an earlier age compared to other study populations. Higher alpha diversity in archaeal-positive samples, both in mothers and in children at the ages of 2 and 5, compared with archaeal-negative samples seen in the present study, is worth further investigation. Additionally, the study suggests a potential role of maternal and perinatal factors, particularly GDM, which was not evident until the age of 5 years, emphasizing the necessity of long-term studies.

Enterococcus faecium is a member of the human gut microbiota that has evolved into a problematic nosocomial pathogen and a leading cause of infections in hospitalized patients. Treatment of E. faecium infections is complicated by antibiotic resistance, making it important to understand resistance mechanisms and their broader consequences in this pathogen. Here, we explored the collateral effects of rifampin resistance-associated mutations in the E. faecium RNA polymerase β-subunit (RpoB). Of 14,384 publicly available E. faecium genomes, nearly one-third carried a mutation in the rifampin resistance-determining region (RRDR) of RpoB. In a local population of 710 E. faecium clinical isolates collected from patients at a single hospital, we found significant associations between the presence of RRDR mutations and prior exposure to rifamycin antibiotics, as well as associations between RRDR mutations and altered daptomycin susceptibility. To investigate the phenotypic impacts of RRDR mutations, we generated and studied four isogenic strains with distinct RRDR mutations (Q473K, G482D, H486Y, and S491L) that overlapped with clinical isolate variants. Transcriptomic and phenotypic analyses revealed allele-specific effects on E. faecium gene expression, growth dynamics, antibiotic susceptibility, isopropanol tolerance, and cell wall physiology. One frequently observed mutation, H486Y, caused minimal transcriptional changes and enhanced bacterial fitness under antibiotic stress. In contrast, the S491L mutation induced extensive transcriptional changes and slowed bacterial growth but also conferred increased isopropanol tolerance, potentially enhancing bacterial survival on hospital surfaces. Overall, our findings highlight the multifaceted impacts of RRDR mutations in shaping E. faecium physiology and antibiotic resistance, two important features of this hospital-associated pathogen.IMPORTANCEUnderstanding how antimicrobial resistance affects bacterial physiology is critical for developing effective therapeutics against bacterial infections. In this study, we found that rifampin resistance-associated mutations in RpoB are widespread in Enterococcus faecium, a leading multidrug-resistant pathogen. By studying isogenic wild-type and RpoB mutant strains, we discovered that RpoB mutations, although conferring resistance to rifampin, have distinct allele-specific effects on other bacterial phenotypes. Some of these collateral effects appear to promote E. faecium resistance to antibiotics and survival in the hospital environment, raising questions about the selective pressures driving their emergence. Overall, our study underscores the importance of examining the collateral effects of resistance-associated mutations in multidrug-resistant pathogens, which could help mitigate their persistence and spread among vulnerable patients.