Natural products have provided most of our modern pharmacopeia, serving as active molecules or scaffolds for active molecules. Their use in drug development is often inspired by their traditional or historical medical use. For many decades, this discovery pipeline has focused on identifying a single molecule responsible for much of the biological activity of a "raw" natural product preparation (e.g., a whole-plant extract) and scoping this molecule for clinical potential. However, it is increasingly realized that historical/traditional remedies with significant biological activity may owe this activity to the combined action of multiple molecules. Concomitantly, microbiologists increasingly argue that effectively fighting antimicrobial-resistant infections will rely on combination therapies that combine multiple antimicrobials and/or adjuvant molecules. We previously reconstructed a complex historical remedy, Bald's eyesalve. Our reconstruction of this remedy had strong antibiofilm activity, which relied on the presence of multiple ingredients. Here, we report that Bald's eyesalve has multiple antibacterial effects on exemplar gram-positive (Staphylococcus aureus) and gram-negative (Acinetobacter baumannii) pathogens. Bald's eyesalve disrupts bacterial membrane integrity; inhibits the expression of genes associated with bacterial adhesins, virulence factors, and efflux pumps in both S. aureus and A. baumannii; inhibits quorum sensing in S. aureus; and causes the downregulation of genes involved in de novo nucleotide biosynthesis in S. aureus. Finally, we show that this multifaceted mechanism of action makes it difficult for S. aureus, A. baumannii, and Pseudomonas aeruginosa to evolve resistance against Bald's eyesalve. Bald's eyesalve could be used to identify a defined cocktail of natural products suitable for preclinical testing as a multi-target antibacterial preparation to which resistance may arise more slowly than current single-molecule antibiotics.IMPORTANCEThe increasing mortality and economic cost of antimicrobial resistance (AMR) have made it one of the biggest threats to global health. Available antibiotics are in short supply due to the slow progress in the discovery of new antibiotics. Hence, there is a need for alternative treatment options against difficult-to-treat infections. We have identified a natural product cocktail based on a historical remedy with broad-spectrum antibacterial activity and a multifaceted mechanism of action. We have shown that there is slower resistance evolution to this cocktail compared to mainline antibiotics, and it could be used as the foundation of an alternative treatment to antibiotics in clinical settings.
Echinocandin antifungal drugs, such as caspofungin, inhibit β-1,3-glucan synthesis to impair cell wall formation. Caspofungin is effective in the clinic against Aspergillus and Candida species but ineffective against Cryptococcus neoformans, the fungus responsible for life-threatening meningoencephalitis in immunocompromised individuals. The mechanisms of caspofungin resistance in C. neoformans are associated with calcineurin signaling and functions that influence membrane and cell wall composition. The synergistic effects of caspofungin have been studied in combination with various inhibitors, but it is not known whether resistance can be overcome by agents that target the endoplasmic reticulum to influence protein glycosylation, as well as cell wall and membrane composition. Additionally, the role of the polysaccharide capsule of C. neoformans in blocking the access of caspofungin to the target Fks1 glucan synthase is not clear. Here, we identified a potential synergistic effect between caspofungin and the glycosylation inhibitor tunicamycin, with a significant reduction in proliferation, capsule attachment, and chitin and β-1,3-glucan levels when the drugs were used in combination. A targeted screen also identified other agents that influence ER function and that potentially display synergy with caspofungin. Overall, our analysis provides evidence that targeting the ER to impair cell wall and membrane integrity and capsule formation is an effective strategy to overcome caspofungin resistance in C. neoformans.IMPORTANCECryptococcus neoformans is the major cause of life-threatening meningoencephalitis in immunocompromised people. Unfortunately, the number of antifungal drugs available to treat cryptococcal disease is quite limited, and unlike other pathogenic fungi, C. neoformans is intrinsically resistant to the echinocandin group of drugs. These drugs target cell wall biosynthesis, and the mechanisms of cryptococcal resistance are not entirely clear. In this study, we demonstrate synergy between the echinocandin drug caspofungin and tunicamycin, an inhibitor of protein glycosylation in the endoplasmic reticulum. Combined treatment impacts cell wall and plasma membrane composition, thus providing insights into approaches to overcome resistance.
DNA-based vaccines have been developed and tested against a range of infectious agents, including influenza A viruses (IAV). Bacterial plasmid DNA is widely used as a vector for delivering genes encoding vaccine immunogens. However, the inclusion of antibiotic resistance genes in conventional plasmid constructs raises safety concerns. Linear DNA expression cassettes produced via PCR that lack antibiotic resistance genes, and bacterial sequences can serve as a safer alternative vector. In this study, we employed a mouse model of IAV to evaluate the protective efficacy of a lipid nanoparticle (LNP)-encapsulated linear DNA expression cassette containing the hemagglutinin gene of IAV H1N1 produced by PCR amplification. For comparison, a circular plasmid DNA containing the same gene was also constructed. The LNP-encapsulated DNA cassette (LNP-L) exhibited similar physicochemical characteristics, such as particle size, polydispersity index, zeta potential, and encapsulation efficiency, compared to the LNP-encapsulated circular plasmid DNA (LNP-P). However, LNP-L produced significantly lower transgene expression than LNP-P in cell culture. Mice immunized with LNP-L developed a delayed onset of antibody response compared to those immunized with LNP-P, but the magnitudes of both humoral and cell-mediated immune responses were comparable between the two groups after booster immunization. Moreover, mice immunized with LNP-L exhibited both Th1 and Th2 responses, similar to those immunized with LNP-P. Importantly, mice immunized with either LNP-L or LNP-P were fully protected upon challenge with a lethal dose of IAV H1N1. These findings suggest that linear DNA expression cassettes encapsulated in LNPs represent a promising strategy for vaccine development.IMPORTANCELipid nanoparticle (LNP)-encapsulated DNA vaccines represent a promising platform for the rapid development of vaccines against infectious pathogens. However, the inclusion of an antibiotic-resistance gene in plasmid DNA vectors used to deliver vaccine immunogens raises potential safety concerns. This study explores the feasibility of using a linear expression cassette produced via PCR amplification as an alternative system for delivering vaccine immunogens. Compared with conventional plasmid DNA, the linear expression cassette contains only essential elements, including a promoter, the gene encoding the vaccine immunogen, and a polyadenylation signal, and can be produced in a cell-free system. Using a mouse model for influenza A virus, we demonstrate that an LNP-encapsulated linear DNA cassette induces humoral and cell-mediated immune responses and protects immunized mice against a lethal influenza virus challenge. Thus, the LNP-encapsulated linear expression cassette may provide a safer approach for rapid responses to infectious diseases in animals.
Acid mine drainage (AMD) is a global pollution problem characterized by low pH and high concentrations of metals. Active remediation is often cost-prohibitive, but Fe(II)-oxidizing microbes may be used for passive bioremediation. To leverage these species, we must understand the factors that control their distribution. Here, we examined the environmental and ecological factors that control these species with the aim of determining if microbial seeding is a viable remediation strategy. Although stochastic processes appeared to control the distribution of the majority of taxa inhabiting AMD ecosystems, the distribution of Fe(II) oxidizers appeared to be driven by environmental filtering and competition. The abundance of all the major Fe(II)-oxidizing genera had significant relationships with pH, with pH explaining 10%-38% of the variation in their abundance. The genera appeared to have pH preferences, with Acidithiobacillus and Leptospirillum preferring environments with pH below 3, Gallionella, Sideroxydans, and Ferritrophicum preferring environments with pH above 3.5, and Ferrovum preferring intermediate-pH environments. Once the effect of pH is removed, genera that share pH preferences were negatively correlated, indicating that they were likely competing for the Fe(II)-oxidizing niche in their preferred environments. Communities were also shaped by dispersal limitation, which suggests that microbial seeding may be possible in these environments. Future seeding attempts should consider species interactions and ecology more generally to inform their efforts. IMPORTANCE:Acid mine drainage (AMD) is a global pollution problem affecting streams worldwide. One method of remediating AMD is by using naturally occurring microbial communities to remove iron and other metals. However, we do not have a complete understanding of the factors that control the distribution of these species or if it is possible to seed species from one environment into another. Here, we examine the factors that control community assembly in AMD ecosystems. We find that individual species appear to be dispersal-limited; thus, microbial seeding may be a viable method for AMD remediation.
An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs) that bind relatively conserved regions of the otherwise highly variable envelope glycoprotein. Among these, VRC01-class bNAbs are a reproducible antibody class that bind the CD4-binding site through genetic features encoded by the VH1-2 heavy chain and a light chain containing a rare five-amino-acid-long complementarity-determining region 3 (CDRL3). We previously developed a germline-targeting immunogen, iv4/iv9, derived from anti-idiotypic monoclonal antibodies (ai-mAbs) that target these genetic signatures of VRC01-class bNAbs. Here, we applied structure-guided modification of iv4/iv9 that improved selective binding to VRC01 precursors in vitro and carried out immunizations in ATX-GK mice. These mice are transgenic for human antibody genes, producing a diverse, genetically human antibody repertoire. We found that ATX-GK mice harbor VRC01 precursors at frequencies lower than those found in humans. Class-switched VRC01 precursors were detected in a minority of mice immunized with a modified iv4/iv9 immunogen. Collectively, these results indicate that the ATX-GK mice have utility to evaluate VRC01-class germline-targeting immunogens but are a stringent model due to a low frequency of VRC01-class B cells. They further suggest that the ai-mAb immunogens evaluated herein will require further optimization to reproducibly prime VRC01-class B cells in ATX-GK mice.IMPORTANCEAn effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs) that bind relatively conserved regions of the otherwise highly variable envelope glycoprotein. Among these, VRC01-class bNAbs are a reproducible antibody class that bind the CD4-binding site through genetic features encoded by the VH1-2 heavy chain and a light chain containing a rare five-amino-acid-long complementarity-determining region 3 (CDRL3). We previously developed a germline-targeting immunogen, iv4/iv9, derived from anti-idiotypic monoclonal antibodies that target these genetic signatures of VRC01-class bNAbs. Here, we evaluate the B-cell response to immunization with iv4/iv9 and with a structure-guided modified iv4/iv9 in ATX-GK mice. These modifications are intended to improve the selectivity of VRC01 precursors in a diverse polyclonal B cell repertoire. These mice are transgenic for human antibody genes, producing a diverse, genetically human antibody repertoire. We found that ATX-GK mice harbor VRC01 precursors at frequencies lower than those found in humans. Class-switched VRC01 precursors were detected in a minority of animals immunized with the modified iv4/iv9. Collectively, these results indicate that although the iv4/iv9 immunogen did not reproducibly elicit VRC01-class B cells, ATX-GK mice have utility to evaluate VRC01-class germline-targeting immunogens but are a stringent model due to a low frequency of VRC01-class B cells.
Post-translational modifications, including ubiquitination, have emerged as important regulators of viral infection and the host innate immune response. We screened the proteins of SARS-CoV-2 for ubiquitination and identified the RNA-dependent RNA polymerase (RdRp) NSP12 as being ubiquitinated, which has not previously been reported. Importantly, we confirmed that NSP12 is ubiquitinated during SARS-CoV-2 infection, and the NSP12 proteins of different coronaviruses are differentially associated with ubiquitin chains. SARS-CoV-2 NSP12 was primarily associated with K63-linked polyubiquitin chains, which increased SARS-CoV-2 NSP12 stability relative to OC43 NSP12, which was associated with K48 chains. Additionally, we identify the atypical E3 ubiquitin ligase MYCBP2 as a mediator of serine ubiquitination of SARS-CoV-2 NSP12 on serine-564 and observe that MYCBP2 promotes SARS-CoV-2 replication in cell-based assays. Substitution of this single residue (S564A) in CoV-2 NSP12 prevented recovery of infectious virus in three independent attempts, precluding direct genetic validation of its role in viral replication. To our knowledge, these findings provide the first evidence of serine ubiquitination of a viral protein and suggest a previously unrecognized role for RdRp ubiquitination in coronavirus biology.IMPORTANCEViruses often hijack host post-translational modification systems, including ubiquitination, to enhance the functions of their proteins and evade host innate immune responses. We screened the viral proteins of SARS-CoV-2 and found that NSP2, NSP5, NSP12, NSP15, and NSP16 are ubiquitinated. Focusing on NSP12, the RNA-dependent RNA polymerase (RdRp), we found that it is associated with multiple types of ubiquitin modifications, and RdRp proteins from other coronaviruses are differentially ubiquitinated. We also found that SARS-CoV-2 RdRp undergoes ubiquitination on a serine, rather than a canonical lysine residue, mediated by the atypical E3 ubiquitin ligase, MYCBP2. Consistent with this, MYCBP2 promotes efficient SARS-CoV-2 replication in cell-based assays. To our knowledge, this study provides the first evidence of non-lysine ubiquitination of a viral protein and demonstrates the serine selectivity of MYCBP2 for the first time in a cellular context. These findings raise important questions about the regulatory function of serine NSP12 ubiquitination in coronavirus replication and its potential contribution to viral pathogenicity.
Vaccines that elicit balanced, protective immunity to all four dengue virus (DENV) types are needed to reduce the global burden of dengue disease. Achieving this goal has been a challenge for dengue vaccine developers, particularly when immunizing dengue immunologically naïve individuals. In this randomized, phase I, open-label study of 40 healthy adults, we evaluated a heterologous, tetravalent purified inactivated vaccine (PIV) prime and a tetravalent live attenuated vaccine (LAV) boost. We compared a 0, 90 day PIV/LAV prime-boost schedule to a 0, 180 day schedule. These two PIV/LAV regimens elicited similar, high, and balanced cellular and humoral immune responses to DENV 1-4, although the condensed schedule was associated with more severe adverse events. In a subsequent clinical trial [K. E. Lyke, J. V. Chua, M. Koren, H. Friberg, et al., Lancet Infect Dis 24:896-908, 2024, https://doi.org/10.1016/S1473-3099(24)00100-2], the PIV/LAV vaccinees were challenged with DENV-1 and most were unprotected and experienced more pronounced dengue fever-like illness than unvaccinated controls. These outcomes underscore critical gaps in our understanding of mechanistic correlates of dengue vaccine-mediated protection and disease risk. IMPORTANCE:Dengue vaccine research and development has been complicated by several factors, including an incomplete understanding of immunologic correlates of protection and immune-mediated risk of severe disease. High and balanced immunity elicited by the tetravalent purified inactivated vaccine (PIV)/live attenuated vaccine (LAV) prime-boost vaccine strategy was predicted to provide protection from dengue virus type 1 (DENV-1) challenge in a subsequent clinical study, but vaccinees were unprotected and had an unexpected increase in symptom severity compared to unvaccinated controls. Evidence of exposure to a specific DENV type by viral genome detection (e.g., by quantitative RT-PCR) in acute samples or measurement of post-exposure, type-specific antibody responses (e.g., by serum antibody depletion) are known correlates of protection. However, current methods of measuring type-specific antibody are not ideal for evaluating groups of vaccinees, even in relatively small clinical trials. Newer, high-throughput methods of determining DENV type-specific immune responses and discovery of other correlates of protection are needed to more reliably evaluate dengue vaccine candidates.
Every year, billions of passengers, pets, and livestock are transported by air, which increases the risk of a global spread of pathogens like severe acute respiratory syndrome coronavirus 1 and 2, H1N1 influenza, the Ebola virus, and foot-and-mouth disease virus. However, due to its complexity and high safety standards, aircraft decontamination is limited to manual surface disinfection, and an approved method for disinfecting otherwise inaccessible areas is still unavailable. To address this issue, we present the development and validation of an effective room disinfection method utilizing low concentrations of aerosolized peroxyacetic acid (aPAA), stabilized by hydrogen peroxide, known as "dry fog." Therefore, we determined the minimal effective antimicrobial concentration of aPAA for a wide range of agents, including bacteria, viruses, and bacterial endospores. Then, we applied the developed disinfection protocol to a large panel of relevant materials multiple times in the context of the official material compatibility tests (MCTs) requested by the European Union Aviation Safety Agency (EASA) to demonstrate that its application will not cause a negative effect on the aircraft airworthiness. After successfully passing these complex material compatibility tests, we finally validated the disinfection protocol within a narrow-body aircraft A320 for its antimicrobial efficacy by using the most resistant microorganisms to aPAA among our test organisms, the bacteriophage MS2, and spores of Geobacillus stearothermophilus. Thus, we are now able to present the first aerosol-based room disinfection method suitable for aircraft that could easily be adopted for other aircraft types and other means of mass transport as well. IMPORTANCE:Despite numerous outbreaks in recent years, particularly the Ebola virus epidemic and the severe acute respiratory syndrome coronavirus 2 pandemic, there is still no approved method for disinfecting contaminated aircraft. Due to the highly complex nature of aircraft material and technology, it is difficult to balance the antimicrobial efficacy and material compatibility of the biocides used in a way that meets high safety standards. Here, we demonstrate an aerosol-based disinfection protocol using a mixture of peracetic acid and hydrogen peroxide. This protocol passed all material compatibility tests according to relevant aircraft certification specifications established by the European Union Aviation Safety Agency, as well as the manufacturer qualification test programs, and proved to be highly effective against viruses, bacteria, and even bacterial endospores without compromising the airworthiness of the aircraft. By targeting both contaminated surfaces and objects, as well as airborne pathogen reservoirs, it minimizes the spillover risk of zoonotic agents and prevents the mechanical transmission of high-consequence agricultural pathogens into disease-free geographical regions. This makes it the first suitable semi-automatic airborne disinfection method that can be used in addition to routine manual surface disinfection following contamination resulting from contact with people, animals, or goods infected or contaminated with high-consequence pathogens.
Temperature is known to affect organismal size, yet the underlying mechanism has been debated. Here, we develop a simple growth model of heterotrophic plankton and explain the temperature-size effect based on the demand and supply of oxygen. The model applies the growth rate-temperature relationship based on protein stability, capturing six data sets of a planktonic ciliate Urotricha. The model then computes the oxygen demand, based on the growth rate, which must be balanced by the diffusive uptake of oxygen. Because the smaller cell size favors oxygen uptake per cell volume, the predicted volume-temperature relationship is roughly the inversion of the growth rate-temperature relationship. With this inversive pattern, models are able to capture the data. This model-data consistency indicates that Urotricha may adjust their body size to meet the oxygen demands. Given that similar inversive relationships have been observed in other organisms, we hypothesize that oxygen demand and uptake are the key factors in constraining the volume of heterotrophic planktonic organisms. IMPORTANCE:The temperature-size relationship is a famous rule in physiological ecology based on common observations, but the mechanism underlying this rule has remained unclear. In this study, we develop a simple growth model of heterotrophic plankton, which explains the temperature-size relationship based on the demand and supply of oxygen. The model predicts temperature change will cause growth rate change; with higher growth rates, the increase of O2 demand causes a smaller cellular size. The model results also capture six datasets of Urotricha ciliates, suggesting that these organisms may adjust their body size to meet oxygen requirements. Given that similar patterns have been observed in other organisms, we hypothesize that oxygen demand and uptake are key factors constraining cell volume. Our study highlights oxygen as an intermediate factor underpinning temperature and cell volume, providing an explanation for the temperature-size relationship.
Antimicrobial resistance (AMR) is a systemic global threat, frequently described as a silent pandemic due to the challenges in attributing mortality to drug-resistant infections. Addressing this crisis necessitates a paradigm shift from viewing AMR as a linear consequence of antibiotic exposure to understanding it as an emergent property of ecological systems and metabolic plasticity. The ASM-IISc Symposium on the One Health Approach to AMR brought together a multidisciplinary cohort to deliberate on the drivers and solutions for resistance at the intersection of human, animal, and environmental health. Key thematic deliberations highlighted the role of technology in predicting resistance evolution and identifying novel targets for drug combinations. Furthermore, the symposium underscored the necessity of ecology-driven surveillance, extending genomic monitoring to environmental reservoirs. Ultimately, these findings establish a roadmap toward proactive, predictive, and integrated One Health strategies. Solutions demand mechanistic research and ecosystem-level interventions that bridge molecular insight with global public health strategy.
Treatment of severe invasive Streptococcus pyogenes infections has historically relied on β-lactam antibiotics with adjunctive clindamycin. Rising rates of lincosamide resistance have led to adoption of linezolid as an alternative adjunctive therapy. This report describes a clinical S. pyogenes isolate carrying a multi-drug resistance gene, cfr(C), known to confer the phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A (PhLOPSA) resistance phenotype in other bacterial species. Strain GAS-390756 is a pharyngeal isolate collected from a child with pharyngitis in the United States. Whole-genome sequence analysis demonstrated that the isolate belonged to emm type 89 and carried an integrative and conjugative element with erm(B) and cfr(C) cargo genes (ICESpy390756). Antimicrobial susceptibility testing demonstrated the isolate was resistant to erythromycin and clindamycin but susceptible to linezolid, consistent with the activity of erm(B) but not cfr(C). Comparative analysis of cfr(C)'s genomic context suggested that the promoter sequence may have been lost during ICESpy390756's acquisition of the gene. While GAS-390756 remains susceptible to linezolid, the expansion of cfr(C)'s host range into S. pyogenes and its presence on a novel mobile element are concerning. In light of known expression mechanisms for other silent antimicrobial resistance genes, this observation carries epidemiologic and public health importance. IMPORTANCE:Treatment of severe invasive Streptococcus pyogenes infection has traditionally relied on the adjunctive use of clindamycin with β-lactam antibiotic therapy. However, rising rates of clindamycin non-susceptibility have led clinicians to adopt linezolid as an alternative therapy. We identified S. pyogenes isolated from a child with pharyngitis that carried the cfr(C) gene. This gene causes resistance to multiple antibiotics, including clindamycin and linezolid, in other bacterial species. At the time of this study, we found no other instances of the cfr(C) gene carried by S. pyogenes in either published reports or the public genome sequence database. While this isolate remained sensitive to linezolid, the expanded host range of cfr(C) found in a novel potentially mobile genomic element may herald the eventual emergence of broader antibiotic resistance in S. pyogenes.
Linda Horianopoulos is a yeast biologist who uses a combination of comparative genomics, evolutionary analyses, and targeted molecular genetics to research the diversity of metabolism and stress responses across different yeast species. In this mSphere of Influence article, she highlights how a research article about Hsp90's ability to potentiate rapid evolution of drug resistance across different fungal species (https://doi.org/10.1126/science.1118370) influenced her work and the lens through which she appreciates biological complexity.
The annual Theobald Smith Society (TSS) spring meeting was convened at Rutgers University in New Brunswick, New Jersey, on 8 May 2026. TSS is the New Jersey branch of ASM and holds two annual meetings, in the fall and spring. These meetings bring together microbiologists, trainees at all levels, and professionals from both academia and industry to share their research, to network, and to engage in discussions on a wide array of topics that fall under ASM's three main units: health, mechanism discovery, and applied and environmental microbiology. The TSS spring meeting brought together more than 140 attendees from institutions across New Jersey. This report highlights the vision and work of TSS and ASM, the breadth of research presented at the meeting through invited talks and posters, and the two keynote lectures on nutritional immunity and the modern decline of human microbiome diversity.
Molecular and microscopy-based diagnostic capacity is often insufficient or unavailable in places where infectious disease burdens are highest, such as in West Africa. Rapid diagnostic testing (RDT) can provide quick and affordable diagnoses of common infections but is an imperfect solution due to limitations around detecting and dealing with false-negative and false-positive results. An alternative to RDT is unbiased metagenomic sequencing for pathogen surveillance. Here, we present data from unbiased metagenomic sequencing used to identify causes of undiagnosed febrile illness in Jos, Plateau State, Nigeria. Proof of concept for this approach has been demonstrated by several groups who have identified epidemic and endemic viral diseases like Lassa fever, yellow fever, and chikungunya. We show that unbiased deep sequencing and metagenomic analysis can be used to identify RNA viruses in clinical samples. We sequenced RNA from sera of patients (n = 343), many of whom were acutely febrile (76%), in a survey of clinics in Jos. We detected five human-infecting viruses in 39 (11 %) specimens. Among these were hepatitis B virus, human pegivirus, and several anelloviruses. While most of the viruses identified are unlikely to cause clinical symptoms in the patients we sampled, their presence demonstrates the validity of our approach. Additionally, our sequencing data allowed us to identify genetic material from potentially pathogenic bacteria, another possible etiological agent of febrile illness. IMPORTANCE:In low-resource areas, fevers due to infectious pathogens are a major source of illness, but tools for detecting and identifying such pathogens are often limited. Unbiased approaches for identifying genetic material from all potentially infectious organisms in a sample represent an opportunity for discovering sources of fever. Metagenomic sequencing can improve insight into pathogen landscapes in low-resource settings, potentially providing early detection of disease outbreaks. However, unbiased metagenomic sequencing (mNGS) is no panacea; it is susceptible to contamination and false positives. We used mNGS to evaluate serum from >300 Nigerian clinic-goers in Jos, Nigeria, most of whom (>70%) had fevers of unknown origin. Our goal was to understand arbovirus prevalence in Jos, Nigeria, and identify the sources of infection not routinely monitored for at clinics. We detected hepatitis B virus, as well as nonpathogenic anelloviruses. Our study provides insight into the utility and limitations of mNGS for pathogen surveillance.
The expression of a group 4 capsule (G4C) has been identified in Shigella sonnei and Shigella flexneri serotype 6 and demonstrated to play a significant role in pathogenic processes in S. sonnei. However, it has not been examined phenotypically in S. flexneri 6. Despite the impact of G4C, no systematic analysis of its expression or the presence of the genomic determinants has been performed across a large collection of S. sonnei and S. flexneri serotypes 2a and 6 or any of the other >46 Shigella strains and serotypes that cause disease. Our examination of the genomes of 1,219 geographically diverse Shigella isolates revealed the presence of intact gfc operons not only in the majority (≥98%) of S. sonnei and S. flexneri 6 strains but also, surprisingly, in the majority (≥97%) of Shigella boydii and Shigella dysenteriae strains. Approximately 95% of non-serotype 6 S. flexneri isolates contained a range of diverse gfc mutations preventing G4C expression; the remaining 5% contained an intact gfc operon yet had single-nucleotide polymorphisms within the promoter that resulted in lack of expression. These findings establish that no S. flexneri serotypes other than 6 express a G4C. Capsule expression was directly linked to reduced epithelial cell invasion for all strains examined, confirming differences in pathogenic processes between capsule-positive and capsule-negative Shigella strains. This work identifies a more widespread expression of capsule among Shigella strains than previously appreciated and identifies critical genetic requirements. IMPORTANCE:Shigella is the leading cause of moderate-to-severe diarrhea in 1- to 5-year-old children in low-resource settings, yet there is no licensed vaccine. Despite the impact of the group 4 capsule (G4C) on Shigella epithelial cell invasion, its expression has not been determined for the majority of Shigella species and serotypes that cause disease. This study investigated the presence of intact genes necessary for G4C expression in 1,219 geographically diverse Shigella isolates and examined phenotypes associated with G4C expression. We report that G4C is much more widespread than previously appreciated; it is expressed in the vast majority of Shigella sonnei and Shigella flexneri 6 clinical isolates, as well as Shigella boydii and Shigella dysenteriae strains. No S. flexneri serotypes outside of serotype 6 expressed a G4C. This study provides evidence that G4C expression in Shigella is much more widespread than previously recognized and emphasizes the importance of capsule expression in host-pathogen interactions.
Burial is a prevalent approach for disposing of human and animal carcass. Carcass decomposition is a key natural disturbance that reshapes microbial communities and modulates biogeochemical cycles. Animal intestines serve as critical natural reservoirs of antibiotic resistance genes (ARGs), and carcass decomposition concurrently remodels intestinal bacterial assemblages and resistome profiles. Most previous studies have characterized microbial succession in surface-exposed carcasses, yet the coordinated temporal shifts of intestinal microbiota, resistomes, and horizontal gene transfer (HGT) signatures within buried carcasses remain underexplored. We established a buried rat carcass model and performed metagenomic sequencing to characterize temporal dynamics of intestinal bacteria, ARGs, mobile genetic elements (MGEs), and HGT events. Bacterial communities underwent directional succession accompanied by reduced α-diversity and stage-specific β-diversity. Proteobacteria gradually outcompeted Firmicutes and Bacteroidetes to become dominant taxa. The resistome followed regular temporal changes: tetracycline and macrolide-lincosamide-streptogramin (MLS) ARGs prevailed in early decomposition, while multidrug, β-lactam, polymyxin, and quinolone ARGs accumulated in mid-late stages, with ARG richness peaking on day 28. Approximately 83% of temporally dynamic ARG subtypes were positively correlated with decomposition duration. Procrustes analysis (R2 = 0.859) revealed strong correlations between bacterial succession and resistome dynamics, with Proteobacteria as potential multidrug-resistant hosts and major HGT donors. This study identifies a correlative cascade of microbial succession that drives MGE functional shift, which in turn increased HGT potential and ultimately leads to resistome accumulation in buried carcass intestinal habitats. These findings expand the theoretical framework of disturbance-driven microbial-resistome co-succession and offer insights into drivers of antibiotic resistance propagation in terrestrial cadaver systems.IMPORTANCEAnimal carcass burial is a ubiquitous natural terrestrial disturbance, and carcass intestinal contents represent a major endogenous reservoir of environmental antibiotic resistance genes (ARGs). Although extensive research has characterized microbial succession of exposed carcasses, the co-occurrence patterns of gut microbiome and resistome together with associated horizontal gene transfer (HGT) under buried conditions remain underexplored. Using metagenomic profiling of decomposing rat intestinal feces, this study explores a potential correlative cascade spanning bacterial succession, mobile genetic element (MGE) functional shifts, increased HGT potential, and gradual ARG enrichment. Our findings expand the ecological data set focused on subsurface buried carcass habitats and advance mechanistic knowledge of coupled microbiome-resistome succession driven by postmortem decomposition.
Monosodium glutamate (MSG; L-glutamate monosodium salt) is among the most widely used flavor enhancers, yet its molecular effects on gut microbial physiology remain poorly understood. Here, we examined the strain-specific transcriptomic and metabolic responses of Clostridium butyricum and Bacteroides thetaiotaomicron grown in pure anaerobic culture with 0.1% (wt/vol) MSG. Although MSG exposure was not associated with major changes in total bacterial biomass dynamics, both species showed a temporal functional reprogramming. In C. butyricum, MSG rapidly intensified metabolic activity to capitalize on exogenous glutamate by activating central carbon metabolism, redox-balancing pathways, and the GABA shunt, thereby collectively enhancing butyrate synthesis, a metabolite associated with gut barrier integrity, energy regulation, and anti-inflammatory signaling. On the other hand, B. thetaiotaomicron adopted a conservative, homeostatic response, suppressing glycan utilization and central carbon and energy metabolism, and maintaining stable SCFA production through controlled regulation that buffered against abrupt dietary perturbations. Overall, MSG functioned as a potent metabolic signal, triggering distinct adaptive strategies in two dominant gut bacteria. IMPORTANCE:The impact of monosodium glutamate (MSG) as a highly consumed food additive on the gut microbiome is often overlooked, and community-level analyses reveal little change, masking distinct phenotypic responses of individual strains. By combining gene expression and metabolite profiling using two key human gut bacteria, we show that MSG is sensed as a metabolic signal. A butyrate-producing gut bacterium increases energy metabolism and butyrate production, and a fiber-degrading gut bacterium transiently moderates metabolism to maintain stable fermentation products. These differences suggest that MSG's physiological effects may depend on which bacterial groups dominate an individual's microbiome.
Early virus-induced cellular responses are a fundamental diagnostic feature of viral infections; however, early virus-host interactions, particularly during viral entry, remain difficult to study in living cells because many commonly used markers and antibodies require fixation or can interfere with cell viability. Although virus-induced cellular responses constitute a fundamental diagnostic feature of viral infections, their detection by microscopy typically relies on later-stage effects. Microscopic observation, therefore, serves as a complementary approach to molecular techniques, but the time required for visible changes to develop limits its utility for rapid assessment. Here, we propose a label-free digital holotomography (DHT) approach for the detection of early virus-induced cellular responses during the first hours of infection, including equine arteritis virus (EAV), equine herpesvirus 1 (EHV-1), and equine rhinitis B virus (ERBV). Using this method, we show that as early as 1-2 h post-infection, holotomography is capable of capturing virus-induced alterations in cellular density based only on changes in the refractive index (RI), which reflects local variations in cellular mass density and biochemical composition. In addition to global RI changes, we focused on subcellular alterations occurring at the early stages of infection, including modifications in lipid droplet-like structures and nucleoli. Our results reveal that these changes become detectable rapidly and can be captured with high sensitivity, displaying both cell type- and virus-dependent patterns. Our findings suggest that DHT may provide a rapid, label-free, and quantitative platform for detecting virus-induced subcellular changes as early as 1-2 h post-infection. IMPORTANCE:Digital holotomography (DHT) facilitates the early, label-free detection of infection and serves as a complementary diagnostic tool, allowing the rapid initiation of treatment. This method is universal and can be broadly applied to the investigation of primary host interactions, including general cellular and biomolecular optical density changes. These applications provide a strong foundation for viral entry inhibitor testing. Moreover, this approach highlights the critical importance of studying viral infections in natural host systems rather than relying solely on well-established experimental models. In this context, we employed equine lung cells (ELCs) as a biologically relevant and appropriate model to better reflect natural host-virus interactions. These findings should be interpreted in the context of an exploratory imaging data set and will require confirmation in replicate-based and longitudinal single-cell studies.
Corynebacterium macginleyi is an opportunistic pathogen linked to ocular infections, but comprehensive genomic studies remain scarce, especially in China. This study characterized its genomic features, virulence potential, and antimicrobial resistance using 13 clinical isolates and 27 public genomes. Methods included matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), average nucleotide identity (ANI), whole-genome sequencing, Clusters of Orthologous Groups/Kyoto Encyclopedia of Genes and Genomes annotation, core-single nucleotide polymorphism phylogeny, virulence and resistance gene analysis, and Clinical and Laboratory Standards Institute susceptibility testing. All isolates were accurately identified as C. macginleyi, with MALDI-TOF MS and ANI outperforming conventional methods. Phylogenetic analysis revealed three clades with geographic clustering. Functional profiles were centered on metabolism. Virulence genes were conserved, mainly mediating adhesion, stress response, and immune evasion. Resistance genes were limited but diverse, dominated by erm(X). Phenotypic testing showed frequent macrolide resistance, while most isolates remained susceptible to the other tested antimicrobials; fluoroquinolone resistance was detected in a subset of isolates. Isolate B64 exhibited a unique phylogenetic position, higher virulence, and resistance gene burden, consistent with multidrug resistance. In conclusion, C. macginleyi shows geographic diversity, conserved virulence, and evolving resistance, supporting precise identification and continuous surveillance.IMPORTANCEThe precise molecular mechanisms underlying the pathogenicity of Corynebacterium macginleyi are not fully elucidated, and large-scale genomic and epidemiological studies are still limited, particularly in certain regions such as China. Therefore, comprehensive investigations integrating whole-genome sequencing with clinical data are urgently needed to better understand its genetic characteristics, virulence-associated traits, and antimicrobial resistance profiles. Such efforts will provide a critical foundation for improving diagnostic accuracy and optimizing therapeutic strategies for ocular infections caused by this organism.
