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 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 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.

Malaria control requires the coordination of different strategies due to the lack of an effective vaccine and the emerging resistance of parasites to drugs and of vectors to insecticides. Therefore, efficient and environmentally safe alternative control strategies are still needed. In this study, we explored the composition of microbiota of the and its variability in the presence of natural infection during the wet and dry seasons, in order to determine their potential as a novel vector control-based approach to fight malaria. An entomological survey of a collection of was conducted in Bankeng. Using 16S ribosomal RNA amplicon-based sequencing, we investigated the bacterial microbiota of mosquitoes naturally infected or uninfected with . A total of 120 mosquitoes were selected randomly corresponding to 60 mosquitoes per infection status. Overall, 99 bacterial taxa were detected across all the samples, with 97 of these shared between uninfected and infected. A total of two were unique to uninfected (, ), while no bacteria were unique to infected mosquitoes. However, there were significant differences in bacterial composition between both groups. Additionally, differential abundance revealed notable variations in microbiota composition, with 14 bacterial genera more abundant in uninfected mosquitoes and only two bacterial genera more abundant in the infected mosquitoes. Observed amplicon sequence variants and Shannon indices revealed a significant difference in bacterial diversity between infected (positive) and uninfected (negative) with higher diversity observed in negative samples during the wet season and in positive samples during the dry season. These findings highlight potential associations between certain bacterial taxa and infection status, suggesting they may be linked to susceptibility or resistance, although causality cannot be determined from this study. Ultimately, this baseline information provides a foundation for studies on the functions and interactions of the microbiota in natural populations of and their susceptibility to natural infection.IMPORTANCEMalaria control faces challenges due to the absence of an effective vaccine and growing resistance to drugs and insecticides, highlighting the need for alternative strategies. This study investigates the microbiota composition of mosquitoes in Bankeng, Cameroon, and its association with natural infection. Using 16S rRNA amplicon sequencing, the bacterial communities of 120 mosquitoes-60 infected and 60 uninfected-were analyzed. A total of 99 bacterial taxa were identified, with 97 shared between both groups. Only two taxa ( and ) were exclusive to uninfected mosquitoes, and none were unique to the infected group. Significant differences in microbiota composition were observed: 14 bacterial genera were more abundant in uninfected mosquitoes, while only two were enriched in infected ones. These findings suggest that specific bacteria may influence susceptibility to infection. This study provides foundational knowledge for exploring microbiota-based or paratransgenic strategies in malaria vector control.

β-Lactam/β-lactamase inhibitor combinations have significantly improved treatment outcomes for infections caused by serine β-lactamase (SBL)-producing pathogens. However, the continued emergence and spread of metallo-β-lactamases (MBLs), for which no clinically approved inhibitors currently exist, poses a serious threat to the long-term effectiveness of β-lactam-based therapies. To bridge this therapeutic gap, the boronic acid transition state analog, taniborbactam (Venatorx Pharmaceuticals), was developed, targeting SBLs and widespread MBLs such as NDM-1 and VIM-2. However, taniborbactam-escape variants have been detected among various MBL enzymes, including members of the NDM and IMP families. Here, we explored whether covalently combining two complementary inhibitor structures, a boronic acid transition state analog and a dipicolyl ethylenediamine-based metal chelator, can restore β-lactam susceptibility in MBL-producing bacterial strains, including taniborbactam-escape variants. APC24-7 successfully sensitized clinical isolates of SBL- and MBL-producing and to meropenem. While APC24-7 demonstrated similarities in resensitization behavior to taniborbactam against a wide range of isogenic expressing single SBLs, APC24-7 reversed NDM-9- or IMP-26-mediated meropenem resistance more efficiently. To investigate the potential role of the chelator motif in the MBL inhibition of APC24-7, susceptibility tests were conducted with an excess of exogenous Zn². APC24-7-mediated resensitization remained unaffected in the presence of Zn² for strains producing NDM-1 and VIM-2. However, its ability to reverse NDM-9- and IMP-26-mediated meropenem resistance was attenuated upon Zn² supplementation. These findings demonstrate that combining functionally complementary chemical structures, such as chelators and boronic acids, can aid in expanding the resensitization ability of existing β-lactamase inhibitors.IMPORTANCEThe ability of bacteria such as and to circumvent antimicrobial chemotherapy has become a global public health crisis. The high prevalence of β-lactamase enzymes capable of rendering our most prescribed antibiotics, the β-lactams (BLs) inactive, has left us with few available treatment options against infections caused by these bacteria. The use of small molecules that inhibit especially serine β-lactamases has substantially prolonged the lifetime of BL antibiotics. Yet, most clinically available inhibitors either do not possess or have limited ability to reverse resistance conferred by metallo-β-lactamase (MBL) enzymes. Combining chelator and transition state analog technology, our hybrid compound restores the effectiveness of BL antibiotics in cases of resistance conferred by both serine β-lactamases (SBLs) and MBLs. Our approach of covalently combining a chelator with an existing SBL inhibitor scaffold offers a promising solution for managing life-threatening infections and prolonging the use of clinically available BLs.

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 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 RNA polymerase β-subunit (RpoB). Of 14,384 publicly available genomes, nearly one-third carried a mutation in the rifampin resistance-determining region (RRDR) of RpoB. In a local population of 710 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 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 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 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 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.

is a growing public health concern, capable of causing long-term contamination of healthcare settings, skin colonization, and life-threatening bloodstream infections. However, pathogenesis is not well understood, which is exacerbated by limitations and discrepancies in existing animal infection models. Further, the effects of growth phase on virulence have not been examined, despite growth phase being linked to virulence in many bacterial species. To address this question, and to develop an immunocompetent murine model of infection, we directly compared log-phase and stationary-phase systemic infection in immunocompetent C57BL/6J mice at high and low doses of infection. Systemic infection with high-dose log-phase results in rapid mortality between 2 h and 1 day post-infection, whereas stationary phase results in significantly extended survival. However, at low doses of infection, there was no difference in mortality kinetics between log-phase and stationary-phase cells. We observed that initially colonizes multiple organs but is rapidly cleared from the lungs and spleen, while kidney fungal burdens remain stable. Mice infected with high-dose log-phase had fibrin-associated blood clotting in multiple organs and decreased serum fibrinogen levels, suggesting that coagulation may drive rapid mortality. This was associated with increased β-glucan exposure and mannan abundance in log-phase . These results will inform the development of a more standardized animal model of systemic infection, which can be used to reveal key aspects of pathogenesis.IMPORTANCEDespite its growing medical importance, there is limited understanding of pathogenesis, due in part to limitations of existing laboratory models of infection. To develop a more complete understanding of factors that contribute to pathogenesis, it will be necessary to establish consistent parameters for animal models of infection. To address this need, we directly compared log and stationary growth phases on pathogenesis in immunocompetent C57BL/6J mice using a single virulent Clade I isolate. At a high dose of infection, host survival was dramatically different between log-phase or stationary-phase , suggesting that growth phase can affect pathogenesis. These differences correlated with increased exposure of pathogen-associated molecular patterns in the cell wall in log-phase cells. These results will be instrumental in the future development of standardized animal models to study pathogenesis.

Enterococci are commensals of the intestinal tract that are intrinsically resistant to cephalosporins, antibiotics that inhibit peptidoglycan synthesis. Prior treatment with cephalosporins is a risk factor for acquiring an enterococcal infection. We previously showed that FtsW, a SEDS (hape, longation, ivision, and porulation) protein, is essential for enterococcal cephalosporin resistance. SEDS proteins catalyze glycosyltransferase reactions to polymerize strands of peptidoglycan. Bacterial genomes typically only encode for two SEDS proteins, FtsW and RodA, that form the core of two different peptidoglycan synthases thought to function at distinct locations in the cell. However, a few bacterial genera, including enterococci, encode homologs of not only FtsW and RodA but also additional SEDS proteins. In general, very little is known about the function of these additional SEDS proteins. The genome of encodes two additional SEDS homologs, whose expression is induced in response to antibiotic-mediated cell wall stress by the CroS/R two-component system. However, nothing was previously known about the function of these SEDS homologs. In this work, we determined that these two additional SEDS homologs in each possess glycosyltransferase activity , preferentially associate with distinct bPBPs in , can functionally substitute for either FtsW or RodA (but not both), and are upregulated in a CroR-dependent manner in response to FtsW depletion, enhancing peptidoglycan synthesis and cephalosporin resistance.IMPORTANCESEDS (hape, longation, ivision, and porulation) proteins are transmembrane glycosyltransferases that play a critical role in synthesis of bacterial peptidoglycan. It is well known that most bacteria possess two SEDS protein homologs, known as FtsW and RodA, that participate in peptidoglycan synthesis at distinct locations in the cell. Some bacterial genomes also encode, in addition to FtsW and RodA, additional SEDS protein homologs whose functions are typically poorly characterized. is a commensal of the human intestinal tract and an important opportunistic pathogen that encodes two such additional SEDS proteins, whose functions have not been reported previously. Our results reveal new insights into the activity and function of these additional SEDS homologs, showing that they are genuine glycosyltransferases that enhance peptidoglycan synthesis and cephalosporin resistance in response to cell wall stress.

Rodent-borne hantaviruses pose a continual public health threat to humans through zoonotic transmission, with case fatality rates of up to 50% in some cases. Human infections can lead to hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome depending on the viral species. Despite the morbidity and mortality associated with this family of viruses, no anti-viral therapeutics or vaccines are available to treat and prevent hantavirus disease. The relative shortage of commercially available reagents to study hantavirus infections and likely contributes to the challenges in developing viral countermeasures. This report describes the generation of a panel of mouse monoclonal antibodies that collectively recognize the four viral proteins of Seoul virus (SEOV, ), an Old World (OW) hantavirus with worldwide distribution, and the causative agent of HFRS. We have validated the specificity and versatility of these antibodies against a subset of OW and New World hantaviruses in assays relying on antigen recognition in denatured or native conformations. We present several antibodies that specifically recognize the SEOV nucleoprotein and polymerase protein in Western blotting and immunostaining assays. We also identified three novel antibodies directed against the glycoprotein complex that are capable of binding to the N-terminal glycoprotein of all hantaviruses tested. These antibodies are freely available to all hantavirus researchers to add to the small, but growing, collection of reliable and available reagents to be used to study hantavirus biology, identify novel antiviral compounds, and measure viral prevalence in the laboratory and the field.IMPORTANCEPathogenic hantaviruses cause severe hemorrhagic disease and pose a significant public health threat worldwide. Insufficient research into the biology of these viruses has slowed the development of effective direct-acting antivirals and vaccines. Here, we describe the generation and validation of novel, specific monoclonal antibodies for the detection of Seoul virus proteins . These reagents can be used to fill in critical gaps in knowledge regarding hantavirus entry, protein expression, and particle generation.

Equine strangles caused by subspecies () remains a significant cause of morbidity and mortality in horses, and there is a need for improved diagnostic and vaccination strategies for addressing this pathogen. ORFeome phage display is a platform that allows for rapid screening for potential antigenic epitopes by construction of phage-displayed peptide libraries. In this study, an ORFeome library was used to screen serum from a panel of 17 horses with known exposure to to identify antigenic bacterial proteins. From this screen, three major proteins were identified: a novel proline-rich repeat domain protein, a serine peptidase, and the M-like protein SeM. These three proteins are predicted to be expressed on the surface of the bacterial cell by the presence of N- and C-terminal signals. The proline-rich repeat protein and serine peptidase were confirmed to be immunogenic by enzyme-linked immunoassay (ELISA) using the recombinant full-length proteins against sera from horses with strangles, horses infected with the related pathogen subsp. , and healthy control horses. Due to the native IgG binding activity of SeM, ELISA against the full-length protein was not conducted, but the specificity of the antibody response against the recovered ORFeome clones was confirmed and an antigenic region identified. Both the proline-rich repeat protein and serine peptidase were found to be highly conserved in global genomes, indicating these proteins may be useful as vaccine candidates against or as diagnostic markers to specifically identify infections in horses.

Carbapenem-resistant (CREC), particularly strains producing New Delhi metallo-β-lactamase-9 (NDM-9), pose a growing threat as agents of nosocomial infections. Despite their emergence since 2013, a comprehensive global phylogeographic and genetic characterization of -carrying CREC is lacking. Through 7 years (2018-2024) of surveillance of CREC strains in a tertiary hospital, we obtained seven -carrying CREC. Antimicrobial susceptibility testing, conjugative transfer experiments, whole-genome sequencing (WGS), and fitness analysis were performed. Publicly available genomes of -carrying CREC from NCBI (curated by July 2025) were integrated for global analysis. All seven -carrying CREC exhibited resistance to most antimicrobials tested, except colistin. WGS revealed diverse -carrying plasmid types (IncB/O/K/Z, IncHI2, IncFIB, and IncC) and sequence types of strains (ST156 predominant). Key mobile genetic elements IS and IS facilitated dissemination. Plasmid structural analysis traced the evolution of the IncB/O/K/Z plasmid, revealing potential intra-hospital persistence and spread. Carriage of -carrying plasmid imposed a significant fitness cost. Global analysis ( = 203 isolates) demonstrated high genetic diversity (56 STs), with ST156 (20.1%) being the most prevalent. Spatially, isolates were concentrated in Asia (China: 85.2%). Primary isolation sources were humans (39.4%) and chickens (34.9%), with a notable shift toward human predominance since 2016. Our findings elucidate the critical role of specific mobile elements in transmission, highlight the significant burden in China, document a shift toward human-associated isolates, and identify ST156 as a globally prevalent lineage. We emphasized the necessity of intensified surveillance to track the dissemination of -carrying CREC.IMPORTANCEThis study provides the first integrative geographic and genomic epidemiology analysis of -carrying carbapenem-resistant (CREC). Our 7-year surveillance and genomic analysis revealed critical insights into the genetic characteristics and transmission dynamics of CREC carrying . The identification of mobile genetic elements, such as IS and IS, underscores their role in the horizontal transfer of resistance genes, facilitating the spread of . Furthermore, given the high frequency of -carrying CREC in China and its likelihood of spreading clonally in hospitals, there is an immediate need to intensify surveillance efforts. Adopting a One Health perspective, our study highlights the interconnected antimicrobial resistance risks spanning human, animal, and environmental health domains, advocating for strengthened global phylogeographic and phylogenetic surveillance alongside clinical interventions to curb the spread of these high-risk epidemic clones.

The present study investigated T and B cell responses following a second heterologous booster dose of BNT162b2 administered after a two-dose CoronaVac regimen for coronavirus disease 2019 (COVID-19) vaccination in 15 healthcare workers. Blood samples were collected 4 weeks after the first booster and at both 4 and 24 weeks after the second BNT162b2 booster. Interferon-γ-secreting CD4+ and CD8+ T cells were detectable 4 weeks after the first booster, whereas only CD4+ T cells remained detectable at both 4 and 24 weeks after the second booster. Seven of the 15 participants (46.7%) were diagnosed with COVID-19 approximately 16 weeks after receiving the second booster. These individuals exhibited significantly higher frequencies of CD4+ T cells at 24 weeks post-booster than at 4 weeks post-booster. In contrast, the non-COVID-19 group exhibited significantly higher CD4+ T cell responses 4 weeks after the second booster. Memory B cells were detected at low frequencies at all three time points. IgG antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein were detectable at all three time points, with a significant decline observed 24 weeks after the second booster. Overall, CD4+ T and B cell responses induced by a heterologous second booster dose of BNT162b2 following a primary two-dose CoronaVac regimen were rapidly elicited and sustained for at least 6 months.IMPORTANCEThere is limited evidence regarding T and B cell responses following a primary COVID-19 vaccination series with CoronaVac and two heterologous BNT162b2 booster doses. This study investigated the longitudinal T and B cell responses induced by a second heterologous BNT162b2 booster following a primary two-dose CoronaVac COVID-19 vaccination regimen. These results demonstrate that CD4+ T cells induced by the second heterologous BNT162b2 booster play a key role in protection against SARS-CoV-2 infection and progression to severe disease. This study suggests the need for the future consideration of repeated emergency vaccine-boosting strategies in response to emerging viral infections.

The emergence of pathogens resistant to antimicrobials has become a forefront concern for clinicians and patients alike. Antimicrobial resistance (AMR) is exacerbated by the misuse and overuse of antibiotics. Pregnant women and their infants are an important area of focus, as antibiotic use during this vulnerable period of development may generate reservoirs of AMR genes, which would contribute to future risk. Identifying the extent of antibiotic use and its association with ARG composition and persistence within this window is crucial. We sought to characterize the gut resistomes of 3-month-old infants ( = 212) and pregnant women in their third trimester ( = 99) to assess ARG burden in these populations. For a subset of women and their infants ( = 33 pairs), we explored overlap of ARG. Preliminary analyses demonstrated that pregnant women and infants had markedly different resistome communities and identified other environmental and demographic characteristics to be associated with univariate differences in infant ARG composition. When controlling for the race of the mother, infant diet, and infant antibiotic exposure since birth, delivery by cesarean section was associated with increased diversity of ARG relative to the diversity of ARG in the samples from vaginally born infants. Cesarean-born infants had increased richness of aminoglycoside ARG and increased diversity of beta-lactamase and tetracycline ARG relative to vaginally born infants. Furthermore, infants consuming any formula had increased overall richness and diversity of ARG in multivariate analyses. This study provides further insight into how diet and method of delivery are associated with resistome composition within the first 3 months of infant microbiome development.Pregnancy and the first 3 months of life are vulnerable periods for antibiotic exposure and subsequent development of antimicrobial resistance (AMR). AMR is an increasingly worrisome problem for global public health. The full repertoire of AMR genes present in the gut collectively is referred to as the resistome. Herein, the associations between a variety of demographic and environmental factors, including race of the pregnant women, sex of the infant, mode of delivery, amount of breast milk consumed in infant diet, and antibiotic exposure during the first 3 months of life, with resistome composition are reported. Infants consuming any formula had a greater richness and diversity of ARG overall, and cesarean-born infants had greater diversity of ARG within their resistomes. These findings give insight into the early seeding of the infant resistome, which is crucial to understanding how the resistome develops throughout life.

Tuberculosis (TB) is one of the most common infectious diseases caused by bacteria worldwide. The increasing prevalence of multidrug-resistant TB (MDR-TB) and latent TB infection (LTBI) has intensified the global TB burden. Therefore, the development of new drugs for MDR-TB and LTBI is urgently required. We have reported that the derivative of dithiocarbamate sugar derivative, 2-acetamido-2-deoxy-β-D-glucopyranosyl -dimethyldithiocarbamate (OCT313), exhibits anti-mycobacterial activity against MDR-MTB. Here, we identified the target of OCT313. In experimentally generated OCT313-resistant bacteria, adenine at position 1,092 in the metabolic enzyme phosphotransacetylase (PTA) gene was replaced with cytosine. This mutation is a nonsynonymous mutation that converts methionine to leucine at position 365 in the PTA protein. OCT313 inhibited the enzymatic activity of recombinant wild-type PTA, but not of the mutant PTA (M365L). PTA is an enzyme that produces acetyl-coenzyme A (acetyl-CoA) from acetyl phosphate and CoA and is involved in metabolic pathways; therefore, it was expected to also be active against dormant bacilli. OCT313 exhibits antibacterial activity in the Wayne model of dormancy using BCG, and overexpression of PTA in OCT313-resistant bacilli restored sensitivity to OCT313. Collectively, the target of OCT313 is PTA, and OCT313 is a promising antimicrobial candidate for MDR-TB and LTBI.IMPORTANCEThrough this study, we propose a new target for the development of medicines to treat multidrug-resistant tuberculosis and latent tuberculosis infection. The target enzyme phosphotransacetylase (PTA) is a key enzyme that functions in major metabolic pathways, and the homologous structures of PTA enzymes vary greatly among bacterial species. Since the treatment of mycobacterial disease is long term, the development of antibiotics targeting PTA is useful for species-specific therapy.

Ecological boundaries between different environments are important for generating and maintaining biodiversity and for understanding community assembly. Classic examples include temperature gradients between upland and lowland habitats and estuaries (brackish) between fresh- and saltwater. While numerous studies have examined community assembly of free-living organisms across ecological boundaries, few studies have considered community assembly of host-associated (HA) organisms, including HA microbiota, across similar ecological boundaries. This is likely because it is unclear what constitutes an ecological boundary for organisms that reside on a host. We identify hybrid hosts (i.e., hosts derived from breeding events between two different genetic lineages) as ecological boundaries for HA organisms. More specifically, we argue that the intermediate genetic compositions of hybrid organisms, often accompanied by traits that are either intermediate to or else combinations of progenitor traits, create transition zones between the environments experienced by microbes on or in progenitor host lineages. This, in turn, paves the way for microbial community coalescence (i.e., mixing of microbial communities) that is directly analogous to community assembly in classic ecological boundaries. Further, because many hybrid hosts reside along ecological boundaries themselves, hybrid microbiota often experience simultaneous boundaries of both their hosts and their hosts' environment-an underexplored phenomenon that we term a "multiscale ecological boundary." By introducing ecological boundaries into HA microbiota literature, our goals are to further understanding of HA microbiota assembly and to propose a framework for studying the effects of ecological boundaries at multiple scales.IMPORTANCEBoundaries between environments provide important insight into how ecological communities are structured across broader landscapes. Of particular interest is how communities assemble within the transition zone constituting the boundary (i.e., where the transition in environmental variables occurs) and whether transitions in community composition parallel transitions in environmental variables. While ecological boundaries have a long history in classic ecology, similar concepts have recently emerged in microbiota literature. Currently, however, most studies of microbial ecological boundaries focus on environmental microbiota, rather than host-associated (HA) microbiota. This is likely because it is unclear what constitutes an ecological boundary in HA microbiota systems. We propose hybrid hosts as an HA analog for environmental ecological boundaries. Specifically, we outline how different types of hybrid hosts serve as models for different types of ecological boundaries. We then outline how the ecological boundary framework can be used to interpret HA microbial community coalescence (i.e., mixing) across host species. Finally, we suggest that many hybrid hosts reside within the transition zones of larger scale ecological boundaries. When this happens, hybrid hosts can be used to examine a novel phenomenon that we term a "multiscale ecological boundary."

is a filamentous fungus found in compost and soil that can cause invasive and/or chronic disease in humans. Diagnosis and treatment of aspergillosis often occur when has formed dense networks of hyphae within the lung. These hyphal networks are multicellular, encased in an extracellular matrix, and have reduced susceptibility to contemporary antifungal drugs, similar to bacterial biofilms. A model of these dense hyphal networks observed can be recapitulated using a static, submerged biofilm culture. The mechanisms underlying filamentous fungal cell physiology at different stages of biofilm development remain to be defined. Here, we utilized RNA sequencing, metabolic modeling, and molecular genetics approaches to identify genes and metabolic pathways critical for biofilm development. These analyses revealed that ethanol and butanediol fermentation pathways are important for the development of a mature biofilm. Correspondingly, a predicted transcription factor () was observed to be required for mature biofilm development. Taken together, these data define key genes and metabolic pathways critical for biofilm development.

() is a predominant non-aureus staphylococcal species colonizing the teat skin and mammary gland of dairy ruminants. Although often linked to mild or subclinical mastitis, specific strains may also play protective roles against major udder pathogens. In this study, we characterized two . isolates (4S77 and 4S90) that displayed antimicrobial activity against Gram-positive bacteria. Complete genome sequencing revealed a conserved, plasmid-encoded biosynthetic gene cluster for the thiopeptide bacteriocin micrococcin P1 (MP1). All genes necessary for MP1 biosynthesis, modification, export, and immunity were identified, and compound production was confirmed by high-performance liquid chromatography and liquid chromatography-mass spectrometry. Comparative analysis with publicly available genomes revealed that the MP1 cluster appears unique to these isolates. Both strains showed full phenotypic susceptibility to tested antibiotics, despite 4S90 carrying the gene, which did not confer detectable resistance under standard conditions. Classical staphylococcal toxin genes were also absent. Virulence gene profiling revealed a conserved repertoire of colonization- and persistence-associated genes, including factors involved in adhesion, capsule formation, and iron acquisition, but no markers of aggressive pathogenicity. Mobile genetic elements, including prophages and genomic islands, were common but did not carry antimicrobial resistance or virulence genes, suggesting a low risk of transmission of new pathogenic traits to the endogenous microbiome, including opportunistic bacteria. These findings suggest that MP1-producing strains combine antimicrobial functionality with low virulence potential, highlighting their potential ecological role as protective commensals on the teat skin and in the broader mammary ecosystem of dairy ruminants.

Human respiratory syncytial virus (HRSV) is a main cause of acute lower respiratory tract infections in infants, the elderly, and immunocompromised patients. Although vaccines have recently been approved for the elderly and for pregnant women, there is no curative treatment for HRSV. HRSV replicates in the cytoplasm of infected cells, and transcription and replication of the viral genome depend on the viral RNA polymerase complex, which recruits cellular factors for RNA synthesis. Among them, the eukaryotic translation elongation factor 1A (eEF1A) was previously shown to be critical for HRSV replication. eEF1A activity can be inhibited by plitidepsin (Aplidin), a cyclopeptide extracted from the ascidian Aplidium albicans, which was shown to be highly potent against SARS-CoV-2, with a 50% inhibitory concentration (IC) of 0.70 to 1.62 nM depending on the cell line. Here, we investigated whether plitidepsin could also inhibit HRSV replication. We found that plitidepsin inhibited HRSV replication with an IC of ≈3 nM in cell cultures. However, further investigation revealed that plitidepsin has pleiotropic effects, affecting the translation of both cellular and viral proteins in a similar manner. Overall, our results show that plitidepsin blocks cellular translation and indicate that plitidepsin can induce a proteasome-mediated degradation of eEF1A, depending on the cell line, also showing the dependence of HRSV replication on cellular factors, such as eEF1A. These results thus highlight an original mechanism of action of plitidepsin on eEF1A, which renders the use of this compound for antiviral therapy very risky.

Synergy between influenza A virus (IAV) and 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 , 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 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 3rd African Microbiome Symposium was held in Cape Town, South Africa, from 20 to 22 November 2024. The symposium featured a diverse range of local and international microbiome research and provided a platform for 79 researchers, students, and industry members to engage in discussions on the microbiome within an African context and focusing on translational research. This meeting review shares highlights, findings, and recommendations derived from the event. Insights from two panel discussions revealed key barriers to microbiome research in Africa, including limited funding, infrastructure gaps, and a shortage of trained local scientists. Recommendations centered on increased investment, institutional training, adherence to ethical guidelines, and the fostering of equitable global partnerships.

Bacterial populations often display remarkable morphological heterogeneity. Fluorescence-activated cell sorting (FACS) is an important tool for understanding this diversity. FACs allows researchers to obtain pure samples of each morphological variant (or morphotype) that is present within a mixed population of cells and thus permits each morphotype to be phenotyped. In FACS, cells are first labeled with fluorescent markers, such as antibodies or transgenic constructs, and then separated out based on their possession of these labels. However, since the development of fluorescent labels requires knowledge of bacterial biology, it is often impossible to apply FACS to understudied and/or unculturable bacteria. This challenge has limited our capacity to investigate the biology of bacterial size and shape in all but a small, largely culturable subset of bacterial taxa. Here, we present an innovative strategy that permits label-free cell sorting of bacterial morphotypes, using an unculturable, pleiomorphic pathogen () as a model bacterium. We show that imaging flow cytometry (IFC) can be used to systematically identify light-scattering and autofluorescence "signatures" of bacterial morphotypes, on which basis cell sorting can be conducted. Critically, our IFC-enabled cell sorting strategy yields samples of sufficient purity (>90%) for common downstream analyses, for example, "-omics" analyses. Our work represents an innovative application of IFC and provides an economical, widely applicable solution to a central problem in the study of bacterial diversity.IMPORTANCEBacteria come in many different shapes and sizes. Why this morphological variation exists is a long-standing question in microbiology, but it remains difficult to answer. To phenotype different morphological variants (morphotypes) within a bacterial population, we need to separate them from one another. This is normally achieved using fluorescence-activated cell sorting, whereby morphotypes are labeled with fluorescent antibodies and separated on the basis of their differential fluorescence. Unfortunately, it is difficult to develop fluorescent labels specific to unculturable or poorly studied bacteria because of the limited availability of appropriate molecular tools. Here, we demonstrate that imaging flow cytometry can be used to design and validate label-free cell sorting strategies. Recently, there has been a resurgence of interest in bacterial morphological diversity and a call to expand its study across the tree of life. Our work will help microbiologists to answer this call.

is a Gram-negative, intracellular, enteric pathogen that primarily causes hemorrhagic septicemia in fish. It survives and replicates within host cells by delivering a subset of effector proteins via the type III secretion system (T3SS). Previous research has identified a novel T3SS effector, EseQ, in however, its function remains unclear. This study reveals that EseQ binds to both α-tubulin and GEF-H1 (Rho guanine nucleotide exchange factor 1), causing microtubule destabilization and the release of activated GEF-H1. Active GEF-H1 then stimulates the conversion of GDP-RhoA (the inactive form) to GTP-RhoA (the active form), which subsequently induces stress fiber formation in a ROCK-dependent manner. Stress fibers induced by EseQ alter the architecture of zonula occludens-1-mediated intercellular junctions. This leads to increased permeability of the epithelial barrier, thereby facilitating the translocation of through epithelial cell layers and its invasion into zebrafish larvae. In conclusion, this study demonstrates that the T3SS effector protein EseQ promotes invasion by manipulating the microtubule and actin cytoskeletons and by disrupting the epithelial barrier.IMPORTANCE causes severe hemorrhagic septicemia in marine and freshwater fish worldwide, resulting in significant economic losses for the aquaculture industry (K. Y. Leung, Q. Wang, Z. Yang, and B. A. Siame, Virulence 10:555-567, 2019, https://doi.org/10.1080/21505594.2019.1621648). Our previous research identified a novel type III secretion system effector, EseQ, in whose function remains to be elucidated. In this work, we showed that EseQ binds to tubulin and GEF-H1 and destabilizes microtubules. GEF-H1 released from microtubules activates the RhoA-ROCK-MLCII signaling pathway, leading to stress fiber formation in epithelial cells. EseQ deforms the epithelial barrier and promotes 's invasion in a stress fiber-dependent manner. This work contributes to the understanding of the mechanism by which invades host cells.

The Profession of Microbiology (POM) embodies the bulk of the American Society for Microbiology (ASM) members and represents the career preparation arm of the ASM for academia, industry, and clinical lab professions. The ASM Council on Microbial Sciences hosted a virtual retreat in 2025 to identify the future of the POM. The retreat presentations centered on workforce development, professional development, innovations in technology, and interdisciplinary collaborations. Various aspects were identified, such as the need to prepare for careers in industry, as an important goal of future training. It was also clear that scientists, in all walks of life, need professional development training throughout their careers, from early trainees to senior scientists. Innovations in technology warrant continual training to keep abreast of global issues. Finally, the need for science advocacy and the ability to effectively communicate science to citizens is important. The ASM is best suited to leading the way in the recruitment of young scientists to the field of microbiology and providing the necessary training to keep them ahead of the changing technologies. As such, the ASM is poised to prepare its members for a quickly changing career workplace, one that will require collaboration between the many sciences and the community.