The emergence of multidrug-resistant Klebsiella pneumoniae (KPN) and antibiotic-tolerant persister cells poses a significant challenge to existing anti-infection therapies. Given the urgent need for sustainable alternatives to antibiotics, phage cocktails are emerging as a promising alternative to control K. pneumoniae infections. We isolated three lytic phages vB_KpnM_NB (1-3) from Ningbo environmental samples, classified them into the Drexlerviridae family, and determined the biological characteristics of two representative phages. Genomic analysis confirmed that these phages are closely related and lack resistance and virulence genes, ensuring biosafety. Subsequently, a stable KPN persister model was established using amikacin, with a biphasic killing pattern observed during treatment. At a multiplicity of infection of 10, the phage cocktail eliminated 99.00% of persister cells, while individual phages were less effective. The phage cocktail also inhibited persister-derived biofilm formation, showing improved results when combined with amikacin. This combination significantly reduced capsule polysaccharide production in persisters, weakening the outer membrane barrier. These findings demonstrate that the phage cocktail-amikacin combination effectively targets planktonic cells, persister cells, and biofilms, providing a promising strategy against persisters and recurrent K. pneumoniae infections. IMPORTANCE:This study fills the critical gap in understanding how phage cocktails synergize with amikacin against K. pneumoniae persister cells. By constructing a highly specific phage vB_KpnM_NB cocktail, establishing a stable persister model, and performing in vitro bactericidal and biofilm assays, we demonstrate that the cocktail effectively eliminates planktonic cells, persisters, and biofilms. We clarify the core synergistic mechanism: inhibiting capsular polysaccharide synthesis, improving phage adsorption, and disrupting the bacterial outer membrane barrier. These findings provide experimental evidence for the prevention and control of multidrug-resistant and carbapenem-resistant K. pneumoniae persister infections, establishing a safe and effective phage-antibiotic combination therapy. The results are crucial for addressing antibiotic tolerance and controlling chronic, recurrent infections. They hold significant theoretical and translational value for the treatment of refractory infections in clinical settings and offer new insights into the development of novel antimicrobial strategies.
Trichoderma reesei is known for its ability to secrete high amounts of cellulases, enzymes of fundamental importance in generating products from lignocellulosic biomass. Diverse signaling pathways and transcription factors (TFs) control the cellulolytic repertoire in T. reesei to ensure correct adaptation to the environment. Here, we analyzed RNA-Seq data and identified a new potential regulator of cellulase production in T. reesei: a novel TF named Nsd3, a homolog of NsdC from Aspergilli. Deletion of nsd3 reduced vegetative growth and conidiation on solid medium. Phenotypic characterization of the Δnsd3 strain showed that it is more sensitive to osmotic stress, but more resistant to cell wall and oxidative stresses. Our results showed that Nsd3 is a repressor of cellulase expression by directly regulating key genes in the cellulolytic pathway, an unreported role for this TF in fungi. Loss of nsd3 leads to a faster and more robust induction of cellulolytic genes, and higher cellulase and hemicellulase activities. Transcriptional profiling by RNA-Seq, chromatin accessibility profiling by ATAC-Seq, and protein-DNA interaction assays showed that sugar transporters are important targets of Nsd3 during cellulase expression regulation. Combined with microscopy and gene expression analyses, the ATAC-Seq data also highlighted Nsd3 as a central regulator of cell wall remodeling and organization. Furthermore, the transcriptomics also showed that Nsd3 regulates genes involved in secondary metabolism. These results showed that Nsd3 regulates several physiological processes and provide novel insights into the regulatory system of cellulases in T. reesei that can be used in the design of high-performance strains for biorefinery.IMPORTANCETrichoderma reesei is a key player in the production of hydrolytic enzymes for the degradation of lignocellulose biomass, and transcription factors are important targets for genetic engineering to construct cellulase-hyperproducing strains. Here, we identified the transcription factor Nsd3 and characterized its role as a regulator of cellulase production in T. reesei. We applied two powerful genomics methods (transcriptome sequencing and chromatin accessibility sequencing) to unravel the global role of Nsd3 and its regulatory mechanism. Nsd3 participates in various biological processes in T. reesei, including cell wall remodeling, calcium metabolism, and secondary metabolism, in addition to regulating the expression of sugar transporters. Protein-DNA interaction assays demonstrate that Nsd3 acts through important genes to regulate cellulase expression, including ace4, crt1, stp1, and cel1b. Our study provides mechanistic insights about how Nsd3 regulates diverse physiological processes in T. reesei. This work also applied ATAC-Seq for the first time to study chromatin accessibility in T. reesei.
Citrus greening disease, associated with the bacterium Candidatus Liberibacter asiaticus (CLas), has severely impacted citrus yields on a global scale. CLas is vectored by the Asian citrus psyllid, Diaphorina citri Kuwayama, in a circulative and propagative manner. Extensive application of chemical insecticides for suppression of psyllid populations has resulted in widespread resistance. Despite considerable effort and expenditure, citrus greening disease continues to impair citrus production. For transmission to occur, CLas must bind and infect the psyllid vector via the gut epithelium, but little is known of the specific molecular interactions involved. By screening a phage display library, we selected ten psyllid gut binding peptides, a subset of which have sequence similarity to CLas surface proteins. Peptide binding to the surface of the psyllid gut epithelium was confirmed by identification of gut proteins bound by specific peptides. We evaluated whether these peptides could interfere with the interaction between CLas and the gut epithelium of D. citri, thereby impeding pathogen transmission. Peptide DcNy2 significantly reduced CLas acquisition and inoculation by D. citri compared to control treatments. Nymphs fed specific peptides exhibited reduced CLas acquisition and infection of both the alimentary canal and salivary glands without negatively impacting psyllid fecundity, development, or survival. These results support peptide-mediated disruption of CLas transmission by D. citri as a potential component of integrated strategies to manage citrus greening disease.IMPORTANCECitrus greening disease, or huanglongbing, causes billions of dollars in losses and represents one of the greatest challenges facing global citrus production. Despite significant investment on multiple fronts, effective disease management strategies are lacking. This study shows for the first time that short peptides that bind to the surface of the gut of the Asian citrus psyllid vector interfere with CLas association with the gut. This critical first gut binding step is required for CLas to infect and propagate within the psyllid vector. The downstream consequence of this peptide-mediated interference is reduced acquisition and transmission of the citrus pathogen. This work provides the foundation for new transgenic or paratransgenic tools for use in the management of citrus greening disease.
Climate change threatens polar ecosystems through rising temperatures and changing light regimes. Phytoplankton release of dissolved organic carbon (DOC) is a major carbon-cycle component, yet it remains unclear how temperature, light, and associated bacteria jointly affect DOC and dissolved organic matter (DOM) composition. Here, we analyzed axenic and xenic cultures of the Arctic diatom Thalassiosira gravida at 9°C and 13.5°C under 16:8 h and 24:0 h light:dark cycles. Extracellular DOC was quantified, and molecular composition characterized by untargeted ultrahigh-resolution mass spectrometry in original culture filtrates. In axenic cultures, growth rates were 266% higher at 13.5°C 24:0 h, and cell-normalized DOC concentrations 52% lower compared to 9°C 16:8 h, suggesting a shift in carbon allocation. Xenic cultures contained 50-80% lower cell-normalized DOC concentrations than axenic cultures and converged at 11.7 ± 0.9 pmol DOC cell-1. Despite similar DOC concentrations in xenic cultures, the 13.5°C 24:0 h treatment showed lower chemodiversity (-8%), H/C ratios (-7%), and higher oxidation (+24%) than 9°C 16:8 h, consistent with more strongly processed DOM. At 9°C, axenic 24:0 h cultures showed higher chemodiversity (+14%) and H/C ratios (+7%) compared to 16:8 h, indicating that prolonged photoperiods altered DOM composition toward signatures associated with greater putative bioavailability. Contrasting axenic-xenic patterns suggest that bacterial presence weakened the treatment-dependent DOM responses observed in axenic cultures. Overall, temperature and photoperiods altered algal DOC quantity and quality, and its microbial transformation, with implications for carbon cycling.IMPORTANCEThe Arctic Ocean is warming rapidly, and changing sea-ice conditions alter light availability. Phytoplankton release dissolved organic carbon (DOC), which can be transformed by associated bacteria, thus playing an essential role in the marine carbon cycle. Still, the fate of such DOC transformations remains difficult to predict in the scope of future climate change scenarios. Using axenic and xenic cultures of the Arctic diatom Thalassiosira gravida as a model, our findings emphasize that algal extracellular release and its microbial transformation must be evaluated not only in the framework of bulk DOC dynamics but also with respect to molecular diversity and composition. Therefore, physiological responses of Arctic phytoplankton to higher temperatures and prolonged photoperiods may affect algal carbon exudation as well as the diversity and composition of microbially processed DOM in a warming Arctic Ocean.
Environmental dissemination of antimicrobial resistance is a growing global concern. This study investigated the occurrence, genetic context, and mobility of the oxazolidinone resistance genes in enterococci isolated from high-altitude river sediments in the Gilgit-Baltistan region of Pakistan. A total of 17 florfenicol-resistant enterococci were recovered from 3 of the 12 sampling sites analyzed. Four multidrug-resistant strains (two Enterococcus faecium, one E. faecalis, and one E. hirae) harboring the optrA gene were selected for whole-genome sequencing using Illumina and Nanopore platforms. Genomic analyses showed that optrA was plasmid-borne in all isolates and co-localized with additional resistance genes, including macrolide, phenicol, and tetracycline resistance determinants. The optrA genetic context was frequently associated with insertion sequences, suggesting their role in the mobilization of multidrug-resistance elements. Mini-translocatable units of the optrA genetic context were detected as circular forms in two of four strains, further supporting their mobility potential. Conjugation assays demonstrated successful horizontal transfer of the optrA gene mediating linezolid resistance, with transfer frequencies ranging from 2.03 × 10⁻⁶ to 6.36 × 10⁻⁴ transconjugants per recipient cell. optrA-carrying plasmids remained stable after serial passages without antibiotic pressure. Phylogenetic analysis showed that isolates belonged to distinct sequence types and clustered with strains of diverse geographic and ecological origins, suggesting dissemination of optrA across different genetic backgrounds rather than clonal expansion. The occurrence of linezolid-resistant enterococci in high-altitude river sediments suggests their widespread distribution and emphasizes the need for broader environmental surveillance programs to monitor clinically relevant resistances even in remote ecosystems. IMPORTANCE:This study provides important insights into the environmental dissemination of antimicrobial resistance by demonstrating the occurrence and genetic diversity of clinically relevant resistance determinants in enterococci isolated from remote high-altitude river sediments. The findings emphasize that natural environments could serve as reservoirs and exchange hubs for antimicrobial resistance, even in ecosystems with limited direct anthropogenic impact. Therefore, integrated One Health surveillance strategies that include environmental compartments alongside human and animal populations are required to better monitor and mitigate the emergence of clinically significant resistant bacteria.
Oysters are a popular raw seafood worldwide, yet their contamination with human pathogens poses substantial public health risks. However, the seasonal dynamics of host-associated microbiota and the environmental drivers of pathogen accumulation in Crassostrea hongkongensis remain poorly understood. Here, we conducted 16S rRNA amplicon sequencing to profile the gill and intestinal microbiota of C. hongkongensis, sampled quarterly over a 1-year period in Beihai and Zhanjiang, southern China. A pronounced divergence in oyster-associated bacterial communities was observed between the two locations in December. Key bacterial families, such as Lachnospiraceae and Muribaculaceae, showed significant temporal fluctuations in abundance, suggesting their potential role in maintaining bacterial community stability within oysters. These microbial shifts were significantly correlated with environmental parameters such as chlorophyll a and pH. In parallel, we used plate counting to quantify five foodborne pathogens and total viable counts. Vibrios were more frequently detected in oyster tissues than in ambient seawater. Pathogen-associated amplicon sequence variants exhibited a strong response to variations in pH and chlorophyll a, whereas the abundance of culturable Vibrio vulnificus was significantly negatively correlated with temperature and nitrate. The integration of high-throughput sequencing and culture-based methods provides comprehensive insights into the dynamics of pathogenic bacteria within oysters under natural mariculture settings. This study offers valuable ecological insights into the interactions within the oyster-associated microbial ecosystem, contributing to a deeper understanding of its dynamics and implications for public health. IMPORTANCE:Mariculture of Crassostrea hongkongensis serves as a critical interface between coastal ecology and public health, yet seasonal dynamics of its microbiome and core stabilizing taxa remain poorly understood in subtropical aquaculture. This study identified Acetobacteraceae, Lachnospiraceae, Prevotellaceae, and Muribaculaceae as key families associated with seasonal microbiome stability in C. hongkongensis, highlighting their potential contribution to community resilience. It further revealed that Vibrio species detection was negatively correlated with temperature. These findings advance our understanding of interactions between the oyster microbiome and the environment, and provide a theoretical basis for improving oyster health management and promoting sustainable aquaculture practices.
Using treated municipal wastewater for crop irrigation is a key strategy to combat drought-induced water scarcity. However, current wastewater reclamation standards systematically underestimate risks from spore-forming pathogens. As highlighted in a recent minireview by A. Mrozinski, C. Le Maréchal, and E. Topp in Applied and Environmental Microbiology (92:e00173-26, 2026, https://doi.org/10.1128/aem.00173-26), Clostridioides difficile and Clostridium perfringens survive conventional disinfection, persist indefinitely in agricultural soils, and harbor critical antibiotic resistance genes. To safeguard the food supply and protect public health, regulatory frameworks must shift from relying solely on standard vegetative bacterial indicators and include monitoring resilient, spore-forming pathogens.
Microbiome prediction models overlook phylogeny or fail to preserve evolutionary structure. In a recent Applied and Environmental Microbiology article (B. Dong, B. Wang, J. Chen, X. Xu, and Z. Z. Xu, Appl Environ Microbiol 92:e00788-26, 2026, https://doi.org/10.1128/aem.00788-26), Dong et al. present PhyloGCNE, a graph-convolutional framework that addresses these limitations by preserving evolutionary topology and learning adaptive edge-aware signal propagation. This commentary evaluates whether and when phylogenetic information improves microbiome-based prediction and considers how biological relevance and transferability can be established across contexts.
Peptidylprolyl isomerase (PPIase) catalyzes the rate-limiting step of proline cis-trans isomerization during protein folding. In bacteria, multiple PPIases are commonly present and participate in diverse physiological processes. Myxococcus xanthus DK1622 possesses as many as 17 PPIase genes. We previously reported the functional divergence of the four trigger factor family PPIases. Here, we systematically assessed the contributions of the remaining PPIases in cell survival, sociality, and stress tolerance. Pin1, localized in the periplasm, was essential for cell growth. Absence of the membrane-bound Pin3 impaired the social motility, predation ability, and sporulation but did not affect the fruiting body formation of M. xanthus. Furthermore, nearly all M. xanthus PPIases contributed to tolerance to environmental stresses. The transcriptional levels and expression patterns of these PPIases varied distinctly, even among members of the same family or with high sequence homology. Our findings provide a comprehensive view of functional divergences and suggest a potential functional cooperation network of PPIases in M. xanthus.IMPORTANCEPeptidylprolyl isomerase (PPIase), a ubiquitous enzyme present across nearly all kingdoms of life, catalyzes the cis-trans isomerization of peptidyl-prolyl bonds in polypeptide chains, thereby significantly accelerating protein folding. As a result, PPIases play critical roles in a wide range of physiological processes mediated by their substrate proteins. Myxobacteria are distinguished by their complex multicellular social behaviors. Among the 17 PPIases belonging to three families in Myxococcus xanthus DK1622, the membrane-bound Pin3, one of the SurA homologs, is involved in social behaviors such as social motility, predation, and sporulation. Given that PPIases are widely recognized as chaperones, our results also indicate that these M. xanthus PPIases extensively contribute to stress tolerance. Our findings underscore the essential functions of PPIases in cellular processes and reveal a correlation between the expansion of cellular functionalities and the functional evolution of PPIase proteins.
Nitrous oxide (N2O) is a potent greenhouse gas and ozone-depleting substance, with a global warming potential 273 times greater than CO2 over a 100-year horizon. Microbial reduction of N2O to dinitrogen represents a key pathway for mitigating emissions under diverse environmental conditions. Here, we report the first comprehensive genomic and physiological characterization of Stutzerimonas frequens strain E49, a newly isolated N2O-reducing bacterium obtained from activated sludge treating landfill leachate. Whole-genome sequencing revealed a 4.51-Mbp circular chromosome and a 35.3-kbp plasmid with high completeness. Functional annotation identified a complete denitrification gene set, including nosZ, as well as the ectABCD-ask gene cluster associated with ectoine biosynthesis, suggesting adaptation to osmotic stress. Strain E49, a uniform rod-shaped bacterium (1.5-2.5 μm), efficiently reduced N2O under anaerobic conditions in the absence of an externally supplied organic carbon source, achieving a biomass-specific rate of 0.70 ± 0.02 µmol-N2O/mg-biomass/h and a cell-specific rate of 7.93 ± 0.23 × 10-10 µmol-N2O/cell/h. Among the cultivation regimens tested, nitrate-free DSMZ 1180 medium supplemented with NH4Cl yielded the highest activity, indicating medium-dependent regulation of N2O respiration. Comparative analysis showed that strain E49 outperformed several reported N2O-reducing isolates under carbon-limited conditions. These findings demonstrate the metabolic versatility of strain E49 and highlight its potential role as a biological sink for N2O in low-nutrient environments.IMPORTANCENitrous oxide is a powerful greenhouse gas that contributes to climate change and ozone depletion. Microorganisms that convert nitrous oxide into nitrogen gas play an essential role in reducing these emissions. In this study, we investigated Stutzerimonas frequens strain E49, a bacterium isolated from wastewater treatment sludge. We found that this organism can efficiently reduce nitrous oxide even without an external supply of organic carbon, which is typically required by most bacteria. This suggests that the bacterium can rely on internal energy reserves to carry out this process. We also identified the genetic basis for its nitrous oxide reduction and its ability to adapt to environmental stress. These findings improve our understanding of how nitrous oxide-reducing bacteria function in nutrient-limited environments and may support the development of strategies to mitigate emissions in wastewater treatment and other engineered systems.
HIV remains among the world's most serious healthcare challenges, with adolescent girls and young women in sub-Saharan Africa at particularly high risk of infection. Bacterial vaginosis (BV) is a key risk factor for HIV acquisition; however, current treatment strategies are limited. Optimal vaginal lactobacilli protect against BV and HIV, largely through immunoregulatory and antimicrobial activities mediated in part by lactic acid. Toward the development of a live biotherapeutic for African women, we sampled 181 isolates of vaginal lactobacilli from 25 BV-negative South African women. Fifty isolates were selected for evaluation of inflammatory responses using vaginal epithelial cells, D- and L-lactate, lactic acid production, and culture acidification. Aside from a single Ligilactobacillus salivarius strain, Lactobacillus crispatus isolates acidified the culture media the most and produced the most D- and L-lactic acid. Inflammatory cytokine responses to different strains of lactobacilli were variable, with Lb. crispatus eliciting the lowest levels of cytokine production, while some strains induced substantial inflammatory responses. When all properties were evaluated collectively, Lb. crispatus strains exhibited the most desirable biotherapeutic characteristics. Whole genome sequence analysis of 10 Lb. crispatus isolates led to the identification of putative bacteriocins and intact prophage sequences in all isolates, while no antimicrobial resistance elements were detected. Importantly, the majority of Lb. crispatus isolates were more closely related to one another than to isolates from other geographical regions. This supports the need for live biotherapeutics to be tailored for the population of intended use.IMPORTANCEHIV remains highly prevalent in sub-Saharan Africa, particularly among adolescent girls and young women. Bacterial vaginosis (BV), characterized by the loss of protective lactobacilli, affects approximately one in four women in this region and increases HIV susceptibility. However, effective and durable therapeutics are lacking. Live biotherapeutics containing beneficial lactobacilli represent a promising treatment strategy, yet there are no approved products including strains isolated from African women, despite well-established geographic variation in vaginal microbiome composition. Through the characterization of 50 vaginal Lactobacillus, Limosilactobacillus, and Ligilactobacillus strains from 25 BV-negative South African women, we demonstrate substantial strain-level variation in live biotherapeutic-relevant properties. Some isolates produced minimal lactic acid or induced marked inflammatory responses, highlighting the importance of rigorous strain selection. Notably, whole genome sequencing revealed that South African Lactobacillus crispatus strains had distinct genomes compared to isolates from other regions, providing evidence that vaginal live biotherapeutics should be tailored to the populations of intended use.
Minerals are known to influence microbial metabolism as nutrient sources or redox partners, yet whether chemically inert, non-nutritive minerals can regulate microbial physiology, and through what mechanisms, remains poorly understood. Here, we used transcriptomics to investigate the response of Enterococcus faecalis to Al2O3 and SiO2 particles spanning nanoscale to millimeter scale. Mineral exposure triggered extensive transcriptional reprogramming across hundreds of differentially expressed genes. Notably, a massive upregulation of iron acquisition genes (log2FC range from 3.8 to 4.2) concurrent with oxidative stress defenses (catalase, thiol peroxidase, and NADH oxidase) is suggestive of an "iron paradox," which is potentially attributable to mineral-mediated nutrient sequestration and steric hindrance of membrane transporters alongside interfacial reactive oxygen species generation. To adapt, E. faecalis orchestrated a coordinated metabolic shift, repressing serine catabolism (log2FC = -2.7) while investing nitrogen into glutathione biosynthesis (cystathionine synthase genes, log2FC = 3.7). Correlation analysis identified an ompR-sigV axis through which E. faecalis discriminates mineral particle size, with nanoscale particles eliciting stronger transcriptional responses than their larger counterparts. This stress response additionally upregulated virulence-associated genes and antibiotic resistance genes without direct antimicrobial selection pressure. These findings suggest that non-nutritive minerals shape microbial physiology through physical and surface-chemical cues independent of their nutritional value, highlighting the need for further exploration of the non-nutritional functions of minerals in microbial ecology.IMPORTANCEEven in the absence of utilizable nutrients, non-nutritive minerals such as Al2O3 and SiO2 can profoundly influence the transcriptional responses of Enterococcus faecalis. Using transcriptomic sequencing, we show that these inert minerals regulate microbial transcription, enhancing iron acquisition, oxidative stress repair, pathogenicity, and antibiotic resistance. Mineral-mediated transcriptional control is driven primarily by physical contact, surface chemistry, and particle size sensing, rather than conventional metabolic interactions. These findings identify inert minerals as signaling molecules that actively modulate microbial transcription, playing a proactive role in microbial evolution and environmental adaptation. This study redefines inert minerals as active carriers of transcriptional regulation, filling a critical gap in geomicrobiology and providing new insights into microbial environmental responses, biogeochemical cycling, and the mechanisms underlying microbial functional evolution and maintenance.
ABSTRACT Passive wastewater sampling has emerged as a cost-effective approach for pathogen monitoring, but its application in very small populations has not been well studied. This study reports the use of tampons as passive samplers in a small-scale nursing home of 24 residents to detect respiratory viruses, including SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus (RSV). Weekly wastewater surveillance by reverse transcription digital PCR (RT-dPCR) for a 1-year period resulted in a cluster of consecutive SARS-CoV-2 detections over 4 weeks. When wastewater SARS-CoV-2 concentrations reached higher levels in the third and fourth weeks, whole-genome sequencing achieved >98% genome coverage, identifying lineage LB.1.5, while lower-concentration samples produced only partial genomes. Clinical testing was initiated in the fourth week when clinical symptoms also emerged, and confirmed SARS-CoV-2 infection in 15 of 24 residents and staff. Sequencing of the clinical samples confirmed LB.1.5 in 11 cases, corroborating the wastewater findings. Influenza A subtypes H3N2 and H1N1 were additionally detected in wastewater by targeted enrichment sequencing, and RSV was detected once by RT-dPCR, neither of which was associated with confirmed clinical cases. These results demonstrate that wastewater surveillance can precede clinical symptoms for early detection, and passive sampling using tampons is a practical and sensitive strategy for outbreak surveillance in small-scale facilities. The study also highlights the need for clear response protocols that include rapid clinical testing and cohorting measures when wastewater viral concentrations exceed defined thresholds.IMPORTANCENursing homes are among the highest-risk settings for infectious disease outbreaks, yet early detection remains challenging due to the high prevalence of asymptomatic infections and limited resources for routine clinical surveillance. This study demonstrates that inexpensive tampon-based passive wastewater samplers can detect SARS-CoV-2 up to 3 weeks before clinical symptoms emerge in a nursing home with only 24 residents, which is the smallest population in which this method has been validated. Wastewater-derived genomes matched clinical sequencing quality, enabling identification of a novel SARS-CoV-2 lineage new to Hawai'i. These findings support passive wastewater surveillance as a practical early warning tool for small congregate care facilities, provided that clear institutional response protocols are in place.
Many cool-season grasses (Poaceae subfam. Poöideae) host seed-transmissible symbionts (endophytes) in the fungal genus Epichloë, which can produce diverse alkaloids that protect against invertebrate and, in some cases, vertebrate herbivores. Rarely have population surveys been conducted to assess comprehensive alkaloid profiles and diversity of Epichloë in wild grasses. In this study, we surveyed Brachyelytrum erectum, which is a woodland grass in an early-diverging lineage of Poöideae, and commonly symbiotic with Epichloë brachyelytri. Analytical methods based on high-resolution UHPLC-MS/MS were refined to provide rapid, comprehensive detection, and quantitation of E. brachyelytri alkaloids, for six B. erectum populations in Kentucky. Chemotypes were identified with two or three of the alkaloids exo-1-acetamidopyrrolizidine (1), chanoclavine (2), and peramine (3). Both 1 and 2 are known as intermediates in biosynthetic pathways to more complex alkaloids, and chemotypes having both 1 and 2 as pathway end-products are novel. Such chemotypes were also identified in other species, and phylogenetic analysis indicated their multiple origins by a combination of convergent evolution, possible horizontal gene transfer, and interspecific hybridization. Alkaloid levels were comparable between most populations and at most plant developmental stages. Levels of 3 were compared between E. brachyelytri variants with and without 1, providing evidence for competition between the pathways in young shoots, but not in older leaves or seeds. Furthermore, levels of 1 and 2 were moderate to high compared with their respective alkaloid classes in other grass-Epichloë symbiotic systems. We conclude that production of the alkaloids likely represents an important metabolic investment by E. brachyelytri.IMPORTANCEDefensive mutualisms, symbioses of hosts with organisms that defend them against parasites or predators, play important ecological roles. A widespread example is protection of cool-season grasses by symbiotic Epichloë species, which are fungi that transmit in seeds and produce several kinds of anti-insect alkaloids. Profiles of alkaloids evolve due to shifting balances of their benefits and the costs of producing them. In this study, Epichloë brachyelytri symbiotic with the wild forest grass Brachyelytrum erectum produced three alkaloids, of which two have been rarely reported. Furthermore, variations in its alkaloid profiles and quantities of each alkaloid at different plant growth stages and tissues suggested that occasional loss of its most abundant alkaloid can be adaptive due to the metabolic load of producing it. Although rare, similar alkaloid profiles were identified in several other species in which they arose by a combination of convergent evolution, possible horizontal gene transfer, and interspecific hybridization.
We investigated the adaptation of the aerotolerant Campylobacter jejuni Bf strain to conditions mimicking the poultry slaughter process. We showed that C. jejuni Bf survives and actively multiplies under combined thermal and oxidative stress. Stress exposure induces cell rounding, loss of motility, remodeling of membrane composition, decreased membrane fluidity, and metabolic reprogramming with increased intracellular ATP levels. While maintaining the lipid composition of its plasma membrane, C. jejuni modulates the lipid composition of its extracellular vesicles when exposed to stress. Notably, stressed C. jejuni cells release extracellular vesicles with increased toxicity toward the epithelial barrier of Caco-2 cells, potentially facilitating invasion of the gut epithelium.IMPORTANCECampylobacter infections are one of the leading causes of foodborne gastroenteritis worldwide. Campylobacter readily enters the food chain and is transmitted to humans, primarily through the consumption of contaminated poultry meat. The high prevalence of aerotolerant human Campylobacter jejuni isolates suggests a correlation between their ability to survive under aerobic conditions, virulence, and resistance to harsh stress conditions. However, the underlying mechanism remains unclear. Our results show that C. jejuni extracellular vesicles are part of a survival strategy that links environmental adaptation with pathogenicity.
In two randomized controlled field studies, we investigated the effect of biochar-amended dairy manure composting on the concentrations and prevalence of generic and antibiotic-resistant E. coli and enterococci. Fresh dairy manure mixed with sawdust and biochar was randomly distributed into plastic reactors and composted for 6 months. Pre-compost (biochar, sawdust, fresh dairy manure), manure mixture, and final compost samples were cultured for enumeration and prevalence. While biochar and sawdust were not significant sources, fresh dairy manure was the major contributor of bacteria to the initial manure mixture. Biochar amendment did not significantly reduce bacterial load beyond that could be achieved by composting alone. Spring-summer composting was more effective than fall-winter composting. While E. coli was more prone, enterococci were more likely to survive composting. Dairy manure composting eliminated bacteria (3rd generation cephalosporin-resistant [3GCr] and extended spectrum beta-lactamase-producing [ESBL]-E. coli) resistant to critically important and highest priority antibiotics for human health and significantly reduced the prevalence and concentrations of other investigated bacteria. 3GCr- and ESBL-E. coli from fresh dairy manure were multidrug resistant and carried blaCTX-M genes, a widely distributed ESBL gene type. By whole-genome sequencing, ESBL-E. coli from experiment 1 (fall-winter) had diverse resistance profiles, sequence types, plasmid profiles, virulence factor genes, and more blaCTX-M genes (CTX-M-1, CTX-M-32, and CTX-M-55). However, those from experiment 2 (spring-summer) were clonal and carried blaCTX-M-55 only. In conclusion, dairy manure composting effectively removes antibiotic resistant bacteria of public health importance, with no added effect of biochar amendment. IMPORTANCE:Composting is a microbially mediated decomposition of organic matter into a nutrient-rich product for use as fertilizer. Biochar, obtained from the pyrolysis of biomass including animal manure, has been used for soil amendment. Beyond its nutrient stabilizing potential, composting has been shown to reduce bacterial load in animal manure. However, its effect with or without a biochar addition was not evaluated under a randomized controlled field trial. Biochar was nearly bacteriologically "sterile," and the sawdust used as a bulking agent had low concentrations of innocuous bacteria indicating that dairy manure was the main source of bacteria to the pre-compost mixture. Biochar did not have an extra effect on the bacterial load beyond composting alone. Composting during spring/summer is more effective than fall/winter. Furthermore, it is more effective against gram-negatives than gram-positive bacteria. Importantly, composting effectively eliminated multidrug-resistant bacteria of critical importance to public health for safer use of dairy manure.
Midurethral slings (MUS) are synthetic mesh implants used to treat stress urinary incontinence in women. Positioned via retropubic or transobturator approaches, they support the mid-urethra to maintain closure during increases in intra-abdominal pressure. Although generally effective, long-term MUS complications have been associated with chronic inflammation, pain, and infection, with explanted slings often showing bacterial colonization. The underlying mechanisms remain unclear, particularly the role of microbial biofilms. We examined the presence and characteristics of biofilms on MUS removed for various complications to better inform clinical management. MUS samples (n = 112) from 52 women were imaged using confocal laser scanning microscopy following application of a eubacterial FISH probe, TOTO-1, and DAPI. Biofilms were detected on all samples, forming dense bacterial aggregates along mesh fibers. The vaginal portions of the MUS demonstrated higher biofilm volumes than retropubic or obturator regions. However, mesh exposed through vaginal tissue did not show increased bacterial load. When all samples were analyzed, no significant differences in biofilm volume were observed between complication types, including chronic pain, vaginal or lower urinary tract perforation, and recurrent incontinence. While biofilms were universally present, greater volumes in the vaginal region did not correlate with worse clinical outcomes. These findings suggest a threshold between benign colonization and clinically significant infection. Variation in bacterial composition, rather than biofilm quantity, may influence host response and the development of mesh-related complications. IMPORTANCE:Biofilms are common on explanted midurethral slings, but their volume does not predict complications. These findings suggest that bacterial composition and host response, rather than bacterial load, drive mesh-related morbidity. Understanding these factors may improve mesh design and guide strategies to prevent infection and chronic complications.
Advances in microscopy have enabled the widespread application of in vivo pathogen tracking approaches to bacterial pathogenesis. Pasteurella multocida (P. multocida) is a major zoonotic pathogen causing severe respiratory diseases in livestock. However, genetic manipulation of clinical swine-derived isolates remains hindered by the lack of stable genetic tools. As a result, no genetically engineered in vivo tracking strain limits research in P. multocida infection dynamics. In this study, we evaluated diverse replication mechanisms and identified that rolling circle replication (RCR)-type replicons exhibit superior transformation robustness in swine isolates. Leveraging the temperature-sensitive pSET4s backbone and an optimized mpheS counter-selection marker, we developed a high-efficiency, markerless genetic manipulation system. The system exhibited high copy numbers and significantly enhanced transformation yields across diverse clinical swine isolates. To validate gene knockout efficiency, we performed markerless deletions of the capsule biosynthesis genes hyaD and hyaE. Phenotypic characterization revealed that hyaluronic acid (HA) deficiency led to complete attenuation of virulence in a murine model. Furthermore, we engineered a 15-Pm::GFP reporter strain via site-specific gene knock-in without detectable effects on bacterial growth or virulence. This strain enables direct visualization of bacterial colonization in the lung and liver by fluorescence imaging and allows real-time, in situ monitoring of pathogen and Kupffer cell interactions. Collectively, our results established an effective genetic toolkit for swine-derived P. multocida and developed fluorescently labeled strains for in vivo tracking. This plasmid provides a useful tool for investigating pathogenic mechanisms and in vivo infection dynamics of P. multocida. IMPORTANCE:P. multocida is a critical threat to global animal health, while the difficulty of genetic modification in swine isolates has interrupted the link of the genomic and pathogenic phenotypes. This study provides a highly efficient, RCR-based markerless manipulation system optimized with cross-serogroup applicability for swine isolates. Validated across 15 clinical swine isolates, this plasmid offers a robust tool for genetic analysis and the rational design of next-generation vaccines. By defining the essential roles of hyaD and hyaE, we demonstrate that the hyaluronic acid capsule is required for virulence and pulmonary colonization. Moreover, the development of a biologically fluorescent reporter strain enables high-resolution, in situ tracking of infection dynamics, providing a powerful tool for real-time visualization during P. multocida infection.
α-Amylases (EC 3.2.1.1) are among the most important industrial enzymes in starch saccharification, detergent, paper, and textile industries. Canonical α-amylases typically contain Ca2+ in their structure, which is not directly involved in catalysis but essential in maintaining the structural integrity of α-amylases. Ca2+ ions are easily removed by chelating reagents, which may affect the activity and stability of α-amylases and thereby hinder their applications in some industries. Here, we identified a metal-free α-amylase, AmyY, from the bacterium Alkalimonas sp. NCh-2 isolated from an alkaline hot spring. AmyY contains a catalytic module of glycoside hydrolase family 13 (GH13) and a carbohydrate-binding module of family 20 (CBM20). AmyY exhibits high activity under alkaline conditions (pH 8.0-11.0) and tolerance to high salinity, chelating reagents, and surfactants. We solved the structures of AmyY and its complex with acarbose. Although the overall topology and active sites of AmyY resemble those of reported GH13 α-amylases, the structure of AmyY is devoid of metal ions. The inability of AmyY to bind metal ions results from replacements of acidic amino acid residues by neutral ones. Combined with biochemical, structural, and bioinformatic data, we demonstrated that AmyY and its homologs represent a class of metal-free α-amylases in GH13. Moreover, structural analyses revealed that the CBM20 of AmyY is highly flexible, and we obtained a mutant with enhanced thermostability by truncating the CBM20. This mutant demonstrates remarkable wash performance and desizing capability at alkaline pH, which underscores its application potential in the detergent and textile industries.IMPORTANCEWhile canonical GH13 α-amylases typically contain Ca2+, our biochemical and structural data reveal that AmyY has evolved a metal-free architecture. This is achieved through the replacement of acidic metal-coordinating residues by neutral ones, abolishing metal binding without compromising catalytic efficiency. This work expands the structural and functional diversity of GH13 by defining a class of metal-free α-amylases, with AmyY and its homologs as representatives. Furthermore, the engineered AmyY with enhanced thermostability offers a candidate biocatalyst for industrial applications, such as the detergent industry. The structure of AmyY also offers a blueprint for engineering other α-amylases into metal-independent forms.
