Endophytic fungi form an integral part of plant microbiomes, influencing host physiology, stress resilience, and secondary metabolism. While next-generation sequencing (NGS) has greatly advanced the identification of endophytes, it often falls short of assigning functional roles, necessitating integration with culture-based approaches for downstream applications. Picrorhiza kurrooa, a critically endangered Himalayan medicinal herb valued for its hepatoprotective picrosides, suffers from reduced metabolite content in tissue culture-derived plants, likely due to microbiome loss in the course of aseptic in-vitro practices. Moreover, the diversity and functional role of fungal endomicrobiome in P. kurrooa remain unexplored. Internal transcribed spacer (ITS)-based amplicon sequencing was performed to assess and compare the endophytic fungal communities of wild-type (Wt) and in-vitro propagated (Tc) P. kurrooa. Fungal taxa unique to Wt-plants were identified and cross-referenced with culturable isolates. A dominant isolate present only in Wt-plants, Trichoderma harzianum PKRF1, was reintroduced into Tc-plants to evaluate its effect on plant growth and picroside biosynthesis. Whole-genome sequencing and comparative genomics of PKRF1 were also conducted to elucidate its functional capabilities and possible candidates for its endophytic nature. Metagenomic analysis revealed a significant reduction in fungal diversity in Tc plants, with several taxa, including Trichoderma, Cyphellophora, and Preussia, exclusively associated with Wt-plants. Inoculation of Tc-plants with PKRF1 led to successful root colonization, enhanced photosynthetic efficiency, biomass, and significantly higher levels of picrosides. Transcript profiling confirmed upregulation of key biosynthetic genes. Genomic analysis of PKRF1 revealed genes associated with multiple plant-beneficial traits, including nutrient acquisition, phytohormone production, stress tolerance, plant colonization, and competitive interactions, distinguishing it from non-endophytic Trichoderma isolates. These findings provide the first comprehensive insight into changes in endophytic fungal diversity of P. kurrooa associated with in-vitro cultivation. Furthermore, the application of cultivated endophytes from wild plants demonstrated the potential to restore microbial functions lost during in-vitro propagation and enhance secondary metabolite production in cultivated plants. Overall, this approach offers a promising strategy to integrate metagenomic information into beneficial plant-microbe interactions for practical applications.

Detection challenges of Escherichia coli O157 in raw meat: Diagnostic discordance between VIDAS, qPCR, and culture-based methods.

Amphibian and reptile microbiomes are important contributors to host health and ecosystem dynamics but understudied compared to microbiomes of other vertebrates. Through study of amphibian and reptile microbiomes, investigators can advance theory and practice in animal health, ecosystem function, conservation, and more. This special issue of Frontiers of Amphibian and Reptile Science features six publications in the field of amphibian and reptile microbiome research, and here, we highlight the contributions of these and other recent publications to four main areas of recent advancement in the field: 1) expanding “microbiome” beyond bacteria, 2) expanding study of host life-history stages and anatomical microbial habitats, 3) characterizing whole microbial communities, particularly in disease ecology studies, and 4) developing and implementing databases and bioinformatics tools. We also discuss several avenues for future research that would bring amphibians and reptiles to the forefront of microbiome research. With the continued implementation of classic culture-based and sequencing approaches paired with cutting-edge tools in vivo , in vitro , and in silico , herpetofauna microbiome research is poised for exciting growth in the near future.

Characterization of Dominant Heterotrophic Flagellates in Anaerobic Digesters Using Combined Culture-based and Metabarcoding Approaches.

This study examines the implementation of career guidance and counseling in high schools using a Systematic Literature Review (SLR) approach. The authors reviewed 26 papers that met the exclusion criteria related to career guidance. Among these, 10 papers were selected based on inclusion criteria relevant to career guidance and counseling in secondary schools during the period 2014-2024. This study aims to identify trends, approaches, and interventions that effectively prepare students for career challenges. The results show that integrating digital technology, culture-based approaches, and hands-on field experience is the primary strategy for improving students' career readiness. However, challenges such as limited resources, gaps in access to technology, and a lack of training for supervisors remain obstacles to implementation. Therefore, cross-sector collaboration is considered essential to create a career guidance system that is adaptive, inclusive, and responsive to global demands. Keywords: career guidance, high school, systematic literature review, digital technology, local culture

Soil salinization and crude oil contamination are critical global threats to ecosystems, agriculture, and human health. Bioremediation is widely recognized as a cost-effective and eco-friendly strategy for removing petroleum pollutants from soil. In this study, we investigated salinity-driven bacterial community dynamics collected from crude oil-contaminated soil in Cam Ranh Bay, Khanh Hoa, over a 21-day enrichment cultivation, using shotgun metagenomic and culture-based approaches. The enrichment cultivation was performed in Bushnell-Haas mineral salts (BHMS) medium supplemented with 5% (v/v) crude oil-diesel mixture (5:95) and 1.5% NaCl. Shotgun metagenomic analysis revealed that after 21 days of enrichment, the relative abundance of crude oil-degrading genera increased markedly in the enriched samples compared to the native samples-for example, Pseudomonas rose from 0.44% to 3.51%, Gordonia from 0.03% to 78.68%, and Achromobacter from 0.03% to 3.77%. Functional analysis further identified metabolic pathways, including hydrocarbon degradation, osmoprotection, and heavy metal detoxification. In addition, 36 representative bacterial strains were isolated from the enriched cultures, predominantly belonging to the genera Pseudomonas, Bacillus, Stenotrophomonas, and Achromobacter. All isolates were able to degrade crude oil under salinity stress conditions of up to 4%. Notably, Rhodococcus sp. KH5 and Gordonia sp. KH53 maintained consistently high degradation efficiencies across 0-4% salinity, ranging from 17.67-35.00% and 28.67-36%, respectively. Overall, our findings demonstrate that saline enrichment shifts the bacterial community toward halotolerant hydrocarbon and crude oil degraders.

Plants in ex situ conservation nurseries acquire diverse fungal associates that may be moved among nurseries or into the wild during outplanting, including fungal endophytes that contribute to a broad range of functions and occur in leaves, sometimes alongside pathogens. To improve understanding of fungal symbionts in a plant of high conservation concern, we characterized foliar fungal endophytes of Torreya taxifolia, one of the world's most threatened conifers, in an ex situ conservation nursery. We used culture-based and culture-free approaches to characterize fungal endophytes in leaves of T. taxifolia over 2 years and evaluated how endophytes varied spatially and as a function of environmental, plant-specific, and edaphic factors. We also contrasted them with fungi in other plants (local species and species cultivated at a regional scale) and with soil fungi. Culture-free methods revealed species-rich and phylogenetically diverse foliar fungal endophytes of T. taxifolia that vary spatially, reflecting symbiont acquisition from nearby plants, environmental factors, and plant stress. Endophyte community composition is subject to both stochasticity and temporal turnover and differs markedly from fungal communities in soils and other plants in the area. Our study provides novel insights into factors that can shape fungal endophyte communities for a critically endangered tree species. In addition to identifying local determinants of endophytic symbioses, our work illustrates that plants in conservation nurseries host rich foliar fungal communities of potential importance in plant germplasm protection.

While industrial-scale dairy fermentations often employ pasteurized milk as the substrate, many farmhouse and traditional production practices apply raw milk derived from a variety of mammals. Certain artisanal production systems rely on the autochthonous microbiota of the milk, fermentation vessels, equipment and/or environment to initiate milk coagulation. While the technological properties of lactic acid bacteria associated with dairy fermentations are well described, their interactions with other organisms during fermentation and cheese ripening are poorly investigated. This study presents an overview of the microbial ecology of raw and pasteurized milk used in the production of Irish farmhouse cheeses using metagenomic and culture-based approaches. Metagenomic analysis of four raw milk-derived cheeses established the dominant presence of either lactococci or Streptococcus spp. and with a secondary population of various lactobacilli. Interestingly, the Brie sample was also demonstrated to possess significant proportion of Hafnia spp. This was corroborated in culture-based analysis where Hafnia isolates were also identified. Furthermore, we report on the motility phenotype, lactose utilization ability and metabolic products of isolates of Hafnia paralvei and Hafnia alvei, and determine that these strains could grow in a non-antagonistic manner on plates with strains of Lactococcus lactis and Streptococcus thermophilus. As artisanal and farmhouse production systems are often associated with protected or regionally significant products, it is essential to develop a clear understanding of the microbial communities within and the complex relationships between the community members.

Advances in sequencing technologies have transformed human microbiome research, yet most analyses still rely on species-level profiles. However, strains rather than species represent the true ecological and functional units of the microbiome. Individual strains can vary substantially in gene content, metabolic capacity, virulence factors, antimicrobial resistance, and host-interaction properties. These differences critically influence immune responses, epithelial barrier integrity, disease susceptibility, and therapeutic outcomes. Here, we synthesize recent human microbiome studies that provide robust strain-resolved evidence, focusing on three major themes: (i) the emergence and long-term persistence of personalized strain repertoires, (ii) strain-specific pathobiont traits that drive host pathology, and (iii) the implications of strain-level ecology for the development of next-generation microbiome therapeutics. We also highlight key methodological innovations including high-resolution amplicon profiling, advanced metagenomic and single-cell genomics, and culture-based functional approaches that collectively enable strain-level resolution and are reshaping the field.

A plant's phenotype is determined by the traits of both the plant itself and its associated microbiome. However, we still have a poor understanding of the extent to which plant microbial recruitment contributes to disease resistance. We conducted a cross-inoculation experiment in which rhizosphere microbiomes from Fusarium wilt-resistant and susceptible banana varieties were collected and used to colonize the next planting cycle, and microbiome dynamics during recruitment and transfer were tracked. Culture-based approaches were used to construct synthetic microbial communities (SynComs) and test the effects of variety-specific metabolites on isolated strains. Transferring the rhizosphere microbiome from a highly resistant variety to a susceptible variety reduced Fusarium wilt pathogen abundance by 37.65% compared with transferring the susceptible plant's microbiome, while sterilized microbiomes had no detectable effect. Constructed SynComs recapitulated the suppressive effects of their source microbiomes, and metabolites derived from the highly resistant variety, characterized by enrichment of shikimic acid, stearic acid, and D-(-)-ribofuranose, promoted the growth of these beneficial microbes. Our results highlight that plant resistance levels are largely determined by the plant's ability to recruit a disease-suppressive microbiome, suggesting that enhancing microbial recruitment may represent an avenue to improve the disease resistance of susceptible varieties.

Infectious keratitis remains a leading cause of corneal blindness and visual impairment worldwide, with bacterial, fungal, amoebic and viral pathogens presenting major diagnostic and therapeutic challenges. Early and precise pathogen identification is essential to initiate early treatment and prevent irreversible ocular damage. Direct visualisation of pathogens by microscopy of corneal scrapings has been one of the most rewarding and time-tested methods; however, ocular microbiology laboratories seem to be losing the expertise for the same. Culture methods also seem to be losing favour for their long turnaround time and low sensitivity. These have paved the way for newer, rapid and sensitive molecular techniques to make a foray into the realm of diagnosis of non-viral keratitis. This review critically evaluates recent advances in molecular diagnostics for infectious keratitis, focusing on DNA/RNA-based sequencing, real-time PCR (qPCR) and mass spectrometry (MS) and examines their clinical relevance, limitations and potential for point-of-care application.An extensive literature analysis was conducted, integrating findings from peer-reviewed studies published between 2000 and 2025. Emerging diagnostic platforms were compared with conventional microbiological methods in terms of diagnostic accuracy, turnaround time and clinical applicability across paediatric and adult cohorts. Traditional culture-based approaches exhibit limited sensitivity (30%-60%) and slow pathogen detection. In contrast, multiplex and quantitative PCR enable rapid, sensitive and specific pathogen identification, including in culture-negative cases. Next-generation sequencing offers broad-spectrum, culture-independent profiling of bacteria, fungi and protozoa, enhancing diagnostic yield and understanding of pathogen diversity. MS has transformed microbial and host proteomic/metabolomic analyses, identifying candidate biomarkers for disease severity and prognosis. Recent innovations such as portable qPCR devices, clustered regularly interspaced short palindromic repeats-based diagnostics and artificial intelligence-assisted data interpretation extend molecular testing to near-patient settings. The integration of these technologies into routine clinical workflows promises earlier intervention, personalised therapy and improved visual outcomes, particularly in resource-limited environments.

Acinetobacter baumannii is widely recognized as a problematic pathogen in healthcare settings due to its ability to acquire resistance to multiple antimicrobial agents. However, less attention has been given to its presence outside hospitals. In this cross-sectional, laboratory-based surveillance study, we investigated the occurrence of A. baumannii in ready-to-eat (RTE) foods sold at retail outlets in four cities of the Al-Qassim region, Saudi Arabia, during a single season. A total of 240 RTE food samples were analyzed using culture-based and molecular approaches for species confirmation, and antimicrobial susceptibility profiles were determined. A. baumannii was identified in 19 samples (7.9%), spanning several food categories. Most isolates showed resistance to multiple antimicrobial classes, and 16 met the criteria for multidrug resistance (MDR). Among the confirmed isolates, blaOXA-23-like was detected in 16 (84.2%), blaOXA-24/40-like in 2 (10.5%), and blaOXA-58-like in 1 (5.3%). Resistance to fluoroquinolones, tetracyclines, and aminoglycosides was common, and OXA-type carbapenemase genes were detected in 16 isolates. These findings indicate that RTE foods can represent non-clinical environments in which MDR A. baumannii may be detected. Including food sources in antimicrobial resistance surveillance may therefore strengthen our understanding of the ecology of this pathogen within a One Health framework.

Back-slopping is widely used in many food fermentations to ensure process continuity and product consistency. The feasibility of using tempeh in back-slopping has not yet been adequately investigated. This study hypothesized the possibility of storing and reusing tempeh as a starter for back-slopping. Tempeh was frozen, dried and lyophilized for up to 6months and then used in back-slopping. The microbiota, proteolytic profile and volatilome of tempeh produced by back-slopping (BS-tempeh) were analyzed using culture-based and metataxonomic approaches, HPLC and GC-MS, respectively. Standard plate count analyses revealed a predominance of Rhizopus spp., whereas Enterobacteriaceae, coliforms and other undesirable microorganisms remained below detection limits in all samples. The microbiota profile showed that BS-tempeh produced with frozen starter better preserved the microbiota, with α-diversity values comparable to the control at T0 (p=0.054), whereas BS-tempeh obtained with dried starter increased α-diversity (T0, p=0.0076; T3, p=0,0061; T6, p=0.015) and introduced low-abundance and potentially opportunistic taxa, including Klebsiella, Enterococcus and other Enterobacteriaceae. No statistically significant differences in proteolysis and amino acid profiles were observed among the samples (p>0.05). Multivariate analysis of volatilome profile revealed that the fermentation process led to a convergence of the volatile profiles, regardless of starter preservation type. Overall, the results demonstrated that frozen tempeh offers the highest microbiological stability compared to drying and lyophilization, highlighting the potential application of tempeh back-slopping in artisanal production and small- to medium-scale industrial settings as an alternative or complement to commercial starters.

Whole-genome shotgun metagenomics reveals environmental perturbations in the gut microbiome and resistome of wild raccoon dogs rescued at a wildlife center.

The insect microbiome profoundly influences host physiology and ecology, yet its composition and evolutionary dynamics in thrips remain poorly understood. Here, we present a systematic characterization of thrips-associated microbiomes through integrated metagenomic and culture-based approaches. Our analysis reveals that thrips microbiomes are dominated by both intracellular symbionts (e.g., Wolbachia and Spiroplasma) and extracellular taxa (e.g., Serratia, Pantoea, and Acinetobacter), with species-specific compositions exhibiting frequent gains and losses of bacterial lineages. We demonstrate that thrips microbiomes exhibit low interspecific microbial sharing, forming host-specific bacterial communities with minimal overlap between species. To address methodological challenges in microbiome research, we developed a dual-sequencing framework combining short-read sequencing (for comprehensive taxonomic detection) and long-read sequencing (for genomic verification), enabling the reconstruction of high-quality metagenome-assembled genomes that validated short-read findings. Furthermore, we isolated and sequenced the complete genomes of two dominant extracellular symbionts-Pantoea dispersa and Serratia marcescens-and performed pan-genome analyses. These revealed small core gene sets and expansive accessory genomes, including host-specific functional genes (e.g., hydrolases and neurotoxic N-acetyltransferases) likely involved in host adaptation. Our study provides a foundational genomic resource and a robust analytical pipeline for dissecting thrips microbiome evolution, with implications for understanding insect-microbe interactions and symbiont-mediated adaptations.

Biosurfactants produced by microorganisms play essential roles in ecosystem function and hold significance promise for biotechnological applications. However, their diversity and distribution remain poorly depicted due to the limitations of culture-based approaches. In this study, we conducted a large-scale genomic data mining of 142,135 microbial genomes of putative biosurfactant-producing taxa, spanning 21 distinct ecosystems, to systematically profile gene association with 10 major biosurfactant classes. Using a list of 18 key functional genes, we mapped their taxonomic and ecological distribution and analyzed patterns of gene co-occurrence. We found that rhamnolipid biosynthesis genes are nearly ubiquitous across microbial lineages, reflecting their fundamental role in microbial adaptation. In contrast, emulsan and serrawettin pathways are more restricted to plant-associated and fungal ecosystems. The highest diversity of biosurfactant-related genes was found in genomes recovered from nutrient-rich habitats, including plant-associated, algal, and wastewater ecosystems. Co-occurrence network analysis revealed two distinct organizational strategies: a rare, conserved core cluster of genes associated with fengycin, surfactin, iturin lichenysin and plipastatin production, and a widespread, modular periphery linked to rhamnolipid, emulsan, and serrawettin W1 pathways, that are likely driven by the need to adapt to environmental complexity. Notably, we identified previously unreported genomes with biosurfactant production potential, significantly expanding the known biodiversity and ecological range of potential producers. Our findings establish biosurfactant production as a key microbial trait shaped by habitat, with broad implications for microbial ecology, ecosystem monitoring and sustainable biotechnology. This work provides comprehensive genomic resource for biosurfactant research, laying the foundation for targeted bioprospecting and integrative functional studies.

This study examines the integration of social capital, local institutions, and cultural values in mangrove ecotourism governance within Berau Regency, East Kalimantan. Employing a mixed-methods approach combining Structural Equation Modeling-Partial Least Squares (SEM-PLS), ecological carrying capacity analysis, and economic valuation, this research reveals how indigenous governance structures interact with modern sustainability frameworks. Primary data from 325 respondents across five coastal communities and secondary ecological data spanning 2022-2025 demonstrate that social capital significantly mediates the relationship between local institutional strength and community welfare outcomes (β=0.687, p<0.001). The ecological carrying capacity assessment indicates sustainable visitor thresholds ranging from 180 to 240 visitors per day across different mangrove zones. Economic analysis reveals that community-based ecotourism generates average monthly household income increases of 42.3% compared to pre-ecotourism baselines. The novelty of this research lies in its quantitative integration of cultural capital metrics, institutional quality indices, and tangible welfare indicators within a single analytical framework. Results demonstrate that communities with stronger traditional governance systems (adat institutions) achieve 34% higher sustainability scores and 28% greater income equity. Policy implications emphasize the necessity of hybrid governance models that formalize indigenous knowledge systems while maintaining cultural authenticity. This study contributes to sustainable development literature by providing empirical evidence for culture-based conservation approaches in tropical coastal ecosystems.

Trichomoniasis, caused by Trichomonas vaginalis (T. vaginalis), is a common sexually transmitted disease (STD) that primarily affects the genital and urinary tracts. Trichomoniasis has a worldwide distribution and is also widely endemic in China. In recent years, due to the continuous increase in the proportion of recurrent or persistent vaginal trichomoniasis, the related clinical diagnosis and treatments are facing new challenges. Currently, clinical diagnosis relies on clinical features and laboratory tests. Microscopic examination of vaginal secretions is a commonly used diagnostic method in which active T. vaginalis can be observed under a microscope in vaginal secretions. Although microscopic examination has a high specificity, the sensitivity is only 50%-60%. Therefore, the development of more sensitive and accurate clinical diagnostic methods are urgently needed for trichomoniasis. In this study, a clinical diagnosis of T. vaginalis infection was conducted at the Maternal and Child Health Hospital of Hubei province, Xiangyang No.1 People's Hospital, The First Hospital of Hunan University of Chinese Medicine, and Jingzhou Central Hospital. Different methods-fluorescent PCR (F-PCR), microscopic examination, sequencing and culture-were employed to diagnose T. vaginalis. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), negative likelihood ratio (NLR), and accuracy of F-PCR are 100% (98-100%), 98% (96%-99%), 97% (93%-98%), 100% (99%-100%), 0.00, and 99% (97%-100%), respectively. Compared with traditional methods such as microscopic examination, F-PCR offers superior sensitivity and accuracy. Additionally, F-PCR has a lower probability of false negatives compared with sequencing and culture-based approaches. Compared with methods such as microscopic examination, sequencing, and culture, it significantly reduces the probability of false negatives; and is more sensitive and accurate, making it a powerful diagnostic method for clinical diagnoses.

From single cells to organotypic cultures: Measuring and analyzing keratinocyte migration.

Detection of fungal contamination on museum books stored under controlled environmental conditions: A discrepancy between culture-based and metagenomic analysis approaches.