Microorganisms

19 pages, 6917 KB

Open AccessArticle

Nanobodies Targeting the GP4 Protein Inhibit PRRSV Replication

by Wenxiang Zhang, Aodi Wu, Honghuan Li, Tao He, Qianqian Dong, Hanwen Zhang, Jie Chen, Song Jiang and Jinliang Sheng

Microorganisms 2025, 13(11), 2524; https://doi.org/10.3390/microorganisms13112524 - 2 Nov 2025

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) infection inflicts enormous economic losses on the global swine industry and imposes significant pressure on agricultural production. However, there are currently no clinically approved effective therapeutics specifically targeting PRRSV. Accordingly, the development of novel antiviral agents [...] Read more.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection inflicts enormous economic losses on the global swine industry and imposes significant pressure on agricultural production. However, there are currently no clinically approved effective therapeutics specifically targeting PRRSV. Accordingly, the development of novel antiviral agents against PRRSV is urgently needed. Notably, the structural glycoprotein 4 (GP4) of PRRSV—which plays a crucial role in viral entry into host cells—represents a promising target for antiviral development. Nanobodies, characterized by their small size, structural stability, high affinity, and excellent solubility, have emerged as attractive candidates for next-generation therapeutic development. Yet, to date, no specific nanobodies targeting PRRSV GP4 have been reported. In this study, we isolated GP4-specific nanobodies using phage display technology and investigated their mechanisms underlying viral suppression through a series of in vitro functional assays. Our results demonstrate that Nb6, Nb31, and Nb85 significantly inhibit PRRSV infection by disrupting both viral attachment to host cells and subsequent internalization processes. Collectively, these findings indicate that Nb6, Nb31, and Nb85 hold substantial potential for development as antiviral agents against PRRSV infection. Full article

(This article belongs to the Section Veterinary Microbiology)

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19 pages, 887 KB

Open AccessArticle

Exploratory In Vitro Evaluation of Maternal–Infant Bifidobacterium Strains for Microbiota Modulation in a Pediatric Cystic Fibrosis Context

by Maria Esteban-Torres, Isabel Blanco, Andrea Asensio-Grau, Nuria Ruiz, Manuel Bernabeu and Joaquim Calvo-Lerma

Microorganisms 2025, 13(11), 2523; https://doi.org/10.3390/microorganisms13112523 - 2 Nov 2025

Abstract

This study explores the potential of novel Bifidobacterium isolates as targeted probiotic supplements for children with cystic fibrosis (CF), a condition often associated with gut dysbiosis. Five strains of Bifidobacterium genus (B. animalis IATA01, B. pseudocatenulatum IATA35, B. longum [...] Read more.

This study explores the potential of novel Bifidobacterium isolates as targeted probiotic supplements for children with cystic fibrosis (CF), a condition often associated with gut dysbiosis. Five strains of Bifidobacterium genus (B. animalis IATA01, B. pseudocatenulatum IATA35, B. longum IATA02, B. bifidum IATA13, and B. longum IATA05) isolated from maternal–infant fecal samples were assessed in vitro following the FAO/WHO guidelines. Their probiotic potential was evaluated through simulated gastrointestinal digestion in the CF context, their adhesion to mucin, and their carbohydrate fermentation capacity. Additionally, their impact on colonic microbiota modulation was analyzed using static in vitro colonic fermentation with fecal inocula from four pediatric patients with CF to assess the presence of different bacterial groups associated with dysbiosis via qPCR and short-chain fatty acid production by GC-MS. Three strains (IATA01, IATA35, and IATA05) demonstrated survival after gastrointestinal digestion, with IATA01 exhibiting the highest adhesion to mucin but limited carbohydrate fermentation capacity. All strains increased the Bifidobacterium levels after colonic fermentation, while their effects on reducing pathogenic groups and promoting beneficial bacteria such as Akkermansia and Faecalibacterium varied depending on the strain and the individual inoculum. These findings highlight the strain-specific effects of Bifidobacterium and evidence a specific impact on colonic microbiota, depending on the composition of the basal inoculum, highlighting individual-specific responses. Full article

(This article belongs to the Section Gut Microbiota)

29 pages, 4911 KB

Open AccessArticle

Heavy Metal and Petroleum Hydrocarbon Contaminants Promote Resistance and Biofilm Formation in Vibrio Species from Shellfish

by Gongshi Lin, Yingpeng Li, Ying Qiao, Theerakamol Pengsakul, Guobin Chen and Lixing Huang

Microorganisms 2025, 13(11), 2522; https://doi.org/10.3390/microorganisms13112522 - 2 Nov 2025

Abstract

Shellfish are an essential component of the human diet, yet their safety is increasingly compromised by contamination with heavy metals, petroleum hydrocarbons, and pathogenic microorganisms, such as Vibrio, which pose significant health risks. This study examined shellfish samples from seafood markets, assessing [...] Read more.

Shellfish are an essential component of the human diet, yet their safety is increasingly compromised by contamination with heavy metals, petroleum hydrocarbons, and pathogenic microorganisms, such as Vibrio, which pose significant health risks. This study examined shellfish samples from seafood markets, assessing the levels of heavy metals (e.g., cadmium, copper) and petroleum hydrocarbons, while isolating and identifying Vibrio species carried by the shellfish. The antimicrobial resistance profiles, resistance genes, and biofilm-forming capacities of these strains were further characterized. Results revealed significant seasonal fluctuations in heavy metal concentrations, with some samples exceeding regulatory limits, indicating potential health risks for long-term consumers. Likewise, Vibrio abundance and resistance varied seasonally, with a notable prevalence of multidrug-resistant strains, likely influenced by antibiotic misuse and environmental pressures in coastal regions. Correlation analyses suggested potential links between heavy metal contamination and Vibrio resistance, as well as biofilm formation, supporting the hypothesis that metal-induced stress may facilitate resistance gene transfer and enhance biofilm-mediated resistance. This study reveals the seasonal dynamics of antimicrobial resistance (AMR) in shellfish-derived Vibrio species and elucidates the dose–response effects of heavy metals and petroleum hydrocarbons, as well as their synergistic selection mechanisms. These findings provide a scientific foundation for assessing shellfish safety, deciphering AMR transmission, and developing ecosystem-based strategies for aquaculture monitoring. Full article

(This article belongs to the Section Biofilm)

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35 pages, 1304 KB

Open AccessReview

Probiotic Potential of Traditional and Emerging Microbial Strains in Functional Foods: From Characterization to Applications and Health Benefits

by Chijioke Christopher Uhegwu and Christian Kosisochukwu Anumudu

Microorganisms 2025, 13(11), 2521; https://doi.org/10.3390/microorganisms13112521 - 2 Nov 2025

Abstract

Global consumer demand for probiotic-enriched functional foods has increased as consumers become increasingly aware of the connection between what they eat and its role in their long-term health. Compared with conventional foods that primarily deliver fundamental nutrients, functional foods include biologically active compounds [...] Read more.

Global consumer demand for probiotic-enriched functional foods has increased as consumers become increasingly aware of the connection between what they eat and its role in their long-term health. Compared with conventional foods that primarily deliver fundamental nutrients, functional foods include biologically active compounds capable of influencing physiological processes. While traditionally used probiotic strains like Lactobacillus and Bifidobacterium are still at the center of this trend, there is growing interest in the exploration of emerging and novel microbial candidates that harbor new functional properties. This review addresses the characterization, modes of action, technological limitations, regulatory guidelines, and prospective health benefits of new probiotic strains in functional foods. The review further highlights the need for precise strain selection, novel encapsulation technologies for viability, and strict safety assessments in accordance with EFSA’s QPS (Qualified Presumption of Safety) and the United States FDA GRAS (Generally Recognized As Safe) specifications. Current research focuses on the classical benefits of probiotics, including gut microbiota modulation, immunomodulation, antimicrobial activity, lowering of cholesterol, and mental health. However, long-term clinical validation, strain specificity, personalized application, and effective communication to consumers are some areas where gaps remain. Addressing these challenges through the incorporation of omics technologies, synthetic biology, and more detailed microbiome–host interaction studies will be the key to unlocking the full potential of next-generation probiotics and sustaining consumer trust in this emerging market. Full article

(This article belongs to the Special Issue Microbial Safety and Beneficial Microorganisms in Foods)

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14 pages, 2457 KB

Open AccessArticle

Effect of High-Temperature Stress on Fatty Acid Composition and Undecylprodiginine Biosynthesis in Streptomyces coelicolor M511

by Youngjong Han, Yujun Park, Kyudong Han and SangJoon Mo

Microorganisms 2025, 13(11), 2520; https://doi.org/10.3390/microorganisms13112520 - 1 Nov 2025

Abstract

Actinomycetes are a representative group of bacteria that inhabit soil; in particular, Streptomyces coelicolor M511 produces actinorhodin and undecylprodiginine. Among them, undecylprodiginine has antibiotic and immunosuppression activity and is a secondary metabolite with high potential applications in biotechnological and pharmaceutical fields. High temperature [...] Read more.

Actinomycetes are a representative group of bacteria that inhabit soil; in particular, Streptomyces coelicolor M511 produces actinorhodin and undecylprodiginine. Among them, undecylprodiginine has antibiotic and immunosuppression activity and is a secondary metabolite with high potential applications in biotechnological and pharmaceutical fields. High temperature stress (37 °C) reduced the biosynthesis of undecylprodiginine and induced specific branched chain alkylprodiginine derivatives, compared with the optimal growth temperature (30 °C). Also, the stress stimulated the synthesis of straight-chain FA for enhancing membrane rigidity. The inhibition of undecylprodiginine biosynthesis under high temperature stress seems to be induced by the heat sensitivity of the RedP enzyme, and this inhibition is compensated by FAS FabH. Since FabH, a homologue of RedP, has a broader substrate specificity, it leads to the production of methylundecylprodiginine and methyldodecylprodiginine. The external addition of isoleucine (as well as that of leucine and valine to a far lesser extent) enhances the synthesis of these derivatives since isoleucine catabolism generates precursors used for the biosynthesis of these compounds. These findings reveal temperature-dependent changes in precursor utilization and prodiginine diversity, providing insights into metabolic plasticity and strategies establishing a foundation for secondary metabolite derivatives engineering strategies through precursor supplementation or temperature regulation. Full article

(This article belongs to the Section Microbial Biotechnology)

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22 pages, 3119 KB

Open AccessArticle

Characterization of Bacterial Communities in Volcanic Soil from Northern Patagonian Area of Chile

by Patricia Aguila-Torres, Mauricio González, Marcela Hernández, Constanza Aguado-Norese, Jonathan E. Maldonado, Richard M. Miranda, Roxana González-Stegmaier, Daniel E. Palma, Luis A. Rojas and Macarena Mellado

Microorganisms 2025, 13(11), 2519; https://doi.org/10.3390/microorganisms13112519 - 1 Nov 2025

Abstract

Osorno volcano (41.1° S, 72° W) is located in the Andean Southern Volcanic Zone. The volcano lies within a national park as part of the protected areas system. This setting provides an opportunity to compare soil microbial communities between sectors with (H) and [...] Read more.

Osorno volcano (41.1° S, 72° W) is located in the Andean Southern Volcanic Zone. The volcano lies within a national park as part of the protected areas system. This setting provides an opportunity to compare soil microbial communities between sectors with (H) and without (NI) anthropogenic activities within a volcanic territory. To do so, we selected one of the most visited volcanoes in Chilean Patagonia to examine composition, diversity (taxonomic and phylogenetic), and co-presence and mutual exclusion interaction networks between members of volcanic soil bacterial communities. Soil DNA was extracted, and the 16S rRNA gene was analyzed by high-throughput DNA sequencing, followed by taxonomic identification. The most prevalent phylum across all sites (H and NI) was Pseudomonadota, followed by Acidobacteriota, Actinobacteriota, and Chloroflexota. Based on taxonomic and phylogenetic indices, we found that the diversity of bacteria was significantly less in the humanized area than in the non-intervened areas. Beta diversity analysis also revealed a clear separation between humanized and non-intervened soils. Additionally, a decrease in network connectivity was observed at NI sites. Our results provide clear evidence that anthropogenic factors, such as tourism, vehicle parking, and combustion processes, are key drivers shaping bacterial community structure in volcanic soils, with potential consequences for ecosystem health and the capacity to provide ecosystem services. Full article

(This article belongs to the Special Issue Earth Systems: Shaped by Microbial Life)

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10 pages, 540 KB

Open AccessArticle

β-Actin as an Endogenous Control Gene in Real-Time PCR for Detection of West Nile and Usutu Virus in Mosquitoes

by Jeanne Lai, Carlotta Tessarolo, Elisabetta Ercole, Marina Gallo, Monica Lo Faro, Claudia Palmitessa, Valerio Carta, Alessio Ferrari, Alessandra Favole, Mattia Begovoeva, Francesco Ingravalle, Simone Peletto, Nicolò Francesco Fiscella, Roberta Irelli, Eugenia Ciarrocchi, Walter Martelli, Andrea Mosca, Giulia Cagnotti, Cristina Casalone and Cristiano Corona

Microorganisms 2025, 13(11), 2518; https://doi.org/10.3390/microorganisms13112518 - 31 Oct 2025

Abstract

Mosquito-borne viruses like West Nile virus (WNV) and Usutu virus (USUV) present growing public health concerns, especially with climate change and expanding vector ranges. This study describes the development and validation of a duplex Real-Time RT-PCR assay targeting β-actin (ACTB) mRNA as an [...] Read more.

Mosquito-borne viruses like West Nile virus (WNV) and Usutu virus (USUV) present growing public health concerns, especially with climate change and expanding vector ranges. This study describes the development and validation of a duplex Real-Time RT-PCR assay targeting β-actin (ACTB) mRNA as an endogenous control and a conserved 92 bp region shared by WNV and USUV genomes. Degenerate primers for ACTB ensure RNA extraction quality and PCR performance while enabling simultaneous detection of both viruses. A total of 1002 mosquito pools collected in Piedmont, Italy, during the 2024 vector season under the National Surveillance Plan for Arboviruses (PNA), were tested. The assay showed 100% accuracy—ACTB mRNA was detected in all pools, and six pools tested positive for WNV or USUV (three each). Diagnostic specificity was confirmed on 40 horse and bovine serum samples. Sanger sequencing confirmed ACTB identity across multiple mosquito species. The assay also demonstrated reproducibility across different operators and thermocyclers. The limit of detection (LOD) evaluation showed that the assay is capable of detecting viral RNA at very low concentrations, confirming its high analytical sensitivity. The duplex RT-PCR here developed is a reliable, sensitive, and specific tool for arbovirus surveillance, combining pathogen detection with internal quality control of RNA extraction and amplification, thus improving early warning and rapid response to mosquito-borne disease threats. Full article

(This article belongs to the Special Issue Interactions between Parasites/Pathogens and Vectors)

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16 pages, 1363 KB

Open AccessArticle

Tackling Conifer Needle Cast and Ash Dieback with Host-Derived Microbial Antagonists Exhibiting Plant Growth-Promoting Traits

by Milana Šilanskienė, Dorotėja Vaitiekūnaitė and Vaida Sirgedaitė-Polikaitienė

Microorganisms 2025, 13(11), 2517; https://doi.org/10.3390/microorganisms13112517 - 31 Oct 2025

Abstract

Needle cast (Lophodermium seditiosum Minter, Staley & Millar) in Scots pine (Pinus sylvestris L.) and European ash (Fraxinus excelsior L.) dieback (Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz & Hosoya) are among the most destructive forest and tree plantation diseases [...] Read more.

Needle cast (Lophodermium seditiosum Minter, Staley & Millar) in Scots pine (Pinus sylvestris L.) and European ash (Fraxinus excelsior L.) dieback (Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz & Hosoya) are among the most destructive forest and tree plantation diseases in Europe, threatening not only targeted plant species but also the whole ecosystem. While considerable research effort has focused on microbial antagonists against ash dieback, comparable investigations into needle cast biocontrol remain virtually absent from the literature. Here, isolated microbial antagonists from European ash and Scots pine were evaluated for their efficacy against respective pathogens. In vitro dual-culture assays revealed bacteria with strong inhibitory effects on pathogen growth, as well as multiple plant growth-promoting traits (PGPTs). It was found that bacteria from the genera of Pantoea, Erwinia, Priestia, and Pseudomonas inhibited the growth of H. fraxineus by ≥70%. Most significantly, our investigation revealed that bacteria isolated from Scots pine, belonging to the genera Pseudomonas, Bacillus, and Priestia, inhibited the growth of L. seditiosum by 50% to 80%, representing one of the first reported bacterial antagonisms for this neglected pathogen. All isolates were positive for at least two PGPTs, primarily due to mineralization of organic phosphate and the production of siderophores. The dual functional traits of isolated bacteria highlight their potential application in integrated forest protection strategies, particularly for the previously overlooked L. seditiosum pathosystem. Full article

(This article belongs to the Special Issue Plant Growth-Promoting Bacteria)

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29 pages, 719 KB

Open AccessReview

Gut Microbiome and Immune System Crosstalk in Chronic Inflammatory Diseases: A Narrative Review of Mechanisms and Therapeutic Opportunities

by Jefferson J. Feng, Nikhil R. Maddirala, Ashley Saint Fleur, Fenfen Zhou, Di Yu, Feng Wei and Yongrong Zhang

Microorganisms 2025, 13(11), 2516; https://doi.org/10.3390/microorganisms13112516 - 31 Oct 2025

Abstract

The gut microbiota, a complex community of trillions of microorganisms residing in the gastrointestinal tract, plays a vital role in maintaining host health and regulating a wide range of physiological functions. Advances in molecular biology have greatly expanded our understanding of the dynamic [...] Read more.

The gut microbiota, a complex community of trillions of microorganisms residing in the gastrointestinal tract, plays a vital role in maintaining host health and regulating a wide range of physiological functions. Advances in molecular biology have greatly expanded our understanding of the dynamic interactions between the gut microbiome and the immune system. Disruption of this microbial community, known as dysbiosis, can compromise epithelial barrier integrity, trigger aberrant immune activation, and lead to the production of proinflammatory metabolites. These changes are increasingly recognized as contributing factors in the pathogenesis of chronic inflammatory diseases. Emerging research highlights the gut microbiota as a key modulator of immune homeostasis, influencing both local and systemic inflammatory processes during the initiation and progression of these diseases. Understanding the mechanisms underlying gut microbiota-immune interactions will offer new avenues for therapeutic interventions. This review focuses on six representative chronic inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, psoriasis, systemic lupus erythematosus, asthma, and vasculitis, all of which are characterized by dysregulated immune responses and persistent inflammation. Our goal is to synthesize the recent research on the role of gut microbiome in the pathogenesis of the diseases listed above and provide insights into the development of microbiota-based therapies, particularly fecal microbiota transplant, dietary modifications, prebiotic and probiotic interventions, for their treatment. Full article

(This article belongs to the Special Issue Correlations Between the Gastrointestinal Microbiome and Diseases)

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28 pages, 1618 KB

Open AccessReview

The Gut Microbiota of Drosophila melanogaster: A Model for Host–Microbe Interactions in Metabolism, Immunity, Behavior, and Disease

by Kyu Hong Cho and Song Ok Kang

Microorganisms 2025, 13(11), 2515; https://doi.org/10.3390/microorganisms13112515 - 31 Oct 2025

Abstract

The gut microbiota of Drosophila melanogaster offers a simplified yet powerful system to study conserved mechanisms of host–microbe interactions. Unlike the highly complex mammalian gut microbiota, which includes hundreds of species, the fly gut harbors a small and defined community dominated by Lactobacillus [...] Read more.

The gut microbiota of Drosophila melanogaster offers a simplified yet powerful system to study conserved mechanisms of host–microbe interactions. Unlike the highly complex mammalian gut microbiota, which includes hundreds of species, the fly gut harbors a small and defined community dominated by Lactobacillus and Acetobacter. Despite its low diversity, this microbiota exerts profound effects on host physiology. Commensal bacteria modulate nutrient acquisition, regulate insulin/TOR signaling, and buffer dietary imbalances to support metabolic homeostasis and growth. They also influence neural and behavioral traits, including feeding preferences, mating, and aggression, through microbial metabolites and interactions with host signaling pathways. At the immune level, microbial molecules such as peptidoglycan, acetate, uracil, and cyclic dinucleotides activate conserved pathways including Imd, Toll, DUOX, and STING, balancing antimicrobial defense with tolerance to commensals. Dysbiosis disrupts this equilibrium, accelerating aging, impairing tissue repair, and contributing to tumorigenesis. Research in Drosophila demonstrates how a low-diversity microbiota can shape systemic host biology, offering mechanistic insights relevant to human health and disease. Full article

(This article belongs to the Special Issue Gut Microbiome in Homeostasis and Disease, 3rd Edition)

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9 pages, 1207 KB

Open AccessCommunication

Recent Trends in Prevalence of HPV Infection in Nasopharyngeal Carcinoma in Japan

by Luyao Liu, Nobuyuki Hirai, Satoru Kondo, Makiko Moriyama-Kita, Ryotaro Nakazawa, Shigetaka Komura, Makoto Kano, Daisuke Uno, Manabu Inaba, Takayoshi Ueno, Yosuke Nakanishi, Kazuhira Endo, Hisashi Sugimoto and Tomokazu Yoshizaki

Microorganisms 2025, 13(11), 2514; https://doi.org/10.3390/microorganisms13112514 - 31 Oct 2025

Abstract

Background: Nasopharyngeal carcinoma (NPC) is a malignant tumor in which the etiologic contribution of Epstein–Barr virus (EBV) is well established. However, similar to that of oropharyngeal carcinoma, some papers reported that human papilloma virus (HPV) contributed to the development of NPC in non-endemic [...] Read more.

Background: Nasopharyngeal carcinoma (NPC) is a malignant tumor in which the etiologic contribution of Epstein–Barr virus (EBV) is well established. However, similar to that of oropharyngeal carcinoma, some papers reported that human papilloma virus (HPV) contributed to the development of NPC in non-endemic regions. Previously, we conducted a study on HPV infection in patients with NPC between 1996 and 2015 in our department. The current study aims to evaluate the incidence and role of HPV infection in NPC pathogenesis using samples of NPC after 2015. Methods: Paraffin-embedded tumor samples from 26 patients with NPC who were treated at our department between 2015 and 2022 were analyzed. HPV polymerase chain reaction, p16 immunohistochemistry, HPV genotyping, and in situ hybridization for EBV-encoded RNA were performed to determine the viral infection status. Results: Of the 26 patients, 19 (73%) were EBV-positive and HPV-negative, 1 (4%) was EBV-negative and HPV-positive, and 6 (23%) were negative for both EBV and HPV. The detection rate of HPV has slightly increased from 3% to 4% over the past decade. Conclusion: Although Japan is a non-endemic region for NPC, HPV infection is exceedingly rare and may have a limited role in NPC development in Japan. However, the detection rate of HPV has not significantly changed in the past decade, further supporting the view that HPV has a relatively small impact on the pathogenesis of NPC in Japan. Full article

(This article belongs to the Section Virology)

19 pages, 2704 KB

Open AccessArticle

Metagenome-Based Functional Differentiation of Gut Microbiota and Ecological Adaptation Among Geographically Distinct Populations of Przewalski’s gazelle (Procapra przewalskii)

by Jingjie Zhang, Feng Jiang, Xiaohuan Li, Pengfei Song and Tongzuo Zhang

Microorganisms 2025, 13(11), 2513; https://doi.org/10.3390/microorganisms13112513 - 31 Oct 2025

Abstract

Przewalski’s gazelle (Procapra przewalskii) is an endangered ungulate endemic to the Qinghai–Tibet Plateau, with a small population size and exposure to multiple ecological pressures. Its gut microbiota may play a crucial role in host environmental adaptation. To investigate the functional divergence [...] Read more.

Przewalski’s gazelle (Procapra przewalskii) is an endangered ungulate endemic to the Qinghai–Tibet Plateau, with a small population size and exposure to multiple ecological pressures. Its gut microbiota may play a crucial role in host environmental adaptation. To investigate the functional divergence of gut microbial communities, we performed high-throughput metagenomic sequencing on 105 wild fecal samples collected from 10 geographic regions around Qinghai Lake. The results revealed significant regional differentiation in key functional modules related to metabolism, antibiotic resistance mechanisms, and virulence-associated pathways. All populations showed enrichment in core metabolic pathways such as carbohydrate and amino acid metabolism, with carbohydrate-active enzymes dominated by glycoside hydrolases (GHs) and glycosyltransferases (GTs), exhibiting overall functional conservation. Although populations shared many antibiotic- and virulence-related reference genetic markers, the marker composition associated with distinct resistance mechanisms and pathogenic processes exhibited clear population-specific patterns, suggesting differential microbial responses to local environmental pressures. Correlation network analysis further identified core taxa (e.g., Arthrobacter and Oscillospiraceae/Bacteroidales lineages) as key genera linking community structure with core metabolic, resistance-related, and virulence-associated marker functions. Overall, the gut microbiota of Przewalski’s gazelle exhibits a complex spatially structured functional differentiation, reflecting host–microbiome co-adaptation under region-specific ecological pressures. These findings provide critical methodological and theoretical support for microecological health assessment and regionally informed conservation management of this endangered species. Full article

(This article belongs to the Section Gut Microbiota)

13 pages, 1385 KB

Open AccessArticle

Genetic Diversity and Clonal Expansion of Pathogenic Leptospira in Brazil: A Multi-Host and Multi-Regional Panorama

by Maria Isabel Nogueira Di Azevedo and Walter Lilenbaum

Microorganisms 2025, 13(11), 2512; https://doi.org/10.3390/microorganisms13112512 - 31 Oct 2025

Abstract

Leptospirosis is a globally distributed zoonosis of major public health and veterinary relevance, caused by pathogenic species of the genus Leptospira. Brazil is a hotspot for transmission due to its ecological diversity and complex host–environment interfaces. This study explored the genetic diversity [...] Read more.

Leptospirosis is a globally distributed zoonosis of major public health and veterinary relevance, caused by pathogenic species of the genus Leptospira. Brazil is a hotspot for transmission due to its ecological diversity and complex host–environment interfaces. This study explored the genetic diversity and structure of circulating pathogenic Leptospira spp. in Brazil through a single-locus sequence typing (SLST) analysis based on the secY gene. A total of 531 sequences were retrieved from GenBank and subjected to phylogenetic and haplotype diversity analyses. Maximum likelihood reconstruction revealed strongly supported clades for seven species, with L. interrogans being the most prevalent and broadly distributed across hosts and regions. This species showed evidence of clonal expansion, with a dominant haplotype (n = 242) shared by humans, domestic animals, and wildlife. In contrast, L. santarosai and L. noguchii exhibited high haplotypic diversity and reticulated network structures, reflecting greater evolutionary variability. The species L. kirschneri and L. borgpetersenii displayed reduced haplotypic variation, the latter mainly associated with cattle, consistent with its host-adapted profile. Host- and biome-based haplotype networks revealed both the broad ecological adaptability of certain lineages and the exclusive presence of haplotypes restricted to specific environments, such as those found in marine mammals from the Atlantic Ocean. Genetic distance analyses confirmed the strong taxonomic resolution of the gene secY, which effectively distinguished closely related species while capturing intraspecific diversity. These findings provide a comprehensive molecular overview of pathogenic Leptospira in Brazil, highlighting ecological connectivity across hosts and biomes, as well as the contrasting evolutionary dynamics among species. Beyond describing genetic patterns, our analyses emphasize evolutionary processes, host–environment connectivity, and the implications for One Health. This integrative framework strengthens the basis for surveillance and control strategies in other endemic regions in the world. Full article

(This article belongs to the Special Issue Microparasites: Diversity, Phylogeny and Molecular Characterization)

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23 pages, 4580 KB

Open AccessArticle

Bacillus velezensis 7-A as a Biocontrol Agent Against Fusarium verticillioides, the Causal Agent of Rice Sheath Rot Disease

by Boyu Liu, Qunying Qin, Jianchao Hu, Jiayi Wang, Juan Gan, Ye Zhuang, Zhengxiang Sun and Yi Zhou

Microorganisms 2025, 13(11), 2511; https://doi.org/10.3390/microorganisms13112511 - 31 Oct 2025

Abstract

Rice sheath rot has progressively developed into a growing threat to global rice production, particularly in intensively managed systems conducive to disease development. Therefore, accurate identification of the causal pathogen and the development of sustainable management strategies represent urgent scientific requirements. In this [...] Read more.

Rice sheath rot has progressively developed into a growing threat to global rice production, particularly in intensively managed systems conducive to disease development. Therefore, accurate identification of the causal pathogen and the development of sustainable management strategies represent urgent scientific requirements. In this study, we isolated the causal organism of rice sheath rot from infected rice tissues and identified it as Fusarium verticillioides based on multi-locus sequence analysis. Eight endophytic bacterial strains were recovered from healthy rice root systems. Among the isolates, Bacillus velezensis isolate 7-A exhibited the strongest antifungal activity against F. verticillioides. This isolate demonstrated broad-spectrum antifungal activity, with inhibition rates ranging from 54.8% to 71.8%. Phylogenetic analysis based on 16S rRNA and gyrB gene sequences identified it as B. velezensis. Further characterization revealed that B. velezensis 7-A is capable of secreting proteases and synthesizing siderophores. The filtered liquid from sterile fermentation markedly inhibited the growth of mycelium in F. verticillioides and induced marked morphological abnormalities. Liquid LC-MS analysis identified multiple antifungal active substances, including camphor, ginkgolides B, salicin, cinnamic acid, hydroxygenkwanin, stearamide, β-carotene, and others. A pot experiment demonstrated that the fermentation broth of B. velezensis 7-A effectively suppressed the occurrence of rice sheath rot, achieving a relative control efficacy of 61.3%, which is comparable to that of a 10% carbendazim water-dispersible granule (WDG). Additionally, isolate 7-A enhances plant disease resistance by activating the activities of key defense enzymes. These findings provide preliminary insights into its potential application in integrated and sustainable disease management programs. Full article

(This article belongs to the Special Issue Beneficial Microorganisms for Sustainable Agriculture)

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19 pages, 3508 KB

Open AccessArticle

Deficiency in the msbB Gene Reduced the Salmonella Typhimurium Virulence Through Mechanisms Beyond LPS Modification

by Ling Yang, Zhuodong Chai, Jiaqian Qi, Yan Zhang, Yuqi Zhou, Zhenyu Li and Yinan Wei

Microorganisms 2025, 13(11), 2510; https://doi.org/10.3390/microorganisms13112510 - 31 Oct 2025

Abstract

The Salmonella enterica serovar Typhimurium (ST) mutant lacking the msbB gene (ΔmsbB) has been widely studied as a candidate for attenuated bacterial vectors in therapeutic applications. Deletion of msbB results in LPS with under-acylated lipid A, which lowers endotoxicity while maintaining [...] Read more.

The Salmonella enterica serovar Typhimurium (ST) mutant lacking the msbB gene (ΔmsbB) has been widely studied as a candidate for attenuated bacterial vectors in therapeutic applications. Deletion of msbB results in LPS with under-acylated lipid A, which lowers endotoxicity while maintaining structural integrity. This attenuation has traditionally been attributed to reduced TLR4 activation due to weaker interaction between the modified lipid A and TLR4. In our study, we confirmed that ΔmsbB ST was less lethal than wild-type (WT) ST in a mouse sepsis model. However, this difference persisted even in TLR4- and caspase-11-deficient mice, suggesting that LPS signaling is not the primary determinant of virulence. In vitro, bone marrow–derived macrophages (BMDMs) from TLR4- or caspase-11-deficient mice showed only modest reductions in ST-induced cell death and cytokine production. Importantly, ΔmsbB ST behaved similarly to WT ST in these assays, further indicating that LPS-mediated signaling is not central to the observed attenuation. Our previous studies showed that ST-induced mortality in mice is primarily mediated through NLRC4 activation. Using qPCR and immunoblotting, we found that expression of NLRC4 activators was diminished in the ΔmsbB strain. Additionally, the mutant exhibited increased outer membrane permeability—likely contributing to its heightened antibiotic sensitivity—and reduced motility due to lower flagellin protein levels. In summary, the attenuation of virulence observed in the ΔmsbB strain is not directly due to altered LPS–TLR4 interactions, but rather an indirect effect of diminished expression of virulence factors that activate the NLRC4 inflammasome. Full article

(This article belongs to the Section Molecular Microbiology and Immunology)

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17 pages, 2149 KB

Open AccessArticle

Substituting Chemical by Organic Fertilizer Improves Soil Quality, Regulates the Soil Microbiota and Increases Yields in Camellia oleifera

by Li Wen, Hanfang Luo, Chao Li, Kaikai Cheng, Lihong Shi, Lingling Liu, Ke Wang and Haiming Tang

Microorganisms 2025, 13(11), 2509; https://doi.org/10.3390/microorganisms13112509 - 31 Oct 2025

Abstract

The partial substitution of chemical fertilizer with organic fertilizer has been regarded as an effective strategy for enhancing crop yield and soil quality. Nevertheless, its effects on soil properties and microbes remain contentious. In this study, we examined the effects of four different [...] Read more.

The partial substitution of chemical fertilizer with organic fertilizer has been regarded as an effective strategy for enhancing crop yield and soil quality. Nevertheless, its effects on soil properties and microbes remain contentious. In this study, we examined the effects of four different fertilization strategies (including without fertilizer (CK), 100% chemical fertilizer (NPK), 30% organic fertilizer + 70% chemical fertilizer (LOM) and 60% organic fertilizer + 40% chemical fertilizer (HOM)) on soil nutrients and microbial communities through metagenomic sequencing in a Camellia oleifera field experiment. Compared to CK and NPK, HOM significantly increased SOC, TN, TP, AK and AN contents. The substitution of organic fertilizer notably increased Camellia oleifera yield, with the highest increase of 93.35% observed in HOM relative to NPK. Soil bacterial and fungal communities responded inconsistently to fertilization patterns. Bacteria predominated as the main soil microorganisms, and higher rates of organic fertilizer substitution facilitated a shift from bacterial to fungal communities. Organic fertilizer substitution significantly increased soil bacteria diversity and fungal richness, particularly in the HOM. Soil bacterial community structure was more sensitive to fertilization regimes than soil fungi. High rates of organic fertilizer substitution substantially suppressed oligotrophic and increased copiotrophic bacterial communities. Mucoromycota emerged as the dominant fungal group, with a considerable increment in HOM soils. SOC and TN were the main factors affecting Camellia oleifera yield and shaping soil bacteria and fungal diversity and composition. This study provided crucial insights into the ecological implications of organic fertilizer application and the potential of managing soil microorganisms for sustainable Camellia oleifera productivity. Full article

(This article belongs to the Special Issue Restoring the Integrated Behaviour of the Soil-Plant-Microbe System, 2nd Edition)

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20 pages, 14107 KB

Open AccessArticle

Unveiling Species Diversity Within Early-Diverging Fungi from China XI: Eight New Species of Cunninghamella (Mucoromycota)

by Yang Jiang, Heng Zhao, Xin-Yu Ji, Zi-Ying Ding, Wen-Xiu Liu, Fei Li, Shi Wang, Xiao-Yong Liu and Zhe Meng

Microorganisms 2025, 13(11), 2508; https://doi.org/10.3390/microorganisms13112508 - 31 Oct 2025

Abstract

The genus Cunninghamella is widely distributed, primarily saprotrophic, occasionally endophytic and phytopathogenic. Analysis based on the internal transcribed spacer (ITS), the large subunit (LSU) of ribosomal DNA, and translation elongation factor 1 alpha (TEF1α), along with morphological comparisons, resulted in a [...] Read more.

The genus Cunninghamella is widely distributed, primarily saprotrophic, occasionally endophytic and phytopathogenic. Analysis based on the internal transcribed spacer (ITS), the large subunit (LSU) of ribosomal DNA, and translation elongation factor 1 alpha (TEF1α), along with morphological comparisons, resulted in a discovery of eight new species. Molecular phylogenetic analyses placed each of these new species within well-supported clades. Cunninghamella crassior sp. nov., with short and thick spines, and C. fusca sp. nov. with brown sporangiola, are sister clades to each other. C. diffundens sp. nov., containing dispersed granules in sporangiola, is closely related to C. irregularis Zhao. C. tuberculata sp. nov., producing sporangiola with nodule-like protrusions. C. fulvicolor sp. nov., sister to C. irregularis, forms yellowish-brown pigmented colonies. C. guttulata sp. nov., with teardrop-shaped sporangiola, and C. inaequalis sp. nov., with uneven sporangiola, are both closely related to C. regularis Zhao. C. monosporangiola sp. nov., characterized by only one sporangiolum on some vesicles, is sister to C. verrucosa Zhao. This study represents the eleventh installment in a series investigating early-diverging fungal diversity in China and expands the number of accepted species in Cunninghamella to 39. Full article

(This article belongs to the Section Environmental Microbiology)

15 pages, 12642 KB

Open AccessArticle

Effect of Earthworm Digestion on Abundance, Composition and Diversity of Bacterial Pathogens in Sewage Sludge from Wastewater Treatment Plants

by Manuel Aira and Jorge Domínguez

Microorganisms 2025, 13(11), 2507; https://doi.org/10.3390/microorganisms13112507 - 31 Oct 2025

Abstract

The increased production of sewage sludge is a major environmental concern as the sludge contains hazardous components, particularly human bacterial pathogens (HBPs). Transit of sewage sludge through the earthworm gut reduce or even eliminate HBPs and modify bacterial taxonomic and functional composition. However, [...] Read more.

The increased production of sewage sludge is a major environmental concern as the sludge contains hazardous components, particularly human bacterial pathogens (HBPs). Transit of sewage sludge through the earthworm gut reduce or even eliminate HBPs and modify bacterial taxonomic and functional composition. However, it is unclear whether the effect is general or dependent on the type of sewage sludge involved. We characterized the taxonomic and functional profiles of bacterial assemblages in sewage sludge from different wastewater treatment plants (WWTPs), before (sludge) and after earthworm gut transit (casts). We found that composition and diversity of both taxonomic and functional bacterial communities of sludge and casts were significantly different. However, these differences varied among WWTPs with both increases and decreases in composition and diversity after gut transit. Interestingly, most bacterial taxa present in earthworm casts were not detected in the original sewage sludge. All sludge samples initially contained low levels of HBPs, which were significantly reduced or eliminated in earthworm casts. Nevertheless, gut transit increased the abundance of some HBPs. Further studies should determine whether vermicomposting effectively eliminates these HBPs and whether the differences in earthworm cast bacterial communities, which are dependent on the sewage sludge source, persist in the final vermicompost. Full article

(This article belongs to the Section Environmental Microbiology)

18 pages, 663 KB

Open AccessArticle

Microbiological Investigation and Clinical Efficacy of Professional Topical Fluoride Application on Streptococcus mutans and Selemonas sputigena in Orthodontic Patients: A Randomized Controlled Clinical Trial

by Alessia Pardo, Stefano Marcoccia, Camilla Montagnini, Annarita Signoriello, Elena Messina, Paolo Gaibani, Gloria Burlacchini, Camillo Salgarelli, Caterina Signoretto and Nicoletta Zerman

Microorganisms 2025, 13(11), 2506; https://doi.org/10.3390/microorganisms13112506 - 31 Oct 2025

Abstract

Fluoride prophylaxis is a cornerstone in preventing dental caries, a disease for which orthodontic patients are at high risk due to the reduced effectiveness of home oral hygiene and increased plaque accumulation. Recent evidence defines caries as polymicrobial, involving Streptococcus mutans, Lactobacilli, [...] Read more.

Fluoride prophylaxis is a cornerstone in preventing dental caries, a disease for which orthodontic patients are at high risk due to the reduced effectiveness of home oral hygiene and increased plaque accumulation. Recent evidence defines caries as polymicrobial, involving Streptococcus mutans, Lactobacilli, and emerging species such as Selenomonas sputigena. This prospective, randomized, controlled study evaluated professional topical fluoride in the form of gel and varnish in 68 patients aged 8–17 years wearing fixed orthodontic appliances. Participants were divided into three equal groups: two intervention groups and one control group. Clinical parameters (DMFT, salivary pH, PCR%) and microbiological analyses of plaque and saliva (oral Streptococci, S. mutans, S. sputigena, Lactobacilli, total bacterial count) were assessed at baseline (T0) and after 4 months (T1), following professional hygiene and fluoride application for the intervention groups. At T1, salivary pH increased in the gel group, and PCR% decreased significantly in all groups, with the most pronounced decrease observed in the varnish group. PCR analysis showed a higher rate of S. mutans and S. sputigena negativization in intervention groups. Culture-based analyses revealed reductions in oral Streptococci and Lactobacilli in intervention groups, while levels increased in controls. Overall, both clinical and microbiological variables indicated improvements in the fluoride-treated groups compared to controls, highlighting the efficacy of professional fluoride prophylaxis in orthodontic patients. Full article

(This article belongs to the Special Issue Oral Microbes and Human Health, Second Edition)

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