Microorganisms

22 pages, 1071 KB

Open AccessArticle

Oral Administration of the Probiotic Lacticaseibacillus rhamnosus CA15 in a Large Cohort of Women with Bacterial Vaginosis and Mixed Vaginitis: Clinical Evidence from a Randomized, Double-Blind, Placebo-Controlled Study

by Alessandra Pino, Amanda Vaccalluzzo, Stefano Cianci, Marco Palumbo, Giuseppe Caruso, Cinzia Caggia and Cinzia L Randazzo

Microorganisms 2025, 13(12), 2651; https://doi.org/10.3390/microorganisms13122651 - 21 Nov 2025

Abstract

Probiotics represent a valuable approach to boost vaginal health. A randomized double-blind placebo-controlled study was conducted to confirm the health benefits of the orally administered probiotic Lacticaseibacillus rhamnosus CA15 strain in a large cohort of women with bacterial vaginosis and mixed vaginitis, mainly [...] Read more.

Probiotics represent a valuable approach to boost vaginal health. A randomized double-blind placebo-controlled study was conducted to confirm the health benefits of the orally administered probiotic Lacticaseibacillus rhamnosus CA15 strain in a large cohort of women with bacterial vaginosis and mixed vaginitis, mainly related to mild aerobic vaginitis and vulvovaginal candidiasis. Recruited women were randomly assigned in a 1:1 ratio to receive, for 10 days, oral capsules containing 1 × 10¹⁰ colony-forming units of the L. rhamnosus CA15 strain (Active group) or a placebo (Placebo group). Two-hundred women completed this study. Clinical and microbiological parameters were assessed at baseline (T0), 10 days (T1), and 30 days post-treatment (T2). In addition, quality of life was evaluated at T0 and T2. The study protocol was registered on clinicaltrials.gov (ID: NCT05796921). The oral administration of the L. rhamnosus CA15 strain resulted in significant improvements in clinical characteristics of vaginal dysbiosis as well as changes in the vaginal microbiota composition. Furthermore, participants allocated to the Active group reported enhancements in physical and psychological health, social relations, environment, and overall quality of life. No significant changes were observed in the Placebo group. The present study highlights the ability of the L. rhamnosus CA15 to manage vaginal dysbiosis, offering new perspectives for the treatment and prevention of common gynecological disorders. Full article

(This article belongs to the Section Antimicrobial Agents and Resistance)

16 pages, 566 KB

Open AccessArticle

Invertebrate-Derived DNA (iDNA) to Identify Sand Flies’ Bloodmeal: A Molecular Approach to Identifying Hosts in Blood-Feeding Vectors of Leishmaniasis

by Bruno Oliveira Cova, Bruno Henrique Saranholi, Carla Cristina Gestich, Paulo Roberto Machado, Adriano Figueiredo Monte-Alegre and Albert Schriefer

Microorganisms 2025, 13(12), 2650; https://doi.org/10.3390/microorganisms13122650 - 21 Nov 2025

Abstract

DNA metabarcoding data obtained by next generation sequencing (NGS) has been used to identify species in mixed biological samples, such as DNA from the gut content of invertebrates that feed on vertebrates (invertebrate-derived DNA, iDNA). This investigation employed DNA metabarcoding approach to determine [...] Read more.

DNA metabarcoding data obtained by next generation sequencing (NGS) has been used to identify species in mixed biological samples, such as DNA from the gut content of invertebrates that feed on vertebrates (invertebrate-derived DNA, iDNA). This investigation employed DNA metabarcoding approach to determine vertebrate hosts of female phlebotomine sand flies, blood-feeding leishmaniasis vectors. We evaluated performance across three mitochondrial markers: a mammal-specific mini-barcode (16S rRNA), a pan-vertebrate mini-barcode (12S rRNA), and a standard CytB barcode region. Phlebotomine sand flies collections occurred in the Cacao Region of Southeastern Bahia, Brazil, an American Tegumentary Leishmaniasis (ATL) endemic zone. Our analysis examined iDNA from forty female specimens pooled in thirteen samples of seven sand fly species, including confirmed ATL vectors. Metabarcoding-derived operational taxonomic units (OTUs) underwent taxonomic assignment through comparison with GenBank NCBI^® reference databases. Results identified twenty vertebrate OTUs: primates (four OTUs), rodents (four), ungulates (five), marsupials (one), plus a domestic dog and a chicken. Notably, non-mammalian taxa, including reptiles (one OTU) and amphibians (three), were detected. The iDNA metabarcoding approach allowed us to accurately sample the diversity of phlebotomine sandflies’ bloodmeals in a single specimen of a non-engorged female sand fly with mixed feeding. Full article

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

22 pages, 962 KB

Open AccessReview

Biofilm as a Key Element in the Bacterial Pathogenesis of Forest Trees: A Review of Mechanisms and Ecological Implications

by Miłosz Tkaczyk

Microorganisms 2025, 13(12), 2649; https://doi.org/10.3390/microorganisms13122649 - 21 Nov 2025

Abstract

Bacterial diseases of forest trees represent an increasing threat to ecosystem health and the sustainability and resilience of forest management, particularly under changing climate conditions. One of the key yet still insufficiently understood adaptive mechanisms of pathogens is biofilm formation—a structured community of [...] Read more.

Bacterial diseases of forest trees represent an increasing threat to ecosystem health and the sustainability and resilience of forest management, particularly under changing climate conditions. One of the key yet still insufficiently understood adaptive mechanisms of pathogens is biofilm formation—a structured community of bacterial cells embedded in a matrix of extracellular polymeric substances (EPS), which provides protection against stress factors, biocides, and the host’s defensive responses such as antimicrobial compounds or immune reactions.. This paper presents a comprehensive review of current knowledge on the role of biofilms in the bacterial pathogenesis of forest trees, covering their formation mechanisms, molecular regulation, and ecological significance. Four key stages of biofilm development are discussed—adhesion, microcolony formation, EPS production, and dispersion—along with the roles of quorum sensing systems and c-di-GMP-based signaling in regulating these processes. Examples of major tree pathogens are presented, including Pseudomonas syringae, Erwinia amylovora, Xylella fastidiosa, the Brenneria–Gibbsiella complex associated with Acute Oak Decline (AOD) andLonsdalea populi. Biofilm formation is shown to play a crucial role in the colonization of xylem, leaf surfaces, and tissues undergoing necrosis, where biofilms may stabilize decomposition zones and support saprophytic–pathogenic transitions. In the applied section, the concept of “biofilm-targeted control” is discussed, encompassing both chemical and biological strategies for disrupting biofilm structure—from quorum-sensing inhibitors and EPS-degrading enzymes to the use of biosurfactants and antagonistic microorganisms. The need for in situ research in forest environments and the adaptation of advanced imaging (CLSM, micro-CT) and metagenomic analyses to tree systems is also emphasized. This review concludes that biofilms are not merely a physiological form of bacterial organization but a complex adaptive system essential for the survival and virulence of pathogens in forest ecosystems. Understanding their functions is fundamental for developing sustainable and ecologically safe phytosanitary strategies for forest protection. Full article

(This article belongs to the Special Issue Beneficial Biofilms: From Mechanisms to Applications)

15 pages, 1786 KB

Open AccessArticle

Effects of Human Trampling on Soil Microbial Community Assembly in Yangzhou Urban Forest Park

by Jingwei Lian, Liwen Li, Xin Wan, Dongmei He, Yingzhou Tang, Wei Xing and Yingdan Yuan

Microorganisms 2025, 13(12), 2648; https://doi.org/10.3390/microorganisms13122648 - 21 Nov 2025

Abstract

Human trampling in urban forest parks has received increasing attention, yet its effects on microbial community assembly remain elusive. This study investigated how trampling influences soil physicochemical properties and microbial communities in Zhuyuwan Scenic Area. Neutral and null community models were used to [...] Read more.

Human trampling in urban forest parks has received increasing attention, yet its effects on microbial community assembly remain elusive. This study investigated how trampling influences soil physicochemical properties and microbial communities in Zhuyuwan Scenic Area. Neutral and null community models were used to analyze the effects of trampling on microbial assembly processes. Trampling altered both soil physicochemical properties and microbial diversity. Fungal richness differed significantly between control and light-trampling plots. Soil bulk density (SD) was strongly negatively correlated with other soil physical properties, which were positively intercorrelated. Model analyses showed that light trampling strengthened stochastic processes in bacterial community assembly, whereas heavy trampling reduced this effect. Increasing trampling intensity intensified the influence of stochastic processes on fungal community assembly. Bacterial communities were mainly shaped by heterogeneous selection, while fungal communities were primarily governed by dispersal limitation. These results enhance understanding of how trampling disturbance influences microbial community assembly and provide a theoretical basis for the ecological management and restoration of urban forest parks. Full article

(This article belongs to the Section Environmental Microbiology)

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14 pages, 2584 KB

Open AccessArticle

Highly Alkaline-Resistant Enterococcus faecalis Induces Compromised M1 Polarization and Phagocytosis of Macrophage via Z-DNA Binding Protein 1

by Yifang Xiao, Runze Liu, Yanling Yang, Xinlu Li, Yi Min and Wei Fan

Microorganisms 2025, 13(12), 2647; https://doi.org/10.3390/microorganisms13122647 - 21 Nov 2025

Abstract

Persistent apical periodontitis (PAP) of human teeth is related to Enterococcus faecalis (E. faecalis) with higher alkaline resistance. This study aimed to investigate how highly alkaline-resistant (HAR) E. faecalis modulates macrophage M1 polarization and phagocytosis via Z-DNA-binding protein 1 (ZBP1). HAR [...] Read more.

Persistent apical periodontitis (PAP) of human teeth is related to Enterococcus faecalis (E. faecalis) with higher alkaline resistance. This study aimed to investigate how highly alkaline-resistant (HAR) E. faecalis modulates macrophage M1 polarization and phagocytosis via Z-DNA-binding protein 1 (ZBP1). HAR E. faecalis was generated through serial alkaline passaging. RAW264.7 macrophages were infected with standard or HAR E. faecalis. M1 polarization markers and Cd274 (Programmed death-ligand 1 (PD-L1)) were profiled by RT-qPCR. ZBP1 was detected by RT-qPCR and immunofluorescence staining, and was silenced using small interfering RNA (siRNA). iNOS, ZBP1 and PD-L1 proteins were analyzed by Western blotting. The phagocytosis of CFDA-SE-labeled bacteria was then quantified by confocal microscopy and flow cytometry. The results showed that HAR E. faecalis induced significantly lower expression of ZBP1, M1 polarization markers and Cd274 in macrophages than the standard strain. After ZBP1 knock-down, expression of these markers decreased. Macrophages phagocytosed much fewer HAR E. faecalis than the standard strain. After ZBP1 knock-down, the differences between the two strains disappeared. In conclusion, HAR E. faecalis induced compromised M1 polarization and phagocytosis of macrophage via ZBP1. These findings may provide new insights into the pathogenesis and treatment of PAP. Full article

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

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18 pages, 1852 KB

Open AccessArticle

Construction of Recombinant Escherichia coli Expressing Ammonia Assimilation Genes and Evaluation of Its Effect on Removing Ammonium Nitrogen (NH₄⁺-N)

by Pan Pan, Yongkun Yang, Runxuan Shi, Yulin Kang, Hanli Xu, Xiyu Cheng, Qiong Yan and Honggang Hu

Microorganisms 2025, 13(12), 2646; https://doi.org/10.3390/microorganisms13122646 - 21 Nov 2025

Abstract

Treating wastewater with high ammonium nitrogen (NH₄⁺-N) is a public and environmental priority. Unlike nitrification–denitrification, ammonia assimilation channels NH₄⁺-N into the glutamate biosynthetic pathway, avoiding gaseous nitrogen species (NOx, N₂). Here we engineered Escherichia coli [...] Read more.

Treating wastewater with high ammonium nitrogen (NH₄⁺-N) is a public and environmental priority. Unlike nitrification–denitrification, ammonia assimilation channels NH₄⁺-N into the glutamate biosynthetic pathway, avoiding gaseous nitrogen species (NOx, N₂). Here we engineered Escherichia coli to enhance ammonia assimilation by co-expressing three key genes, gdhA, glnA, and guaA. The genes were synthesized, cloned into expression plasmids, and introduced into E. coli BL21 for IPTG-inducible expression. Expression of the target proteins at expected sizes was observed, and NH₄⁺-N removal was assessed in flask fermentations. Recombinant strains exhibited significantly higher NH₄⁺-N reduction than the empty vector control; among them, BL21(pET-gdhA-glnA-guaA) performed best, achieving a maximum removal efficiency of 90.09% under the tested conditions. These results indicate that reinforcing the glutamate pathway through multi-gene co-expression can effectively lower NH₄⁺-N in culture and provide a basis for developing recombinant bacteria for practical sewage treatment. Full article

(This article belongs to the Section Environmental Microbiology)

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15 pages, 414 KB

Open AccessReview

Biotic and Abiotic Factors on Rhizosphere Microorganisms in Grassland Ecosystems

by Bademu Qiqige, Yuzhen Liu, Yu Tian, Li Liu, Weiwei Guo, Ping Wang, Dayou Zhou, Hui Wen, Qiuying Zhi, Yuxuan Wu, Xiaosheng Hu, Ming Li and Junsheng Li

Microorganisms 2025, 13(12), 2645; https://doi.org/10.3390/microorganisms13122645 - 21 Nov 2025

Abstract

Rhizosphere microbiota, serving as pivotal drivers of multifunctionality in grassland ecosystems, are jointly shaped by the dual influences of biotic and abiotic factors. Among biotic components, plant functional types selectively modulate microbial communities through root exudate specificity, while soil fauna (e.g., nematodes and [...] Read more.

Rhizosphere microbiota, serving as pivotal drivers of multifunctionality in grassland ecosystems, are jointly shaped by the dual influences of biotic and abiotic factors. Among biotic components, plant functional types selectively modulate microbial communities through root exudate specificity, while soil fauna (e.g., nematodes and earthworms) drive microbial interaction networks via biophysical disturbances and trophic cascades. However, excessive nematode grazing suppresses the hyphal extension of arbuscular mycorrhizal fungi (AMF). Moderate grazing facilitates the proliferation of ammonia-oxidizing bacteria through fecal input, whereas intensive grazing induces topsoil compaction, leading to a dramatic 40–60% reduction in lipopolysaccharide content in Gram-negative bacteria. Long-term chemical fertilization significantly decreases the fungal-to-bacterial ratio, while organic amendments enhance microbial carbon use efficiency by activating extracellular enzymatic activities. Regarding abiotic factors, the stoichiometric characteristics of soil carbon, nitrogen, and phosphorus directly regulate microbial metabolic strategies. Hydrological dynamics influence microbial respiratory pathways through oxygen partial pressure shifts—drought stress inhibits mycelial network development. Future research should focus on predicting the emissions of gases such as N₂O (ozone monomer) and optimizing nitrogen fertilizer management to significantly reduce greenhouse gas emissions at the source. The soil organic carbon storage in grassland ecosystems is extremely large. Effective prediction and management can make these soils become important carbon “sinks”, offsetting the carbon dioxide in the atmosphere. At the same time, transcriptomics and metabolic flux analysis should be combined with multi-omics technologies and in situ labeling methods to provide theoretical basis and technical support for developing mechanism-based and predictable grassland restoration and adaptive management strategies from both macroscopic and microscopic perspectives. Full article

(This article belongs to the Section Environmental Microbiology)

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14 pages, 1532 KB

Open AccessArticle

Downregulation of oar-miR-125b Drives Blood–Brain Barrier Breakdown Through the TNFSF4–NF-κB Inflammatory Axis in Enterococcus Faecalis Meningitis

by Longling Jiao, You Wu, Borui Qi, Pengfei Zhao, Ming Zhou, Runze Zhang, Yongjian Li, Jingjing Ren, Shuzhu Cao and Yayin Qi

Microorganisms 2025, 13(12), 2644; https://doi.org/10.3390/microorganisms13122644 - 21 Nov 2025

Abstract

Bacterial meningitis involves complex molecular networks, including microRNA-mediated regulation of inflammatory responses; however, the specific role of Ovis aries microRNA-125b (oar-miR-125b) in this process remains poorly understood. In this study, using a lamb model of Enterococcus faecalis-induced meningitis, we observed significant downregulation of [...] Read more.

Bacterial meningitis involves complex molecular networks, including microRNA-mediated regulation of inflammatory responses; however, the specific role of Ovis aries microRNA-125b (oar-miR-125b) in this process remains poorly understood. In this study, using a lamb model of Enterococcus faecalis-induced meningitis, we observed significant downregulation of oar-miR-125b, which inversely correlated with its newly identified target, Tumor Necrosis Factor Superfamily Member 4 (TNFSF4). Dual-luciferase reporter assays confirmed that oar-miR-125b directly binds to the 3′ Untranslated Region (3′UTR) of TNFSF4 but not to the 3′UTRs of Kelch Like Family Member 31 (KLHL31) or NF-κB Inhibitor Interacting Ras Like 2 (NKIRAS2). Mechanistically, decreased oar-miR-125b expression relieves its repression of TNFSF4, leading to NF-κB pathway activation and blood–brain barrier disruption. Collectively, our results demonstrate that oar-miR-125b serves as a key anti-inflammatory regulator in bacterial meningitis by targeting TNFSF4 and constraining NF-κB signaling, highlighting its potential as a therapeutic target for attenuating neuroinflammation in meningitis. Full article

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

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Microorganisms, Volume 13, Issue 12 (December 2025) – 8 articles

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