Microorganisms

13 pages, 3265 KB

Open AccessArticle

Seasonal Variations in the Occurrence of SARS-CoV-2 RNA Recovered from Wastewater Treatment Facilities (WWTFs) Within South Africa

by Kingsley Ehi Ebomah, Luyanda Msolo, Velisa Vuyolwethu Qongwe, Okuhle Mayoyo, Piwe Athi Ntlati, Balisa Ngqwala, Nolonwabo Nontongana, Renee Street, Rabia Johnson and Anthony Ifeanyi Okoh

Microorganisms 2026, 14(2), 495; https://doi.org/10.3390/microorganisms14020495 - 18 Feb 2026

Abstract

Several researchers have documented the occurrence of the unfamiliar severe acute respiratory syndrome coronavirus 2 ribonucleic acid (also known as SARS-CoV-2 RNA) in various raw wastewater (WW) samples analyzed globally. The efficiency of strategic WW-based epidemiology (WBE) approach as a timely cautioning tool [...] Read more.

Several researchers have documented the occurrence of the unfamiliar severe acute respiratory syndrome coronavirus 2 ribonucleic acid (also known as SARS-CoV-2 RNA) in various raw wastewater (WW) samples analyzed globally. The efficiency of strategic WW-based epidemiology (WBE) approach as a timely cautioning tool for human coronavirus disease-2019 (COVID) and other similar outbreaks is highly promising. This strategy offers a cost-effective, population-wide surveillance tool that can detect rising case trends, from days to weeks before clinical reports, thus enabling proactive public health interventions. This study aimed to detect the occurrence of the viral genome in WW over four seasons, which contributes to the database for multi-plant surveillance research in South Africa. About 480 WW influent samples were amassed from ten sampling points situated in nine wastewater treatment facilities (WWTFs) in Amathole District Municipality (ADM) located in the Province of Eastern Cape (EC), South Africa (SA). The study was carried out for a period of one year. Quantitative real-time polymerase chain reaction (i.e., RT-PCR) was operated to identify the viral genomes in the respective total RNA samples. Of the 480 extracted RNA samples, 210 (44%) were positive with viral genome copies (gc) that ranged from 700 to 40,000 GC/mL. Our results were contrasted with existing COVID-19-positive cases throughout the COVID omicron wave in the ECP. Variations in gc were observed across different seasons, with the highest GC observed in winter. In contrast, there were significant inconsistencies in the existing data of COVID-19 clinical cases, thus indicating no connection between both data. However, with more similar studies, advanced innovative WBE strategies could possibly act as prompt warning tools to signal public health officials about potential future outbreaks. Full article

(This article belongs to the Special Issue Pathogen Surveillance in Wastewater)

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15 pages, 3366 KB

Open AccessArticle

Polycyclic Aromatic Hydrocarbon Pollution Stress Impairs Soil Enzyme Activity and Microbial Community

by Yuancheng Wang, Donglei Wu, Junxiang Liu and Haolong Xu

Microorganisms 2026, 14(2), 494; https://doi.org/10.3390/microorganisms14020494 - 18 Feb 2026

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are widely prevalent harmful organic pollutants. Enzymatic activities (such as those of dehydrogenases, catalase, protease and urease), as well as the microbial community structure and assembly (through 16S and ITS amplicon sequencing), were evaluated 90 days after PAH contamination [...] Read more.

Polycyclic aromatic hydrocarbons (PAHs) are widely prevalent harmful organic pollutants. Enzymatic activities (such as those of dehydrogenases, catalase, protease and urease), as well as the microbial community structure and assembly (through 16S and ITS amplicon sequencing), were evaluated 90 days after PAH contamination and compared to those in normal soils. Microbial activity, as indicated by soil urease, catalase, and protease activities, was inhibited under PAH stress. Furthermore, PAH stress exerted significant impacts on the soil microbial community structure. Notably, PAH stress reduced soil bacterial and fungal biomass and inhibited the abundance of microbial taxa involved in soil carbon and nitrogen cycling (e.g., Marmoricola, Pedobacter, and Streptomyces), along with the majority of predicted responsive metabolic functions, particularly those related to amino acid and carbohydrate metabolism. PAH stress enriched PAH-degrading microorganisms, including Pseudomonas, Mycobacterium, Bacillus, Cycloclasticus, and Flavobacterium. The niche breadth of bacterial and fungal communities decreased significantly under PAH stress (51.5 and 14.1, respectively) compared to that in normal soil (63.7 and 22.3), which was further supported by Beta Nearest Taxon Index and co-occurrence network analysis. PAH stress increased the contribution of heterogeneous selection to soil microbial assembly (100%) compared to that in normal soil (80%). Thus, the majority of microbial community responses to PAH stress were adversely affected. These results suggest that PAH contamination may profoundly affect the soil quality by restricting the survival space of bacteria and fungi. Full article

(This article belongs to the Section Environmental Microbiology)

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18 pages, 1424 KB

Open AccessArticle

Unraveling the Coevolutionary Dynamics of Phage and Bacterial Protein Warfare Occurring in the Drains of Beef-Processing Plants

by Vignesh Palanisamy, Joseph M. Bosilevac, Darryll A. Barkhouse, Sarah E. Velez and Sapna Chitlapilly Dass

Microorganisms 2026, 14(2), 493; https://doi.org/10.3390/microorganisms14020493 - 18 Feb 2026

Abstract

Phages, the most abundant entities on Earth, exhibit a complex interplay with bacteria, especially within environmental biofilms, resulting in an ecological arms race. This study investigates the interaction between phages and bacteria in the drains of beef-processing plants using high-throughput sequencing and metagenomic [...] Read more.

Phages, the most abundant entities on Earth, exhibit a complex interplay with bacteria, especially within environmental biofilms, resulting in an ecological arms race. This study investigates the interaction between phages and bacteria in the drains of beef-processing plants using high-throughput sequencing and metagenomic analysis. Metagenomic data collected from 75 drain samples from beef-processing plants were analyzed to investigate phage–bacterial interactions. First, assembled contigs were screened to identify viral sequences, which were then taxonomically annotated to determine the viral composition, including phages. Functional annotation of these viral sequences provided information about the viral genes and their roles in bacterial interactions specifically associated with attack and counterattack of bacteria. In parallel, bacterial contigs were examined to identify genes associated with antiphage defense systems, providing insights into the strategies adapted by bacteria to resist phage infection. Taxonomic annotation of viral sequences from the bulk metagenomic data revealed the presence of phages targeting Pseudomonas, Klebsiella, and Enterococcus. The higher abundance of Pseudomonas phages aligns with our previous study, where Pseudomonas was identified as the dominant bacterial genus, suggesting potential copersistence of phages and their hosts. Functional annotation of phage contigs revealed infective and lysis-related genes, highlighting their potential role in bacterial attack. Conversely, bacterial contigs encoded antiphage defense systems, including CRISPR-Cas, restriction–modification, and other defense-related genes. The study also uncovered the presence of anti-CRISPR proteins in phages, suggesting a counterattack on the bacterial defense. These findings provide evidence for phage attack, bacterial defense, and phage counterattack and may showcase the ongoing coevolutionary arms race between phages and bacteria. While this evidence looks promising, these results remain preliminary and further studies are needed to validate these findings. Still, this study provides a foundational understanding of bacteria–phage coexistence in beef-processing plant drains and paves the way for further explorations of these intricate interactions and their possible applications in controlling pathogenic microorganisms within biofilms. Full article

(This article belongs to the Section Environmental Microbiology)

19 pages, 1157 KB

Open AccessReview

Current Applications and Immunological Considerations of Salmonella enterica Serovar Typhimurium as a Vaccine Vector

by Adam S. Hassan, Kaitlin Winter, Charles M. Dozois, Brian J. Ward and Momar Ndao

Microorganisms 2026, 14(2), 492; https://doi.org/10.3390/microorganisms14020492 - 18 Feb 2026

Abstract

Live attenuated Salmonella enterica serovar Typhimurium has been investigated for decades as an orally delivered vaccine vector due to its ability to target the intestinal mucosa and engage both innate and adaptive immune responses. In humans, S. Typhimurium infection is largely restricted to [...] Read more.

Live attenuated Salmonella enterica serovar Typhimurium has been investigated for decades as an orally delivered vaccine vector due to its ability to target the intestinal mucosa and engage both innate and adaptive immune responses. In humans, S. Typhimurium infection is largely restricted to the gastrointestinal tract, distinguishing it from Salmonella Typhi and providing a rationale for its use in mucosal vaccine strategies. In this review, we discuss the biological features of S. Typhimurium that support its use as a vaccine vector and summarize current understanding of the immune responses generated during wild-type infection, including innate activation and downstream T cell and B cell responses. We compare key biological differences between Salmonella Typhi and S. Typhimurium and outline emerging vector design strategies, including delayed attenuation and chromosomal integration of heterologous antigens. We then review applications of attenuated S. Typhimurium vectors targeting viral, bacterial, and parasitic pathogens, highlighting shared immunological outcomes and design principles across platforms. Finally, we discuss recent advances in vector engineering, including chromosomal integration of heterologous antigens, as well as remaining gaps in knowledge related to the durability of immune responses and translational considerations. Full article

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

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17 pages, 2040 KB

Open AccessArticle

Use of Thermal and Emerging Non-Thermal Treatments Reveal Biomolecular and Morphological Changes in Pathogenic E. coli

by Maxsueli Machado, Jelmir Craveiro Andrade, Eduardo Eustáquio de Souza Figueiredo and Carlos Adam Conte-Junior

Microorganisms 2026, 14(2), 491; https://doi.org/10.3390/microorganisms14020491 - 18 Feb 2026

Abstract

(1) Background: We sought to explore the changes in the biomolecular profile and morphology of Pathogenic heat-resistant E. coli isolated from animal-based food. (2) Methods: Six strains underwent heat (60 °C for 6 min), ultrasound (US; 299 W), UVC (4950 mJ/cm²), [...] Read more.

(1) Background: We sought to explore the changes in the biomolecular profile and morphology of Pathogenic heat-resistant E. coli isolated from animal-based food. (2) Methods: Six strains underwent heat (60 °C for 6 min), ultrasound (US; 299 W), UVC (4950 mJ/cm²), and combined treatments (UVC+US and heat+UVC). Afterwards, biomolecular characterization across four spectral regions was evaluated by Fourier transform infrared (FT-IR) spectroscopy and analyzed by principal component analysis (PCA) for treated and non-treated strains (control group). These regions are fatty acids (3010–2800 cm⁻¹), proteins and peptides (1700–1200 cm⁻¹), carbohydrates (1200–900 cm⁻¹), and amide A (3280–3120 cm⁻¹). Additionally, treated and untreated strains were assessed for surface damage using scanning electron microscopy (SEM). (3) Results: Among all the regions studied, the amide A and fatty acids regions exhibited the most significant variations in absorbance for treated strains compared to the control. Treatments such as US, heat, and UVC+US tended to increase Principal Components (PCs) and, consequently, absorbance. On the other hand, UVC and heat+UVC showed the opposite trend in these regions. SEM images showed filamentous cells for strains treated with UVC and UVC+US, indicating that cells continued to replicate under these conditions. These results highlight how thermal and non-thermal treatments influence specific biomolecular and morphological regions of E. coli. The methodologies used provide reliable data for understanding stress responses, which can guide the development of more effective technologies for eliminating multi-resistant pathogens. Full article

(This article belongs to the Special Issue The Evolution of Foodborne Pathogens in the 4.0 Era: A Food Safety Perspective)

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18 pages, 2595 KB

Open AccessArticle

Characteristics, Whole Genome Analysis of a Virulent Phage from Avian-Derived Enterococcus faecalis and Its Application in Poultry Product Processing Safety

by Xiaoming Li, Mengli Zhao, Lei Zhang, Guobo Sun, Xiujun Duan and Guoshun Chen

Microorganisms 2026, 14(2), 490; https://doi.org/10.3390/microorganisms14020490 - 18 Feb 2026

Abstract

To explore high-quality phage resources for controlling Enterococcus faecalis (E. faecalis) contamination, a virulent phage vB-Efa1 was isolated and purified from poultry slaughterhouse sewage in this study. Its biological characteristics, whole-genome features, and potential in ensuring poultry product processing safety were [...] Read more.

To explore high-quality phage resources for controlling Enterococcus faecalis (E. faecalis) contamination, a virulent phage vB-Efa1 was isolated and purified from poultry slaughterhouse sewage in this study. Its biological characteristics, whole-genome features, and potential in ensuring poultry product processing safety were systematically investigated. The phage belongs to the Siphoviridae family, with an optimal multiplicity of infection (MOI) of 0.1 and a titer of 8.87 lg PFU/mL; it has a 30 min latent period and stable lytic activity, retaining good stability at 25–37 °C, pH 6–8, and 4 °C. Its circular whole genome is 166,586 bp in length with a GC content of 35.46%, encoding 276 genes; no antibiotic resistance genes were detected, and only one low-pathogenic-risk virulence-related sequence was identified. Application tests in poultry products revealed that temperature is the key factor regulating phage titer: the titer stably maintained 5.5–6.6 lg PFU/mL at 4 °C, while proliferating significantly at 25 °C, reaching 7.55–8.38 lg PFU/mL at 12 h. Collectively, vB-Efa1 exhibits superior biological traits, environmental adaptability, and biosafety, making it a promising biocontrol candidate for mitigating E. faecalis contamination in poultry products. Full article

(This article belongs to the Section Microbial Biotechnology)

26 pages, 4050 KB

Open AccessArticle

Uncovering the Prokaryotic Diversity of Hypersaline Soils of Odiel Saltmarshes Natural Area Through Metagenome-Assembled Genomes

by Cristina Galisteo, Fernando Puente-Sánchez, Rafael R. de la Haba, Stefan Bertilsson, Antonio Ventosa and Cristina Sánchez-Porro

Microorganisms 2026, 14(2), 489; https://doi.org/10.3390/microorganisms14020489 - 18 Feb 2026

Abstract

The hypersaline soils of the Odiel Saltmarshes Natural Area in Southwest Spain harbor highly diverse microbial communities adapted to extreme conditions. However, their genomic diversity remains largely unexplored. In addition to high salinity, these soils are contaminated with heavy metals, creating a hostile [...] Read more.

The hypersaline soils of the Odiel Saltmarshes Natural Area in Southwest Spain harbor highly diverse microbial communities adapted to extreme conditions. However, their genomic diversity remains largely unexplored. In addition to high salinity, these soils are contaminated with heavy metals, creating a hostile environment of great interest for studying extremophilic microorganisms and their metabolic adaptations. This study aims to characterize the uncovered prokaryotic taxa as Candidatus species inhabiting the hypersaline soils of the Odiel Saltmarshes, based on their metagenomic assembled genomic sequences. The reconstructed genomes were assessed for quality based on completeness and contamination thresholds and subsequently taxonomically classified. Comparative genomic analysis of six high-quality MAGs revealed key metabolic traits related to survival under extreme salinity and heavy metal conditions. The findings provide new insights about microbial diversity of hypersaline environments and expand the catalog of known prokaryotic genomes. Detailed characterization of six novel Candidatus taxa highlights the unique adaptations of these microorganisms, enhancing our understanding of life in extreme habitats. Full article

(This article belongs to the Special Issue Halophiles)

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14 pages, 1140 KB

Open AccessArticle

Paenibacillus–Pseudomonas Consortium Improves Barley Performance with Minimal Impact on Native Rhizobacterial Community

by Jakub Dobrzyński, Aleksandra Naziębło, Iryna Kulkova, Magdalena Szpytma, Adrianna Antosik, Monika Sitarek-Andrzejczyk and Barbara Wróbel

Microorganisms 2026, 14(2), 488; https://doi.org/10.3390/microorganisms14020488 - 18 Feb 2026

Abstract

The intensive use of mineral nitrogen fertilizers in cereal production contributes to environmental degradation, highlighting the need for more sustainable crop management strategies. Plant growth-promoting bacteria (PGPB) offer a promising alternative; however, their effects on native rhizosphere communities remain underexplored, particularly in barley. [...] Read more.

The intensive use of mineral nitrogen fertilizers in cereal production contributes to environmental degradation, highlighting the need for more sustainable crop management strategies. Plant growth-promoting bacteria (PGPB) offer a promising alternative; however, their effects on native rhizosphere communities remain underexplored, particularly in barley. This study evaluates the impact of a bacterial consortium composed of Paenibacillus sp. Z15 and Pseudomonas sp. KR227 on barley growth, yield, and rhizosphere bacteria under field conditions in temperate climate (2025). Plant biometric traits, photosynthetic pigment content, and soil properties were measured, and rhizobacterial communities were analyzed using 16S rRNA gene (V3–V4) sequencing. The PGPB consortium significantly increased early root biomass (120%), shoot height (7.8%), and grain yield (15.5%), while no significant effects were observed on soil chemistry or photosynthetic pigments. Sequencing revealed no major changes in alpha or beta diversity; however, transient shifts in the relative abundance of specific taxa were detected relatively shortly after inoculation and mostly disappeared by harvest. These findings indicate that the Paenibacillus–Pseudomonas consortium can enhance barley performance without disrupting native rhizobacterial communities. Overall, the results support the potential of PGPB as a sustainable agronomic tool and provide new insights into PGPB–microbiome interactions in barley under field conditions. Full article

(This article belongs to the Special Issue Advances in Agro-Microbiology)

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18 pages, 3423 KB

Open AccessArticle

Responses of Biofilm-Forming Halophilic Calothrix and Coelastrella Strains to Environmental Stressors Associated with Climate Change

by Gabrielle Zammit, Kristina Fenech and Emmanuel Sinagra

Microorganisms 2026, 14(2), 487; https://doi.org/10.3390/microorganisms14020487 - 17 Feb 2026

Abstract

Research into the effects of environmental stressors associated with global climate change (GCC) on cyanobacteria and microalgae is scarce, with bloom-forming planktonic cyanobacteria being the exception. This study aimed to address the issue by assessing morphological and biochemical changes in cyanobacterial and microalgal [...] Read more.

Research into the effects of environmental stressors associated with global climate change (GCC) on cyanobacteria and microalgae is scarce, with bloom-forming planktonic cyanobacteria being the exception. This study aimed to address the issue by assessing morphological and biochemical changes in cyanobacterial and microalgal cells exposed to an increased temperature (T), ultraviolet radiation (UVR) and carbon dioxide (CO₂) concentration. The strains selected were Calothrix sp. SLM0211 and Coelastrella sp. SLM0503, which were isolated from a coastal environment in the central Mediterranean island of Malta. Elevated UVR had a pronounced effect on Calothrix sp. filaments, which produced screening compounds and resorted to trichome coiling to enhance self-shading. Enhanced growth was observed in cultures of Calothrix sp. grown at an increased CO₂ concentration, which produced significantly high amounts of biomass, chlorophylls and carotenoids. An increased T resulted in stunted growth and low biomass accumulation in both strains. Each strain exhibited a unique response to T and UVR stressors, which stimulated the production of exopolymeric substances (EPS) and mycosporine-like amino acids (MAAs) in cultures of Calothrix sp. and lipid production in Coelastrella sp. cells. Our findings indicate that the effects of stressors related to GCC on cyanobacterial and microalgal cells are strain-specific, making changes at community and ecosystem levels difficult to predict. Full article

(This article belongs to the Special Issue Microorganisms: Climate Change and Terrestrial Ecosystems)

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11 pages, 787 KB

Open AccessArticle

Role of Next-Generation Sequencing in Excluding the Nosocomial Origin of a Case of Legionnaires’ Disease Integrating Environmental Surveillance and Clinical Diagnosis

by Francesco Paglione, Cataldo Maria Mannavola, Marilena La Sorda, Maria Luisa Ricci, Maria Scaturro, Silvia Laura Bosello, Roberta Masnata, Francesca Romana Monzo, Sara Vincenti, Patrizia Laurenti, Maurizio Sanguinetti and Flavio De Maio

Microorganisms 2026, 14(2), 486; https://doi.org/10.3390/microorganisms14020486 - 17 Feb 2026

Abstract

Legionella pneumophila (Lp) remains one of the major causes of community- and hospital-acquired pneumonia, yet its diagnosis and source attribution continue to pose significant challenges. Here, we describe the case of an immunocompromised patient who developed Legionnaires’ disease during hospitalization. Following [...] Read more.

Legionella pneumophila (Lp) remains one of the major causes of community- and hospital-acquired pneumonia, yet its diagnosis and source attribution continue to pose significant challenges. Here, we describe the case of an immunocompromised patient who developed Legionnaires’ disease during hospitalization. Following activation of the hospital’s internal surveillance system, Lp and Legionella anisa (L. anisa) were recovered from multiple water distribution points using a simplified culture-based protocol. Whole-genome sequencing (WGS) demonstrated that all environmental isolates belonged to a single clonal strain, whereas the clinical isolate was genetically unrelated, thereby excluding the hospital water system as the source of infection. Although not implicated in the patient’s disease, the detection of both Lp and L. anisa within the plumbing system highlighted underlying structural contamination and the potential masking effect of non-L. pneumophila species during culture-based surveillance. These findings support the integration of conventional microbiological methods with high-resolution genomic tools to enhance surveillance accuracy, support outbreak investigations, and strengthen public health responses. Overall, this case underscores the value of WGS as a decisive tool for source attribution, including the robust exclusion of a suspected nosocomial source, in complex clinical and environmental scenarios. Full article

(This article belongs to the Special Issue Legionella pneumophila: A Microorganism with Thousand Faces, 2nd Edition)

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21 pages, 1855 KB

Open AccessArticle

Draft Genome Sequence of Bacillus sp. Strain 11B20, a Promising Plant-Growth Promoting Bacterium Associated with Maize (Zea mays L.) in the Yaqui Valley, Mexico

by Alina Escalante-Beltrán, Pamela Helué Morales-Sandoval, Amelia Cristina Montoya-Martínez, Edgar A. Cubedo-Ruíz, Rubén Félix-Gastélum, Fannie Isela Parra-Cota and Sergio de los Santos-Villalobos

Microorganisms 2026, 14(2), 485; https://doi.org/10.3390/microorganisms14020485 - 17 Feb 2026

Abstract

Strain 11B20 was isolated from a commercial field of maize (Zea mays L.) located in the Yaqui Valley, Mexico. The draft genome sequence revealed a genomic size of 3,759,824 bp, 41.6% G + C content, 973,288 bp N50, 2 L50, and 29 [...] Read more.

Strain 11B20 was isolated from a commercial field of maize (Zea mays L.) located in the Yaqui Valley, Mexico. The draft genome sequence revealed a genomic size of 3,759,824 bp, 41.6% G + C content, 973,288 bp N50, 2 L50, and 29 contigs. According to the 16S rRNA gene, strain 11B20 belongs to the genus Bacillus. Genome annotation revealed 3952 coding DNA sequences (CDSs) grouped into 319 subsystems. Among these, several CDSs were associated with traits related to plant growth promotion, including (i) virulence, disease, and defense (33 CDSs); (ii) iron acquisition and metabolism (28 CDSs); and (iii) secondary metabolism (6 CDSs), among others. In vitro, metabolic analysis (IAA, siderophore biosynthesis; phosphorus solubilization; and tolerance to thermal, hydric, and saline stress) confirmed the genomic background of this strain. Finally, in planta assays showed that the inoculation of Bacillus sp. 11B20 significantly (p ≤ 0.05) increased the root length (48.2%) and root dry weight (35.4%) versus non-inoculated maize plants. Thus, this is the first report of Bacillus sp. 11B20 as a promising beneficial strain for sustainable corn production, and further research is needed to ensure the success of the application of this strain in agriculture. Full article

(This article belongs to the Special Issue Advances in Plant–Soil–Microbe Interactions)

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24 pages, 4072 KB

Open AccessArticle

Multi-Kingdom Fecal Microbiota Alterations in Horses with Severe Equine Asthma

by Rafaela Santos, Laszlo Hunyadi, Emily Sundman, Luis Morales Luna, Sarah Cate Hyde, Makala Cain, Kagan Migl, Jacob Ancira, Craig Tipton and Fernanda Rosa

Microorganisms 2026, 14(2), 484; https://doi.org/10.3390/microorganisms14020484 - 17 Feb 2026

Abstract

Severe equine asthma (SEA) is a chronic inflammation of airways affecting ~14–20% of adult horses in the Northern Hemisphere. SEA is characterized by a mixed phenotype of T helper cell responses with marked neutrophilia in the bronchoalveolar lavage fluid (BALF) of affected horses. [...] Read more.

Severe equine asthma (SEA) is a chronic inflammation of airways affecting ~14–20% of adult horses in the Northern Hemisphere. SEA is characterized by a mixed phenotype of T helper cell responses with marked neutrophilia in the bronchoalveolar lavage fluid (BALF) of affected horses. Human studies have demonstrated the impact of gut microbiota in many diseases, including asthma susceptibility and severity. However, the potential role of the gut–lung axis in the development and persistence of SEA remains to be determined. This study aimed to identify key bacterial, archaeal, and fungal microbiota alterations in the feces of horses with severe neutrophilic asthma (n = 4) compared to healthy horses (n = 8). Archaea alpha diversity was lower in the feces of SEA-affected horses, but with high abundance of archaea genus Candidatus Nitrosocosmicus, which impacts hydrogen metabolism in horses with SEA. Other key bacterial and fungi species differences lower in SEA included Blautia and Alternaria, respectively. Blautia is associated with positive metabolic health due to its fibrolytic capabilities. Overall, our findings indicate that horses experiencing severe neutrophilic asthma have an imbalance in the intestinal microbiota that may exacerbate systemic inflammatory responses through the gut–lung axis. Full article

(This article belongs to the Section Gut Microbiota)

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16 pages, 1106 KB

Open AccessArticle

Enhanced Biomass, Paramylon, and Lipids Production by Non-Axenic Cultivation of Euglena gracilis in Anaerobically Digested Livestock Wastewater

by Yun-Ju Kang, Hyun-Jin Lim, Min-Su Kang, Yeong-Jun Lee and Jong-Hee Kwon

Microorganisms 2026, 14(2), 483; https://doi.org/10.3390/microorganisms14020483 - 17 Feb 2026

Abstract

Wastewater-based microalgal cultivation enables coupling environmental remediation with the production of sustainable, value-added biomass. In this study, Euglena gracilis was cultivated under non-axenic conditions in a 2% anaerobically digested livestock wastewater (LSWW)-based medium to enhance biomass accumulation, paramylon storage, and biodiesel precursor production, [...] Read more.

Wastewater-based microalgal cultivation enables coupling environmental remediation with the production of sustainable, value-added biomass. In this study, Euglena gracilis was cultivated under non-axenic conditions in a 2% anaerobically digested livestock wastewater (LSWW)-based medium to enhance biomass accumulation, paramylon storage, and biodiesel precursor production, while simultaneously removing residual nitrogen and phosphorus. The LSWW medium was strongly phosphate-limited relative to ammoniacal nitrogen (N:P mass ratio ~39:1), which constrained growth. Adjustment of the N:P ratio to ~10:1 by NaH₂PO₄ supplementation, together with MgSO₄·7H₂O addition, significantly enhanced biomass production, whereas trace metals and CaCl₂ provided minimal benefit. Cultivation at an initial pH of 3 resulted in substantially higher biomass accumulation than at pH 7 under xenic conditions. Under these optimized conditions, total phosphate and ammonia were efficiently removed, decreasing from 5.27 to 0.009 mg/L (99.8%) and from 57.40 to 2.11 mg/L (96.3%), respectively. Although paramylon accumulation was low in LSWW alone (~4% dry weight), short-term ethanol supplementation (0.095%, v/v, 24 h) enhanced paramylon content to ~20% dry weight. Subsequent anaerobic treatment further enhanced lipid conversion, increasing fatty acid methyl ester (FAME) content to ~45% dry weight. Collectively, low-pH non-axenic cultivation of E. gracilis in LSWW, combined with minimal nutrient supplementation, provides an integrated platform for enhanced biomass, paramylon, and biodiesel precursor production with efficient nutrient removal. Full article

(This article belongs to the Special Issue Microalgae at the Nexus of Sustainability: From Ecosystem Health to Biotechnological Innovation)

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21 pages, 6803 KB

Open AccessArticle

Microbial Ecology of Rotten Sea Ice: Implications for Arctic Carbon Cycling with Global Warming

by Carie M. Frantz, Byron C. Crump, Shelly Carpenter, Erin Firth, Mónica V. Orellana, Bonnie Light and Karen Junge

Microorganisms 2026, 14(2), 482; https://doi.org/10.3390/microorganisms14020482 - 16 Feb 2026

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Abstract

“Rotten” sea ice, ice in an advanced stage of melt, represents an important but understudied habitat in the rapidly changing Arctic. As Arctic warming accelerates, this late-season ice type will become more prevalent, yet little is known about its microbial inhabitants or their [...] Read more.

“Rotten” sea ice, ice in an advanced stage of melt, represents an important but understudied habitat in the rapidly changing Arctic. As Arctic warming accelerates, this late-season ice type will become more prevalent, yet little is known about its microbial inhabitants or their roles in Arctic marine biogeochemical cycles. We examined microbial communities (prokaryote and algal abundance, 16S and 18S rRNA gene and transcript sequencing) and biogeochemical properties of rotten sea ice and earlier-season ice near Utqiaġvik, Alaska, USA. Rotten ice was comparatively warm, isothermal, and largely drained of brine, with extensive, interconnected pore networks linked to melt ponds above and seawater below. Unlike earlier-season ice, fluids saturating rotten ice were vertically homogeneous in pH, dissolved inorganic carbon, prokaryote and phytoplankton abundance, and microbial community composition. However, particulate carbon and nitrogen exhibited strong vertical gradients, with the highest concentrations near the surface. Microbial communities in rotten ice were significantly different from those in earlier-season ice and varied between individual floes. These findings indicate that rotten ice constitutes a distinct microbial habitat and may serve as an important source of nutrient-rich particulate matter in the future Arctic Ocean during the summer melt season. Full article

(This article belongs to the Special Issue Polar Microbiome Facing Climate Change)

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24 pages, 3758 KB

Open AccessArticle

Two Novel Thiosulfate-Oxidizing Species from Coastal Sediments Reveal Distinct Ecological Strategies: Pseudothioclava alba sp. nov. and Terasakiella sediminum sp. nov

by Hui Zhou, Jieni Qu, Xu Lin, Ning Wang, Zihan Jiang, Qiliang Lai and Hong Xu

Microorganisms 2026, 14(2), 481; https://doi.org/10.3390/microorganisms14020481 - 16 Feb 2026

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Abstract

Two sulfur-oxidizing bacterial strains, FCG-A2^T and FCG-A23^T, were isolated from coastal sediments collected in Fangchenggang, Guangxi Province, China. Phylogenetic analyses based on 16S rRNA gene and whole-genome sequences placed strain FCG-A2^T within the genus Pseudothioclava and strain FCG-A23^T [...] Read more.

Two sulfur-oxidizing bacterial strains, FCG-A2^T and FCG-A23^T, were isolated from coastal sediments collected in Fangchenggang, Guangxi Province, China. Phylogenetic analyses based on 16S rRNA gene and whole-genome sequences placed strain FCG-A2^T within the genus Pseudothioclava and strain FCG-A23^T within the genus Terasakiella. Genomic relatedness (ANI, AAI, dDDH, and POCP) to the closest described taxa was below the accepted species thresholds, demonstrating that both isolates represent novel species. Strain FCG-A2^T grew at 15–35 °C (optimum 25–30 °C), at pH 5.0–10.0 (optimum pH 8.0), and with 1–4% (w/v) NaCl concentrations (optimum 3%). Strain FCG-A23^T grew at 20–33 °C (optimum 25–30 °C), at pH 6.0–9.0 (optimum, pH 8.0), and with 2–6% (w/v) NaCl (optimum 2%). For both strains, ubiquinone-10 was the major respiratory quinone, and the predominant fatty acids were summed feature 3 (C_16:1ω7c and/or C_16:1ω6c) and summed feature 8 (C_18:1ω7c and/or C_18:1ω6c); strain FCG-A2^T additionally contained C_16:0 as a major fatty acid. Both strains oxidized thiosulfate to sulfate, consistent with the presence of genes encoding the Sox system and assimilatory sulfate reduction pathways. Comparative genome annotation further suggested a broader carbohydrate-degradation potential in FCG-A2^T than in FCG-A23^T, implying a wider ecological distribution and greater opportunities for FCG-A2^T to perform sulfur oxidation across habitats. The draft genomes had G + C contents of 62.09% (FCG-A2^T) and 49.06% (FCG-A23^T). Based on these results, we propose Pseudothioclava alba sp. nov. (type strain FCG-A2^T = MCCC 1K08969^T = KCTC 8462^T) and Terasakiella sediminum sp. nov. (type strain FCG-A23^T = MCCC 1K08972^T = KCTC 8464^T). Full article

(This article belongs to the Section Environmental Microbiology)

22 pages, 1975 KB

Open AccessArticle

Root-Driven Filtering Overrides Biochar and Microbial Inoculants in Structuring Bacterial Assemblages of Seawater Rice Cultivation Ecosystem in a Saline–Alkali Soil

by Fangjing Hu, Pengjun Chen, Jiao Zhang, Yudi Guo, Kaihua Li, Su Liu, Lingzhi Li, Xu Chen, Jun Cui and Xi-En Long

Microorganisms 2026, 14(2), 480; https://doi.org/10.3390/microorganisms14020480 - 16 Feb 2026

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Abstract

Saline–alkali soils significantly hinder agricultural productivity in China’s coastal areas. Although both plant growth-promoting rhizobacteria (PGPR) and biochar have individually demonstrated the capacity to boost crop yield and soil fertility, their synergistic effects on seawater rice and soil ecosystems remain uncertain. In this [...] Read more.

Saline–alkali soils significantly hinder agricultural productivity in China’s coastal areas. Although both plant growth-promoting rhizobacteria (PGPR) and biochar have individually demonstrated the capacity to boost crop yield and soil fertility, their synergistic effects on seawater rice and soil ecosystems remain uncertain. In this study, we examined the individual and interactive influences of lychee biochar (2.5% and 5% w/w) and PGPR inoculation on soil physicochemical properties and bacterial community assembly along a soil–root continuum, encompassing bulk soil, rhizosphere soil, rhizoplane, and root endosphere, in a controlled pot experiment with seawater rice. The application of biochar significantly altered soil pH, electrical conductivity, and nutrient availability in both bulk and rhizosphere soils, resulting in pronounced changes in bacterial community composition. The effects generated by biochar were partially mitigated when PGPR was co-applied. The relative abundances of Bacillota and Bacteroidota grew progressively from bulk soil to the root endosphere across all treatments, indicating a significant compartment-dependent selection. Co-occurrence network analysis and FAPROTAX-based functional predictions revealed several taxa and functions that were progressively enriched toward the root, including the halotolerant genera Exiguobacterium and Chryseobacterium, highlighting a significant host-mediated filtration process that functioned independently of the inoculated strains. Multivariate analyses further demonstrated that soil pH was the primary driver of bacterial community structure in bulk and rhizosphere soils, whereas plant-root selection dominated in the rhizoplane and endosphere. Overall, our results demonstrate that, within a seawater-rice and soil ecosystem, the selective influence of the host plant on root-associated microbiomes exceeds that of either biochar amendment or PGPR inoculation. This work improves our understanding of biochar–PGPR–plant interactions in saline–alkali soils and provides insight into sustainable strategies for enhancing rice production under salinity stress. Full article

(This article belongs to the Topic New Challenges on Plant–Microbe Interactions)

22 pages, 4818 KB

Open AccessArticle

Distinctive Microbial Processes and Controlling Factors of Nitrous Oxide Emission in an Agricultural River Network: Perspective in Riparian Zone Type and Season

by Zhangmu Jing, Shengqiang Tu, Hongjie Gao and Qingqian Li

Microorganisms 2026, 14(2), 479; https://doi.org/10.3390/microorganisms14020479 - 16 Feb 2026

Viewed by 45

Abstract

The emission of nitrogen oxides (N₂O) in rivers is an important source of potent greenhouse gases. However, the mechanism at the interface between rivers and riverbanks remains unclear. This study quantified N₂O emissions from natural and artificial riparian zones [...] Read more.

The emission of nitrogen oxides (N₂O) in rivers is an important source of potent greenhouse gases. However, the mechanism at the interface between rivers and riverbanks remains unclear. This study quantified N₂O emissions from natural and artificial riparian zones across seasons and explored the microbial mechanisms affecting N₂O production and consumption in an intensive agricultural river network in China. Significant seasonal variability in N₂O emission rates was observed (p < 0.05), with mean values of 0.56 ± 0.09 mmol·m⁻²·h⁻¹ in autumn and 1.13 ± 0.32 mmol·m⁻²·h⁻¹ in spring. In spring, emissions from natural riparian zones (1.38 ± 0.28 mmol·m⁻²·h⁻¹) were significantly higher than those from artificial riparian zones (0.89 ± 0.05 mmol·m⁻²·h⁻¹). All wind-based models significantly overestimated N₂O emissions (p < 0.05) due to inflated IPCC emission factors (EF_5r), exceeding measured values by 1.76–3.09 times. Dissolved organic carbon and nitrite nitrogen were identified as key environmental drivers of N₂O emissions. Nitrogen fixation and ammonification accounted for 82.3% of N₂O production. Network analysis revealed a dominant microbial niche containing nitrifiers, sulfate-reducing bacteria, and carbohydrate-degrading taxa. Partial least squares path modeling indicated that riparian zone type altered DOC and NO₂⁻ availability, regulated nifH and ureC gene abundances, and enhanced N₂O production. These findings underscore the importance of riparian-zone-specific microbial regulation of riverine N₂O emissions and demonstrate the necessity of refining EF_5r estimates for agricultural river networks. Full article

(This article belongs to the Section Environmental Microbiology)

20 pages, 11450 KB

Open AccessArticle

UspF Regulates Type III Pili-Mediated Adhesion, Oxidative Stress Resistance, and Virulence in Klebsiella pneumoniae

by Yinyan Yin, Yiran Jiang, Wangxin Wu, Jing Zhu, Feng Zhang, Wenqing Luo, Chuang Meng, Yang Yang, Xinyu Miao, Tao Qin and Qingqing Gao

Microorganisms 2026, 14(2), 478; https://doi.org/10.3390/microorganisms14020478 - 15 Feb 2026

Viewed by 156

Abstract

Klebsiella pneumoniae (K. pneumoniae, KP) is a significant opportunistic pathogen responsible for both nosocomial and community-acquired infections. Bacterial adhesion is the critical initial step for host colonization and the establishment of disease. In this study, we utilized a mariner transposon [...] Read more.

Klebsiella pneumoniae (K. pneumoniae, KP) is a significant opportunistic pathogen responsible for both nosocomial and community-acquired infections. Bacterial adhesion is the critical initial step for host colonization and the establishment of disease. In this study, we utilized a mariner transposon mutagenesis system to construct a mutant library from the clinical KP strain KP20, identifying a mutant with significantly impaired epithelial cell adhesion due to an insertion in the uspF gene. Genetic knockout experiments confirmed that uspF deletion markedly reduced the adhesion to human airway epithelial cells (Calu-3) and downregulated the transcription of type III pili-encoding genes (mrkABDF). Furthermore, uspF deficiency compromised antioxidant stress and serum resistance and increased susceptibility to dendritic cell and macrophage phagocytosis. In vivo challenge experiments further demonstrated that uspF deletion significantly attenuated K. pneumoniae virulence in mice. These findings provide important insights into the molecular pathogenesis of K. pneumoniae and identify UspF as a potential target for therapeutic intervention. Full article

(This article belongs to the Section Medical Microbiology)

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10 pages, 536 KB

Open AccessArticle

Evidence of in Utero Anti-Neospora caninum Antibody Production in Paired Sow and Umbilical Cord Blood Samples

by Labrini V. Athanasiou, Eleni G. Katsogiannou, Constantina N. Tsokana, Dimitrios Gougoulis, Stavros M. Papadakis and Vasileios G. Papatsiros

Microorganisms 2026, 14(2), 477; https://doi.org/10.3390/microorganisms14020477 - 15 Feb 2026

Viewed by 104

Abstract

Neosporosis, caused by Neospora caninum, is a major protozoal disease responsible for reproductive disorders and economic losses in livestock. Swine are susceptible to N. caninum infection, as evidenced by serological and experimental studies, but the impact of natural infection on reproduction failure [...] Read more.

Neosporosis, caused by Neospora caninum, is a major protozoal disease responsible for reproductive disorders and economic losses in livestock. Swine are susceptible to N. caninum infection, as evidenced by serological and experimental studies, but the impact of natural infection on reproduction failure remains poorly defined. The objective of this study was to investigate N. caninum transplacental transmission in naturally infected sows by detecting an active fetal immune response in their stillborn piglets. Paired maternal blood and umbilical cord blood (UCB) samples were collected from 247 sows and stillborn piglets across 39 farrow-to-finish farms in mainland Greece. Sera were tested for anti-N. caninum IgG and IgM antibodies using an indirect fluorescence antibody test. An IgG and IgM seropositivity for N. caninum of 8.91% and 3.64%, respectively, was reported in sows, while lower percentages of IgG and IgM antibodies (3.24% and 0.81%, respectively) were detected in UCB samples. Overall, antibodies were detected in 4.05% of UCB samples, indicative of in utero antibody production. Positive samples were more frequently encountered on smaller farms with up to 250 sows, possibly due to lower biosecurity standards. The detection of antibodies in UCB resulting from the fetal immune response to intrauterine N. caninum infection is indicative of the potential involvement of N. caninum parasitism in reproductive system disorders. Testing of UCB for the presence of anti-Neospora antibodies elucidates the dynamics of parasite transmission within the farm and provides evidence for the implementation of more efficient biosecurity and preventative measures. Full article

(This article belongs to the Section Veterinary Microbiology)

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13 pages, 3417 KB

Open AccessArticle

Taxonomic and Functional Diversity of Leaves and Stem Endophytes of Eight Agave Species

by Natalia Ysabel Labrín-Sotomayor, Patricia Alejandra Becerra-Lucio, Hugo Ruiz-González and Yuri Jorge Peña-Ramírez

Microorganisms 2026, 14(2), 476; https://doi.org/10.3390/microorganisms14020476 - 15 Feb 2026

Viewed by 233

Abstract

More than 63% of Mexico’s territory is classified as arid or semiarid, where plants belonging to the genus Agave have evolved. Adaptation to drylands resulted from biochemical, physiological, and anatomical properties shared with other crassulacean plants; however, microbial symbionts also play critical roles [...] Read more.

More than 63% of Mexico’s territory is classified as arid or semiarid, where plants belonging to the genus Agave have evolved. Adaptation to drylands resulted from biochemical, physiological, and anatomical properties shared with other crassulacean plants; however, microbial symbionts also play critical roles in plants’ growth, health, and drought tolerance. To explore endophytic communities in Agave plants, we used a shotgun metagenomic approach. The taxonomic and functional diversity of endophytes were studied in the leaves and stem organs of Agave americana, A.angustifolia, A. fourcroydes, A. karwinskii, A. potatorum, A. tequilana, A. cupreata, and A. rodacantha. The microbial community structure did not differ significantly among species, regardless of geographic origin or local environmental conditions, whereas significant differences were observed between organs. We found 4058 genera shared among organs, of which 957 genera are exclusive to the stem and 492 to the leaves. The community analysis of stems and leaves identified bacterial genera, including Acinetobacter, Klebsiella, Escherichia, Corynebacterium, and Streptomyces. Significant differences were also observed between organs in the functional annotations. The dominant functional categories were associated with cell signaling and protein metabolism in both organs. Full article

(This article belongs to the Special Issue Advances in Genomics and Ecology of Environmental Microorganisms)

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14 pages, 3177 KB

Open AccessArticle

Development and Validation of a Multiplex TaqMan Real-Time PCR Assay for Simultaneous Detection of PEDV Genotypes G1, S-INDEL, and G2

by Chuan-Hao Fan, Hai-Xia Li, Hui-Qiang Zhen, Ye-Qing Zhu, Li-Fan Liu, Lu-Lu Zhang, Yao-Wei Huang and Yang-Yang Li

Microorganisms 2026, 14(2), 475; https://doi.org/10.3390/microorganisms14020475 - 14 Feb 2026

Viewed by 145

Abstract

Porcine epidemic diarrhea virus (PEDV) is a major pathogen responsible for severe diarrhea, dehydration, and high mortality in neonatal piglets, continually threatening global swine production. Rapid differentiation of its major genotypes (classical G1, variant G2, and recombinant S-INDEL) is vital for molecular epidemiology [...] Read more.

Porcine epidemic diarrhea virus (PEDV) is a major pathogen responsible for severe diarrhea, dehydration, and high mortality in neonatal piglets, continually threatening global swine production. Rapid differentiation of its major genotypes (classical G1, variant G2, and recombinant S-INDEL) is vital for molecular epidemiology and effective disease control, yet existing approaches rely mainly on time-consuming sequencing and phylogenetic analysis of the S gene. To overcome this limitation, we developed a novel triplex TaqMan-based real-time PCR assay for rapid detection and differentiation of the three PEDV genotypes. The assay demonstrated high sensitivity, with the lowest detection limit of 10² copies/μL, and strong specificity, showing no cross-reactivity with six other common swine pathogens (TGEV, PDCoV, PoRV, PRRSV, CSFV, and PRV). It also exhibited excellent reproducibility, with both intra- and inter-assay coefficients of variation maintained below 1.5%. In clinical validation, the assay showed 100% concordance with results obtained from S gene sequencing and phylogenetic analysis. Furthermore, testing of 160 clinical samples revealed cases of co-infection involving G2 and S-INDEL strains. In conclusion, this rapid, specific, and reproducible assay provides a reliable tool for routine molecular diagnosis, facilitating large-scale epidemiological surveillance and enabling genotype-informed control strategies against PEDV. Full article

(This article belongs to the Special Issue Viral Infection on Swine: Pathogenesis, Diagnosis and Control)

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16 pages, 1975 KB

Open AccessArticle

MtrR Regulates a Major Lytic Transglycosylase (ltgA) Responsible for Peptidoglycan-Derived Cytotoxin Release and Autolysis in Neisseria gonorrhoeae

by Alaa I. Telchy, Tia Morgan, Kathleen T. Hackett, Ronald K. McMillan, Robert A. Nicholas, Joseph P. Dillard and Daniel Williams

Microorganisms 2026, 14(2), 474; https://doi.org/10.3390/microorganisms14020474 - 14 Feb 2026

Viewed by 120

Abstract

The multiple-transferable resistance protein (MtrR) is a transcriptional repressor of the mtrCDE-encoded drug efflux pump and Type IV pilus biosynthesis (pilM), and an activator of penicillin-binding protein 1 (ponA) expression in Neisseria gonorrhoeae. Previously published microarray data [...] Read more.

The multiple-transferable resistance protein (MtrR) is a transcriptional repressor of the mtrCDE-encoded drug efflux pump and Type IV pilus biosynthesis (pilM), and an activator of penicillin-binding protein 1 (ponA) expression in Neisseria gonorrhoeae. Previously published microarray data suggested that MtrR is also an activator of ltgA expression in the gonococcus. LtgA is a lytic transglycosylase responsible for approximately half of recycled peptidoglycan fragments and released peptidoglycan-derived cytotoxins, which cause ciliary damage and induce specific inflammatory responses. The fragments generated by LtgA during peptidoglycan remodeling can either be recognized by the permease AmpG for uptake into the bacterial cytoplasm and recycled for new cell wall growth and general metabolism or released into the external milieu. Therefore, we sought to define the capacity of MtrR to regulate LtgA expression in gonococci. We show that MtrR binds to the ltgA promoter region in a concentration-dependent manner, and that this binding results both in increased ltgA mRNA transcription and LtgA protein levels during exponential growth. Deletion of mtrR in N. gonorrhoeae decreased peptidoglycan monomer release from growing cells and increased autolysis. These results suggest that MtrR regulation of ltgA impacts peptidoglycan-derived cytotoxin release and autolysis in the gonococcus. This study suggests a central role of MtrR in coordinating aspects of the cellular envelope that may contribute to gonococcal pathogenesis. Full article

(This article belongs to the Special Issue Transcriptional Regulation in Bacteria, 2nd Edition)

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16 pages, 1498 KB

Open AccessArticle

Mutations and Metabolic Factors Affecting Interaction of Toxigenic and Atoxigenic Aspergillus flavus

by Szilvia Kovács, Barbara Brendzsák, Walter P. Pfliegler, Lajos Nagy, Kamirán Áron Hamow, István Pócsi and Tünde Pusztahelyi

Microorganisms 2026, 14(2), 473; https://doi.org/10.3390/microorganisms14020473 - 14 Feb 2026

Viewed by 205

Abstract

Aflatoxin B₁ (AFB₁) produced by Aspergillus flavus poses severe food safety risks. Competitive exclusion using atoxigenic A. flavus strains offers a promising biological control approach to managing agricultural contamination by reducing populations of toxigenic strains and aflatoxin levels. However, reliable [...] Read more.

Aflatoxin B₁ (AFB₁) produced by Aspergillus flavus poses severe food safety risks. Competitive exclusion using atoxigenic A. flavus strains offers a promising biological control approach to managing agricultural contamination by reducing populations of toxigenic strains and aflatoxin levels. However, reliable identification of atoxigenic strains remains challenging, and the mechanisms underlying competitive interactions between toxigenic and atoxigenic strains require clarification for effective implementation. Therefore, this study systematically analysed A. flavus strains for aflatoxin gene clusters and AFB₁ production to address these critical gaps. Our analysis revealed that atoxigenic strains had intron losses and high-impact mutations in several genes, particularly aflL and aflLa, which affect aflatoxin biosynthesis. Key genes norA/aflE, verA/aflN, and omtA/aflP emerged as mutation hotspots, sometimes causing false-negative PCR results that complicate strain identification. Also, AFB₁ production was inversely related to spore concentration on MEA medium, with fewer spores resulting in higher toxin levels. Interaction tests demonstrated that toxigenic and atoxigenic strains exhibited morphological changes only when co-cultured without physical separation, suggesting that this was mediated by diffusible molecules. Furthermore, differences in the levels of linoleic acid reduction products distinguished toxigenic from atoxigenic strains. These findings thus illuminate the complex genetic and metabolic factors influencing aflatoxin production and fungal interactions. Full article

(This article belongs to the Special Issue Fungal Biology and Interactions—3rd Edition)

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20 pages, 3986 KB

Open AccessArticle

Investigation of the Mechanisms of Transition of Gram-Negative Bacterial Cells into Induced Anabiosis Using Computational Methods of Classical Molecular Dynamics

by Ksenia Tereshkina, Eduard Tereshkin, Licheng Zhang, Petr Zaytsev, Vladislav Kovalenko, Yuriy Litti, Olga S. Sokolova, Yurii Krupyanskii and Nataliya Loiko

Microorganisms 2026, 14(2), 472; https://doi.org/10.3390/microorganisms14020472 - 14 Feb 2026

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Abstract

Studying the mechanisms by which Gram-negative heterotrophic bacteria transition from active metabolism to dormancy is an important task, as it is directly related to the problem of bacterial antibiotic resistance and the spread of nosocomial infections. Using electron microscopy, microbiology, and molecular modeling, [...] Read more.

Studying the mechanisms by which Gram-negative heterotrophic bacteria transition from active metabolism to dormancy is an important task, as it is directly related to the problem of bacterial antibiotic resistance and the spread of nosocomial infections. Using electron microscopy, microbiology, and molecular modeling, we investigated the dose-dependent mechanisms of action of 4-hexylresorcinol (4HR), a chemical analog of the anabiosis autoinducer, on the cell membranes of Gram-negative bacteria (using Escherichia coli as an example), leading to the formation of stressed, dormant, and mummified cells. It was shown that 4HR penetrates membranes equally easily both as single molecules and as micelles, distributing itself across the membrane so that the hydrocarbon radicals are aligned parallel to the lipid tails. When micelles penetrate the membrane, uneven distribution of 4HR within and between leaflets occurs, as well as lipid redistribution within the membrane, leading to the appearance of a third peak on the phospholipid electron density profile and a third black band in the membrane region in TEM images of such cells. At 4HR concentrations in solution of 200 µM, its micelles cover the cell membranes in a thick layer, penetrate into the membrane, and completely saturate it. Even higher concentrations create agglomerates or actually micellar arrays within the cell membranes, leading to cell death through mummification. Full article

(This article belongs to the Special Issue Antimicrobial Research: In Silico Strategies for Drug Discovery Against Microbial Infections)

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30 pages, 6324 KB

Open AccessReview

The Gut–Liver Axis in MASLD: From Host–Microbiome Crosstalk to Precision Therapeutics

by Ji Zhou, Bowen Zhu, Ziqian Bing, Tingting Wang and Yue Zhao

Microorganisms 2026, 14(2), 471; https://doi.org/10.3390/microorganisms14020471 - 14 Feb 2026

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Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) is an emerging global health challenge with limited effective therapeutic options. The gut microbiota, at the interface of host metabolism and immunity, acts as a critical disease modifier via the gut–liver axis. This review goes beyond cataloging [...] Read more.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is an emerging global health challenge with limited effective therapeutic options. The gut microbiota, at the interface of host metabolism and immunity, acts as a critical disease modifier via the gut–liver axis. This review goes beyond cataloging its associations and synthesizes how intrinsic and extrinsic factors sculpt a permissive microbial ecosystem. These factors likely converge to establish a state of “metabolic dysbiosis”, fueling MASLD progression through three core mechanisms: compromised intestinal barrier integrity with immune activation, dysregulation of key microbial metabolite axes, and direct hepatic insult from gut-derived products. Next, we evaluate the translational landscape through a mechanism-informed precision framework, with an emphasis on how microbiome-based interventions could be aligned with non-invasive biomarkers increasingly used for MASLD risk stratification and treatment monitoring. By integrating evidence across scales, this review aims to frame a roadmap from microbiome correlations to causality-driven, personalized therapeutic strategies for MASLD. Full article

(This article belongs to the Section Gut Microbiota)

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20 pages, 1348 KB

Open AccessCommentary

Why Clinical Trials of Microbiome-Targeted Interventions Often Fail to Support Health Claims: A Commentary on Probiotics and Translational Design

by Raul de Jesus Cano and Gissel García Menéndez

Microorganisms 2026, 14(2), 470; https://doi.org/10.3390/microorganisms14020470 - 14 Feb 2026

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Abstract

The rapid expansion of probiotics and other microbiome-modulating interventions has been accompanied by a growing number of human clinical trials. However, despite frequent reports of statistically significant microbiome changes, relatively few studies generate evidence that convincingly supports health claims or translates into reproducible, [...] Read more.

The rapid expansion of probiotics and other microbiome-modulating interventions has been accompanied by a growing number of human clinical trials. However, despite frequent reports of statistically significant microbiome changes, relatively few studies generate evidence that convincingly supports health claims or translates into reproducible, clinically meaningful outcomes. This gap is often attributed to the inherent complexity and inter-individual variability of the gut microbiome; however, recurring shortcomings in trial design and interpretation likely play an equally important role. In this Commentary, we examine common failure modes that weaken the clinical validation of microbiome-mediated interventions. These include overreliance on descriptive microbiome metrics (e.g., alpha diversity and taxonomic shifts) as surrogate endpoints, misalignment between prespecified endpoints and the claims ultimately advanced, and excessive dependence on symptom-only outcomes in settings characterized by substantial placebo responsiveness. We further highlight how inadequate control of key confounders—particularly diet, antibiotic exposure, and concomitant medications—combined with endpoint overload and underpowered study designs, can obscure true biological signal and increase the risk of irreproducible findings. We argue that stronger evidence emerges when the microbiome is treated as a mechanistic mediator rather than a clinical endpoint. Trials are most interpretable when intended claims are prospectively defined, linked to explicit biological mechanisms, and evaluated using a hierarchy of endpoints that prioritizes host-relevant outcomes and objective biomarkers, with microbiome measures integrated to support mechanistic plausibility. Adoption of staged development pathways disciplined statistical planning, and transparent management of confounding variables can further improve reproducibility and clinical relevance. Full article

(This article belongs to the Special Issue Clinical Applications and Validation of Probiotics: Exploring Therapeutic and Preventive Potential Across Diverse Medical Conditions)

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20 pages, 2405 KB

Open AccessArticle

A Marine Alkaline Protease from Bacillus safensis DL12: Heterologous Expression, Purification and Preliminary Application in Animal Feed

by Mingchen Ma, Zhaohui Liu, Wei Zheng, Nilu Yang, Yue Guo, Jinlong Ma and Chunshan Quan

Microorganisms 2026, 14(2), 469; https://doi.org/10.3390/microorganisms14020469 - 14 Feb 2026

Viewed by 95

Abstract

Cottonseed meal (CSM) is a cost-effective protein source, but its application is limited by the toxicity of free gossypol. Traditional physical and chemical detoxification methods are costly, energy-intensive, and cause nutrient loss, while microbial fermentation-based biological detoxification is considered more sustainable than chemical [...] Read more.

Cottonseed meal (CSM) is a cost-effective protein source, but its application is limited by the toxicity of free gossypol. Traditional physical and chemical detoxification methods are costly, energy-intensive, and cause nutrient loss, while microbial fermentation-based biological detoxification is considered more sustainable than chemical or physical approaches. This study reports an alkaline protease from the marine strain Bacillus safensis DL12 isolated from Yellow Sea sediments. Following cloning of its encoding gene and heterologous expression, enzymatic characterization of the purified enzyme revealed optimal activity at pH 8.0 and 50 °C, with Fe²⁺, Cu²⁺, Ni²⁺, and dithiothreitol (DTT) significantly enhancing its activity. Substrate hydrolysis analysis using the purified enzyme on soybean meal, peanut meal, rapeseed meal, and cottonseed meal demonstrated that, compared to the control group, cottonseed meal hydrolysates exhibited a 55.6% relative increase in peptide content and a 41.5% relative improvement in the degree of hydrolysis (DH), indicating higher hydrolysis efficiency among the four substrates. Notably, when hydrolyzing cottonseed meal with purified enzyme versus crude enzyme preparation at equivalent activity, the purified enzyme effectively reduced free gossypol content by 70% compared to the control, achieving more efficient detoxification than the crude enzyme preparation and most reported microbial treatments. These results highlight the potential of B. safensis DL12 protease as a marine-derived enzyme, offering promising prospects for enhancing protein digestibility and addressing the long-standing challenge of gossypol toxicity in cottonseed meal utilization. Full article

(This article belongs to the Section Microbial Biotechnology)

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17 pages, 1553 KB

Open AccessArticle

Microplastics Facilitate Protozoan Pathogen Contamination in Shellfish

by Minji Kim, Colleen A. Burge, Chelsea M. Rochman, Elizabeth VanWormer, Chloe Resngit, Lezlie Rueda, Blythe Marshman, James Moore, Darrielle Williams and Karen Shapiro

Microorganisms 2026, 14(2), 468; https://doi.org/10.3390/microorganisms14020468 - 14 Feb 2026

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Abstract

Concerns about microplastic pollution have risen as numerous studies have reported detection of microplastics in foods, including seafood. One emerging concern is the ability of microplastics to vector pathogens that can adhere to biofilms on microplastic surfaces. Here, we investigated whether microplastics can [...] Read more.

Concerns about microplastic pollution have risen as numerous studies have reported detection of microplastics in foods, including seafood. One emerging concern is the ability of microplastics to vector pathogens that can adhere to biofilms on microplastic surfaces. Here, we investigated whether microplastics can facilitate zoonotic protozoan parasite contamination in shellfish. Oysters were selected for this study because they are commonly eaten raw and can harbor zoonotic protozoan pathogens. Acclimated live oysters were exposed in closed aquaria to Cryptosporidium, Giardia, and Toxoplasma (oo)cysts that had been incubated in seawater either as protozoa alone (P treatment) or with preconditioned polyester microfibers (P + M treatment). After overnight exposure, oysters were transferred to clean seawater flow-through aquaria for depuration. Over the experimental period, oysters exposed to both protozoa and microfibers had significantly higher numbers of protozoan pathogens than oysters exposed to protozoa alone. Our study provides experimental evidence that microplastics may facilitate protozoan pathogen contamination in shellfish. These results demonstrate how anthropogenic pollution may have unintended consequences on infectious disease transmission in coastal ecosystems, with potential risk to wildlife populations and human public health. Full article

(This article belongs to the Special Issue Advances in Research on Waterborne Pathogens)

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30 pages, 1135 KB

Open AccessReview

Genetically Modified Microorganisms: Risks and Regulatory Considerations for Human and Environmental Health

by Aaron Lerner, Arnon D. Lieber, Cass Nelson-Dooley, Andre Leu, Michelle Perro, Geoffrey Koch, Carina Benzvi and Jeffrey Smith

Microorganisms 2026, 14(2), 467; https://doi.org/10.3390/microorganisms14020467 - 14 Feb 2026

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Abstract

Advances in affordable genetic engineering have accelerated the creation and large-scale environmental release of genetically modified microorganisms (GMMs). While beneficial applications exist, GMMs may present unique, long-term risks to human and environmental health. Unlike static chemicals, GMMs are biologically active, self-replicating entities capable [...] Read more.

Advances in affordable genetic engineering have accelerated the creation and large-scale environmental release of genetically modified microorganisms (GMMs). While beneficial applications exist, GMMs may present unique, long-term risks to human and environmental health. Unlike static chemicals, GMMs are biologically active, self-replicating entities capable of rapid mutation and global dispersal. Current regulatory frameworks place responsibility on each country to regulate GMMs, without a clear, coordinated international policy. This review details critical risk scenarios, including horizontal gene transfer to native species and the possible disruption of vital human microbiomes (gut, oral, and infant), which could increase resistance to degradation, promote traits that expand a microbe’s range of hosts or ecological niches, and enhance the production of novel metabolites with unexpected biological activity. In soil, GMMs may support the emergence of “super bugs” or destabilize carbon sequestration cycles, potentially impacting climate resilience. Engineered microbial enzymes in the food supply may also act as environmental drivers of autoimmunity. Given the limited understanding of microbial ecology, we propose a decision-based biosafety workflow emphasizing pre-release risk assessment and continuous post-release monitoring. We urge national and international regulators to adopt the precautionary principle to better protect human health and the environment from the potential negative outcomes of GMMs. Full article

(This article belongs to the Section Microbiomes)

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22 pages, 8707 KB

Open AccessArticle

Isolation and Characterization of a Novel Sulfur-Oxidizing Stutzerimonas Species from Hydrothermal Sediments and Its Adaptation to the Hydrothermal Environment

by Yi Ding, Ming-Hua Liu, Yu-Kang Li, Tao Wang, Xue-Wei Xu and Yue-Hong Wu

Microorganisms 2026, 14(2), 466; https://doi.org/10.3390/microorganisms14020466 - 14 Feb 2026

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Abstract

Stutzerimonas, a genus newly separated from the Pseudomonadaceae family in 2022, has attracted considerable attention due to its diverse metabolic capabilities and environmental adaptability. However, the mechanisms underlying its sulfur-oxidizing capacity and survival strategies in extreme environments remain poorly understood. Clarifying potential [...] Read more.

Stutzerimonas, a genus newly separated from the Pseudomonadaceae family in 2022, has attracted considerable attention due to its diverse metabolic capabilities and environmental adaptability. However, the mechanisms underlying its sulfur-oxidizing capacity and survival strategies in extreme environments remain poorly understood. Clarifying potential sulfur-oxidizing microbial groups contributes to a more accurate understanding of energy flow and elemental cycling in hydrothermal ecosystems. In this study, we isolated and identified a sulfur-oxidizing strain, designated 381-2^T, from sediments in the Tianxiu hydrothermal field of the northwest Indian Ocean, and proposed it as a new species of Stutzerimonas. Physiological characterizations demonstrated that strain 381-2^T could oxidize thiosulfate to tetrathionate and encoded the key sulfur oxidation gene tsdA. Cultivation with sulfide minerals showed that strain 381-2^T could influence sulfide mineral weathering through metabolic activities, such as pH regulation, and potentially promote the reprecipitation of metal ions on the microbial surface. Comparative genomic analysis of 322 Stutzerimonas genomes further revealed the widespread presence of the tsdA gene and metal resistance genes, suggesting potential adaptive strategies for survival in hydrothermal environments. This study expands the understanding of Stutzerimonas species and provides insights into their ecological roles in hydrothermal systems. Full article

(This article belongs to the Section Environmental Microbiology)

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Microorganisms, Volume 14, Issue 2 (February 2026) – 234 articles

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