Microorganisms

20 pages, 11062 KiB

Open AccessArticle

The Effect of the swnR Gene on Swainsonine Biosynthesis in Alternaria oxytropis OW7.8, an Endophytic Fungus of Oxytropis glabra

by Ning Ding, Chang Liu, Ping Lu, Lu Bai and Bo Yuan

Microorganisms 2025, 13(6), 1326; https://doi.org/10.3390/microorganisms13061326 - 6 Jun 2025

Abstract

The swnR gene was cloned in the endophytic fungus Alternaria oxytropis OW 7.8 isolated from Oxytropis glabra, and the gene knockout mutant ΔswnR was first constructed in this study. Compared with A. oxytropis OW 7.8, the ΔswnR exhibited distinct morphological [...] Read more.

The swnR gene was cloned in the endophytic fungus Alternaria oxytropis OW 7.8 isolated from Oxytropis glabra, and the gene knockout mutant ΔswnR was first constructed in this study. Compared with A. oxytropis OW 7.8, the ΔswnR exhibited distinct morphological alterations in both colony and mycelial structure, a slower growth rate, and significant reductions in swainsonine (SW) levels, indicating that the function of the swnR gene promoted SW biosynthesis. Six differentially expressed genes (DEGs) closely associated with SW synthesis were identified by transcriptomic analysis of A. oxytropis OW 7.8 and ΔswnR, with P5CR, swnR, swnK, swnH2, and swnH1 downregulating, and sac upregulating. The expression levels of the six genes were consistent with the transcriptomic analysis results. Five differential metabolites (DEMs) closely associated with SW synthesis were identified by metabolomic analysis, with L-Lys, L-Glutamic acid, Saccharopine, and L-Proline upregulating, and L-PA downregulating. The results lay the foundation for the in-depth elucidation of molecular mechanisms and SW synthesis pathways in fungi, and are also of importance for the prevention of locoism in livestock, the control and utilization of locoweeds, and the protection and sustainable development of grassland ecosystems. Full article

(This article belongs to the Special Issue Plant–Fungal Interactions in Biocontrol of Plant Diseases)

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16 pages, 1432 KiB

Open AccessArticle

Quorum-Quenching Activity of Myrtus communis Corsican Essential Oil Against the Marine Bacterium Aliivibrio fischeri

by Elisa Hardy, Jean-Pierre Poli, Ange Bighelli, Mathieu Paoli, Thomas Maroselli, Liliane Berti and Elodie Guinoiseau

Microorganisms 2025, 13(6), 1325; https://doi.org/10.3390/microorganisms13061325 - 6 Jun 2025

Abstract

The quorum-quenching activity of essential oils (EOs) from Corsican aromatic plants was evaluated using the marine bacterium Aliivibrio fischeri as a model system. Among the eleven EOs screened, Myrtus communis EO showed significant interference with QS-regulated phenotypes (swimming motility, bioluminescence, and biofilm formation). [...] Read more.

The quorum-quenching activity of essential oils (EOs) from Corsican aromatic plants was evaluated using the marine bacterium Aliivibrio fischeri as a model system. Among the eleven EOs screened, Myrtus communis EO showed significant interference with QS-regulated phenotypes (swimming motility, bioluminescence, and biofilm formation). Its activity was compared to Origanum vulgaris EO, known for its high carvacrol content and potent QS inhibition. The fractionation of M. communis EO revealed that its most polar fractions exhibited comparable levels of QS-disrupting activity. These chromatographic fractions significantly affected QS-controlled traits, indicating that minor or less volatile compounds may contribute to, or enhance, the overall bioactivity. Furthermore, M. communis EO and its polar fractions displayed stronger anti-QS effects against A. fischeri than O. vulgaris EO. These results highlight M. communis EO as a promising source of natural QS inhibitors and underscore the importance of exploring both complete EOs and their active fractions. This study supports the valorization of Mediterranean endemic flora as a reservoir of bioactive compounds, tested on a model system A. fischeri, and encourages future research on the potential of Myrtus communis against clinical bacterial isolates and the development of novel anti-virulence strategies. Full article

(This article belongs to the Special Issue Research on Antimicrobial Activity of Natural Products, Second Edition)

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16 pages, 9902 KiB

Open AccessArticle

Genome Sequences of the First Phages Infecting Limnohabitans Reveal Their Global Distribution and Metabolic Potential

by Boxuan Deng, Raoqiong Che, Pinxin Zhu, Yongxia Wang, Zhiying Li, Shiying Zhang and Wei Xiao

Microorganisms 2025, 13(6), 1324; https://doi.org/10.3390/microorganisms13061324 - 6 Jun 2025

Abstract

Bacteriophages (phages) are one of the critical biotic drivers of prokaryotic community dynamics, functions, and evolution. Despite their importance in aquatic ecosystems, very few phages have been isolated from freshwater lakes, hampering our understanding of their ecological importance and usage in a variety [...] Read more.

Bacteriophages (phages) are one of the critical biotic drivers of prokaryotic community dynamics, functions, and evolution. Despite their importance in aquatic ecosystems, very few phages have been isolated from freshwater lakes, hampering our understanding of their ecological importance and usage in a variety of biotechnological applications. Limnohabitans, with a ubiquitous distribution, is a metabolically versatile, fast-growing, morphologically diverse freshwater lake bacterial genera. It is especially abundant in pH-neutral and alkaline aquatic habitats, where it represents an average of 12% of freshwater bacterioplankton and plays an important role in funneling carbon from primary producers to higher trophic levels. However, no phages infecting Limnohabitans have been reported to date. Here, we describe, for the first time, three phages infecting Limnohabitans, DC31, DC33, and YIMV22061, isolated from two freshwater lakes in China and characterized using genome content analysis and comparative genomics. DC31 and DC33, recovered from the eutrophic Dianchi Lake, with auxiliary metabolic genes (AMGs), associated with nucleotide metabolism, whereas YIMV22061, isolated from the oligotrophic Fuxian Lake, carried AMGs involved in antibiotic resistance. The AMGs they carried highlight their impacts on Limnohabitans in different environments. Comparative genomic analyses indicate that DC31, DC33, and YIMV22061 represent three novel species in the Caudoviricetes class. IMG/VR database alignment further reveal that these phages are widely distributed across diverse aquatic and terrestrial ecosystems globally, suggesting their ecological significance. This study provides a basis for better understanding Limnohabitans–phage interactions. Full article

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

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17 pages, 18361 KiB

Open AccessArticle

A Comprehensive Safety Assessment of Ralstonia eutropha H16 for Food Applications: Integrating Genomic, Phenotypic, and Toxicological Analyzes

by Xiaoyan You, Shuxia Song, Bing Li, Hui Wang, Le Zhang, Xiangyang Li, Junliang Chen, Zhiguang Zhu and Guoping Zhao

Microorganisms 2025, 13(6), 1323; https://doi.org/10.3390/microorganisms13061323 - 6 Jun 2025

Abstract

Ralstonia eutropha H16, a metabolically versatile bacterium, has gained prominence as a microbial platform for sustainable bioproduction. While its capabilities in synthesizing single-cell proteins and biodegradable materials are well documented, comprehensive strain-level safety evaluations remain insufficient for food-grade applications. This study systematically assessed [...] Read more.

Ralstonia eutropha H16, a metabolically versatile bacterium, has gained prominence as a microbial platform for sustainable bioproduction. While its capabilities in synthesizing single-cell proteins and biodegradable materials are well documented, comprehensive strain-level safety evaluations remain insufficient for food-grade applications. This study systematically assessed the safety of R. eutropha H16 through genomic, phenotypic, and toxicological analyzes. Genomic analyzes revealed the absence or minimal presence of virulence factors and antibiotic resistance genes, aligning with microbiological safety standards. Phenotypic investigations demonstrated a limited gastric fluid tolerance (pH 2.5, survival rate 25.70% after 3 h) and intestinal fluid persistence (pH 8, 44.67% viability after 3 h), coupled with an exceptional bile salt tolerance (0.2% w/v). Antioxidant assays confirmed the fermentation broth specifically scavenges DPPH free radicals (14.60 ± 1.24 μg Trolox/mL), whereas bacterial suspensions and cell-free supernatants exhibited a strong hydroxyl radical scavenging (>90 U/mL) and superoxide anion inhibition (>100 U/L). Acute toxicity testing indicated no mortality or histopathological abnormalities, with an LD₅₀ value exceeding 1 × 10¹¹ CFU/kg. Subacute toxicity studies (28-day, 1 × 10⁸–1 × 10¹⁰ CFU/kg) revealed no significant effects on growth, hematology, or organ function. Minor alterations in serum biochemistry might be attributed to physiological adaptation. Subacute exposure induced transient serum ALT fluctuations without hepatorenal dysfunction, while maintaining hematological parameters within physiological ranges. Collectively, these results substantiate the safety of R. eutropha H16 for food-related applications while underscoring the necessity of strain-specific risk assessments for industrial microbial platforms. Full article

(This article belongs to the Section Food Microbiology)

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22 pages, 4398 KiB

Open AccessArticle

Genome-Driven Functional Validation of Bacillus amyloliquefaciens Strain MEPW12: A Multifunctional Endophyte for Sustainable Sweet Potato Cultivation

by Yiming Wang, Jingwen Hao, Jingsheng Gu, Jiaying Wu, Yongjing Zhang, Ting Liang, Haimeng Bai, Qinghe Cao, Jihong Jiang, Ludan Li and Xiaoying Cao

Microorganisms 2025, 13(6), 1322; https://doi.org/10.3390/microorganisms13061322 - 6 Jun 2025

Abstract

Sweet potato (Ipomoea batatas (L.) Lam.), as an important crop, is rich in polyphenols, vitamins, minerals, and other nutrients in its roots and leaves and is gradually gaining popularity. The use of endophytic bacteria to improve the quality of sweet potato can [...] Read more.

Sweet potato (Ipomoea batatas (L.) Lam.), as an important crop, is rich in polyphenols, vitamins, minerals, and other nutrients in its roots and leaves and is gradually gaining popularity. The use of endophytic bacteria to improve the quality of sweet potato can protect the environment and effectively promote the sustainable development of the sweet potato industry. In this study, 12 strains of endophytic bacteria were isolated from sweet potato. Through nitrogen fixation, phosphorus solubilization, indoleacetic acid production, siderophore production, ACC deaminase production, and carboxymethyl cellulose production, three strains with multiple biological activities were screened out. Among them, MEPW12 had the most plant growth-promoting functions. In addition, MEPW12 promoted host chlorophyll accumulation and inhibited pathogen growth and colonization in sweet potato roots and can utilize various carbon sources and salts for growth. It can also grow in extreme environments of high salt and weak acid. MEPW12 was identified as Bacillus amyloliquefaciens with a genome size of 3,928,046 bp and a GC content of 46.59%. After the annotation of multiple databases, it was found that MEPW12 had multiple enzymatic activities and metabolic potential. Comparative genomics and pan-genomics analyses revealed that other Bacillus sp. strains of MEPW12 have similar functions. However, due to adaptation to different growth environments, there are still genomic differences and changes. Inoculation with MEPW12 induced the high expression of IbGH3.10, IbERF1, and other genes, thereby promoting the growth of sweet potatoes. Bacillus amyloliquefaciens strain MEPW12 is a sweet potato endophyte with multiple growth-promoting functions, which can promote the growth of sweet potato seedlings. This study provides new microbial resources for developing microbial agents and improving the quality of sweet potatoes. Full article

(This article belongs to the Special Issue Plant Growth—Promoting Bacteria and Plant—Soil Interactions in Harsh Environments, 2nd Edition)

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21 pages, 2707 KiB

Open AccessArticle

Distribution of Genetic Determinants Associated with CRISPR-Cas Systems and Resistance to Antibiotics in the Genomes of Archaea and Bacteria

by Laura Antequera-Zambrano, Ángel Parra-Sánchez, Lenin González-Paz, Eduardo Fernandez and Gema Martinez-Navarrete

Microorganisms 2025, 13(6), 1321; https://doi.org/10.3390/microorganisms13061321 - 6 Jun 2025

Abstract

The CRISPR-Cas system represents an adaptive immune mechanism found across diverse Archaea and Bacteria, allowing them to defend against invading genetic elements such as viruses and plasmids. Despite its broad distribution, the prevalence and complexity of CRISPR-Cas systems differ significantly between these domains. [...] Read more.

The CRISPR-Cas system represents an adaptive immune mechanism found across diverse Archaea and Bacteria, allowing them to defend against invading genetic elements such as viruses and plasmids. Despite its broad distribution, the prevalence and complexity of CRISPR-Cas systems differ significantly between these domains. This study aimed to characterize and compare the genomic distribution, structural features, and functional implications of CRISPR-Cas systems and associated antibiotic resistance genes in 30 archaeal and 30 bacterial genomes. Through bioinformatic analyses of CRISPR arrays, cas gene architectures, direct repeats (DRs), and thermodynamic properties, we observed that Archaea exhibit a higher number and greater complexity of CRISPR loci, with more diverse cas gene subtypes exclusively of Class 1. Bacteria, in contrast, showed fewer CRISPR loci, comprising a mix of Class 1 and Class 2 systems, with Class 1 representing the majority (~75%) of the detected systems. Notably, Bacteria lacking CRISPR-Cas systems displayed a higher prevalence of antibiotic resistance genes, suggesting a possible inverse correlation between the presence of these immune systems and the acquisition of such genes. Phylogenetic and thermodynamic analyses further highlighted domain-specific adaptations and conservation patterns. These findings support the hypothesis that CRISPR-Cas systems play a dual role: first, as a defense mechanism preventing the integration of foreign genetic material—reflected in the higher complexity and diversity of CRISPR loci in Archaea—and second, as a regulator of horizontal gene transfer, evidenced by the lower frequency of antibiotic resistance genes in organisms with active CRISPR-Cas systems. Together, these results underscore the evolutionary and functional diversification of CRISPR-Cas systems in response to environmental and selective pressures. Full article

(This article belongs to the Special Issue Microbial Evolutionary Genomics and Bioinformatics)

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10 pages, 799 KiB

Open AccessBrief Report

Heterologous Expression of the Nitrogen-Fixing Gene Cluster from Paenibacillus polymyxa in Bacillus subtilis

by Xiuling Wang, Shiqing Gao, Jun Fu and Ruijuan Li

Microorganisms 2025, 13(6), 1320; https://doi.org/10.3390/microorganisms13061320 - 6 Jun 2025

Abstract

Microbially mediated biological nitrogen fixation is pivotal to sustainable agricultural development. However, optimizing nitrogenase activity in native biological nitrogen-fixing bacteria has been hindered by the complexities of genetic manipulation. Heterologous expression has served as a foundational strategy for engineering next-generation nitrogen-fixing microbial agents. [...] Read more.

Microbially mediated biological nitrogen fixation is pivotal to sustainable agricultural development. However, optimizing nitrogenase activity in native biological nitrogen-fixing bacteria has been hindered by the complexities of genetic manipulation. Heterologous expression has served as a foundational strategy for engineering next-generation nitrogen-fixing microbial agents. In this study, genomic analysis of Paenibacillus polymyxa CR1 revealed an 11 kb nitrogen-fixing (nif) gene cluster. The nif cluster was first synthesized and then assembled using ExoCET technology and finally integrated into the genome of Bacillus subtilis 168 via double-exchange recombination. RT-PCR confirmed the transcription of the nif cluster; however, no nitrogenase activity was detected in the acetylene reduction assay. A promoter replacement strategy (replacing the native promoter with P_veg) enabled B. subtilis to produce active nitrogenase. However, stronger promoters—namely, P₄₃ and P_tp2—did not further enhance nitrogenase activity. This demonstrates that promoter selection requires balancing transcriptional strength with systemic compatibility, particularly for metalloenzymes demanding precise cofactor assembly. This is the first report describing the heterologous expression of the nif gene cluster in B. subtilis, establishing a foundation for engineering high-efficiency nitrogen-fixing biofertilizers. Full article

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

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12 pages, 1648 KiB

Open AccessArticle

The Isolation and Construction of an Infectious Clone for a Duck Adenovirus Type 3 Strain

by Haipeng Lu, Mei Tang, Zhifei Zhang, Mi Wu, Chunxiu Yuan, Xue Pan, Qinfang Liu, Qiaoyang Teng, Bangfeng Xu, Minghao Yan, Dawei Yan, Fenglong Wang and Zejun Li

Microorganisms 2025, 13(6), 1319; https://doi.org/10.3390/microorganisms13061319 - 5 Jun 2025

Abstract

A duck adenovirus type 3 strain, SD2019, was isolated from sick Muscovy ducks in our laboratory in 2019. To study the biological properties of the virus, an infectious clone of the SD2019 strain was successfully established. The plasmid containing the whole genome of [...] Read more.

A duck adenovirus type 3 strain, SD2019, was isolated from sick Muscovy ducks in our laboratory in 2019. To study the biological properties of the virus, an infectious clone of the SD2019 strain was successfully established. The plasmid containing the whole genome of DAdV-3 was digested with Pac I and the linearized DNAs were electortransfected into LMH cells; the cells showed cytopathic effects (CPEs) at 96 h post transfection and the rescued virus (rSD2019) was identified by PCR and indirect immunofluorescence assays (IFAs). The biological characteristics of strain rSD2019 were studied in vitro and in vivo and the results show that rSD2019 grew to similar titers as compared with the wild-type SD2019 strain in LMH cells, as well as showing similar replication and virulence in Muscovy ducks. The establishment of a reliable reverse genetics system for DAdV-3 provides a foundation for future studies of DAdV-3. Full article

(This article belongs to the Section Virology)

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19 pages, 2685 KiB

Open AccessArticle

Thresholds and Trade-Offs: Fire Severity Modulates Soil Microbial Biomass-Function Coupling in Taiga Forests, Northeast of China

by Huijiao Qu, Siyu Jiang, Zhichao Cheng, Dan Wei, Libin Yang and Jia Zhou

Microorganisms 2025, 13(6), 1318; https://doi.org/10.3390/microorganisms13061318 - 5 Jun 2025

Abstract

Forest fires critically disrupt soil ecosystems by altering physicochemical properties and microbial structure-function dynamics. This study assessed short-term impacts of fire intensities (light/moderate/heavy) on microbial communities in Larix gmelinii forests one year post-fire. Using phospholipid fatty acid (PLFA) and Biolog EcoPlate analyses, we [...] Read more.

Forest fires critically disrupt soil ecosystems by altering physicochemical properties and microbial structure-function dynamics. This study assessed short-term impacts of fire intensities (light/moderate/heavy) on microbial communities in Larix gmelinii forests one year post-fire. Using phospholipid fatty acid (PLFA) and Biolog EcoPlate analyses, we found the following: (1) fire reduced soil organic carbon (SOC), dissolved organic carbon (DOC), total nitrogen (TN), and available nitrogen/potassium (AN/AK) via pyrolytic carbon release, while heavy-intensity fires enriched available phosphorus (AP), AN, and AK through ash deposition. (2) Thermal mortality and nutrient-pH-moisture stress persistently suppressed microbial biomass and metabolic activity. Moderate fires increased taxonomic richness but reduced functional diversity, confirming “functional redundancy.” (3) Neither soil microbial biomass nor metabolic activity at the fire site reached pre-fire levels after one year of recovery. Our findings advance post-fire soil restoration frameworks and advocate multi-omics integration to decode fire-adapted functional gene networks, guiding climate-resilient forest management. Full article

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

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21 pages, 1078 KiB

Open AccessArticle

The Vaginal Microbiome: Associations with Vaginal pH, Menopause and Metabolic Parameters

by Yi-Chun Chen, Yi-Fen Chiang, Ko-Chieh Huang, Kai-Lee Wang, Yun-Ju Huang, Tzong-Ming Shieh, Mohamed Ali and Shih-Min Hsia

Microorganisms 2025, 13(6), 1317; https://doi.org/10.3390/microorganisms13061317 - 5 Jun 2025

Abstract

Background: The vaginal microbiota, a critical determinant of women’s health, is influenced by hormonal and metabolic parameters across the lifespan. While Lactobacillus species are beneficial markers of vaginal health, microbial composition undergoes pronounced alterations after menopause. This study aimed to elucidate the associations [...] Read more.

Background: The vaginal microbiota, a critical determinant of women’s health, is influenced by hormonal and metabolic parameters across the lifespan. While Lactobacillus species are beneficial markers of vaginal health, microbial composition undergoes pronounced alterations after menopause. This study aimed to elucidate the associations between vaginal microbiota composition, vaginal pH, menopausal status, and metabolic parameters in Asian women. Methods: Vaginal secretion samples were collected from 40 women (20 premenopausal, 20 postmenopausal). Full-length 16S rRNA gene sequencing was used to characterize the microbiota, categorized into Community State Types (CSTs): CST-I + II (Lactobacillus crispatus/gasseri, protective), CST-III (Lactobacillus iners, neutral), and CST-IV (anaerobic bacteria, harmful). Vaginal pH and clinical data were assessed in relation to microbial profiles. Results: CST distribution differed significantly by menopausal status and vaginal pH. Harmful-type CST-IV was more prevalent in postmenopausal women (70% vs. 40%, p < 0.05), while CST-III was dominant in premenopausal women (45% vs. 5%). CST-IV was associated with elevated pH (median 6.00, p = 0.026) and increased abundance of anaerobes including Bacteroides, Fusobacterium, Porphyromonas, Prevotella, and Streptococcus. Oral antibiotic use reduced both beneficial and harmful CSTs, shifting toward neutral CST-III (75%, p = 0.048). Use of sodium–glucose cotransporter-2 (SGLT2) inhibitors in postmenopausal women was associated with a higher prevalence of protective CST-I + II (57.14% vs. 8.33%, p < 0.05), though no significant impact on pathogen presence was observed. Conclusions: This study highlights the dynamic interplay between menopausal status, metabolic interventions, and vaginal microbiota composition. Findings may inform targeted strategies to maintain vaginal health in aging populations. Full article

(This article belongs to the Section Microbiomes)

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22 pages, 4149 KiB

Open AccessArticle

Profiling of Bacterial Communities of Hospital Wastewater Reveals Clinically Relevant Genera and Antimicrobial Resistance Genes

by Clemente Cruz-Cruz, Javier Gaytán-Cervantes, Carolina González-Torres, Andres Emmanuel Nolasco-Rojas, Miguel Ángel Loyola-Cruz, Laura Delgado-Balbuena, Josué Delgado-Balbuena, Marianela Paredes-Mendoza, María Concepción Tamayo-Ordóñez, Yahaira de Jesús Tamayo-Ordoñez, Emilio Mariano Durán-Manuel, Araceli Rojas-Bernabé, Carlos Alberto Jiménez-Zamarripa, Oscar Sosa-Hernández, Omar Agni García-Hernández, Esther Ocharan-Hernández, Paola Berenice Zárate-Segura, Elizabeth González-Terreros, Daniel Alejandro Ramírez-Villanueva, Claudia Camelia Calzada-Mendoza and Juan Manuel Bello-López Show full author list Hide full author list

Microorganisms 2025, 13(6), 1316; https://doi.org/10.3390/microorganisms13061316 - 5 Jun 2025

Abstract

In Mexico, hospital wastewater (HWW) is a source of chemical and microbiological contamination, and it is released into the municipal sewage system without prior treatment. This water may contain pathogenic bacteria and antimicrobial resistance genes, which represent a risk to Public Health and [...] Read more.

In Mexico, hospital wastewater (HWW) is a source of chemical and microbiological contamination, and it is released into the municipal sewage system without prior treatment. This water may contain pathogenic bacteria and antimicrobial resistance genes, which represent a risk to Public Health and the environment. So far, there are no studies that analyse this problem comprehensively, relating bacterial population structures, chemical contaminants, and seasonality. The aim of this work was to seasonally characterise the bacterial communities of HWW, including clinically relevant bacteria and resistance genes in Hospital Juárez de México (HJM), and to evaluate the impact of physicochemical factors on their composition. A one-year observational, cross-sectional study was conducted at five HWW discharge points of HJM. Fourteen physicochemical parameters were determined by using standard methodologies, and statistical differences between discharges and seasons were evaluated. Bacterial communities were analysed by targeted amplicon sequencing of the V3-V4 region of the 16S rRNA gene. In addition, the presence of eight antimicrobial resistance genes of local epidemiological importance was assessed. Data were analysed using alpha and beta diversity indices, principal component analysis, and multivariate statistical tests. HWW showed high taxonomic diversity, with Proteobacteria, Firmicutes, and Bacteroidetes standing out. Clinically relevant bacteria were identified in 73.3% of the analyses, with Enterobacter and Escherichia-Shigella predominating. Total and dissolved solids, temperature, nitrate, and pH significantly influenced the bacterial composition of HWW. Seven out of the eight genes evaluated were identified, with bla_KPC, bla_OXA-40, and mcr-1 being the most frequent, showing significant seasonal differences. This study underlines the microbiological and chemical complexity of HWW, highlighting the impact of clinically relevant bacteria and antimicrobial resistance genes on Public Health. The findings emphasise the need to implement hospital waste management programmes and ideally specific treatment plants to minimise the associated risks and protect the environment and human health. Full article

(This article belongs to the Section Environmental Microbiology)

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16 pages, 3004 KiB

Open AccessArticle

Unveiling Species Diversity Within Early-Diverging Fungi from China VI: Four Absidia sp. nov. (Mucorales) in Guizhou and Hainan

by Yi-Xin Wang, Zi-Ying Ding, Xin-Yu Ji, Zhe Meng and Xiao-Yong Liu

Microorganisms 2025, 13(6), 1315; https://doi.org/10.3390/microorganisms13061315 - 5 Jun 2025

Abstract

Absidia is the most species-rich genus within the family Cunninghamellaceae, with its members commonly isolated from diverse substrates, particularly rhizosphere soil. In this study, four novel Absidia species, A. irregularis sp. nov., A. multiformis sp. nov., A. ovoidospora sp. nov., and A. verticilliformis [...] Read more.

Absidia is the most species-rich genus within the family Cunninghamellaceae, with its members commonly isolated from diverse substrates, particularly rhizosphere soil. In this study, four novel Absidia species, A. irregularis sp. nov., A. multiformis sp. nov., A. ovoidospora sp. nov., and A. verticilliformis sp. nov., were discovered from soil samples collected in southern and southwestern China, using integrated morphological and molecular analyses. Phylogenetic analyses based on concatenated ITS, SSU, LSU, Act, and TEF1α sequence data reconstructed trees that strongly supported the monophyly of each of these four new taxa. Key diagnostic features include A. irregularis (closely related to A. oblongispora) exhibiting irregular colony morphology, A. multiformis (sister to A. heterospora) demonstrating polymorphic sporangiospores, A. ovoidospora (forming a clade with A. panacisoli and A. abundans) producing distinctive ovoid sporangiospores, and A. verticilliformis (next to A. edaphica) displaying verticillately branched sporangiophores. Each novel species is formally described with comprehensive documentation, including morphological descriptions, illustrations, Fungal Names registration identifiers, designated type specimens, etymological explanations, maximum growth temperatures, and taxonomic comparisons. This work constitutes the sixth instalment in a series investigating early-diverging fungal diversity in China aiming to enhance our understanding of the diversity of fungi in tropical and subtropical ecosystems in Asia. In this paper, the known species of Absidia are expanded to 71. Full article

(This article belongs to the Special Issue Advancing Microbial Taxonomy and Genomics: Bridging Diversity and Functionality)

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16 pages, 1472 KiB

Open AccessArticle

Effects of Pineapple Peel on the Nutritional and Microbial Profiles of Napier Grass–Sugarcane Top Silage

by Huade Xie, Zhenhua Tang, Fanquan Zeng, Xianqing Luo, Fang Xie, Li Liang, Jingzhen Li, Pinfeng Liao, Lijuan Peng, Zhipei Li, Haiyu Bai, Xiaoqiang Guo and Chengjian Yang

Microorganisms 2025, 13(6), 1314; https://doi.org/10.3390/microorganisms13061314 - 5 Jun 2025

Abstract

Agricultural byproducts, including pineapple peel (PP), are valuable feed additives which support the livestock industry. However, conflicting evidence exists regarding the optimal amount of PP required to achieve optimal fermentation in silage. This study examines the impact of ensiling mixtures of equal proportions [...] Read more.

Agricultural byproducts, including pineapple peel (PP), are valuable feed additives which support the livestock industry. However, conflicting evidence exists regarding the optimal amount of PP required to achieve optimal fermentation in silage. This study examines the impact of ensiling mixtures of equal proportions of fresh Napier grass (NG) and sugarcane top (ST) with varying levels of PP (0% [C], 10% [P1], 20% [P2], and 30% [P3]) on fermentation quality, microbiological profiles, and in vitro ruminal digestion. Compared to the C silage, the dry matter, crude protein, and neutral detergent fiber contents decreased in the silage treated with increasing PP (p < 0.05). P1 exhibited lower (p > 0.05) pH, higher (p > 0.05) lactic acid content, and lower (p < 0.05) NH₃-N content than other silage. The Chao 1, ACE index, and relative abundance of Lacticaseibacillus and Lactobacillales were decreased following the order of C > P1 > P2 > P3 (p < 0.05). Although there were no significant differences observed in most vitro ruminal fermentation parameters among four silages (p > 0.05), P1 exhibited higher total gas production, total volatile fatty acid, acetate acid, acetate-to-propionate ratio, and lower pH than the other silages. These results demonstrated that a NG and ST mixture co-ensiling with appropriate PP enhances the NG and ST mixture silage quality, and the optimum addition ratio for PP was 10% on a fresh matter basis. Full article

(This article belongs to the Section Food Microbiology)

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16 pages, 2668 KiB

Open AccessArticle

Fungal β-Glucans Enhance Lactic Acid Bacteria Growth by Shortening Their Lag Phase and Increasing Growth Rate

by Andrea Bukša, Filip Petrović and Željka Maglica

Microorganisms 2025, 13(6), 1313; https://doi.org/10.3390/microorganisms13061313 - 5 Jun 2025

Abstract

The gut microbiome has a significant role in general health and well-being. Novel types of prebiotics, such as fungal polysaccharides, show potential for the formulation of new synbiotic formulations. However, little is known about the underlying mechanisms of the prebiotic effects of such [...] Read more.

The gut microbiome has a significant role in general health and well-being. Novel types of prebiotics, such as fungal polysaccharides, show potential for the formulation of new synbiotic formulations. However, little is known about the underlying mechanisms of the prebiotic effects of such compounds. This study investigated the prebiotic properties of fungal glucan extracts from Pleurotus ostreatus, Lentinula edodes, and Saccharomyces cerevisiae, employing a novel high-throughput method based on optical density measurements. This approach enabled the simultaneous screening of the effects of multiple extracts on six different strains of probiotic bacteria. Experiments were conducted to evaluate the effect of the extracts on the growth dynamics (the duration of the lag phase and the growth rate) of probiotic strains of the genera Lactobacillus and Lacticaseibacillus and on pathogenic bacteria. Fungal polysaccharide supplementation, particularly with their β-glucans, significantly shortened the lag phase by an average of 7–8 h in all tested strains and increased the growth rate by 2-fold in four strains of lactic acid bacteria. Different magnitudes of effects were observed across the various strain–extract combinations. This study lays the groundwork for elucidating the mechanism by which fungal β-glucans stimulate growth in probiotic bacteria and for the rapid screening of optimal combinations for formulating innovative synbiotics. Full article

(This article belongs to the Special Issue Fungal Metabolites: Powering Pharmacological and Agricultural Frontiers)

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15 pages, 2785 KiB

Open AccessArticle

Temperature-Driven Divergence in Microbial Consortia and Physicochemical Functionality: A Comparative Study of High- and Medium-Temperature Daqu

by Huawei Yuan, Jia Zheng, Liping Ding, Hong Wang, Qin Jiang, Chao Zhang, Tingna Xie, Guohui Nan, Li Li and Kai Lou

Microorganisms 2025, 13(6), 1312; https://doi.org/10.3390/microorganisms13061312 - 5 Jun 2025

Abstract

Daqu, a crucial fermentation starter for Chinese Baijiu, develops distinct microbial and physicochemical profiles depending on fermentation temperature, which significantly influence enzymatic activity and flavor formation. While high-temperature (HT-Daqu, 65 °C) and medium-temperature (MT-Daqu, 60 °C) variants [...] Read more.

Daqu, a crucial fermentation starter for Chinese Baijiu, develops distinct microbial and physicochemical profiles depending on fermentation temperature, which significantly influence enzymatic activity and flavor formation. While high-temperature (HT-Daqu, 65 °C) and medium-temperature (MT-Daqu, 60 °C) variants are known to produce different liquor aromas, systematic comparisons of their microbial and physicochemical dynamics remain limited. This study integrated physicochemical assays (moisture, starch, acidity, enzymatic activity) with 16S rRNA and ITS (Internal Transcribed Spacer) sequencing to analyze HT-Daqu (HQ1–HQ3) and MT-Daqu (MQ1–MQ3) from Sichuan breweries. Results revealed that HT-Daqu exhibited significantly lower moisture (p < 0.05) and starch content (p < 0.05) but higher acidity (p < 0.05) compared to MT-Daqu. Enzymatic activities were generally reduced in HT-Daqu, except for neutral protease. Microbial profiling revealed distinct microbial dynamics between HT-Daqu and MT-Daqu: HT-Daqu harbored thermophilic Bacillus (40–60% relative abundance) with reduced fungal diversity, while MT-Daqu prioritized fungal consortia—Aspergillus dominated MQ1 (78%) and Saccharomyces transiently peaked in MQ2 (35%)—which correlated with enhanced saccharification enzyme activities and esterification potential. Alpha-diversity indices confirmed higher bacterial diversity in HT-Daqu and greater fungal richness in MT-Daqu. Correlation networks highlighted temperature-driven linkages, such as Bacillus positively associating with acidity. These findings elucidate the trade-offs between microbial stress adaptation and metabolic efficiency under different thermal regimes, providing actionable insights for optimizing Daqu production through targeted microbial management and temperature control to enhance liquor quality. Full article

(This article belongs to the Special Issue Microbial Fermentation, Food and Food Sustainability)

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14 pages, 649 KiB

Open AccessArticle

PCR-Based Legionella Risk Evaluation of Drinking Water Systems—An Empirical Field Evaluation

by Markus Petzold, Nicole Zacharias, Sarah Uhle, Laurine Kieper, Nico Tom Mutters, Thomas Kistemann and Christiane Schreiber

Microorganisms 2025, 13(6), 1311; https://doi.org/10.3390/microorganisms13061311 - 4 Jun 2025

Abstract

Pathogens in water systems pose potential health risks. Several countries provide guidelines and risk management strategies for clean water systems. Regarding legionellae, culture-based methods are still the gold standard, whereas molecular methods such as quantitative real-time PCR (qPCR) are controversially discussed among experts [...] Read more.

Pathogens in water systems pose potential health risks. Several countries provide guidelines and risk management strategies for clean water systems. Regarding legionellae, culture-based methods are still the gold standard, whereas molecular methods such as quantitative real-time PCR (qPCR) are controversially discussed among experts as an alternative. It remains questionable as to whether monitoring by qPCR contributes to sustainable water hygiene and effective health prevention. Drinking water samples from 101 buildings were culture-based analyzed to determine the legionellae concentration, along with qPCR tests. The negative predictive values for Legionella spp. and L. pneumophila qPCR regarding the cultivation method were 100% and 98%, respectively. As Legionella spp. DNA is ubiquitously detected, the positive predictive value was low. L. pneumophila DNA was in 18% of the drinking water samples detected by qPCR, among which only 7% was quantifiable. Neither gold-standard methods of cultivation nor qPCR methods alone are suitable to monitor the risk to health by legionellae in water environments adequately. To overcome methodical difficulties, the benefits of a strategic integration of qPCR alongside cultivation methods should be applied to develop a comprehensive protocol for the stepwise analysis of water samples, which can be implemented in international regulatory frameworks in the future. Full article

(This article belongs to the Special Issue Clinical and Environmental Surveillance for the Prevention of Legionellosis, 2nd Edition)

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25 pages, 3666 KiB

Open AccessArticle

Validation of Core and Whole-Genome Multi-Locus Sequence Typing Schemes for Shiga-Toxin-Producing E. coli (STEC) Outbreak Detection in a National Surveillance Network, PulseNet 2.0, USA

by Molly M. Leeper, Morgan N. Schroeder, Taylor Griswold, Mohit Thakur, Krittika Krishnan, Lee S. Katz, Kelley B. Hise, Grant M. Williams, Steven G. Stroika, Sung B. Im, Rebecca L. Lindsey, Peyton A. Smith, Jasmine Huffman, Alyssa Kelley, Sara Cleland, Alan J. Collins, Shruti Gautam, Eishita Tyagi, Subin Park, João A. Carriço, Miguel P. Machado, Hannes Pouseele, Dolf Michielsen and Heather A. Carleton Show full author list Hide full author list

Microorganisms 2025, 13(6), 1310; https://doi.org/10.3390/microorganisms13061310 - 4 Jun 2025

Abstract

Shiga-toxin-producing E. coli (STEC) is a leading causing of bacterial foodborne and zoonotic illnesses in the USA. Whole-genome sequencing (WGS) is a powerful tool used in public health and microbiology for the detection, surveillance, and outbreak investigation of STEC. In this study, we [...] Read more.

Shiga-toxin-producing E. coli (STEC) is a leading causing of bacterial foodborne and zoonotic illnesses in the USA. Whole-genome sequencing (WGS) is a powerful tool used in public health and microbiology for the detection, surveillance, and outbreak investigation of STEC. In this study, we applied three WGS-based subtyping methods, high quality single-nucleotide polymorphism (hqSNP) analysis, whole genome multi-locus sequence typing using chromosome-associated loci [wgMLST (chrom)], and core genome multi-locus sequence typing (cgMLST), to isolate sequences from 11 STEC outbreaks. For each outbreak, we evaluated the concordance between subtyping methods using pairwise genomic differences (number of SNPs or alleles), linear regression models, and tanglegrams. Pairwise genomic differences were highly concordant between methods for all but one outbreak, which was associated with international travel. The slopes of the regressions for hqSNP vs. allele differences were 0.432 (cgMLST) and 0.966 wgMLST (chrom); the slope was 1.914 for cgMLST vs. wgMLST (chrom) differences. Tanglegrams comprised of outbreak and sporadic sequences showed moderate clustering concordance between methods, where Baker’s Gamma Indices (BGIs) ranged between 0.35 and 0.99 and Cophenetic Correlation Coefficients (CCCs) were ≥0.88 across all outbreaks. The K-means analysis using the Silhouette method showed the clear separation of outbreak groups with average silhouette widths ≥0.87 across all methods. This study validates the use of cgMLST for the national surveillance of STEC illness clusters using the PulseNet 2.0 system and demonstrates that hqSNP or wgMLST can be used for further resolution. Full article

(This article belongs to the Special Issue The Molecular Epidemiology of Infectious Diseases)

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13 pages, 1991 KiB

Open AccessArticle

Host Serum Amyloid A1 Facilitates Streptococcus pneumoniae Adaptation to Acidic Stress Induced by Pneumococcal Anaerobic Metabolism

by Weichen Gong, Masayuki Ono, Tomoko Sumitomo, Momoko Kobayashi, Yujiro Hirose and Shigetada Kawabata

Microorganisms 2025, 13(6), 1309; https://doi.org/10.3390/microorganisms13061309 - 4 Jun 2025

Abstract

Streptococcus pneumoniae (S. pneumoniae) is a leading cause of pneumonia, and its interaction with host acute-phase proteins remains underexplored. Serum amyloid A1 (SAA1), an acute-phase protein, plays a crucial role in immune modulation. This study investigates the role of SAA1 in [...] Read more.

Streptococcus pneumoniae (S. pneumoniae) is a leading cause of pneumonia, and its interaction with host acute-phase proteins remains underexplored. Serum amyloid A1 (SAA1), an acute-phase protein, plays a crucial role in immune modulation. This study investigates the role of SAA1 in the early stages of respiratory infection by S. pneumoniae and its potential contribution to bacterial adaptation under acidic stress. We used a murine nasal infection model to simulate the early phase of S. pneumoniae invasion into the lower respiratory tract. Levels of SAA1 and C-reactive protein (CRP) in bronchoalveolar lavage fluid (BALF) and serum were quantified using ELISA. In vitro assays examined the effect of serum and recombinant SAA1 on bacterial survival under acidic conditions. Fluorescence-labeled recombinant SAA1 and microscopy were utilized to assess SAA1 internalized by S. pneumoniae. Following nasal infection, SAA1 levels in BALF were significantly reduced, whereas CRP levels remained unchanged. In vitro, serum enhanced S. pneumoniae’s resistance to acidic byproducts including formic, lactic, and acetic acids. Specifically, formic acid promoted bacterial uptake of SAA1, and this internalization improved bacterial tolerance to acidic conditions. Fluorescence microscopy confirmed that SAA1 is internalized by S. pneumoniae. S. pneumoniae can internalize SAA1 to bolster resistance to acid stress, particularly formic acid. This study reveals a novel host–pathogen interaction mechanism wherein S. pneumoniae exploits host acute-phase proteins for environmental adaptation, offering new insights into bacterial survival strategies during infection. Full article

(This article belongs to the Section Medical Microbiology)

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15 pages, 507 KiB

Open AccessArticle

Microbial Community and Functional Analysis of Regionally Produced Traditional Korean Grain Vinegar

by Su Jeong Lee, Sun Hee Kim, Hee-Min Gwon and Jinju Park

Microorganisms 2025, 13(6), 1308; https://doi.org/10.3390/microorganisms13061308 - 4 Jun 2025

Abstract

This study investigated changes in microbial communities and functional components during the fermentation of traditional Korean grain vinegars collected from various regions as well as the correlations among these components. Microbial community analysis revealed that Lactobacillus acetotolerans was the dominant microorganism, while Acetobacter [...] Read more.

This study investigated changes in microbial communities and functional components during the fermentation of traditional Korean grain vinegars collected from various regions as well as the correlations among these components. Microbial community analysis revealed that Lactobacillus acetotolerans was the dominant microorganism, while Acetobacter pasteurianus numbers gradually increased during fermentation, playing a key role in acetic acid production. L. acetotolerans, known to thrive in acidic environments, contributed to increasing the acidity of the vinegar and enhanced its preservative properties. The rise in the levels of organic acids, particularly acetic acid, was influenced by the activity of these microorganisms. Additionally, the production of free amino acids, such as alanine, was influenced by interactions between the fermentation medium and microbial communities, significantly contributing to the vinegar’s sweetness. Volatile flavor compounds exhibited considerable diversity due to changes in microbial communities driven by raw-material differences. In particular, five-grain vinegar (YO) tended to generate more complex and intense flavor compounds, with uniformly elevated levels of aldehydes, acids, and ketones. These findings suggest that raw-material selection and fermentation conditions significantly influence the flavor and functional properties of grain vinegars, providing valuable foundational data for improving vinegar production processes to enhance flavor and functionality. Full article

(This article belongs to the Special Issue Food Microbiota in the Center Place: Detection, Antibiotic Resistance, Biofilms, the Impact on Hosts and Targeted Interventions)

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