Microorganisms

16 pages, 2620 KiB

Open AccessArticle

Comparison of the Transformation Ability of the Major Saponins in Panax notoginseng by Penicillum fimorum Enzyme and Commercial β-glucosidase

by Feixing Li, Ruixue Zhang, Dongmei Lin, Jin Yang, Ye Yang, Xiuming Cui and Xiaoyan Yang

Microorganisms 2025, 13(3), 495; https://doi.org/10.3390/microorganisms13030495 (registering DOI) - 23 Feb 2025

Abstract

Ginsenosides with less sugar groups, which are called minor ginsenosides, might have a greater pharmacological activity and better adsorptive ability, but their content in nature is extremely low. In this study, a strain of Penicillium fimorum with a strong saponin transformation ability was [...] Read more.

Ginsenosides with less sugar groups, which are called minor ginsenosides, might have a greater pharmacological activity and better adsorptive ability, but their content in nature is extremely low. In this study, a strain of Penicillium fimorum with a strong saponin transformation ability was isolated from fresh Gastrodia elata. A comparative biotransformation experiment of the major saponins from Panax notoginseng root were conducted using crude enzymes from P. fimorum and commercial β-glucosidase to produce minor ginsenosides. Specifically, the crude enzyme from P. fimorum was able to transform the major saponins from P. notoginseng root into 13 minor saponins in 72 h, while commercial β-glucosidase was able to transform the same major saponins into 15 minor saponins in 72 h. The most significant difference between these two enzymes is their ability to transform Rb₁. To the best of our knowledge, the biotransformation ability of crude enzymes from P. fimorum is reported here for the first time. These two enzymes have the potential to improve the economic value of P. notoginseng root and expand the methods for preparing minor saponins by transforming major saponins in the total saponins of P. notoginseng root. Full article

(This article belongs to the Topic Fermented Food: Health and Benefit)

19 pages, 3437 KiB

Open AccessArticle

The Performance of a Multi-Stage Surface Flow Constructed Wetland for the Treatment of Aquaculture Wastewater and Changes in Epiphytic Biofilm Formation

by Chuanxin Chao, Shen Gong and Yonghong Xie

Microorganisms 2025, 13(3), 494; https://doi.org/10.3390/microorganisms13030494 (registering DOI) - 22 Feb 2025

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Abstract

Constructed wetlands play a critical role in mitigating aquaculture wastewater pollution. However, the comprehensive treatment performance of aquatic plants and microorganisms under various water treatment processes remains insufficiently understood. Here, a multi-stage surface flow constructed wetland (SFCW) comprising four different aquatic plant species, [...] Read more.

Constructed wetlands play a critical role in mitigating aquaculture wastewater pollution. However, the comprehensive treatment performance of aquatic plants and microorganisms under various water treatment processes remains insufficiently understood. Here, a multi-stage surface flow constructed wetland (SFCW) comprising four different aquatic plant species, along with aeration and biofiltration membrane technologies, was investigated to explore the combined effects of aquatic plants and epiphytic biofilms on wastewater removal efficiency across different vegetation periods and treatment processes. The results demonstrated that the total removal efficiency consistently exceeded 60% in both vegetation periods, effectively intercepting a range of pollutants present in aquaculture wastewater. Changes in the vegetation period influenced the performance of the SFCW, with the system’s ability to treat total nitrogen becoming more stable over time. The removal efficiency of the treatment pond planted with submerged plants was highest in July, while the pond planted with emergent plants showed an increased removal rate in November. The aeration pond played a significant role in enhancing dissolved oxygen levels, thereby improving phosphorus removal in July and nitrogen removal in November. Additionally, the α-diversity of epiphytic bacteria in the aeration and biofiltration ponds was significantly higher compared to other ponds. In terms of bacterial composition, the abundance of Firmicutes was notably higher in July, whereas Nitrospirota and Acidobacteriota exhibited a significant increase in November. Furthermore, the functional genes associated with sulfur metabolism, nitrogen fixation, and oxidative phosphorylation displayed significant temporal variations in the aeration pond, highlighting that both growth period changes and treatment processes influence the expression of functional genes within biofilms. Our findings suggest that the integration of water treatment processes in SFCWs enhances the synergistic effects between aquatic plants and microorganisms, helping to mitigate the adverse impacts of vegetation period changes and ensuring stable and efficient wastewater treatment performance. Full article

(This article belongs to the Special Issue Microbial Ecology and Assembly Dynamics in the Aquaculture Environment)

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Location of sampling points of constructed wetland. The blue arrow shows the direction of the water flow; different capital letters (T1–T6) indicate the treatment pond (a); different lowercase letters (a–g) indicate the sampling point site (b). Full article ">Figure 2
The contribution of each treatment pond to pollutant removal. TN, total nitrogen; TDP, total dissolved phosphorus; COD, chemical oxygen demand; TP, total phosphorus; PO4−, orthophosphate; NO3−-N, nitrate nitrogen; NH4+-N, ammonia nitrogen. The asterisk indicates a significant difference between the removal efficiency of the same treatment pond in July and November (Wilcoxon test); * p < 0.05; ** p < 0.01; *** p < 0.001, ns: not significant. Full article ">Figure 3
Total removal efficiency of pollutants in the SFCW system in July and November. Asterisks indicate a significant difference between July and November; (Wilcoxon test); * p < 0.05; ** p < 0.01; ns: not significant. Full article ">Figure 4
Community compositions of bacteria (relative abundance > 1%) at the phylum level across different treatment ponds in July and November (a). Venn diagram of epiphytic bacterial OTUs based on different treatment ponds in July and November (b). Full article ">Figure 5
Nonmetric multidimensional scaling diagram showed the differences in epiphytic bacterial community structure at the OTUs level (calculated using Bray–Curtis) between two periods (a), six treatment ponds in July (b) and six treatment ponds in November (c). Hierarchical clustering of the samples based on Bray–Curtis similarity and the average sample on OTU level (d). Full article ">Figure 6
Redundancy analysis of the bacterial community structure and environmental variables in the epiphytic bacteria in July (a) and November (b). TN, total nitrogen; TDP, total dissolved phosphorus; COD, chemical oxygen demand; TP, total phosphorus; PO4−, orthophosphate; NO3−-N, nitrate nitrogen; NH4+-N, ammonia nitrogen; pH, water pH; DO, dissolved oxygen; ORP, oxidation reduction potential. Full article ">Figure 7
The bar chart of bacterial energy metabolism function predicted by different ponds in two vegetation periods at the level of the second KEGG pathway. T1 to T6 represent the six treatment ponds, J for July and N for November. Full article ">

15 pages, 1567 KiB

Open AccessArticle

Phenotypic and Molecular Characterization of Pyomelanin-Producing Acinetobacter baumannii ST2_Pas;ST1816/ST195_Oxf Causing the First European Nosocomial Outbreak

by Alessandro Leonildi, Alfredo Rosellini, Giulia Gemignani, Giusy Tiseo, Marco Falcone, Cesira Giordano and Simona Barnini

Microorganisms 2025, 13(3), 493; https://doi.org/10.3390/microorganisms13030493 (registering DOI) - 22 Feb 2025

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Abstract

Acinetobacter baumannii is one of the most successful and feared nosocomial pathogens. A. baumannii is considered a global threat in the healthcare setting, mainly owing to its ability to acquire multidrug resistance phenotypes. The A. baumannii pathogenesis is guided by its environmental persistence, [...] Read more.

Acinetobacter baumannii is one of the most successful and feared nosocomial pathogens. A. baumannii is considered a global threat in the healthcare setting, mainly owing to its ability to acquire multidrug resistance phenotypes. The A. baumannii pathogenesis is guided by its environmental persistence, as well as the production of numerous virulence factors. In several bacteria, the production of pigments, such as melanin, has indeed been linked with virulence and pathogenicity. Melanin is a brownish pigment, rarely observed in A. baumannii, that potentially reduces the susceptibility of the bacteria to host defense mechanisms and environmental insults. This study reports the first outbreak in Europe by pyomelanin-producing A. baumannii strains, in a tertiary-care university hospital in Pisa, Italy. Phenotypic and molecular analyses were performed. Full article

(This article belongs to the Collection Feature Papers in Medical Microbiology)

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18 pages, 4936 KiB

Open AccessArticle

Metabolic Reprogramming in Response to Freund’s Adjuvants: Insights from Serum Metabolomics

by Kiruthiga Mone, Eloy Jose Torres Garcia, Fatema Abdullatif, Mahima T. Rasquinha, Meghna Sur, Mostafa Hanafy, Denise K. Zinniel, Shraddha Singh, Raymond Thomas, Raul G. Barletta, Teklab Gebregiworgis and Jay Reddy

Microorganisms 2025, 13(3), 492; https://doi.org/10.3390/microorganisms13030492 (registering DOI) - 22 Feb 2025

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Abstract

Freund’s adjuvants have been used in vaccine and autoimmune settings, and their effects can be overlapping or unique to each. While both incomplete Freund’s adjuvants (IFA) and complete Freund’s adjuvants (CFA) influence antibody and T cell responses, the robust T helper 1 cytokines [...] Read more.

Freund’s adjuvants have been used in vaccine and autoimmune settings, and their effects can be overlapping or unique to each. While both incomplete Freund’s adjuvants (IFA) and complete Freund’s adjuvants (CFA) influence antibody and T cell responses, the robust T helper 1 cytokines induced by the mycobacterial components make CFA the powerful immunostimulating adjuvant. In these studies, the adjuvant effects are investigated in a select population of cells, and the changes, if any, with the metabolic alterations in the systemic compartment are unclear. We investigated whether the effects of IFA and CFA can be influenced by the metabolic shifts in mice immunized with saline, IFA, or CFA using Mycobacterium tuberculosis var. bovis Bacillus Calmette–Guérin (BCG) as a positive control. After seven days of immunization, we analyzed the serum metabolite profiles using liquid chromatography coupled with high-resolution mass spectrometry and multivariate statistical analysis to identify metabolic features between the groups. The data revealed that, in the scores space, the CFA and BCG groups were more closely aligned compared to the saline group, while the IFA group displayed an intermediate profile. Furthermore, comparisons between the CFA and BCG groups showed more significant perturbations in lipid and amino acid metabolism, particularly involving glycerophospholipids, cysteine, and aromatic amino acids. In contrast, comparisons between the BCG and IFA groups indicated a more pronounced disruption in central energy metabolism pathways, such as the citric acid cycle and pyruvate metabolism. Together, the data suggest that the serum metabolite profiles in response to IFA and CFA might play a role in modulating the immune responses. Full article

(This article belongs to the Special Issue Crosstalk Between Host and Pathogen: Innate Immunity, Infection and Metabolic Reprogramming)

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20 pages, 2473 KiB

Open AccessArticle

Antimicrobial-Resistance and Virulence-Associated Genes of Pasteurella multocida and Mannheimia haemolytica Isolated from Polish Dairy Calves with Symptoms of Bovine Respiratory Disease

by Agnieszka Lachowicz-Wolak, Aleksandra Chmielina, Iwona Przychodniak, Magdalena Karwańska, Magdalena Siedlecka, Małgorzata Klimowicz-Bodys, Kamil Dyba and Krzysztof Rypuła

Microorganisms 2025, 13(3), 491; https://doi.org/10.3390/microorganisms13030491 (registering DOI) - 22 Feb 2025

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Abstract

Bovine respiratory disease causes significant economic losses in cattle farming due to mortality, treatment costs, and reduced productivity. It involves viral and bacterial infections, with Pasteurella multocida and Mannheimia haemolytica key bacterial pathogens. These bacteria contribute to severe pneumonia and are often found [...] Read more.

Bovine respiratory disease causes significant economic losses in cattle farming due to mortality, treatment costs, and reduced productivity. It involves viral and bacterial infections, with Pasteurella multocida and Mannheimia haemolytica key bacterial pathogens. These bacteria contribute to severe pneumonia and are often found together. Poland has one of the highest levels of antimicrobial use in food-producing animals among European Union countries. A total of 70 bacterial strains were analyzed, 48 P. multocida and 22 M. haemolytica, collected from affected calves’ respiratory tracts. The bacterial species were confirmed molecularly using PCR, which was also employed to detect antimicrobial resistance and virulence-associated genes. Antimicrobial susceptibility was determined using the broth microdilution method. Antimicrobial resistance varied between the two bacterial species studied. The highest resistance in P. multocida was to chlortetracycline 79.2% (38/48) and oxytetracycline 81.3% (39/48), while M. haemolytica showed 63.6% (14/22) resistance to penicillin and tilmicosin. The highest susceptibility was found for fluoroquinolones: P. multocida demonstrated 91.7% (44/48) susceptibility to enrofloxacin and 87.5% (42/48) to danofloxacin, while 77.3% (17/22) of M. haemolytica were susceptible to both tested fluoroquinolones. The tetH and tetR genes were observed only in P. multocida, at frequencies of 20.8% (10/48) and 16.7% (8/48), respectively. Both species carried the mphE and msrE genes, though at lower frequencies. All M. haemolytica contained the lkt, gs60, and gcp genes. All P. multocida carried the sodA gene, while the hgbB and ompH genes were present in 37.5% (18/48) and 20.8% (10/48) of strains, respectively. The highest resistance was observed against the most commonly used antibiotics in the European Union, although the resistance differed between the studied bacterial species and each strain exhibited the presence of at least one virulence gene. Full article

(This article belongs to the Special Issue Research on Infections and Veterinary Medicine)

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Percentages of insusceptibility to antimicrobials among the tested strains of Pasteurella multocida (P. multocida) and Mannheimia haemolytica (M. haemolytica). * Statistically significant difference in resistance to the given antimicrobial agent between bacterial species (p < 0.05). CTET—chlortetracycline, OXY—oxytetracycline, TUL—tulathromycin, TIL—tilmicosin, PEN—penicillin, ENRO—enrofloxacin, DANO—danofloxacin, XNL—ceftiofur, FFN—florfenicol, SPE—spectinomycin. Full article ">Figure 2
Number of bacterial strains of P. multocida (Pm) and M. haemolytica (Mh) showing growth inhibition at the specified concentrations of the tested antimicrobial agents. The range of tested concentrations is indicated by the frames. The colored fields indicate the concentrations at which bacterial growth was inhibited. Darker shades represent a higher number of inhibited isolates at a given concentration. Green corresponds to P. multocida, while red represents M. haemolytica. The number of strains not inhibited at the tested concentrations was also included. MIC₅₀ and MIC₉₀ values marked with “>” indicate that bacterial growth inhibition was not achieved at the tested concentrations. CTET—chlortetracycline, OXY—oxytetracycline, TUL—tulathromycin, TIL—tilmicosin, PEN—penicillin, AMP—ampicillin, ENRO—enrofloxacin, DANO—danofloxacin, XNL—ceftiofur, FFN—florfenicol, SPE—spectinomycin, CLI—clindamycin, GEN—gentamicin, NEO—neomycin, SDM—sulfadimethoxine, TIA—tiamulin, SXT—trimethoprim–sulfamethoxazole, TYLT—tylosin tartrate. Full article ">Figure 3
Venn diagrams showing the cooccurrence of virulence-associated genes in the studied strains of P. multocida and M. haemolytica. The common parts indicate the types of configurations, while the numbers in the respective fields represent the frequency of each configuration. Full article ">Figure 4
Venn diagrams showing the cooccurrence of antimicrobial resistance genes and phenotypic resistance in the studied strains of P. multocida and M. haemolytica. The common parts indicate the types of configurations, while the numbers in the respective fields represent the frequency of each configuration. CTET—chlortetracycline, OXY—oxytetracycline, TUL—tulathromycin, TIL—tilmicosin. Full article ">

15 pages, 2972 KiB

Open AccessArticle

Soil Fungal Diversity and Community Structure of Russula griseocarnosa from Different Sites

by Zhen Li, Ruoxi Liang and Fei Yu

Microorganisms 2025, 13(3), 490; https://doi.org/10.3390/microorganisms13030490 (registering DOI) - 22 Feb 2025

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Abstract

Russula griseocarnosa is an important ectomycorrhizal edible fungus whose economic and nutritional value are both high. To better understand which abiotic and biotic factors affect the growth of R. griseocarnosa, this study examined the mycosphere soil of R. griseocarnosa growing in five [...] Read more.

Russula griseocarnosa is an important ectomycorrhizal edible fungus whose economic and nutritional value are both high. To better understand which abiotic and biotic factors affect the growth of R. griseocarnosa, this study examined the mycosphere soil of R. griseocarnosa growing in five sites. The soil fungal communities of R. griseocarnosa from five sites of Fujian, Guangxi, and Yunnan Provinces were sequenced by Illumina MiSeq technology, and their community structure comprehensively analyzed in combination with a suite of soil physicochemical properties. The results revealed significantly greater levels of available potassium (AK), available nitrogen (AN), and available phosphorus (AP) in mycosphere soil than bulk soil, and that R. griseocarnosa prefers acidic soil, with Penicillium, Trichoderma, Talaromyces, Mortierella, Tolypocladium, Chloridium, Oidiodendron, and Umbelopsis being the main dominant fungal taxa. Different geographical sites had different indicator fungal genera, and the similarity of fungal communities in the mycosphere decreased with increasing geographical distance among them. Soil pH was the major abiotic factor influencing the structure of the mycosphere fungal communities. Management strategies such as nitrogen, potassium, phosphorus mixed fertilizer, and fungal fertilizer can promote the conservation and sustainable utilization of R. griseocarnosa. Full article

(This article belongs to the Special Issue Soil Microbial Communities and Ecosystem Functions, 2nd Edition)

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Comparison of Chao (A) and Shannon (B) diversity indexes between mycosphere and bulk soil. Significant differences by * p < 0.05; ** p < 0.01. DPF, HTCF, JJF, YYF, and ZPF represent mycosphere soil in different geographical areas, and HTCFCK, JJFCK, YYFCK, and ZPCK represent bulk soil. Full article ">Figure 2
Comparison of fungi community composition between mycosphere and bulk soil. (A) Phylum level; (B) the top 30 genera. DPF, HTCF, JJF, YYF, and ZPF represent mycosphere soil in different geographical areas, and HTCFCK, JJFCK, YYFCK, and ZPCK represent bulk soil. Full article ">Figure 3
LEfSe analysis of mycosphere fungi at genus level. DPF, HTCF, JJF, YYF, and ZPF represent mycosphere soil in different geographical areas. Full article ">Figure 4
Canonical correspondence analysis (CCA) based on the relative abundance of fungal taxa at the OTU level and environmental factors. DPF, HTCF, JJF, YYF, and ZPF represent mycosphere soil in different geographical areas, and HTCFCK, JJFCK, YYFCK, and ZPCK represent bulk soil. AK, AN, AP, and SOC represent available potassium, available nitrogen, available phosphorus, and soil organic carbon, respectively. Full article ">Figure 5
Relationships between mycosphere fungal communities and environmental factors. (A) Variation partition analysis (VPA) of soil/site properties on fungal community. (B) Distance–decay curves of fungal communities. Full article ">Figure 6
Spearman correlation between the LEfSe-significant fungal genera and soil/site properties. Significant correlation was noted when * p < 0.05 and ** p < 0.01. Full article ">Figure 7
Functional classification of the LEfSe-significant fungal genera. DPF, HTCF, JJF, YYF, and ZPF represent mycosphere soil in different geographical areas, and HTCFCK, JJFCK, YYFCK, and ZPCK represent bulk soil. Full article ">

19 pages, 2294 KiB

Open AccessReview

Prevalence and Distribution of Salmonella in Water Bodies in South America: A Systematic Review

by Makarena Sofia Gonzalez Reyes, Rayana Santos Araujo Palharini, Felipe Ferreira Monteiro, Salvador Ayala and Eduardo A. Undurraga

Microorganisms 2025, 13(3), 489; https://doi.org/10.3390/microorganisms13030489 (registering DOI) - 22 Feb 2025

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Abstract

The presence of Salmonella in rivers, lakes, or beaches in South America represents a challenge to public health and aquatic ecosystems. This review explores the distribution, prevalence, and the main factors contributing to the survival and spread of Salmonella, including wastewater discharge, agricultural [...] Read more.

The presence of Salmonella in rivers, lakes, or beaches in South America represents a challenge to public health and aquatic ecosystems. This review explores the distribution, prevalence, and the main factors contributing to the survival and spread of Salmonella, including wastewater discharge, agricultural runoff, and climatic variables such as high temperatures and precipitation. These factors also facilitate the distribution of multidrug-resistant strains in water. The review is based on bibliographic searches in various databases, focusing on Salmonella species, South American countries, and types of water bodies. Predominant serovars include S. Enteritidis and S. Typhimurium, with S. Typhi and S. Panama frequently detected in Chile, S. Enteritidis in Argentina, and S. Typhimurium in Brazil. Less common serovars, including S. Dublin and S. Paratyphi B, were identified, along with subspecies such as diarizonae and houtenae. These findings highlight the role of environmental, physicochemical, and anthropogenic factors influencing Salmonella dynamics. The review identifies research gaps, advocating for further studies to better understand the interactions between Salmonella, climate change, and human activity. Strengthening surveillance and mitigation strategies is crucial to protect water resources and public health in South America. Full article

(This article belongs to the Section Environmental Microbiology)

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14 pages, 3409 KiB

Open AccessArticle

Genome-Wide Survey of Donor Chromosomal Genes Involved in Trans-Kingdom Conjugation via the RP4-T4SS Machinery

by Kazuki Moriguchi, Kazuyuki Nakamura, Yudai Takahashi, Kyoko Higo-Moriguchi, Kazuya Kiyokawa and Katsunori Suzuki

Microorganisms 2025, 13(3), 488; https://doi.org/10.3390/microorganisms13030488 (registering DOI) - 22 Feb 2025

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Abstract

Trans-kingdom conjugation (TKC)/inter-domain conjugation is a horizontal gene transfer phenomenon that transfers DNA from eubacteria to eukaryotes and archaebacteria via a type IV secretion system encoded in IncP1-type broad-host-range plasmids. Although TKC is considered a potential gene introduction tool, donor chromosomal genes that [...] Read more.

Trans-kingdom conjugation (TKC)/inter-domain conjugation is a horizontal gene transfer phenomenon that transfers DNA from eubacteria to eukaryotes and archaebacteria via a type IV secretion system encoded in IncP1-type broad-host-range plasmids. Although TKC is considered a potential gene introduction tool, donor chromosomal genes that influence TKC efficiency have rarely been analyzed, hindering targeted donor breeding. To identify potential TKC-related genes on a donor chromosome, a genome-wide screening of TKC-deficient mutants was performed using a comprehensive collection of Escherichia coli gene knockout mutants (Keio collection) as donors and a Saccharomyces cerevisiae strain as a recipient. Out of 3884 mutants, two mutants (∆aceE, ∆priA) showed a severe decrease in TKC efficiency by more than two orders of magnitude but not in bacterial conjugation. The effect on TKC efficiency by the two mutants was partly recovered by a preculture with a fresh culture medium before the TKC reaction, regardless of the presence of antibiotics. These results suggest that no single chromosomal target gene is solely responsible for universally blocking IncP1-type conjugation by impeding its function. The results also suggest the existence of an unidentified recognition or transfer mechanism distinct from bacterial conjugation, highlighting the novel roles of aceE and priA. Full article

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

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Overall process of the genome-wide screening. The screening process is illustrated in a flow chart. From 3884 knockout mutants, two mutants (∆aceE, ∆priA) were isolated. The details are described in the <a href="#app1-microorganisms-13-00488" class="html-app">Supplementary Materials</a>. Full article ">Figure 2
∆aceE and ∆priA mutants are severely defective in TKC. The result of the final screening step in group 1 is shown as an example of the screening results. Data are expressed as the mean ± standard deviation (SD) of at least three independent experimental replicates. An asterisk (*) indicates a statistically significant difference against the control at p < 0.05 (two-tailed t-test). Full article ">Figure 3
Confirmation analyses that aceE and priA genes are the chromosomal genes responsible for TKC. (A) Normality check of TKC ability in strains that had reintroduced the helper and TKC vector plasmids collected from the two defective mutants into the parental strain BW25113. (B,C) Recovery check of TKC ability in each defective strain by complementing the respective knockout genes (aceE in B and priA in C). The combinations “∆aceE + aceE” and “∆priA + priA” represent complemented strains. Three, four, and four independent experimental replicates were performed in A, B, and C, respectively. Data are presented as the mean ± SD. An asterisk (*) indicates a statistically significant difference against the control at p < 0.05 (two-tailed t-test). Full article ">Figure 4
Analyses of conjugation ability of the two mutants in bacterial conjugation. (A) Conjugal transfer analysis from ∆aceE and ∆priA mutants to E. coli SY327 (λpir). (B,C) Bacterial conjugation ability check between ∆aceE mutants (B) and between ∆priA mutants (C). Donors are BW25113 (shown as aceE+ in (B) and priA+ in (C)) and its ∆aceE and ∆priA mutants, while recipients are SY327 (λpir) (shown as aceE+ in (B) and priA+ in (C)) and its ∆aceE and ∆priA mutants. Three or four, four, and four independent experimental replicates were performed in A, B, and C, respectively. Data are presented as the mean ± SD. Full article ">Figure 5
Analyses of TKC ability in mutants included in the ace operon (A) and primosome genes (B). Each analysis was performed in triplicate. Data are presented as the mean ± SD. Asterisks (**) indicate a statistically significant difference against the control at p < 0.01 (two-tailed t-test). Full article ">Figure 6
Effect of preculture with fresh medium before TKC reaction on ∆aceE and ∆priA mutants. TKC analysis of the two mutants with a preculture of donor cells by substituting the same amount of fresh medium (A) or by resuspending and diluting the collected donor cells at OD600 = 0.09 (B). Shaded and white bars indicate the presence or absence of antibiotics (chloramphenicol and ampicillin), respectively, in the subculture medium. Each analysis was performed in triplicate. Data are presented as the mean ± SD. Different letters indicate significant differences at p < 0.05 using Tukey HSD multiple comparison analysis. Full article ">Figure 7
Proposed TKC models involving the novel functions of aceE and priA genes. (A) Model illustrating the scenario where fresh medium contains compounds that promote TKC. (B) Model illustrating the scenario where overnight culture accumulates compounds that inhibit TKC. These mechanisms are independent of those involved in basic conjugation, because bacterial conjugation ability remains normal in ∆aceE and ∆priA mutants. Full article ">

21 pages, 2053 KiB

Open AccessArticle

The Composition and Function of Bacterial Communities Associated with the Northern Root-Knot Nematode (Meloidogyne hapla) Populations Showing Parasitic Variability

by Isaac Lartey, Gian M. N. Benucci, Terence L. Marsh, Gregory M. Bonito and Haddish Melakeberhan

Microorganisms 2025, 13(3), 487; https://doi.org/10.3390/microorganisms13030487 (registering DOI) - 22 Feb 2025

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Abstract

The co-existence of microbial communities and Meloidogyne hapla populations showing high, medium, and low levels of parasitic variability (PV) in mineral and muck soils with different soil health conditions in Michigan vegetable production fields is established. However, if PV relates or not to [...] Read more.

The co-existence of microbial communities and Meloidogyne hapla populations showing high, medium, and low levels of parasitic variability (PV) in mineral and muck soils with different soil health conditions in Michigan vegetable production fields is established. However, if PV relates or not to bacterial communities is unknown. This study characterized bacterial communities present on and in the body of nine M. hapla field and greenhouse sub-populations isolated from the mineral and muck fields. We utilized a high throughput sequencing of 16S rDNA. Results showed a variable composition (or abundance) of 65 genera in the field and 61 genera in the greenhouse isolates, with 12 genera of unknown and the rest belonging to 14 known functional groups. The medium- and low-PV populations shared more bacterial composition than either one with the high-PV population. Thus, laying a foundation for an in-depth understanding of if the observed associations have any role in cause-and-effect relationships with M. hapla PV. Full article

(This article belongs to the Special Issue Feature Papers in Microbiomes)

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9 pages, 2180 KiB

Open AccessCommunication

Virus Infection of a Freshwater Cyanobacterium Contributes Significantly to the Release of Toxins Through Cell Lysis

by Victoria Lee, Isaac Meza-Padilla and Jozef I. Nissimov

Microorganisms 2025, 13(3), 486; https://doi.org/10.3390/microorganisms13030486 (registering DOI) - 22 Feb 2025

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Abstract

Toxic algal-bloom-forming cyanobacteria are a persistent problem globally for many aquatic environments. Their occurrence is attributed to eutrophication and rising temperatures due to climate change. The result of these blooms is often the loss of biodiversity, economic impacts on tourism and fisheries, and [...] Read more.

Toxic algal-bloom-forming cyanobacteria are a persistent problem globally for many aquatic environments. Their occurrence is attributed to eutrophication and rising temperatures due to climate change. The result of these blooms is often the loss of biodiversity, economic impacts on tourism and fisheries, and risks to human and animal health. Of emerging interest is the poorly understood interplay between viruses and toxic species that form blooms. This is because recent studies have suggested that viruses may exacerbate the harmful effects of these blooms by contributing to the release of toxins into a dissolved phase upon cell lysis. However, to date, there is no experimental evidence that explicitly implicates viruses in microcystin release. Here, we show experimentally that a virus infection of the toxin-producing, harmful, algal-bloom-forming cyanobacterium Microcystis aeruginosa results in a 4-fold increase in the toxin microcystin-LR two days post-infection (dpi). We also show that the concentrations of microcystin remain high after culture discoloration and host cell lysis. Collectively, our results directly implicate viruses as major contributors to microcystin release from cyanobacteria and emphasize the importance of taking viruses into account in predictive models and in the assessment of water quality and safety. Full article

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

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Extracellular cyanotoxin release by cyanobacteria such as Microcystis aeruginosa. (A) The measurable extracellular fraction of cyanotoxins in the absence of a virus infection includes toxins that are typically released upon senescence and/or cell death, with some cyanobacterial species being able to release toxins without cell rupture or death. This is what we measured in our control, uninfected M. aeruginosa NIES-298 treatments. (B) The measurable extracellular fraction of cyanotoxins in the presence of viruses includes intracellular toxins that are typically contained within the cyanobacterial cells and are released upon cell lysis. This is what we measured in our virus-infected M. aeruginosa NIES-298 treatments. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. Full article ">Figure 2
Cyanophage-infected and uninfected M. aeruginosa NIES-298 cyanobacterial cultures and the subsequent analysis of extracellular microcystin-LR dynamics during infection. (a) 1—Cyanobacterial cells were incubated until the late exponential growth phase (i.e., 2.95 × 107 cells mL−1); 2—the culture was then split into six replicates at day 0 (dashed line in (b)), three of which were infected with a cyanophage Ma-LMM01 stock that was at a virus particle density of 1.35 × 107 mL−1 and three of which were inoculated with an equal volume of a 0.02 µm filtrate of the Ma-LMM01 stock; and 4—ELISA essays and total NIES-298 cell abundance measurements were performed using spectrophotometry and haemocytometry, respectively. (b) M. aeruginosa NIES-298 growth dynamics (n = 3, ±SD) of Ma-LMM01-infected (grey line) and uninfected (green line) treatments up to seven days post-infection on day 0 (indicated as a dashed line). (c) Average (± SD, n = 3) extracellular microcystin-LR concentrations in parts per billion (ppb) in cyanophage Ma-LMM01-infected (dark grey bars) and uninfected (green bars) treatments. ** and * denote significant differences (p < 0.01 and p < 0.05, respectively; ANOVA) between infected and uninfected treatments at individual time points. (d) Average daily rate of extracellular microcystin-LR decrease in infected treatments, calculated between the highest measured concentration on day 2 and the last day of the experiment on day seven. (e) Culture pigmentation (photographed) of a representative triplicate treatment, which was either infected (V) or uninfected (C) by viruses, 0–7 dpi. Panel (a) was created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. Full article ">

33 pages, 1646 KiB

Open AccessReview

Effects of Microorganisms in Fish Aquaculture from a Sustainable Approach: A Review

by Jesús Mateo Amillano-Cisneros, María Anel Fuentes-Valencia, José Belisario Leyva-Morales, Macario Savín-Amador, Henri Márquez-Pacheco, Pedro de Jesús Bastidas-Bastidas, Lucía Leyva-Camacho, Zamaria Yoselin De la Torre-Espinosa and César Noé Badilla-Medina

Microorganisms 2025, 13(3), 485; https://doi.org/10.3390/microorganisms13030485 - 21 Feb 2025

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Abstract

Aquaculture is the fastest-growing food production sector. However, it faces significant challenges, including demand from a growing global population, which is estimated to reach 10.4 billion by the year 2100, disease outbreaks, environmental impacts, and the overuse of antibiotics. To address these issues, [...] Read more.

Aquaculture is the fastest-growing food production sector. However, it faces significant challenges, including demand from a growing global population, which is estimated to reach 10.4 billion by the year 2100, disease outbreaks, environmental impacts, and the overuse of antibiotics. To address these issues, sustainable alternatives such as the use of microorganisms (probiotics, bacteriophages, and genetically modified microorganisms) have gained attention. This review examines the effects of these microorganisms on fish aquaculture, focusing on their potential to improve growth, health, and disease resistance while reducing environmental impacts. Probiotics, particularly lactic acid bacteria and yeasts, have been shown to enhance immune responses, digestive enzyme activity, and nutrient absorption in fish. Bacteriophages offer a promising alternative to antibiotics for controlling bacterial pathogens, with studies demonstrating their efficacy in reducing mortality rates in infected fish. Additionally, genetically modified microorganisms (GMMs) have been explored for their ability to produce beneficial compounds, such as enzymes and antimicrobial peptides, which can improve fish health and reduce the need for chemical treatments. Despite their potential, challenges such as regulatory hurdles, public acceptance, and environmental risks must be addressed. This review highlights the importance of further research to optimize the use of microorganisms in aquaculture and underscores their role in promoting sustainable practices. By integrating these biological tools, the aquaculture industry can move towards a more sustainable and environmentally friendly future. Full article

(This article belongs to the Special Issue Aquatic Microorganisms and Their Application in Aquaculture)

13 pages, 784 KiB

Open AccessArticle

Bacteriophage Resistance, Adhesin’s and Toxin’s Genes Profile of Staphylococcus aureus Causing Infections in Children and Adolescents

by Nikolaos Giormezis, Assimina Rechenioti, Konstantinos Doumanas, Christos Sotiropoulos, Fotini Paliogianni and Fevronia Kolonitsiou

Microorganisms 2025, 13(3), 484; https://doi.org/10.3390/microorganisms13030484 - 21 Feb 2025

Viewed by 196

Abstract

Staphylococcus aureus is a common pathogen, often recovered from children’s infections. Βiofilm formation, antimicrobial resistance and production of adhesins and toxins contribute to its virulence. As resistance to antimicrobials rises worldwide, alternative therapies like bacteriophages (among them the well-studied Bacteriophage K) can be [...] Read more.

Staphylococcus aureus is a common pathogen, often recovered from children’s infections. Βiofilm formation, antimicrobial resistance and production of adhesins and toxins contribute to its virulence. As resistance to antimicrobials rises worldwide, alternative therapies like bacteriophages (among them the well-studied Bacteriophage K) can be helpful. The aim of this study was to determine the bacteriophage and antimicrobial susceptibility and the presence of virulence genes among S. aureus from infections in children and adolescents. Eighty S. aureus isolates were tested for biofilm formation and antimicrobial susceptibility. The presence of two genes of the ica operon (icaA, icaD), adhesin’s (fnbA, fnbB, sasG) and toxin’s genes (PVL, tst, eta, etb) was tested by PCRs. Susceptibility to Bacteriophage K was determined using a spot assay. Thirteen isolates were methicillin-resistant (MRSA) and 41 were multi-resistant. Twenty-five S. aureus (31.3%) were resistant to Bacteriophage K, mostly from ocular and ear infections. Twelve S. aureus (15%) were PVL-positive, seven (8.8%) positive for tst, 18 (22.5%) were eta-positive and 46 were (57.5%) etb-positive. A total of 66 (82.5%) isolates carried fnbA, 16 (20%) fnbB and 26 (32.5%) sasG. PVL, tst and sasG carriage were more frequent in MRSA. Bacteriophage-susceptible isolates carried more frequently eta (32.7%) and etb (69.1%) compared to phage-resistant S. aureus (0% and 32%, respectively). Although mainly methicillin-sensitive, S. aureus from pediatric infections exhibited high antimicrobial resistance and carriage of virulence genes (especially for exfoliative toxins and fnbA). MRSA was associated with PVL, tst and sasG carriage, whereas Bacteriophage susceptibility was associated with eta and etb. The high level of Bacteriophage K susceptibility highlights its potential use against staphylococcal infections. Full article

(This article belongs to the Special Issue Combating Antimicrobial Resistance: Innovations and Strategies)

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21 pages, 1894 KiB

Open AccessArticle

Putting Laccase Gene Differences on Genomic Level into Context: An Analysis of Botrytis cinerea Strains from Grapes

by Louis Backmann, Kim Marie Umberath, Pascal Wegmann-Herr, Fabian Weber, Andreas Jürgens and Maren Scharfenberger-Schmeer

Microorganisms 2025, 13(3), 483; https://doi.org/10.3390/microorganisms13030483 - 21 Feb 2025

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Abstract

One of the most important crop pathogens is Botrytis cinerea. It overcomes plant defenses using laccase, an enzyme which is frequently researched. Yet the differences between strains regarding their laccase activity is poorly understood. The aim of this study was to analyze [...] Read more.

One of the most important crop pathogens is Botrytis cinerea. It overcomes plant defenses using laccase, an enzyme which is frequently researched. Yet the differences between strains regarding their laccase activity is poorly understood. The aim of this study was to analyze laccase genes in the context of the regionality, vintage, and laccase activity of the strains. Eight strains were analyzed using whole genome sequencing, and the laccase activity was assessed. The strains were differentiated by SSR-PCR. We looked at all 14 known laccase genome regions as well as the promoter and terminator regions using variant metrics and phylogenetic trees. The laccase genes seem to be correlated with the regionality of the strains rather than the laccase activity, which provides new understanding to the study of pathogen adaption in specific environments. Some of the laccase gene regions showed little to no evolutionary change, while other regions showed a great variety of changes. This research highlights taking different laccase gene regions into context. We provide fundamental information for further research. Further studies, especially on gene expression, could provide insightful information regarding the potential of pathogen infection. Full article

(This article belongs to the Special Issue Fungal Biology and Interactions, 2nd Edition)

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11 pages, 1028 KiB

Open AccessCommunication

Molecular Detection of bla_TEM and bla_SHV Genes in ESBL-Producing Acinetobacter baumannii Isolated from Antarctic Soil

by Clara Pazos, Miguel Gualoto, Tania Oña, Elizabeth Velarde, Karen Portilla, Santiago Cabrera-García, Carlos Banchón, Gabriela Dávila, Fernanda Hernández-Alomia and Carlos Bastidas-Caldes

Microorganisms 2025, 13(3), 482; https://doi.org/10.3390/microorganisms13030482 - 21 Feb 2025

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Abstract

The phenomenon of antimicrobial resistance (AMR) in cold environments, exemplified by the Antarctic, calls into question the assumption that pristine ecosystems lack clinically significant resistance genes. This study examines the molecular basis of AMR in Acinetobacter spp. Isolated from Antarctic soil, focusing on [...] Read more.

The phenomenon of antimicrobial resistance (AMR) in cold environments, exemplified by the Antarctic, calls into question the assumption that pristine ecosystems lack clinically significant resistance genes. This study examines the molecular basis of AMR in Acinetobacter spp. Isolated from Antarctic soil, focusing on the bla_TEM and bla_SHV genes associated with extended-spectrum beta-lactamase (ESBL) production; Soil samples were collected and processed to isolate Antarctic soil bacteria. Molecular detection was then conducted using polymerase chain reaction (PCR) to identify the bacteria species by 16S rRNA/rpoB and 10 different beta-lactamase-producing genes. PCR amplicons were sequenced to confirm gene identity and analyze genetic variability. Acinetobacter baumannii were identified by both microbiological and molecular tests. Notably, both the bla_TEM and bla_SHV genes encoding the enzymes responsible for resistance to penicillins and cephalosporins were identified, indicating the presence of resistance determinants in bacteria from extreme cold ecosystems. The nucleotide sequence analysis indicated the presence of conserved ARGs, which suggest stability and the potential for horizontal gene transfer within microbial communities. These findings emphasize that AMR is not confined to human-impacted environments but can emerge and persist in remote, cold habitats, potentially facilitated by natural reservoirs and global microbial dispersal. Understanding the presence and role of AMR in extreme environments provides insights into its global dissemination and supports the development of strategies to mitigate the spread of resistance genes in both environmental and clinical contexts. Full article

(This article belongs to the Special Issue Antibiotic and Resistance Gene Pollution in the Environment)

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20 pages, 1231 KiB

Open AccessArticle

Cryptococcus neoformans: Brain Preference, Gender Bias, and Interactions with Mycobacterium tuberculosis and Toxoplasma gondii in HIV-Positive Patients

by Ruxandra Moroti, Adriana Hristea, Georgiana Neagu, Irina Penescu, Dragos Florea, Catalin Tiliscan and Serban Nicolae Benea

Microorganisms 2025, 13(3), 481; https://doi.org/10.3390/microorganisms13030481 - 21 Feb 2025

Viewed by 223

Abstract

Background: Cryptococcus neoformans, a high-priority pathogen (WHO, 2022) and ubiquitous fungus, is responsible for hundreds of thousands of meningoencephalitis cases annually, with a high fatality rate. Its distribution is uneven: it primarily affects immunocompromised individuals (especially HIV-positive patients). Our study aims to [...] Read more.

Background: Cryptococcus neoformans, a high-priority pathogen (WHO, 2022) and ubiquitous fungus, is responsible for hundreds of thousands of meningoencephalitis cases annually, with a high fatality rate. Its distribution is uneven: it primarily affects immunocompromised individuals (especially HIV-positive patients). Our study aims to explore the Cryptococcus’ brain tropism in immunosuppressed patients, its gender preference and the possible interactions with other opportunistic neurotropic microorganisms, such as Mycobacterium tuberculosis (MTB) and the brain microbiota, with a particular focus on Toxoplasma gondii (T. gondii). Methods: We conducted a retrospective descriptive analysis of all cases diagnosed with central nervous system cryptococcosis (Crypto-CNS) in HIV-positive patients admitted over 10 years (2010–2019) in a tertiary Romanian hospital. We examined their demographic, clinical, immunobiological, and imaging data, as well as their medical history, comorbidities, and coinfections. Results: Forty-two cases were admitted, with a male predominance (3.6:1) and a mean age of 33.3 years; 24% were diagnosed concomitantly with HIV infection and Crypto-CNS. All patients were severely immunosuppressed, with CD4 counts <200 cells/mm³ (median = 20.5 [1–163], mean = 31.6). Recent/concomitant tuberculosis was found in 10 (27.7%). T. gondii-seropositive patients developed Crypto-CNS at a lower immunological state than seronegative ones (27.1 CD4 cells/mm³ vs. 46.7 cells/mm³, means). Of 25 cases with available brain imagery, 28% had high intracranial pressure. Twelve patients (28.5%) died during the hospitalization within 26.3 days (mean, SD = 21.4); 1-year mortality increased to 50%. In-hospital mortality was associated with lower CD4 counts, increased intracranial pressure, and T. gondii-seropositivity. Conclusions: Crypto-CNS in HIV-positive patients mainly affects men and may be promoted by concomitant or recent tuberculosis. T. gondii may confer some protection even at low immune levels but increases mortality when immunity is critically low. Full article

(This article belongs to the Special Issue Infectious Disease Surveillance in Romania)

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Schematical immune defense against Cryptococcus, in the CNS of an advanced HIV disease and latent toxoplasmosis. Legend: (A) The immune level > 30 (50) CD4 count/mm3. (1) Toxoplasma elicits Th1 responses, to be contained dormant in the cysts. (2) Th1 responses elevate local levels of gamma interferon (IFNg). (3) High local IFNg levels activate local macrophages. (4) If Cryptococcus enters the brain, it is destroyed by the activated macrophages. (B) The immune level < 50 (30) CD4 count/mm3. (5) The immune system is critically impaired, resulting in insufficient T lymphocytes to produce IFNg, even if the stimulus for this activation exists (Toxoplasma is there); consequently, local macrophages are not stimulated (activated). (6) in case of Cryptococcus invasion there is no defense and Cryptococcus flourishes. (7) Additionally, there is no containment for Toxoplasma which can reactivate and produce dopamine, potentially enhancing the aggressiveness of Cryptococcus. Created in BioRender 2025. Moroti, R. (2025) <a href="https://BioRender.com/o98i548" target="_blank">https://BioRender.com/o98i548</a> (Created on 9 February 2025). Full article ">Figure 2
Dopamine, Toxoplasma and Cryptococcus in the Brain. Legend: (1). T. gondii’s tyrosine-hydroxylase contributes to dopamine synthesis (2) Cryptococcus reaches the brain’s neuromelanin (derived from dopamine) and undergoes melanization of the wall and capsule (3) Melanized Cryptococcus is protected from oxidants and proliferates exuberantly; the conglomerates of fungal bodies and their capsules could block the CSF circulation. Created in BioRender. Moroti, R. (2025) <a href="https://BioRender.com/l13l522" target="_blank">https://BioRender.com/l13l522</a> (Created on 9 February 2025). Full article ">

18 pages, 11899 KiB

Open AccessArticle

Investigation of Eumelanin Biosynthesis in Gluconacetobacter tumulisoli FBFS 97: A Novel Insight into a Bacterial Melanin Producer

by Jiayun Song, Yanqin Ma, Zhenzhen Xie and Fusheng Chen

Microorganisms 2025, 13(3), 480; https://doi.org/10.3390/microorganisms13030480 - 21 Feb 2025

Viewed by 183

Abstract

Acetic acid bacteria (AAB) are a group of bacteria, most of which can produce pigments. However, the mechanism of pigment production by AAB is unclear. A strain of AAB, Gluconacetobacter tumulisoli FBFS 97, which can produce a large amount of brown pigment (BP), [...] Read more.

Acetic acid bacteria (AAB) are a group of bacteria, most of which can produce pigments. However, the mechanism of pigment production by AAB is unclear. A strain of AAB, Gluconacetobacter tumulisoli FBFS 97, which can produce a large amount of brown pigment (BP), was isolated in our previous research. In the current study, it was found that the BP yield of the FBFS 97 strain was enhanced in the presence of tyrosine, and an intermediate of melanin, L-3,4-dihydroxyphenylalanine (L-DOPA), was identified using ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The structural properties of BP were analyzed by pyrolysis gas chromatography–mass spectrometry (Py-GC-MS). All these analyses suggest that BP may be eumelanin, a type of melanin. Then, the eumelanin biosynthetic pathway was investigated in the FBFS 97 strain, and three related genes with eumelanin including pheA, yfiH, and phhB in its genome were found and knocked out, respectively. The results showed that eumelanin production increased 1.3-fold in the pheA deletion mutant compared to the wild-type FBFS 97 strain, but when either yfiH or phhB was knocked out, the eumelanin production in the mutants was the same as that in the wild-type FBFS 97 strain. Finally, a possible biosynthetic pathway for eumelanin in the FBFS 97 strain is proposed. Full article

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

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Extraction and purification of BP produced by the FBFS 97 strain. This figure was created using Figdraw 2.0. Full article ">Figure 2
BP yield and biomass of FBFS 97 strain under different carbon sources (A), different glucose concentrations (B), and different fermentation days (C), as well as the effects of different concentrations of tyrosine and phenylalanine (D) and vitamin C (Vc) (E) on BP production by FBFS 97 strain. Full article ">Figure 3
Detection of L-DOPA in FBFS 97 culture by UPLC-Q-TOF-MS. L-DOPA standard (A) and L-DOPA in fermentation broth on the fourth day (B). Full article ">Figure 4
Extraction, purification, and ultraviolet–visible (UV-Vis) absorption spectra of BP. The extracted and purified BP from fermentation broth (A), the UV-Vis spectra of purified BP (B), and the linear curve obtained after plotting the wavelengths against the logarithmic values of absorbances (C). Full article ">Figure 5
TG curve (black curve) and DTG curve (red curve) of BP. Full article ">Figure 6
The schematic diagram of gene traceless modification system (A), agarose gel electrophoresis of the target gene to be knocked out (B): (a) agarose gel electrophoresis of the upstream and downstream flanking regions of the target gene to be knocked out (M: DL8000 DNA marker; 1, 2, 3, 4, 5, 6: PCR products of upstream and downstream regions of yfiH, phhB, and pheA), (b) agarose gel electrophoresis of yfiH, phhB, and pheA single-crossover mutants, (c) agarose gel electrophoresis of ΔyfiH, ΔphhB, and ΔpheA mutants, cell growth (C) and melanin yield (D) of wild-type FBFS 97 and the mutant strains. *: p < 0.05. Full article ">Figure 7
Pathway for biosynthesis of phenyllactic acid. Full article ">Figure 8
Proposed eumelanin biosynthesis pathway in the FBFS 97 strain. (The red cross indicates the knockout of pheA, which increases tyrosine flux and promotes eumelanin production. The red dashed box indicates that eumelanin formation in FBFS 97 may involve enzymes that function like tyrosinase, requiring further study). Full article ">

21 pages, 3251 KiB

Open AccessArticle

Internalization of Lactobacillus crispatus Through Caveolin-1-Mediated Endocytosis Boosts Cellular Uptake but Blocks the Transcellular Passage of Neisseria meningitidis

by Kenny Lidberg, Sarah Pilheden, Mikel Relloso Ortiz de Uriarte and Ann-Beth Jonsson

Microorganisms 2025, 13(3), 479; https://doi.org/10.3390/microorganisms13030479 - 21 Feb 2025

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Abstract

Neisseria meningitidis is a human-specific pathogen that colonizes the nasopharyngeal epithelium, which is populated by a dynamic microbiota that includes Lactobacillus species. Currently, little is known about the interaction between commensal lactobacilli and pathogenic Neisseria, emphasizing a need for deeper studies into [...] Read more.

Neisseria meningitidis is a human-specific pathogen that colonizes the nasopharyngeal epithelium, which is populated by a dynamic microbiota that includes Lactobacillus species. Currently, little is known about the interaction between commensal lactobacilli and pathogenic Neisseria, emphasizing a need for deeper studies into the molecular interactions between the two bacteria species. This, in turn, could add clinical and therapeutic value to existing treatments against an N. meningitidis infection. In this work, we explored how lactobacilli affect the interplay between N. meningitidis and host cells. We report that Lactobacillus crispatus, but not other tested Lactobacillus species, efficiently enters pharyngeal cells via caveolin-mediated lipid raft endocytosis and simultaneously enhances the uptake of N. meningitidis, as well as uptake of other pathogenic and non-pathogenic microbes. After promoting internalization, L. crispatus then prevented N. meningitidis from being released and transcytozed from a confluent cell layer on microporous transwell membranes. Infected cells increased the level of acidic vacuoles and pathogen clearance over time, while lactobacilli survived inside the cells. Taken together, the data suggest a possible route through which the cellular uptake of lactobacilli can increase the uptake of pathogens for destruction. Full article

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

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14 pages, 1103 KiB

Open AccessArticle

Pathotypes and Simple Sequence Repeat (SSR)-Based Genetic Diversity of Phytophthora sojae Isolates in the Republic of Korea

by Ngoc Ha Luong, In-Jeong Kang, Hee Jin You and Sungwoo Lee

Microorganisms 2025, 13(3), 478; https://doi.org/10.3390/microorganisms13030478 - 21 Feb 2025

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Abstract

Phytophthora sojae is the causal agent of the Phytophthora root and stem rot in soybean, which has resulted in a significant increase in the incidence of the disease and substantial yield losses on a global scale. The proliferation of Phytophthora sojae can be mitigated [...] Read more.

Phytophthora sojae is the causal agent of the Phytophthora root and stem rot in soybean, which has resulted in a significant increase in the incidence of the disease and substantial yield losses on a global scale. The proliferation of Phytophthora sojae can be mitigated through the development of Phytophthora-resistant soybean cultivars. A fundamental understanding of the genetic diversity and dynamic changes within the P. sojae population is essential for disease management and the development of new P. sojae-resistant varieties. Although a large number of pathogen samples can lead to more comprehensive interpretations and better conclusions, only six indigenous P. sojae isolates were available in the Republic of Korea at the time of the experiments. Due to the limited availability, this study preliminarily aimed to assess the pathotypes and genetic variation of the six P. sojae isolates collected in the Republic of Korea. The virulence patterns of all the six P. sojae isolates differed based on the 15 soybean differentials known for P. sojae resistance. The six isolates displayed high levels of pathotype complexities, ranging from 8 to 15, which is notably higher than those observed in other countries. Furthermore, 18 of the 21 simple sequence repeat markers used exhibited polymorphisms. The mean allele number (3.8) shows higher genetic variability compared to that (2.5) of isolates from the USA. The gene diversity (0.624) and the mean polymorphic information content (0.579) also displayed high levels of variation among the six isolates. A low mean heterozygosity (0.019) indicated a rare but possible outcrossing between the isolates, which was detected by the SSR marker PS07. Genetic dissimilarity assessments were employed to categorize the six P. sojae isolates into three groups using a neighbor-joining phylogenetic tree and principal component analysis. Although on a small scale, the phenotypic and genotypic assay results obtained indicated a significant variability in the pathotypes and genetic variation within the P. sojae isolates in the Republic of Korea. Though limited in scope, these results will be a cornerstone for elucidating the virulence pathotype and genetic diversity of the P. sojae population in future analyses. These findings also have the potential to improve the soybean breeding strategies aimed at enhancing resistance to P. sojae in the Republic of Korea. Full article

(This article belongs to the Special Issue Plant Pathogenic Fungi: Genetics and Genomics)

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19 pages, 1914 KiB

Open AccessReview

The Metabolic Pathways of Yeast and Acetic Acid Bacteria During Fruit Vinegar Fermentation and Their Influence on Flavor Development

by Yinggang Ge, Yifei Wu, Aihemaitijiang Aihaiti, Liang Wang, Yu Wang, Jun Xing, Min Zhu and Jingyang Hong

Microorganisms 2025, 13(3), 477; https://doi.org/10.3390/microorganisms13030477 - 21 Feb 2025

Viewed by 252

Abstract

Fruit vinegar is a beverage derived from fruits or fruit processing by-products through microbial fermentation. This vinegar possesses a distinctive flavor profile and contains bioactive compounds. It is typically produced using liquid fermentation technology. As consumer demand for the flavor quality of fruit [...] Read more.

Fruit vinegar is a beverage derived from fruits or fruit processing by-products through microbial fermentation. This vinegar possesses a distinctive flavor profile and contains bioactive compounds. It is typically produced using liquid fermentation technology. As consumer demand for the flavor quality of fruit vinegar has increased, precise control over flavor compounds has become crucial for enhancing the quality of fermentation products. Vinegar contains numerous characteristic flavor compounds, including esters, aldehydes, alcohols, and organic acids. These unique flavors primarily result from the accumulation of flavor compounds generated by different raw materials and microorganisms during fermentation. Specifically, yeast and acetobacter promote the formation of distinct fruit vinegar flavors by facilitating the breakdown of carbohydrates, amino acids, and proteins in fruits, as well as the redox and esterification reactions involving alcohols. This paper reviews the metabolic pathways of yeast and acetic acid bacteria during fruit vinegar fermentation and discusses key volatile compounds that influence the flavor of fruit vinegar and their potential relationships, providing theoretical support for regulating flavor quality. Full article

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

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7 pages, 2982 KiB

Open AccessCase Report

A Rare Case Report of a Human Dirofilaria repens Infection

by Christoph Schatz, Magdalena Füßl, Yasemin Caf, Katja Schmitz, Daniela Kresse, Wilhelm Ludwig, Julia Walochnik and Ludwig Knabl

Microorganisms 2025, 13(3), 476; https://doi.org/10.3390/microorganisms13030476 - 21 Feb 2025

Viewed by 160

Abstract

In June 2024, a 41 year-old woman presented to the infectious diseases outpatient clinic with a left inguinal mass progressing in size. The patient had previously been on vacation in Greece. When a tumor was initially suspected, the mass was surgically removed. Staining [...] Read more.

In June 2024, a 41 year-old woman presented to the infectious diseases outpatient clinic with a left inguinal mass progressing in size. The patient had previously been on vacation in Greece. When a tumor was initially suspected, the mass was surgically removed. Staining with Grocott methenamine silver and Alzian blue were inconspicuous, but histopathologic examination revealed a clear histiocytic demarcation, followed by a confirmation of the suspected diagnosis of dirofilariasis caused by Dirofilaria repens by PCR. Even though still a rare event in Austria, the number of human D. repens cases has been continuously increasing in recent years. This is partly due to the increased spread of the parasite due to climate change and globalization. Full article

(This article belongs to the Section Medical Microbiology)

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31 pages, 3494 KiB

Open AccessArticle

Age-Dependent Pleomorphism in Mycobacterium monacense Cultures

by Malavika Ramesh, Phani Rama Krishna Behra, B. M. Fredrik Pettersson, Santanu Dasgupta and Leif A. Kirsebom

Microorganisms 2025, 13(3), 475; https://doi.org/10.3390/microorganisms13030475 - 20 Feb 2025

Viewed by 85

Abstract

Changes in cell shape have been shown to be an integral part of the mycobacterial life cycle; however, systematic investigations into its patterns of pleomorphic behaviour in connection with stages or conditions of growth are scarce. We have studied the complete growth cycle [...] Read more.

Changes in cell shape have been shown to be an integral part of the mycobacterial life cycle; however, systematic investigations into its patterns of pleomorphic behaviour in connection with stages or conditions of growth are scarce. We have studied the complete growth cycle of Mycobacterium monacense cultures, a Non-Tuberculous Mycobacterium (NTM), in solid as well as in liquid media. We provide data showing changes in cell shape from rod to coccoid and occurrence of refractive cells ranging from Phase Grey to phase Bright (PGB) in appearance upon ageing. Changes in cell shape could be correlated to the bi-phasic nature of the growth curves for M. monacense (and the NTM Mycobacterium boenickei) as measured by the absorbance of liquid cultures while growth measured by colony-forming units (CFU) on solid media showed a uniform exponential growth. Based on the complete M. monacense genome we identified genes involved in cell morphology, and analyses of their mRNA levels revealed changes at different stages of growth. One gene, dnaK_3 (encoding a chaperone), showed significantly increased transcript levels in stationary phase cells relative to exponentially growing cells. Based on protein domain architecture, we identified that the DnaK_3 N-terminus domain is an MreB-like homolog. Endogenous overexpression of M. monacense dnaK_3 in M. monacense was unsuccessful (appears to be lethal) while exogenous overexpression in Mycobacterium marinum resulted in morphological changes with an impact on the frequency of appearance of PGB cells. However, the introduction of an anti-sense “gene” targeting the M. marinum dnaK_3 did not show significant effects. Using dnaK_3-lacZ reporter constructs we also provide data suggesting that the morphological differences could be due to differences in the regulation of dnaK_3 in the two species. Together these data suggest that, although its regulation may vary between mycobacterial species, the dnaK_3 might have a direct or indirect role in the processes influencing mycobacterial cell shape. Full article

(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Bacterial Infection)

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The representative growth curves for MmonT and MmonRFPHyg cultivated under various conditions. (A) The growth curve for MmonT (marked with squares) and MmonRFPHyg (marked with circles) cultivated on 7H10 (supplemented with hygromycin in the case of MmonRFPHyg) plates with average generation times ± deviations in hours (h). WT_Expo and RFP_Expo represent exponential growth phases and the trend lines representing the slope of the growth curves are marked in red. The growth was generated by plotting the average values for each time point. The average CFU/mL was calculated from two biological replicates for each strain (see <a href="#sec2-microorganisms-13-00475" class="html-sec">Section 2</a>) and the generation times are given in hours ± error range. (B) The growth curve for MmonRFPHyg in liquid 7H9 media with two exponential growth phases, Expo I and Expo II (highlighted dark) and trend lines representing the slope of the curve in red used to calculate the generation times (see <a href="#sec2-microorganisms-13-00475" class="html-sec">Section 2</a>) ± error range as indicated. (C) The “normal” growth curve for MmonRFPHyg (in grey) and the growth curves after subjecting Expo II cells to various conditions, “re-suspended” in orange, addition of “Tween” in green, and addition of “glycerol” in red (for details see <a href="#sec2-microorganisms-13-00475" class="html-sec">Section 2</a>). The red arrow marks the time when cells were harvested and subjected to various conditions as outlined in see <a href="#sec2-microorganisms-13-00475" class="html-sec">Section 2</a>. (C,D) show representative best fit curves (with R2-values ≈ 1) to the observed bi-phasic growth pattern (see also <a href="#app1-microorganisms-13-00475" class="html-app">Table S3</a>). (D) The growth curves for Expo I cells (MmonRFPHyg) inoculated into “fresh media” (blue triangles) and “spent media” (blue circles) and for Expo II cells inoculated into “fresh media” (red triangles) and “spent media” (red circles). Full article ">Figure 2
Microscopy of MmonT and MmonRFPHyg grown on 7H10 media and 37 °C. (A) Cells stained with FM4-64 (red) and DAPI (blue). Yellow arrows represent the PGB morphologies observed at different time points. Scale bar = 1 µm. (B) MmonRFPHyg cells expressing RFP (red) stained with MTG (green) and DAPI (blue). Yellow arrows indicate the PGB cell morphology. Scale bar = 1 µm. (C,D) Statistical representation of average percentage of occurrence of the various cell morphology types, MmonT (C) and MmonRFPHyg (D). A minimum of 350 cells were analysed for each time point. “d” = number of days. (E) One day old MmonT cells (rods and coccoids, yellow arrows mark the division site) stained with FM4-64 and DAPI depicting asymmetric septum formation (see top panel). Scale bar = 1 mm. (F) TEM image of a two days old culture showing dividing cells in the top panel. The bottom panel shows a TEM image of 2 and 17 days old coccoid MmonT cells showing asymmetric division sites. For each time point and condition, a minimum of 350 cells were counted. Error bars represent standard deviation. Plots represent the final averages of percentage of occurrence (see <a href="#sec2-microorganisms-13-00475" class="html-sec">Section 2</a> for details). Full article ">Figure 3
Visualisation of old MmonT and MmonRFPHyg cells grown on 7H10 media at 37 °C by phase contrast and transmission electron microscopy as indicated. (A) Phase contrast microscopy of 28 days old MmonT cells after enrichment for spores (see <a href="#sec2-microorganisms-13-00475" class="html-sec">Section 2</a>). Yellow arrows mark refractive PGB cells. Scale bar = 1 mm. (B) TEM images of 28 days old MmonT cells after spore enrichment. Red arrows mark internal membrane structures. (C) Internal structures (yellow arrows) observed in PGB cells detected in old MmonRFPHyg cultures (48 days old, top row; 14 days old, middle and bottom rows). Cells were stained with MTG (green) while red is the result of the presence of rfp. Scale bar = 1 mm. (D) MmonT cells one week after growth of enriched PGB cells on fresh 7H10 media. Phase contrast microscopy (top panels; Scale bar = 1 mm) and TEM (bottom panel; Scale bar = 2 mm). Pink arrows mark appearance of rod-shaped cells, see (A) for comparison. Full article ">Figure 4
MmonT genome and functional classification of genes. (A) Overview of the MmonT complete genome. From the outer to inner circle: Green track illustrates the genome overlapping with scale along the genome length and position of genes (red and blue arrow heads mark the direction of transcription) listed in <a href="#app1-microorganisms-13-00475" class="html-app">Table S4</a>. The next two circles represent genes in forward (brown) and reversed (purple) strands. The next circle shows the GC-content distribution calculated with a sliding window of 1000 bp, blue (higher than mean value) and grey (lower than mean value) “spikes” correspond to variations of the mean GC-content 68.4% in ±10 and ±20 units, i.e., outer grey circle = 88.4% and inner grey circle = 48.4%. The inner circle, red (positive) and green (negative) correspond to the GC-skew obtained using a sliding window of 1000 bp. Generation of circus plot, see <a href="http://circos.ca" target="_blank">http://circos.ca</a> (last accessed on 9 July 2019). (B) Subsystem classification of 3557 MmonT genes as indicated. Full article ">Figure 5
Analysis of mRNA levels of selected genes and dnaK paralogs in MmonT. (A–D) Change in mRNA levels as indicated comparing MmonRFPHyg cells of different ages (6, 14 and 48 days) relative to 3 days old cells. The changes are expressed as log2-fold change and the selected genes were grouped as indicated. (A) Peptidoglycan-associated genes, (B) divisome- and elongation-associated genes, (C) arabinogalactan-associated genes and (D) dnaK homologs, the highlighted bars correspond to the change observed for dnaK_3. (E) Illustration of the domain architecture of the four DnaK proteins in MmonT. Along with the expected Hsp70 domain, DnaK_3 (highlighted) is suggested to carry a MreB-Mbl domain while the other three lack this element. Notably, according to the Pfam database the average length of MreB-Mbl proteins encompass 313 amino acids. (F) Gene synteny for dnaK_3, grpE, dnaJ and hspR in MmonT, MmarT and MtbH37Rv. The arrows represent the genes as indicated. (G) Change in DnaK_3, GrpE, DnaJ and HspR mRNA levels expressed as log2-fold change as indicated. For MmonRFPHyg, mRNA levels at different time points (6, 14 and 48 days of growth) relative to levels in exponentially growing cells (3 days of growth). In the case of MmarRFPHyg mRNA levels in exponentially growing cells (OD600 = 0.5) compared to levels in stationary cells (OD600 ≈ 3). Statistical significance, ** p < 0.01; *** p < 0.001. Full article ">Figure 6
Analysis of the over-expression of dnaK_3Mmon and dnaK_3Mmar in MmarT. (A) Represents time course microscopy of MmarRFPHyg (top panel) with the corresponding average frequencies of occurrence of the different cell morphotypes (bottom panel). Scale bar = 1 mm. (B) Statistical distribution of the different cell morphologies in MmarpBS401-dnaK3Mmon (un-induced and induced conditions) observed in the cell cultures. (C) Staining and microscopy of samples corresponding to (B). Left panel: microscopy of MmarpBS401-dnaK3Mmon at two time points (1 day and 3 days) exhibiting occurrence of PGB cells (yellow arrows) and coccoids (black arrows) under un-induced condition. Scale bar = 1 mm. Middle panel: MTG and FM4-64 staining of MmarpBS401-dnaK3Mmon cells showing internal membrane formation and compartmentalisation (red arrows). Scale bar = 1 mm. Right panel: TEM images of 4 days old MmarpBS401-dnaK3Mmon cells (un-induced and after 24 h induction). Red arrows mark the internal membrane formation and presence of internal structures. All cultures were grown on 7H10 media supplemented with hygromycin (100 μg mL−1) at 30 °C. (D) Statistical distribution of the different cell morphologies in MmarpBS401-dnaK3Mmar (un-induced and ‘Tet’ induced conditions) observed in the cell cultures. (E) Expression of dnaK_3Mmon carried on pBS401 in MmarT as determined by qPCR. Cell extracts from three different time points (1 day, 3 days and 5 days) with and without tetracycline induction were considered, “d” = days. The log2-fold change was normalised to the “1 day-induction”. (F) Analysis of the expression of DnaK_3Mmar and DnaK_3Mmon in MmarT by β-galactosidase assay. β-galactosidase activity (DnaK_3-LacZ fused protein) upon addition of the CPRG substrate with (substrate) was measured using a spectrophotometer at 595 nm. The measured absorbance normalised with the total protein content for each sample as shown in the plots. The samples were protein extracts from Mmar cells carrying pIGN vector with dnaK3Mmar-lacZ or dnaK3Mmon-lacZ. The empty vector was used as control (see <a href="#sec2-microorganisms-13-00475" class="html-sec">Section 2</a> for details). The numbers represent an average based on two independent experiments (round 1 and round 2) with two biological replicates. Full article ">

19 pages, 5523 KiB

Open AccessArticle

Erwinia plantamica sp. nov., a Non-Phytopathogenic Bacterium Isolated from the Seedlings of Spring Wheat (Triticum aestivum L.)

by Anna Egorshina, Mikhail Lukyantsev, Sergey Golubev, Eugenia Boulygina, Irina Khilyas and Anna Muratova

Microorganisms 2025, 13(3), 474; https://doi.org/10.3390/microorganisms13030474 - 20 Feb 2025

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Abstract

Erwinia are widely known as phytopathogenic bacteria, but among them, there are also plant-friendly strains that can promote plant growth (PGPR). The Erwinia-like strain OPT-41 was isolated from Triticum aestivum seedlings as a potential PGPR. The cells (0.9–1.3 × 1.5–3.1 µm) of [...] Read more.

Erwinia are widely known as phytopathogenic bacteria, but among them, there are also plant-friendly strains that can promote plant growth (PGPR). The Erwinia-like strain OPT-41 was isolated from Triticum aestivum seedlings as a potential PGPR. The cells (0.9–1.3 × 1.5–3.1 µm) of this microorganism are Gram-negative, rod-shaped, motile (with peritrichous flagella), and non-spore- and non-capsule-forming. The 16S rRNA gene sequence analyses showed it is located in the Erwiniaceae family and has a pairwise similarity above the species delineation threshold of 98.65% with several of its members: Erwinia tasmaniensis (99.21%), Candidatus Pantoea bathycoeliae (98.93%), Pantoea agglomerans (98.87%), Erwinia endophytica (98.83%), Erwinia persicina (98.82%), Erwinia billingiae (98.76%) and Erwinia aphidicola (98.75%). Whole genome-based taxonomy performed on the Type (Strain) Genome Server clarified the status of strain OPT-41, detecting it as a potential new species in the genus Erwinia. The microorganism under study was the most closely related to the type strain of E. phyllosphaerae, demonstrating 27.2% similarity in dDDH, 83.44% similarity in OrthoANIu, and 1.9% difference in G+C content. The major fatty acids of strain OPT-41 were 9 C_16:1, C_14:0, and C_16:0. A combination of genome-based taxonomy and traditional polyphasic taxonomy clearly indicated that strain OPT-41 belongs to a novel Erwinia species, for which the name E. plantamica sp. nov was proposed. OPT-41 (=IBPPM 712=VKM B-3874D=CCTCC AB 2024361) has been designated as the type strain. In addition, OPT-41 was found to have low degradation potential for host plant pectins and proteins and be friendly in Triticum aestivum and Hordeum vulgare crops. Full article

(This article belongs to the Section Plant Microbe Interactions)

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Figure 1
Midpoint-rooted maximum-likelihood tree based on the 16S rRNA gene sequences from strain OPT-41 and its phylogenetic neighbors. The evolutionary model used is GTR+GAMMA. Maximum-likelihood (left) and maximum parsimony (right) bootstrap values (>60%) are shown near the branches. GenBank accession numbers are in parentheses. Bar, 0.009 substitutions per nucleotide position. Full article ">Figure 2
Midpoint-rooted minimum evolution tree based on the whole-genome sequences from strain OPT-41 and its phylogenetic neighbors. The intergenomic distances are calculated by using the GBDP approach. Pseudo-bootstrap values (>60%) based on 100 replications are shown at branch nodes (average branch support, 96.4%). Full article ">Figure 3
Colonies and cell morphology of strain OPT-41: (a) 24 h colonies on TSA medium; (b) Gram-negative rods in 24 h culture; (c) transmission electron microscopy of single cell grown on R2A for 24 h; (d) transmission electron microscopy of cell division; peritrichous flagella is also seen. Full article ">Figure 4
The extracellular pectate lyase (a), polygalacturonase (b), protease (c), and cellulase (d) activities measured in the cultural supernatants of Erwinia plantamica OPT-41 and Pectobacterium atroseptcium SCRI1043 after one day (white box charts) and two days (gray box charts) of cultivation in minimal medium supplemented with wheat extract. Asterisks (*) show the significance of the difference (two-tailed Mann–Whitney test, p < 0.05) between variants designated by brackets. Full article ">Figure 5
Morphological and physiological parameters of Triticum aestivum L. non-inoculated (<img alt="Microorganisms 13 00474 i001" src="/microorganisms/microorganisms-13-00474/article_deploy/html/images/microorganisms-13-00474-i001.png"/>) and inoculated (<img alt="Microorganisms 13 00474 i002" src="/microorganisms/microorganisms-13-00474/article_deploy/html/images/microorganisms-13-00474-i002.png"/>) with Erwinia plantamica strain OPT-41: (a) shoot length, (b) root length, (c) shoot fresh weight, (d) root fresh weight, (e) shoot dry weight, (f) root dry weight, (g) germination energy, (h) final germination, and (i) fungal disease incidence. The data in the figure are the mean ± SD for shoot and root length and the median ± SD for all others. Full article ">Figure 6
Morphological and physiological parameters of Hordeum vulgare L. non-inoculated (<img alt="Microorganisms 13 00474 i003" src="/microorganisms/microorganisms-13-00474/article_deploy/html/images/microorganisms-13-00474-i003.png"/>) and inoculated (<img alt="Microorganisms 13 00474 i004" src="/microorganisms/microorganisms-13-00474/article_deploy/html/images/microorganisms-13-00474-i004.png"/>) with Erwinia plantamica strain OPT-41: (a) shoot length, (b) root length, (c) shoot fresh weight, (d) root fresh weight, (e) shoot dry weight, (f) root dry weight, (g) germination energy, (h) final germination, and (i) fungal disease incidence. The data in the figure are the mean ± SD for shoot and root length and the median ± SD for all others. Asterisks (*) show the significance of the difference between variants designated by brackets (unpaired t test, p < 0.05, for shoot and root length; two-tailed Mann–Whitney test, p < 0.05, for all others). Full article ">

16 pages, 788 KiB

Open AccessArticle

Comparative Analysis of Bacterial Conjunctivitis in the Adult and Pediatric Inpatient vs. Outpatient Population

by Adela Voinescu, Corina Musuroi, Monica Licker, Delia Muntean, Silvia-Ioana Musuroi, Luminita Mirela Baditoiu, Dorina Dugaesescu, Romanita Jumanca, Mihnea Munteanu and Andrei Cosnita

Microorganisms 2025, 13(3), 473; https://doi.org/10.3390/microorganisms13030473 - 20 Feb 2025

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Abstract

The etiology and resistance pattern of bacterial conjunctivitis varies depending on the patient’s care setting and age. A retrospective, observational study was conducted in a tertiary care teaching hospital. A total of 126 patients—76 adults and 50 children—diagnosed with conjunctival infection during inpatient [...] Read more.

The etiology and resistance pattern of bacterial conjunctivitis varies depending on the patient’s care setting and age. A retrospective, observational study was conducted in a tertiary care teaching hospital. A total of 126 patients—76 adults and 50 children—diagnosed with conjunctival infection during inpatient or ambulatory care were analyzed. In the samples of adult patients, isolates were represented by Gram-positive cocci (57.7%; Staphylococcus spp., S. pneumoniae) followed by Enterobacterales (17.97%; P. mirabilis, E. coli, Klebsiella spp.), and non-fermenters (7.69%; Pseudomonas spp., A. baumannii). Multidrug-resistant (52.17%) and extensively drug-resistant (21.73%) pathogens (predominantly Gram-negative bacilli) were identified in conjunctival swabs of hospitalized adult patients. The main isolates (55.77%) identified in children’s conjunctival swabs belonged to S. aureus, H. influenzae, and S. pneumoniae, followed by Enterobacterales (19.22%; E. coli, P. mirabilis, M. morganii) and fungi (3.48%). Methicillin-resistant S. aureus (35.71%) and extended-spectrum beta-lactamase-producing K. pneumoniae (8.7%) were identified in the pediatric subgroup of patients. In critically ill adult patients assisted in the intensive care or burn functional units, bacterial conjunctivitis followed the pattern of infections and antimicrobial resistance specific to these categories of patients. In the case of hospitalized children, conjunctivitis was an integral part of the age-related pathology. Full article

(This article belongs to the Special Issue Healthcare-Associated Infections and Antimicrobial Therapy—2nd Edition)

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19 pages, 2326 KiB

Open AccessArticle

Investigation of Comorbidity and Risk Factors Analysis During Lumpy Skin Disease Outbreaks in India

by Gundallahalli Bayyappa Manjunatha Reddy, Shraddha Bijalwan, Siju Susan Jacob, Sunil Tadakod, Snigdha Madhaba Maharana, Sudeep Nagaraj, Sai Mounica Pabbineedi, Chandana Ramesh Uma, Viveka Prabhu Balappa, Chethan Kumar Harlipura Basavarajappa, Pinaki Prasad Sengupta, Sharanagouda Shiddanagouda Patil and Baldev Raj Gulati

Microorganisms 2025, 13(3), 472; https://doi.org/10.3390/microorganisms13030472 - 20 Feb 2025

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Abstract

Lumpy skin disease (LSD) is a re-emerging viral transboundary disease affecting cattle and buffaloes, resulting in a significant socio-economic impact on the affected regions. LSD is primarily transmitted among susceptible livestock through hematophagous vectors, including ticks and flies. Ticks also function as reservoirs [...] Read more.

Lumpy skin disease (LSD) is a re-emerging viral transboundary disease affecting cattle and buffaloes, resulting in a significant socio-economic impact on the affected regions. LSD is primarily transmitted among susceptible livestock through hematophagous vectors, including ticks and flies. Ticks also function as reservoirs for various haemoprotozoan parasites, increasing the likelihood of coinfections in affected animals. This study investigates the comorbidity of LSD and associated risk factors using diverse datasets. A total of 414 samples from LSD-suspected animals were screened for LSD, infectious bovine rhinotracheitis (IBR), malignant catarrhal fever (MCF), babesiosis, and theileriosis (Theileria annulata and Theileria orientalis), as well as anaplasmosis. Among these, 214 (51.6%) tested positive for LSD. A strong correlation was identified between LSD and oriental theileriosis caused by Theileria orientalis (50.9%). Other significant associations were observed with IBR (34.1%), anaplasmosis (24.7%), tropical theileriosis (15.4%), babesiosis (12.6%), and MCF (12.1%). The transmission dynamics of LSD revealed that hematophagous vectors, particularly Stomoxys, Haematobia, and Rhipicephalus, play a crucial role in its spread, especially in unorganised farming systems. Additionally, Haematobia and Stomoxys flies were implicated in the high transmission rate of oriental theileriosis (39%) in conjunction with LSD. Notably, ticks (Rhipicephalus) facilitated the concurrent transmission of one, two, or three infections alongside LSD. While Musca, a non-hematophagous fly, was found to carry LSD virus (LSDV), it did not test positive for other pathogens. This study highlights the potential for cattle to harbour multiple diseases simultaneously with LSD, emphasising the necessity for integrated transmission studies and comprehensive disease screening in affected livestock. These findings underscore the importance of implementing targeted prevention and control strategies to mitigate disease impact in livestock populations. Full article

(This article belongs to the Section Veterinary Microbiology)

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Figure 1
Study design. The schematic diagram representing the processing for the detection of various pathogens in the clinical samples collected from cattle. Full article ">Figure 2
Demographic distribution. Map showing density-wise distribution of (A) cattle population shown in gradient of blue and (B) LSD-affected regions in gradient of brown in different states of India. Full article ">Figure 3
Distribution of different diseases in cattle. The bar diagram representing the percentage positivity of LSD, IBR, MCF, babesiosis, anaplasmosis, oriental theileriosis, and tropical theileriosis on the X-axis and the percentage positivity on the Y-axis. Full article ">Figure 4
LSD risk factors. The bar diagram showing the association of risk factors (age, breed, sex, and type of farm) with occurrence of lumpy skin disease using univariate regression analysis. Full article ">Figure 5
Risk of comorbidity in LSD. The univariate regression analysis showing different diseases and their associated risk factors (age: (A); gender: (B); breed: (C); and farm type: (D) in LSD-positive cases. Full article ">Figure 6
Transmission of LSD and risk factors. The chi-square analysis showing transmission of LSD and its associated risk factors. Full article ">

17 pages, 2936 KiB

Open AccessArticle

Influence of Cover Crop Root Functional Traits on Sweet Potato Yield and Soil Microbial Communities

by Xinyi Chen, Jie Zhang, Wangbiao Xia, Yangyang Shao, Zhirong Liu, Jian Guo, Wenjing Qin, Li Wan, Jia Liu, Ying Liu and Juntong Zhang

Microorganisms 2025, 13(3), 471; https://doi.org/10.3390/microorganisms13030471 - 20 Feb 2025

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Abstract

The symbiotic relationship between cover crops and soil microorganisms is closely linked to nutrient cycling and crop growth within agroecosystems. However, how cover crops with different root functional traits influence soil microbial communities, soil properties, and crop yields has remained understudied. This study [...] Read more.

The symbiotic relationship between cover crops and soil microorganisms is closely linked to nutrient cycling and crop growth within agroecosystems. However, how cover crops with different root functional traits influence soil microbial communities, soil properties, and crop yields has remained understudied. This study assessed the root traits of hairy vetch (HV) and rapeseed (RP), along with soil properties, sweet potato yield, and microbial enzyme activity under red soil dryland conditions. High-throughput sequencing was also employed to characterize the diversity, composition, and network structure of soil bacterial and fungal communities. According to the plant economic spectrum theory and our research results on plant root traits, HV can be identified as a resource-acquisitive cover crop, and RP treatment can be identified as a resource-conservative cover crop. Although RP treatment did not significantly increase the sweet potato yield, the increase rate reached 8.49%. Resource-conservative cover crops were associated with increased pH, SOC, and TP, which enhanced bacterial species diversity and boosted the populations of Chloroflexi and Alphaproteobacteria. In contrast, resource-acquisitive cover crops promoted the proliferation of Gammaproteobacteria. Network analysis indicated that resource-conservative cover crops facilitated network complexity through intensified intra-community competition. Resource-acquisitive cover crops enhanced the stability of microbial communities. Collectively, these findings underscore the distinct advantages of cover crops with varying root functional traits in shaping soil microbial communities. Appropriate cover crop rotations can effectively regulate microbial communities and hold the potential to enhance crop yield. Full article

(This article belongs to the Special Issue Effect of Microbial Communities for Environmental Protection and Development of Agriculture)

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Figure 1
(a) Principal component analysis (PCA) of functional traits for two distinct cover crops. The figure displays two distinct clusters representing cover crop ecological strategies. One species on the left represents the acquisitive end of the plant economic spectrum, while the other on the right represents the conservative end. The green and blue ovals represent the conservative and acquisitive strategies, respectively. HV, hairy vetch; RP, rapeseed; D, root diameter; C/N, carbon-to-nitrogen ratio; CC, carbon content; NC, nitrogen content; V, volume; SA, surface area; T, number of root tips; L, length. (b) Variations in sweet potato yield under different cropping treatments. The same lowercase letter above the box indicates no significant difference (p > 0.05). Full article ">Figure 2
CK, winter fallow; HV, hairy vetch; RP, rapeseed. AN, available nitrogen; TN, total nitrogen; SOC, soil organic carbon; AP, available phosphorus; TP, total phosphorus. Different letters within the same row indicate significant differences among the treatments (p < 0.05). Full article ">Figure 3
Influence of various cover crops on microbial community diversity and composition. The impact of different cover crop treatments on bacterial (a) and fungal (b) richness indices is depicted. CAP biplots display the differences in bacterial (c) and fungal (d) consortia associated with various cover crops. Cover crops accounted for 20.989% of the total variance in bacterial communities and 27.31% in fungal communities. Different lowercase letters above the boxes indicate significant difference among the treatments (p < 0.05). Full article ">Figure 4
Relative abundance of soil bacterial communities at the phylum (a) and fungal (b) communities at the class level under different treatments. Full article ">Figure 5
Manhattan plots of ASV enrichment in the HV or RP treatments relative to the CK treatment. The plots highlight bacterial species differences between the CK and HV (a) and the CK and RP (b) treatments. The enrichment of ASVs are shown in the HV (c) and RP (d) treatments in contrast to the CK treatment at the fungal level. Each dot or triangle represents an individual ASV, with the colors indicating different phyla. The solid triangles denote the ASVs enriched in the CK treatment, whereas the empty triangles represent those enriched in the HV or RP treatments. The circles positioned below the dashed line signify noise (FDR adjusted p < 0.05, Wilcoxon rank sum test). CPM, counts per million. Full article ">Figure 6
Symbiotic network structures in different cover crops with diverse functional traits. (a) Co-occurrence network patterns of soil fungi. (b) Co-occurrence network patterns of soil bacteria. Distinct colors represent different ecological clusters, and circle sizes correspond to the relative abundance of ASVs. (c) Topological characteristics of soil bacteria co-occurrence networks, including the number of nodes, number of edges, average degree, and linkage density. Different lowercase letters above the boxes indicate significant difference among the treatments. Full article ">

13 pages, 2910 KiB

Open AccessArticle

Nitric Oxide-Mediated Regulation of Chitinase Activity and Cadmium Sequestration in the Response of Schizophyllum commune to Cadmium Stress

by Dongxu Li, Chen Chu, Mengshi Zhao, Suying Hou, Rong Ji and Changhong Liu

Microorganisms 2025, 13(3), 470; https://doi.org/10.3390/microorganisms13030470 - 20 Feb 2025

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Abstract

Schizophyllum commune is an edible fungus with high medicinal value, but exposure to heavy-metal pollution poses significant health risks. Cadmium (Cd) toxicity inhibits fungal growth and leads to Cd accumulation in the mycelium. However, the regulatory mechanisms of Cd-induced growth inhibition and Cd [...] Read more.

Schizophyllum commune is an edible fungus with high medicinal value, but exposure to heavy-metal pollution poses significant health risks. Cadmium (Cd) toxicity inhibits fungal growth and leads to Cd accumulation in the mycelium. However, the regulatory mechanisms of Cd-induced growth inhibition and Cd accumulation remain poorly understood. Here, S. commune 20R-7-F01 was cultured in Cd-supplemented minimal medium (MM) to investigate the response of S. commune 20R-7-F01 to Cd exposure. We found that Cd exposure resulted in growth inhibition and a Cd-dependent increase in endogenous nitric oxide (NO) levels. NO production was primarily mediated by the nitrate reductase (NR) pathway. Cd-induced growth inhibition was alleviated by inhibiting NR activity or scavenging NO, highlighting the role of NO in stress responses. Furthermore, NO was found to enhance chitinase activity, thereby promoting Cd accumulation in the fungal cell wall and leading to growth inhibition. These results reveal a novel mechanism by which S. commune copes with Cd stress. This study highlights the potential of manipulating NO levels as a strategy to enhance fungal tolerance to heavy-metal pollution, providing a new avenue for managing environmental stresses in edible fungi and protecting human health. Full article

(This article belongs to the Section Environmental Microbiology)

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Figure 1
Cd inhibits the growth rate (A) and biomass (B) of Schizophyllum commune. Mycelial growth on solid (A) or liquid (B) MM containing 0–100 μM CdCl2. Relative growth rates are relative to the growth rates of mycelial in the presence of 0 μM Cd. All treatments were incubated at 30 °C for 96 h. Values are mean ± S.E (A, n = 20; B, n = 3). Different letters indicate statistically significant differences (p ≤ 0.05). Full article ">Figure 2
NO production in S. commune mycelium under exposure to various concentrations of CdCl2. (A) Fluorescence intensity diagram of NO production detected by NO-specific fluorescent probe DAF-2DA in mycelium treated with different Cd concentrations. (B) Quantitative analysis of NO content in mycelium. All mycelium were exposed to 0, 10, 20, 50, or 100 μM CdCl2 in solid liquid MM medium at 30 °C for 96 h. μmol mg−1 protein indicates the NO content per mg of protein. Values are mean ± S.E (n = 3). Different letters indicate significant differences (p ≤ 0.05). Bar = 10 μm. Full article ">Figure 3
NO content and nitrate reductase (NR) activity in S. commune mycelium cultured with Cd in the presence and absence of NOS and NR inhibitors. (A) Fluorescence intensity image of NO in mycelium under different treatments detected by the NO-specific fluorescent probe DAF-2DA, and (B) digital fluorescence intensity. (C) NO content. (D) NR activity. Mycelium was cultured in liquid MM medium with 0 or 100 μM Cd, in the presence or absence of 100 μM L-NAME (NOS inhibitor) or 300 μM tungstate (NR inhibitor), at 30 °C for 96 h. Con with the control (0 μM Cd). μmol mg−1 protein indicates the NO content per mg of protein. U represents the amount of enzyme required to convert 1 μmol of substrate in 1 min. U μmol mg−1 protein represents the enzyme activity per mg of protein. Values are mean ± S.E (n = 3). Different letters indicate significant differences (p ≤ 0.05). Bar = 10 μm. Full article ">Figure 4
Effect of NO modulation on Cd accumulation in mycelium. (A) Image of Cd distribution in mycelium, measured using the Cd-specific fluorescent probe Leadmium TM green AM dye; (B) relative fluorescence intensity in the images; (C) NO content. Mycelium was cultured in liquid MM medium with 0 or 100 μM Cd, in the presence or absence of 300 μM SNP (NO donor) or 200 μM cPTIO (NO scavenger), at 30 °C for 96 h. Con with the control (0 μM Cd). μmol mg−1 protein indicates the NO content per mg of protein. Values are mean ± S.E (n = 6). Different letters indicate significant differences (p ≤ 0.05). Bar = 10 μm. Full article ">Figure 5
Effect of Cd and Cd-induced NO on fungal chitinase activity (A) and the relative expression of chitinase-encoding genes (B). Mycelium was cultured in liquid MM medium with 0 or 100 μM Cd, in the presence or absence of 300 μM SNP (NO donor), 200 μM cPTIO (NO scavenger), or 300 μM tungstate (NR inhibitor), at 30 °C for 96 h. Con with the control (0 μM Cd). U represents the amount of enzyme required to convert 1 μmol of substrate in 1 min. U μmol mg−1 protein represents the enzyme activity per mg of protein. Values are mean ± S.E (n = 3). Different letters indicate significant differences (p ≤ 0.05). Full article ">Figure 6
Accumulation of Cd in the cell wall (A) and mycelial biomass (B) of S. commune under exposure to Cd in the presence of NO modulators. Mycelium was cultured in liquid MM medium with 0 or 100 μM Cd, in the presence or absence of 300 μM SNP (NO donor), 200 μM cPTIO (NO scavenger), or 300 μM tungstate (NR inhibitor), at 30 °C for 96 h. Con with the control (0 μM Cd). μg g−1 cell wall represents the Cd content per g of cell wall. Values are mean ± S.E (n = 3). Different letters indicate significant differences (p ≤ 0.05). Full article ">Figure 7
Proposed mechanism of Cd toxicity in inhibiting S. commune 20R-7-F01 growth. Cd exposure activates NR (1), leading to the production of NO (2). Elevated endogenous NO levels subsequently enhance the expression of chitinase genes (3) and increase chitinase activity (4), which facilitates the accumulation of Cd in the fungal cell wall (5). This process ultimately contributes to the inhibition of mycelial growth (6). Green arrows denote positive regulatory effects, while red-capped line indicates negative regulatory effects. Full article ">

11 pages, 1361 KiB

Open AccessArticle

Epidemiological Transitions in Influenza Dynamics in the United States: Insights from Recent Pandemic Challenges

by Marta Giovanetti, Sobur Ali, Svetoslav Nanev Slavov, Taj Azarian and Eleonora Cella

Microorganisms 2025, 13(3), 469; https://doi.org/10.3390/microorganisms13030469 - 20 Feb 2025

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Abstract

The SARS-CoV-2 pandemic has reshaped the epidemiological landscape of respiratory diseases, with profound implications for seasonal influenza. Nonpharmaceutical interventions implemented globally during the pandemic significantly altered human behavior and reduced the prevalence of respiratory pathogens, including influenza. However, the post-pandemic resurgence of influenza [...] Read more.

The SARS-CoV-2 pandemic has reshaped the epidemiological landscape of respiratory diseases, with profound implications for seasonal influenza. Nonpharmaceutical interventions implemented globally during the pandemic significantly altered human behavior and reduced the prevalence of respiratory pathogens, including influenza. However, the post-pandemic resurgence of influenza activity to pre-pandemic levels highlights the persistent challenges posed by this virus. During the 2023–2024 influenza season in the United States, an estimated 40 million individuals contracted influenza, resulting in 470,000 hospitalizations and 28,000 deaths, with the elderly disproportionately affected. Pediatric mortality was also notable, with 724 deaths reported among children. This study examines trends in influenza incidence, vaccination rates, and mortality in the United States from the 2018–2019 through to the 2023–2024 influenza seasons. Additionally, it evaluates the interplay between influenza and SARS-CoV-2 during the pandemic, considering the impact of disrupted air travel, public health measures, and altered virus circulation dynamics. By integrating these insights, the study underscores the critical need for sustained vaccination campaigns and innovative public health strategies to mitigate the dual burden of respiratory diseases. Findings from this analysis highlight the urgency of strengthening prevention and surveillance systems to enhance pandemic preparedness and reduce the impact of respiratory pathogens in an evolving epidemiological landscape. Full article

(This article belongs to the Special Issue Human Infectious Diseases)

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Figure 1

Figure 1
Seasonal influenza and SARS-CoV-2 dynamics before, during, and after the COVID-19 pandemic. Trends in weekly reported cases of influenza (top panel) and SARS-CoV-2 (bottom panel) in the United States from the 2018–2019 to 2023–2024 seasons. The graph is divided into three distinct periods: pre-pandemic (2018–2019), COVID-19 pandemic (2020–2022, shaded in red), and post-pandemic (2023–2024, shaded in gray). The red line in the top panel indicates the COVID-19 Containment and Health Index (obtained from <a href="https://ourworldindata.org/" target="_blank">https://ourworldindata.org/</a>), reflecting the intensity of nonpharmaceutical interventions implemented during the pandemic. The gray vertical lines delimit the different influenza seasons. Major federal containment initiatives for SARS CoV-2 are showed in the bottom panel. Full article ">Figure 2
Geographic trends in influenza cases across six seasons (2018–2024). Heatmaps of influenza cases rate per 100,000 population across U.S. states for the 2018–2019 to 2023–2024 seasons. The 2020–2021 season shows a marked decline in activity during peak COVID-19 measures, while later seasons display a return to pre-pandemic levels, emphasizing regional variability and public health impact. States with low cases or missing data are colored in white. Full article ">Figure 3
Geographic distribution of influenza-related deaths in the United States (2018–2024). Heatmaps showing influenza-related death rate per 1M population across U.S. states during the 2018–2019 to 2022–2023 influenza seasons. Data for the 2020–2021 and 2023–2024 seasons were unavailable. Darker shades represent higher mortality rates, with notable variability in the geographic burden of influenza-related deaths. The absence of data for certain seasons highlights the challenges in consistent surveillance and reporting during and after the COVID-19 pandemic. States with low deaths or missing data are colored in white. Full article ">Figure 4
Seasonal influenza vaccination coverage across the United States (2018–2024). Heatmaps illustrating vaccination coverage for influenza across U.S. states during the 2018–2019 to 2023–2024 influenza seasons. Darker shades represent higher vaccination coverage, with variations observed between seasons and regions. The 2020–2021 season, marked by the COVID-19 pandemic, shows a moderate increase in vaccination coverage compared to prior seasons, likely influenced by heightened public health awareness. Subsequent seasons demonstrate variability in coverage, emphasizing the need for sustained vaccination efforts to achieve optimal influenza prevention. States with missing data are colored in white. Full article ">

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