Microorganisms

14 pages, 1503 KiB

Open AccessArticle

Socioeconomic and Eco-Environmental Drivers Differentially Trigger and Amplify Bacterial and Viral Outbreaks of Zoonotic Pathogens

by Payton Phillips, Negin Nazari, Sneha Dharwadkar, Antoine Filion, Benedicta Essuon Akaribo, Patrick Stephens and Mekala Sundaram

Microorganisms 2025, 13(3), 621; https://doi.org/10.3390/microorganisms13030621 (registering DOI) - 7 Mar 2025

Abstract

The frequency of infectious disease outbreaks and pandemics is rising, demanding an understanding of their drivers. Common wisdom suggests that increases in outbreak frequency are driven by socioeconomic factors such as globalization and urbanization, yet, the majority of disease outbreaks are caused by [...] Read more.

The frequency of infectious disease outbreaks and pandemics is rising, demanding an understanding of their drivers. Common wisdom suggests that increases in outbreak frequency are driven by socioeconomic factors such as globalization and urbanization, yet, the majority of disease outbreaks are caused by zoonotic pathogens that can be transmitted from animals to humans, suggesting the important role of ecological and environmental drivers. Previous studies of outbreak drivers have also failed to quantify the differences between major classes of pathogens, such as bacterial and viral pathogens. Here, we reconsider the observed drivers of a global sample of 300 zoonotic outbreaks, including the 100 largest outbreaks that occurred between 1977 and 2017. We show that socioeconomic factors more often trigger outbreaks of bacterial pathogens, whereas ecological and environmental factors trigger viral outbreaks. However, socioeconomic factors also act as amplifiers of viral outbreaks, with higher case numbers in viral outbreaks driven by a larger proportion of socioeconomic factors. Our results demonstrate that it is useful to consider the drivers of global disease patterns in aggregate due to commonalities that cross disease systems. However, our work also identifies important differences between the driver profiles of bacterial and viral diseases in aggregate. Full article

(This article belongs to the Special Issue Pandemics and Infectious Diseases)

13 pages, 696 KiB

Open AccessArticle

Anthropogenic Blastocystis from Drinking Well and Coastal Water in Guinea-Bissau (West Africa)

by Sara Gomes-Gonçalves, Ana Machado, Adriano Bordalo and João R. Mesquita

Microorganisms 2025, 13(3), 620; https://doi.org/10.3390/microorganisms13030620 - 7 Mar 2025

Abstract

This study provides the first comprehensive investigation of Blastocystis sp. contamination in the drinking well and coastal water sources in Guinea-Bissau, a region grappling with severe water quality challenges. Of the forty-five water samples analyzed (34 well and 9 coastal water sources), Blastocystis [...] Read more.

This study provides the first comprehensive investigation of Blastocystis sp. contamination in the drinking well and coastal water sources in Guinea-Bissau, a region grappling with severe water quality challenges. Of the forty-five water samples analyzed (34 well and 9 coastal water sources), Blastocystis sp. was detected in five (11%, 95% CI: 3.71–24.05) of the wells, which serve as a critical and primary drinking source for local communities. The detection of human-associated Blastocystis sp. subtype (ST)2 and ST3 raises concerns about the potential of fecal contamination as a transmission route for Blastocystis sp., underscoring the public health risks associated with an inadequate WASH (water, sanitation, and hygiene) infrastructure. These findings highlight the urgent need for improved water management and further research on waterborne parasitic infections in resource-limited settings. Full article

(This article belongs to the Special Issue Water Microorganisms Associated with Human Health, 2nd Edition)

20 pages, 1584 KiB

Open AccessReview

A Potential Link Between Oral Microbiota and Female Reproductive Health

by Justyna Marcickiewicz, Małgorzata Jamka and Jarosław Walkowiak

Microorganisms 2025, 13(3), 619; https://doi.org/10.3390/microorganisms13030619 - 7 Mar 2025

Abstract

Oral cavity dysbiosis is associated with numerous inflammatory diseases, including diabetes, inflammatory bowel diseases, and periodontal disease. Changes in the oral microenvironment lead to bidirectional interactions between pathogens and individual host systems, which may induce systemic inflammation. There is increasing evidence linking the [...] Read more.

Oral cavity dysbiosis is associated with numerous inflammatory diseases, including diabetes, inflammatory bowel diseases, and periodontal disease. Changes in the oral microenvironment lead to bidirectional interactions between pathogens and individual host systems, which may induce systemic inflammation. There is increasing evidence linking the condition of the oral cavity with the most common causes of female infertility, such as polycystic ovary syndrome and endometriosis, as well as gestational complications, e.g., low birth weight, preterm delivery, and miscarriages. This review highlights the composition of the female oral microbiome in relation to infertility-related disorders, such as endometriosis and polycystic ovary syndrome, and provides a comprehensive overview of the current state of knowledge on the relationship between a dysbiotic oral microbiome, pregnancy, and its impact on the female reproductive tract. Full article

(This article belongs to the Section Medical Microbiology)

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21 pages, 5216 KiB

Open AccessArticle

Factors Affecting D-Lactic Acid Production by Flocculant Saccharomyces cerevisiae Under Non-Neutralizing Conditions

by Dianti Rahmasari, Prihardi Kahar, Arthur Vinícius de Oliveira, Filemon Jalu Nusantara Putra, Akihiko Kondo and Chiaki Ogino

Microorganisms 2025, 13(3), 618; https://doi.org/10.3390/microorganisms13030618 - 7 Mar 2025

Abstract

Integrating heterogeneous genes is widely used in metabolic engineering to produce D-lactic acid (D-LA), an essential compound in bioplastics and pharmaceuticals. However, research on the effects of integrating various loci on gene expression, especially regarding flocculation behavior, remains limited. This study constructed Saccharomyces [...] Read more.

Integrating heterogeneous genes is widely used in metabolic engineering to produce D-lactic acid (D-LA), an essential compound in bioplastics and pharmaceuticals. However, research on the effects of integrating various loci on gene expression, especially regarding flocculation behavior, remains limited. This study constructed Saccharomyces cerevisiae strains by incorporating a codon-optimized D-LDH gene from Leuconostoc pseudomesenteroides (LpDLDH) into the specific genomic loci of the CYB2, PDC1, MPC1, PDC6, ADH1, and PDC5 genes to redirect pyruvate toward lactic acid. Strains with the LpDLDH gene integrated at the PDC1 locus achieved the highest D-LA titers (51 g/L) with minimal ethanol byproduct, followed by strains with integrations into the CYB2 locus at 31.92 g/L, the MPC1 locus at 10 g/L, and the PDC6 locus at 0.026 g/L. In contrast, strains with LpDLDH integrated at the ADH1 and PDC5 loci failed to produce detectable levels of D-LA and exhibited a complete loss of flocculation. Gene expression analysis revealed a significant expression of genes related to flocculation (FLO5), stress adaptation (HSP150), and cell wall integrity (YGP1, SED1, and SCW11). The CYB2-integrating strain showed strong flocculant properties, contributing to its robustness. These findings highlight the influence of genomic locus selection on metabolic flux and stress adaptation, offering insights into optimizing D-LA production in flocculant S. cerevisiae yeast. Full article

(This article belongs to the Special Issue Yeasts Biochemistry and Biotechnology, 2nd Edition)

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14 pages, 2524 KiB

Open AccessArticle

Diversity of Endophytic Fungi Isolated from Prunus yedoensis and Their Antifungal Activity Against Wood Decay Fungi

by Misong Kim, Manh Ha Nguyen, Sanggon Lee, Wonjong Han, Minyoung Kim, Hyeongguk Jeon, Jinheung Lee, Sangtea Seo, Namkyu Kim and Keumchul Shin

Microorganisms 2025, 13(3), 617; https://doi.org/10.3390/microorganisms13030617 - 7 Mar 2025

Abstract

Endophytic fungi play a vital role in protecting and promoting the growth of their host plants. The diversity of fungal endophytes has been documented across different host plant species and varies depending on factors such as the species of the host, ecological conditions, [...] Read more.

Endophytic fungi play a vital role in protecting and promoting the growth of their host plants. The diversity of fungal endophytes has been documented across different host plant species and varies depending on factors such as the species of the host, ecological conditions, and the health status of the plant. We isolated endophytic fungi from Prunus yedoensis trees with different decay rates. A total of 31 species were isolated from decayed trees, while 33 species were obtained from healthy trees. The number of endophytic fungi exhibiting antifungal activities against wood decay fungi was higher in healthy trees, with 10 species showing activity compared to only 1 species from decayed trees. Endophytic fungus Fusarium acuminatum (BEN48) had the highest inhibition rates against Trametes versicolor, Ganoderma gibbosum, and Vanderbylia fraxinea. Heating conditions did not significantly affect the inhibitory ability of the culture filtrate from BEN48 on wood decay fungi. At 50% concentration, the inhibitory abilities of the culture filtrates against Trametes versicolor, Ganoderma gibbosum, and Vanderbylia fraxinea were 96.5%, 64.1%, and 92.7%, respectively. The inhibitory effects against Trametes versicolor decreased at concentrations of 30% and 10%, resulting in inhibition rates of 83.7% and 50.8%, respectively. For Ganoderma gibbosum, the inhibition rate reduced to 52.6% at 30% concentration and 24.5% at 10% concentration. For Vanderbylia fraxinea, there was no significant difference between the 30% and 10% concentrations, and the inhibition rates for both concentrations were high, measuring 89.9% and 88.8%, respectively. Hence, Fusarium acuminatum (BEN48) has promise as a biocontrol agent for managing wood decay fungi. Full article

(This article belongs to the Special Issue Endophytic Fungus as Producers of New and/or Bioactive Substances)

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Venn diagram illustrating number of endophytic fungal isolates overlapped and non-overlapped from decayed and healthy P. yedoensis trees. Full article ">Figure 2
Antifungal activity of endophytic fungal isolates (left) against wood decay fungi (right) in dual culture assay. BEN07: Paraconiothyrium brasiliense; BEN36: Paraconiothyrium sp.; BEN37: Paraboeremia putaminum; BEN48: Fusarium acuminatum; BEN97: Nigrograna acericola. Full article ">Figure 3
Antifungal activity of heated and unheated culture filtrates from endophytic fungal isolates against Trametes versicolor. Bars represent standard error of mean (n = 5). Bars labeled with same letter are not significantly different between temperature treatments at p < 0.05 using Tukey’s honestly significant difference test. Full article ">Figure 4
Antifungal activity of heated and unheated culture filtrates from endophytic fungal isolates against Ganoderma gibbosum. Bars represent standard error of mean (n = 5). Bars labeled with same letter are not significantly different between temperature treatments at p < 0.05 using Tukey’s honestly significant difference test. Full article ">Figure 5
Antifungal activity of heated and unheated culture filtrates from endophytic fungal isolates against Vanderbylia fraxinea. Bars represent standard error of mean (n = 5). Bars labeled with same letter are not significantly different between temperature treatments at p < 0.05 using Tukey’s honestly significant difference test. Full article ">Figure 6
Antifungal activity of endophytic fungus Fusarium acuminatum (BEN48) against three wood decay fungi at different concentrations of culture filtrates. Bars labeled with same letter are not significantly different between temperature treatments at p < 0.05 using Tukey’s honestly significant difference test. Full article ">Figure 7
Mycelial growth of wood decay fungi on PDA medium amended with different concentrations of culture filtrate from endophytic fungus Fusarium acuminatum (BEN48). Full article ">

16 pages, 249 KiB

Open AccessArticle

Evaluation of the Microbiological Performance and Potential Clinical Impact of New Rapid Molecular Assays for the Diagnosis of Bloodstream Infections

by Mateo Tićac, Tanja Grubić Kezele, Maja Abram and Marina Bubonja-Šonje

Microorganisms 2025, 13(3), 616; https://doi.org/10.3390/microorganisms13030616 - 7 Mar 2025

Abstract

Bloodstream infection (BSI) is a critical medical emergency associated with a high mortality rate. Rapid and accurate identification of the causative pathogen and the results of antimicrobial susceptibility testing are crucial for initiating appropriate antimicrobial therapy. The aim of this study was to [...] Read more.

Bloodstream infection (BSI) is a critical medical emergency associated with a high mortality rate. Rapid and accurate identification of the causative pathogen and the results of antimicrobial susceptibility testing are crucial for initiating appropriate antimicrobial therapy. The aim of this study was to evaluate the performance of a new rapid PCR Molecular Mouse System (MMS) for the identification of Gram-negative bacteria (GNB) and GNB resistance genes directly from a positive blood culture (BC). The validation of these rapid multiplex assays was carried out in a real hospital setting. A total of 80 BSI episodes were included in our study and the results were compared with culture-based methods. BC samples in which GNB had previously been detected microscopically and which originated from different hospital wards were analysed. The MMS GNB identification assay achieved a sensitivity of 98.7% and a specificity of 100% for the covered pathogens. In one BC sample, Klebsiella aerogenes was identified at the family level (Enterobacteriaceae) with MMS. However, in three polymicrobial samples, MMS identified bacteria that were not detected by culture-based methods (Klebsiella pneumoniae, K. aerogenes and Stenotrophomonas maltophilia). MMS also showed excellent overall performance in the detection of GNB resistance markers (100% sensitivity and 100% specificity). The type of extended-spectrum beta-lactamase (ESBL) resistance gene identified correctly with MMS was CTX-M-1/9 (n = 17/20), alone or in combination with SHV-type β-lactamase or with the different types of carbapenemase genes. MMS detected one carbapenemase gene of each type (KPC, NDM and OXA-23) and six OXA-48 genes. In addition, the colistin resistance gene mcr-1 was detected in one positive BC with Escherichia coli (E. coli). The time to result was significantly shorter for MMS than for routine culture methods. A retrospective analysis of the patients’ medical records revealed that a change in empirical antimicrobial therapy would have been made in around half of the patients following the MMS results. These results support the use of MMS as a valuable complement to conventional culture methods for more rapid BSI diagnosis and adjustment of empirical therapy. Full article

(This article belongs to the Special Issue Novel Approaches in the Diagnosis and Control of Emerging Pathogens)

15 pages, 863 KiB

Open AccessArticle

Specific Detection of African Swine Fever Virus Variants: Novel Quadplex Real-Time PCR Assay with Internal Control

by Lihua Wang, Yuzhen Li, Xirui Zhang, Rachel Madera, Homer Pantua, Aidan Craig, Nina Muro, Danqin Li, Jamie Retallick, Franco Matias Ferreyra, Quang Lam Truong, Lan Thi Nguyen and Jishu Shi

Microorganisms 2025, 13(3), 615; https://doi.org/10.3390/microorganisms13030615 - 7 Mar 2025

Abstract

African swine fever (ASF), a highly contagious and lethal viral disease, continues to devastate the global swine industry. The emergence of ASF virus (ASFV) variants with varying genomic deletions poses significant challenges for ASF control. This study presents a novel, sensitive, and reliable [...] Read more.

African swine fever (ASF), a highly contagious and lethal viral disease, continues to devastate the global swine industry. The emergence of ASF virus (ASFV) variants with varying genomic deletions poses significant challenges for ASF control. This study presents a novel, sensitive, and reliable quadplex real-time PCR assay for detecting ASFV variants lacking key genes (I177L, EP402R, and MGF360-14L), either individually or in combination. The assay targets conserved regions within these genes, ensuring broad coverage of diverse ASFV genotypes. A porcine beta-actin (ACTB) internal control was incorporated to minimize false-negative results. Optimization and evaluation using spike-in tests demonstrated high sensitivity, with a limit of detection (LOD) ranging from 1 to 10 plasmid copies or 0.1 TCID₅₀ of ASFV isolates per reaction. No cross-reactivity was observed when testing serum samples from pigs infected with other common swine viruses. Further validation across a diverse panel of samples, including those from naturally ASFV-infected field pigs (n = 54), experimentally ASFV-infected pigs (n = 50), PBS-inoculated pigs (n = 50), ASFV-free field pigs (n = 100), and feral pigs (n = 6), confirmed 100% specificity. This robust assay provides a valuable tool for rapid and accurate ASF surveillance and control efforts, facilitating the timely detection and mitigation of outbreaks caused by emerging ASFV variants. Full article

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

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9 pages, 629 KiB

Open AccessCommunication

Serogroup Distribution of Leptospira Among Humans and Rodents in Zakarpattia Oblast, Ukraine (2018–2023)

by Pavlo Petakh and Oleksandr Kamyshnyi

Microorganisms 2025, 13(3), 614; https://doi.org/10.3390/microorganisms13030614 - 7 Mar 2025

Abstract

Leptospirosis is a zoonotic disease caused by Leptospira bacteria, which affects both humans and animals. This study investigated the prevalence of Leptospira serogroups in human and rodent reservoirs across Zakarpattia Oblast, Ukraine, from 2018 to 2023. The objective was to identify epidemiological patterns [...] Read more.

Leptospirosis is a zoonotic disease caused by Leptospira bacteria, which affects both humans and animals. This study investigated the prevalence of Leptospira serogroups in human and rodent reservoirs across Zakarpattia Oblast, Ukraine, from 2018 to 2023. The objective was to identify epidemiological patterns and assess potential public health risks. Data were sourced from the Public Health Center of Ukraine and regional surveillance initiatives, encompassing reported human cases and rodent-carrier detection. Six main serogroups—Icterohaemorrhagiae, Pomona, Grippotyphosa, Hebdomadis, Canicola, and Tarassovi—were included in the analysis. The results showed that Pomona and Hebdomadis serogroups became more common in 2023. The data from different districts also showed regional differences, with Icterohaemorrhagiae and Pomona being the most frequent serogroups in both humans and rodents. Other serogroups, like Grippotyphosa and Canicola, were found less often. Rodents may be an important source of leptospirosis in Zakarpattia. The growing number of cases in recent years shows the need for improved monitoring, control, and prevention in the region. Full article

(This article belongs to the Special Issue Epidemiology of Leptospira sp. Infection: Current Status, Insights and Future Prospects: 2nd Edition)

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18 pages, 10057 KiB

Open AccessArticle

Effects of NatureKnit™, a Blend of Fruit and Vegetable Fibers Rich in Naturally Occurring Bound Polyphenols, on the Metabolic Activity and Community Composition of the Human Gut Microbiome Using the M-SHIME^® Gastrointestinal Model

by Marlies Govaert, Cindy Duysburgh, Brendan Kesler and Massimo Marzorati

Microorganisms 2025, 13(3), 613; https://doi.org/10.3390/microorganisms13030613 - 7 Mar 2025

Abstract

This study evaluated the impact of a proprietary blend of fruit and vegetable fibers rich in naturally occurring bound polyphenols (commercially marketed as NatureKnit^TM), compared to purified fibers (inulin and psyllium), on the human gut microbiome using the validated M-SHIME^® [...] Read more.

This study evaluated the impact of a proprietary blend of fruit and vegetable fibers rich in naturally occurring bound polyphenols (commercially marketed as NatureKnit^TM), compared to purified fibers (inulin and psyllium), on the human gut microbiome using the validated M-SHIME^® gastrointestinal model. A short-term single-stage colonic M-SHIME^® experiment (with fecal inoculum from three healthy human donors) was used to evaluate the test products compared to a negative control. Samples were assessed for pH, gas pressure, short-chain fatty acid (SCFA) production, lactate, and ammonium from 0 h to 48 h. Microbial community composition was assessed at 0 h (negative control only), 24 h, and 48 h (lumen) or 48 h (mucosal). All test products were fermented well in the colon as demonstrated by decreases in pH and increases in gas pressure over time; these changes occurred faster with the purified fibers, whereas NatureKnit™ demonstrated slow, steady changes, potentially indicating a gentler fermentation process. SCFA production significantly increased over the course of the 48 h experiment with all test products versus negative control. SCFA production was significantly greater with NatureKnit™ versus the purified fibers. Shifts in the microbial community composition were observed with all test products versus negative control. At the conclusion of the 48 h experiment, the absolute bacterial abundance and the richness of observed bacterial taxa in the lumen compartment was significantly greater with NatureKnit™ compared with inulin, psyllium, and negative control. Overall, NatureKnit™ demonstrated greater or similar prebiotic effects on study measures compared with established prebiotic fibers. Full article

(This article belongs to the Section Gut Microbiota)

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Changes in (a) pH and (b) gas pressure over time following test product administration in M-SHIME® short-term colonic incubations. Incubations included the negative control (colonic incubation blank medium), NatureKnit™ (1.667 g fiber/L, 3.333 g/L total), inulin (1.667 g fiber/L), and psyllium (1.667 g fiber/L). Donors A, B, and C represent three individual healthy human fecal donors, and average donor represents the average of the three donors. Incubations were performed in triplicate (n = 3) and the results are presented as mean ± standard deviation. Statistical analysis was performed over the course of the entire colonic incubation phase (i.e., between 0 h and 48 h). Paired student’s t-tests were used to compare changes observed for the test products versus negative control. A p-value of <0.05 was considered statistically significant. Different letters above the bars indicate statistically significant differences between test conditions, while no significant differences were observed between test conditions that share the same letter. M-SHIME® = Mucosal Simulator of the Human Intestinal Microbial Ecosystem. Full article ">Figure 2
Changes in (a) total SCFA, (b) lactate, (c) branched SCFA, and (d) ammonium-N over time following test product administration in M-SHIME® short-term colonic incubations. Incubations included the negative control (colonic incubation blank medium), NatureKnit™ (1.667 g fiber/L, 3.333 g/L total), inulin (1.667 g fiber/L), and psyllium (1.667 g fiber/L). Donors A, B, and C represent three individual healthy human fecal donors, and average donor represents the average of the three donors. Incubations were performed in triplicate (n = 3) and the results are presented as mean ± standard deviation. Statistical analysis was performed over the course of the entire colonic incubation phase (i.e., between 0 h and 48 h) or over the course of the initial time interval (i.e., between 0 h and 6 h; lactate only). Paired student’s t-tests were used to compare changes observed for the test products versus negative control. A p-value of <0.05 was considered statistically significant. Different letters above the bars indicate statistically significant differences between test conditions, while no significant differences were observed between test conditions that share the same letter. M-SHIME® = Mucosal Simulator of the Human Intestinal Microbial Ecosystem; SCFA = short chain fatty acid. Full article ">Figure 2 Cont.
Changes in (a) total SCFA, (b) lactate, (c) branched SCFA, and (d) ammonium-N over time following test product administration in M-SHIME® short-term colonic incubations. Incubations included the negative control (colonic incubation blank medium), NatureKnit™ (1.667 g fiber/L, 3.333 g/L total), inulin (1.667 g fiber/L), and psyllium (1.667 g fiber/L). Donors A, B, and C represent three individual healthy human fecal donors, and average donor represents the average of the three donors. Incubations were performed in triplicate (n = 3) and the results are presented as mean ± standard deviation. Statistical analysis was performed over the course of the entire colonic incubation phase (i.e., between 0 h and 48 h) or over the course of the initial time interval (i.e., between 0 h and 6 h; lactate only). Paired student’s t-tests were used to compare changes observed for the test products versus negative control. A p-value of <0.05 was considered statistically significant. Different letters above the bars indicate statistically significant differences between test conditions, while no significant differences were observed between test conditions that share the same letter. M-SHIME® = Mucosal Simulator of the Human Intestinal Microbial Ecosystem; SCFA = short chain fatty acid. Full article ">Figure 3
Stacked bar plots showing (a) absolute phyla abundances (cells/mL) in the lumen compartment and (b) relative phyla abundances in the mucosal compartment. Incubations included the negative control (colonic incubation blank medium), NatureKnit™ (1.667 g fiber/L, 3.333 g/L total), inulin (1.667 g fiber/L), and psyllium (1.667 g fiber/L). Donors A, B, and C represent three individual healthy human fecal donors. Incubations were performed in triplicate (n = 3). Flow cytometry was used to determine the total number of bacterial cells in the luminal samples. Full article ">Figure 4
Jitter plots showing (a) the absolute abundance (cells/mL) of the top 20 most abundant phyla and families in the lumen compartment after 24 h, (b) the absolute abundance (cells/mL) of the top 20 most abundant phyla and families in the lumen compartment after 48 h, and (c) the relative abundance of the top 20 most abundant phyla and families in the mucosal compartment after 48 h. Incubations included the negative control (colonic incubation blank medium), NatureKnit™ (1.667 g fiber/L, 3.333 g/L total), inulin (1.667 g fiber/L), and psyllium (1.667 g fiber/L). Incubations were performed for each donor in triplicate (per donor, n = 3; total, n = 9). Each dot represents the average across donors. Full article ">Figure 5
Effect of the test products on alpha diversity as calculated by the observed taxa, Shannon, and inverse Simpson indexes in the (a) lumen compartment (24 h and 48 h) and (b) mucosal compartment (48 h). Incubations included the negative control (colonic incubation blank medium), NatureKnit™ (1.667 g fiber/L, 3.333 g/L total), inulin (1.667 g fiber/L), and psyllium (1.667 g fiber/L). Donors A, B, and C represent three individual healthy human fecal donors. Incubations were performed in triplicate (n = 3). Paired student’s t-tests were used to compare each test product with the negative control. A p-value of <0.05 was considered statistically significant; the asterisk represents a significant difference versus negative control. Full article ">

13 pages, 4078 KiB

Open AccessArticle

An Engineered Yeast Expressing an Artificial Heavy Metal-Binding Protein Enhances the Phytoremediation of Alum Mine Soils

by Wenming Wang, Liling Xie, Lin Zhao and Qilin Yu

Microorganisms 2025, 13(3), 612; https://doi.org/10.3390/microorganisms13030612 - 7 Mar 2025

Abstract

Alum mining leads to significant heavy metal and acid pollution within soils. Phytoremediation is a common strategy used to treat alum mine soils, but its efficiency is frequently compromised by the alum-mining-induced impairment of plant growth. To improve the strength of plants against [...] Read more.

Alum mining leads to significant heavy metal and acid pollution within soils. Phytoremediation is a common strategy used to treat alum mine soils, but its efficiency is frequently compromised by the alum-mining-induced impairment of plant growth. To improve the strength of plants against mine pollution, this study constructed the artificial yeast strain ScHB (heavy metal-binding protein-containing Saccharomyces cerevisiae) expressing the de novo designed protein HBGFP (heavy metal-binding green fluorescence protein) and investigated its effect on the phytoremediation of alum mine soils with soil physiochemical assays and heavy metal quantification. This protein was composed of an N-terminal signal peptide, an HB (heavy metal-binding) domain, and a GFP (green fluorescence protein) domain, as well as a C-terminal glycolphosphatidylinositol-anchoring fragment. The exposure of the HBGFP on the ScHB surface increased the growth rate of the yeast cells and enhanced cadmium capture from the cadmium-containing medium. After culturing Medicago sativa in the alum mine soils for 30 days, ScHB remarkably increased the plants’ average height from 17.5 cm to 27.9 cm and their biomass from 3.03 g/plant to 4.35 g/plant, as well as increasing the accumulation of antioxidant agents in the plants. Moreover, the ScHB cells strongly improved the soil quality, with an increase in the soil pH values from 5.47 to 6.21 to 6.9, and increased the levels of soil organic matter, total nitrogen, available phosphorus, and living bacteria. Furthermore, ScHB efficiently improved the plants’ abilities to remove soil heavy metals, decreasing the levels of cadmium, lead, chromium, and copper by 90%, 86%, 97%, and 88%, respectively. This study developed a genetic engineering method to improve the efficiency of phytoremediation against pollution from alum mining. Full article

(This article belongs to the Special Issue Advances on Molecular Microbial Ecology)

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Construction of the artificial heavy metal-binding yeast ScHB. (a) A scheme illustrating the construction of ScHB. (b) Confocal observation of the control Sc0 cells and the ScHB cells. The white arrows indicate the exposure of HBGFP on the yeast cells. (c) Fluorescence intensity of the yeast cells. The asterisk (*) indicates significant difference between the groups (p < 0.05). Full article ">Figure 2
Growth curves (a) and cadmium-capturing capacity (b) of Sc0 and ScHB. The asterisks (*) indicate significant differences between the two strains (p < 0.05). Full article ">Figure 3
EDS mapping analysis of element distribution in the yeast cells. The yeast cells were cultured in YPD medium containing 5 mg/L of Cd2+ for 24 h, followed by centrifugation, drying, and EDS mapping analysis. (a) The EDS mapping images of the yeast cells. (b) The C mass percent. (c) The S mass percent. (d) The Cd mass percent. The asterisks (*) indicate significant differences between the two strains (p < 0.05). Full article ">Figure 4
The growth of M. sativa plants cultured in alum mine soils. (a) Image of the plants. The young plants (height = 10 cm ± 1 cm) were cultured in the alum mine soils with no addition of the yeast cells (Ms), with the addition of Sc0 cells (1 × 108 yeast cells/kg soil, Ms + Sc0), or the addition of Sc0 cells (1 × 108 yeast cells/kg soil, Ms + Sc0). The plants were further cultured for 30 days and then photographed. (b) The height of the plants after 30 days. (c) The biomass of the plants. (d) The chlorophyl contents in the plant leaves. (e) The GSH contents in the leaves. (f) The ascorbic acid contents in the plant leaves. Different letters indicate significant differences between the groups (p < 0.05). Full article ">Figure 5
The quality of alum mine soils cultured with M. sativa (Ms, Ms + Sc0, Ms + ScHB) or not (control) after 30 days: (a) pH values. (b) Organic matter contents. (c) Total nitrogen (TN) contents. (d) Available phosphorus (AvP) contents. Different letters indicate significant differences between the groups (p < 0.05). Full article ">Figure 6
The number of total (a) and cadmium-tolerant (b) bacteria in the rhizosphere alum mine soils of M. sativa. The rhizosphere soils were sampled after 30 days of culturing to determine the bacterial numbers using CFU assays. Different letters indicate significant differences between the groups (p < 0.05). Full article ">Figure 7
Heavy metal contents in the rhizosphere alum mine soils of M. sativa after 30 days of culturing. (a) Cadmium contents. (b) Lead contents. (c) Chromium contents. (d) Copper contents. Different letters indicate significant differences between the groups (p < 0.05). Full article ">

18 pages, 1348 KiB

Open AccessArticle

Phyllanthus emblica: Phytochemistry, Antimicrobial Potential with Antibiotic Enhancement, and Toxicity Insights

by Gagan Tiwana, Ian Edwin Cock and Matthew James Cheesman

Microorganisms 2025, 13(3), 611; https://doi.org/10.3390/microorganisms13030611 - 6 Mar 2025

Abstract

Phyllanthus emblica Linn. (commonly known as Amla or Indian Gooseberry) is commonly used in Ayurvedic medicine to treat respiratory infections, skin disorders, and gastrointestinal issues. The fruit contains an abundance of polyphenols, which contribute to its strong antioxidant properties. The antibacterial activity of [...] Read more.

Phyllanthus emblica Linn. (commonly known as Amla or Indian Gooseberry) is commonly used in Ayurvedic medicine to treat respiratory infections, skin disorders, and gastrointestinal issues. The fruit contains an abundance of polyphenols, which contribute to its strong antioxidant properties. The antibacterial activity of fruit extracts derived from P. emblica against Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae was determined along with the antibiotic-resistant variants extended-spectrum β-lactamase (ESBL) E. coli, methicillin-resistant S. aureus (MRSA), and ESBL K. pneumoniae. Disc diffusion and broth dilution assays were conducted to assess the activity of aqueous, methanolic, and ethyl acetate extracts, with large zones of inhibition of up to 15 mm on agar observed for S. aureus and MRSA. Minimum inhibitory concentration (MIC) values ranging from 158 to 1725 µg/mL were calculated. The aqueous and methanolic extracts of P. emblica were less active against E. coli, ESBL E. coli, K. pneumoniae, and ESBL K. pneumoniae, with the only noteworthy MIC (633 µg/mL) observed for the aqueous extract against K. pneumoniae. Interestingly, a lack of inhibition was observed on agar for any of the extracts against these bacteria. Liquid chromatography–mass spectrometry (LC-MS) analysis identified several notable flavonoids, phenolic acids, terpenoids, and tannins. Notably, Artemia nauplii bioassays indicated that all extracts were nontoxic. The antibacterial activity and absence of toxicity in P. emblica extracts suggest their potential as candidates for antibiotic development, highlighting the need for further mechanistic and phytochemical investigations. Full article

(This article belongs to the Special Issue Plant Extracts and Antimicrobials, Second Edition)

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17 pages, 3968 KiB

Open AccessArticle

Vertical Distribution and Drivers of Antibiotic Resistance Genes in Agricultural Soil Irrigated with Livestock Wastewater

by Ming Shang, Yongchao Gao, Liwen Zheng, Lei Ji, Jianhua Du, Xue Kong, Hui Wang, Feng Shi, Hailun Wang, Jianhui Liu, Xiaodong Yang and Zeyu Wang

Microorganisms 2025, 13(3), 610; https://doi.org/10.3390/microorganisms13030610 - 6 Mar 2025

Abstract

Livestock wastewater reuse could be a potential source for the distribution of antibiotics, antibiotic resistance bacteria (ARB), and antibiotic resistance genes (ARGs) in agricultural soil. In this study, soil samples were collected from different depths (0–60 cm) of farmland that has been subjected [...] Read more.

Livestock wastewater reuse could be a potential source for the distribution of antibiotics, antibiotic resistance bacteria (ARB), and antibiotic resistance genes (ARGs) in agricultural soil. In this study, soil samples were collected from different depths (0–60 cm) of farmland that has been subjected to long-term application of livestock wastewater. The vertical distribution of antibiotics, bacterial communities, and ARGs were assessed to identify the driving factors that could potentially influence the distribution of ARB and ARGs. The results demonstrated distinguished distributions of antibiotics along the soil depths, with tetracyclines (TCs) mainly found in the top 10 cm of the soil (0.11–0.31 μg/kg), while quinolones (QNs), sulfonamides (SAs), and macrolides (MLs) were detected in all 60 cm of soil depth (0.01–0.22 μg/kg). The selection pressure of antibiotics to microorganisms led to the proliferation of ARB, especially tetracycline-resistant bacteria and erythromycin-resistant bacteria. In terms of the distribution/abundance of ARGs, novA and tetA (58) were relatively higher in 0–10 cm surface soil, while vanRM and vanRF were mainly detected in the deeper soil. Different ARGs may have the same host bacteria, which lead to the emergence of multidrug resistant bacteria, such as Ilumatobacter sp., Aggregatilinea sp., Rhabdothermincola sp., and Ornithinimicrobium sp. Soil pH, electrical conductivity (EC), and moisture content (MC) could affect the distribution and proliferation of ARB and were found negatively correlated with most of the ARGs except macB. Therefore, it is potentially possible to eliminate/inhibit the spread of ARGs by adjusting these soil parameters. These findings provide insights into the distribution and dissemination of antibiotics, ARB, and ARGs in agricultural practices of livestock wastewater irrigation and provide effective mitigation strategies to ensure the safe use of livestock wastewater in agriculture. Full article

(This article belongs to the Special Issue Bacterial and Antibiotic Resistance in the Environment (Second Edition))

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Figure 1
The distribution profile of antibiotics in soil under different soil depths. (a) The concentration of total antibiotics. Error bars represent the standard errors (n = 3). Different lowercase letters indicate significant differences in antibiotic concentration among different soil layers (p < 0.05, Tukey HSD test); (b) the concentration of four main antibiotic subcategories, error bars represent the standard errors (n = 3); (c) the concentration of specific antibiotics. CL1-CL5 represent the 0–5 cm, 5–10 cm, 10–20 cm, 20–40 cm, 40–60 cm soil depths of long-term livestock wastewater-applied soils; CK1-CK5 represent the 0–5 cm, 5–10 cm, 10–20 cm, 20–40 cm, 40–60 cm soil depths of control soil. The gray lines in the left side and top are the clustering analysis for the specific antibiotics and the sampling sites, respectively. Full article ">Figure 2
The bacterial community composition in different soil depths. (a) Relative abundance of top 15 bacteria at the phylum level. (b) Relative abundance of top 15 bacteria at the genus level. (c) The composition of ARB in soil under different depths. Error bars represent the standard errors (n = 3). Full article ">Figure 3
Dendrogram of the most differentially expressed bacterial taxa in different soil depths obtained by LEfSe analysis (LDA score > 3.5). Full article ">Figure 4
The number and abundance of ARGs in different soil depths. (a) The shared and unique ARGs presented by Wayne diagram. (b) Relative abundance of the top 10 ARGs types. (c) Relative abundance of the top 30 specific ARGs, the grey lines in left side and top are clustering analysis for the top 30 specific ARGs and the sampling sites, respectively. Full article ">Figure 5
The α-diversity of the ARGs in different soil depths. (a) The Shannon index of the ARGs. (b) The Pielou’s evenness index of the ARGs. Different lowercase letters indicate significant differences of α-diversity among different soil depths (p < 0.05, Tukey HSD test). Error bars represent the standard errors (n = 3). Full article ">Figure 6
Heat map of the correlation between the top 15 bacteria genus and the top 15 ARGs in the soil samples of (a) CL and (b) CK based on Spearman analysis. (* p ≤ 0.05, *** p ≤ 0.001). Red/blue color in each cell indicate positive/negative correlations, and color gradient indicate the strength of correlations between the abundance of the specific bacteria genus and the ARG. Full article ">Figure 7
Redundancy analysis (RDA) of the correlation between the main environmental factors and the ARGs in different soil depths. The blue arrows represent the ARGs, and the red arrows represent the environmental factors. Full article ">Figure A1
PCoA analysis of bacteria communities at the phylum level in different soil depths based on Bray–Curtis distances. Full article ">Figure A2
The α-diversity of the bacterial communities at genus level under different depths. (a) The Shannon index of bacterial communities. (b) The Pielou’s evenness index of the bacterial communities. Different lowercase letters indicate significant differences in α-diversity at different soil depths (p < 0.05, Tukey HSD test). Error bars represent the standard errors (n = 3). Full article ">

20 pages, 3001 KiB

Open AccessArticle

Isolation and Identification of Bacterial Strains Colonizing the Surface of Biodegradable Polymers

by Roberta Esposito, Serena Federico, Amalia Amato, Thomas Viel, Davide Caramiello, Alberto Macina, Marco Miralto, Luca Ambrosino, Maria Luisa Chiusano, Mariacristina Cocca, Loredana Manfra, Giovanni Libralato, Valerio Zupo and Maria Costantini

Microorganisms 2025, 13(3), 609; https://doi.org/10.3390/microorganisms13030609 - 6 Mar 2025

Abstract

Plastics play a key role in every sector of the economy, being used in the manufacturing of products in the fields of health, food packaging, and agriculture. Their mismanagement poses a serious threat to ecosystems and, in general, to human life. For this [...] Read more.

Plastics play a key role in every sector of the economy, being used in the manufacturing of products in the fields of health, food packaging, and agriculture. Their mismanagement poses a serious threat to ecosystems and, in general, to human life. For this reason, particular attention has been paid in the last decade to the use of biodegradable polymers (BPs) as an alternative to classic plastics. In this study, we aimed to identify bacterial strains able to colonize the surface of five BPs: poly(butylene succinate) (PBS), poly(butylene succinate-co-butylene adipate) (PBSA), poly(ε-caprolactone), (PCL), poly(3-hydroxybutyrate) (PHB), and poly(lactic acid) (PLA). For this experiment, mesocosms were designed ad hoc to mimic the conditions in which the polymers can be found in marine environments: i. suspended in the water column; ii. laying over gravel; and iii. under gravel. Four bacterial samples were taken (3, 4, 10, and 12 months from the start of the experiment) from five BPs incubated in the above-mentioned three conditions. Our results demonstrated that bacteria belonging to the Proteobacteria, Actinobacteria, Firmicutes, Bacillota, Bacteroidota, and Cyanobacteria phyla were the most frequent colonizers of the surfaces of the five polymers under analysis, and could be responsible for their degradation, resulting in the evolution of strategies to degrade plastics through the secretion of specific enzymes. Full article

(This article belongs to the Special Issue Genomics Approaches in Microbial Ecology)

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15 pages, 5317 KiB

Open AccessArticle

Metabolomics Provides New Insights into the Mechanisms of Wolbachia-Induced Plant Defense in Cotton Mites

by Xinlei Wang, Sha Wang, Ali Basit, Qianchen Wei, Kedi Zhao, Feng Liu and Yiying Zhao

Microorganisms 2025, 13(3), 608; https://doi.org/10.3390/microorganisms13030608 - 6 Mar 2025

Abstract

Endosymbiotic bacteria play a significant role in the co-evolution of insects and plants. However, whether they induce or inhibit host plant defense responses remains unclear. In this study, non-targeted metabolomic sequencing was performed on cotton leaves fed with Wolbachia-infected and uninfected spider [...] Read more.

Endosymbiotic bacteria play a significant role in the co-evolution of insects and plants. However, whether they induce or inhibit host plant defense responses remains unclear. In this study, non-targeted metabolomic sequencing was performed on cotton leaves fed with Wolbachia-infected and uninfected spider mites using parthenogenetic backcrossing and antibiotic treatment methods. A total of 55 differential metabolites were identified, which involved lipids, phenylpropanoids, and polyketides. KEGG pathway enrichment analysis revealed seven significantly enriched metabolic pathways. Among them, flavonoid and flavonol biosynthesis, glycerophospholipid metabolism, and ether lipid metabolism showed extremely significant differences. In Wolbachia-infected cotton leaves, the flavonoid biosynthesis pathway was significantly up-regulated, including quercetin and myricetin, suggesting that the plant produces more secondary metabolites to enhance its defense capability. Glycerophosphocholine (GPC) and sn-glycerol-3-phosphoethanolamine (PE) were significantly down-regulated, suggesting that Wolbachia may impair the integrity and function of plant cell membranes. The downregulation of lysine and the upregulation of L-malic acid indicated that Wolbachia infection may shorten the lifespan of spider mites. At various developmental stages of the spider mites, Wolbachia infection increased the expression of detoxification metabolism-related genes, including gene families such as cytochrome P450, glutathione S-transferase, carboxylesterase, and ABC transporters, thereby enhancing the detoxification capability of the host spider mites. This study provides a theoretical basis for further elucidating the mechanisms by which endosymbiotic bacteria induce plant defense responses and expands the theoretical framework of insect–plant co-evolution. Full article

(This article belongs to the Special Issue Plant-Microbe Interaction State-of-the-Art Research in China)

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Phenotype of cotton leaves infected by mites with different Wolbachia infection statuses: (A) cotton leaves after YJ infection, (B) cotton leaves after WJ infection, and (C) comparison of the damaged leaf area. ns: not significant. Full article ">Figure 2
PCA of YJ and WJ metabolomic profiles: (A) PCA of variance-stabilized counts in the positive ion mode for YJ vs. WJ, (B) PCA of variance-stabilized counts in the negative ion mode for YJ vs. WJ, (C) OPLS-DA of the positive ion mode in YJ vs. WJ, (D) OPLS-DA of the negative ion mode in YJ vs. WJ, and (E) classification of differential metabolites. Full article ">Figure 3
Differential metabolite analysis: (A) differential metabolite volcano plot of YJ vs. WJ in the positive ion mode, (B) differential metabolite volcano plot of YJ vs. WJ in the negative ion mode, (C) fold changes in significant differential metabolites in the positive ion mode of YJ vs. WJ, (D) fold changes in significant differential metabolites in the negative ion mode of YJ vs. WJ, (E) hierarchical clustering heatmap in the positive ion mode of YJ vs. WJ, and (F) hierarchical clustering heatmap in the negative ion mode of YJ vs. WJ. Full article ">Figure 4
KEGG enrichment analysis of differential metabolites: (A) pathway enrichment map of differential metabolites in YJ vs. WJ and (B) abundance difference score plot for differential metabolite enrichment pathways in YJ vs. WJ. Full article ">Figure 5
KEGG enrichment pathway analysis for differential metabolites. This figure illustrates lipid metabolism, secondary metabolism, and protein and amino acid metabolism within cotton cells. Lysine biosynthesis connected lysine degradation to alkaloid synthesis, with lysine degradation entering the citric acid cycle via the pipecolic acid pathway. Glycerophospholipid metabolism interacted with ether lipid metabolism through 1-acylglycerol-3-phosphate, and both glycerophosphocholine (GPC) and sn-glycerol-3-phosphoethanolamine (PE) were significantly downregulated in both glycerophospholipid metabolism and ether lipid metabolism pathways. Flavonoids such as quercetin and myricetin were significantly upregulated among secondary metabolites, while L-lysine and pantothenic acid were significantly downregulated in the protein digestion and absorption pathway. Full article ">Figure 6
Effects of Wolbachia on the reproductive capacity and fitness of spider mites: (A) survival rate and (B) duration of development. ns: not significant. ****: p < 0.001. Full article ">Figure 7
Differential expression of detoxification metabolism genes in different developmental stages of spider mite. E_W vs. E: egg stage; L_W vs. L: larval stage; N_W vs. N: nymphal stage; A_W_F vs. A_F: adult female; A_W_M vs. A_M: adult male: (A) cytochrome P450, (B) glutathione S-transferase, (C) carboxylesterase, and (D) ABC transporter. Full article ">Figure 8
Metabolic profile of cotton leaves infected by Wolbachia-infected mites: Wolbachia infection that induced secondary metabolites in cotton led to the activation of flavonoid and flavonol biosynthesis and tropane, piperidine, and pyridine alkaloid biosynthesis, resulting in a significant upregulation of flavonoid compounds such as quercetin, enhancing the plant defense. It impairs lipid metabolism, resulting in a significant downregulation of lipid metabolites, such as sn-glycerol-3-phosphoethanolamine and glycerophosphocholine, and disrupting the integrity and functionality of plant cell membranes, potentially facilitating mite feeding. It also disrupts the growth and reproduction of mites via the down-regulation of lysine, leading to a deficiency in essential amino acid supply and a significant increase in L-malic acid, which damaged the fat storage process in mites and shortened their lifespan. Wolbachia infection induced the upregulation of numerous detoxification metabolic genes in mites, enhancing their ability to detoxify plant secondary metabolites. Full article ">

16 pages, 1559 KiB

Open AccessArticle

Etiology and Oral Antibiotic Susceptibility Patterns of the First Urinary Tract Infection Episode in Infants Under 6 Months of Age: A 17-Year, Retrospective, Single-Center Study in Italy

by Francesca Bagnasco, Francesca Lorenzini Ceradelli, Alessio Mesini, Carolina Saffioti, Erica Ricci, Chiara Russo, Marcello Mariani, Elisabetta Ugolotti, Emanuela Caci, Debora Contu, Elisa Tavella, Alessia Cafaro, Giorgio Piaggio, Enrico Verrina, Roberto Bandettini and Elio Castagnola

Microorganisms 2025, 13(3), 607; https://doi.org/10.3390/microorganisms13030607 - 6 Mar 2025

Abstract

Background: Urinary tract infections (UTIs) are among the most common bacterial infections in children, and the antibiotic susceptibility in the youngest patients remains poorly understood. This study aimed to describe the distribution of uropathogens and their antibiotic susceptibility, focusing on oral formulations. Methods: [...] Read more.

Background: Urinary tract infections (UTIs) are among the most common bacterial infections in children, and the antibiotic susceptibility in the youngest patients remains poorly understood. This study aimed to describe the distribution of uropathogens and their antibiotic susceptibility, focusing on oral formulations. Methods: Data from the first microbiological isolation, between January 2007 and December 2023, at Istituto Gaslini, in young infants (aged <6 months), were analyzed. Results: We isolated 2473 infants’ first pathogen, with a median age in the sample of 2.8 months and 62.6% male. A total of 2498 bacterial isolates were identified, of which 88.8% were Gram-negative and 11.2% were Gram-positive. Escherichia coli (53%) was the most frequent isolate, followed by Klebsiella pneumoniae (12.3%) and Enterococcus spp. (9.6%). No significant differences were observed between males and females, but infants younger than 3 months exhibited a significantly different pathogen distribution compared to older infants. The pathogen distribution showed significant changes before and after 2015, with a marked increase in Klebsiella pneumoniae isolates post-2015. Escherichia coli showed increases in resistance to amoxicillin-clavulanate and ciprofloxacin after 2015. Conclusions: Escherichia coli remains the most common uropathogen; however, Klebsiella pneumoniae has not only shown a high prevalence but also significant resistance, particularly in recent years. Full article

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

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18 pages, 4115 KiB

Open AccessArticle

Novel Co-Cultivation Bioprocess with Immobilized Paenibacillus polymyxa and Scenedesmus obliquus for Lipid and Butanediol Production

by Jnanada Shrikant Joshi, Laura Fladung, Olaf Kruse and Anant Patel

Microorganisms 2025, 13(3), 606; https://doi.org/10.3390/microorganisms13030606 - 5 Mar 2025

Abstract

Microalgal biotechnology is gaining attention due to its potential to produce pigments, lipids, biofuels, and value-added products. However, challenges persist in terms of the economic viability of microalgal lipid production in photobioreactors due to slow growth rates, expensive media, complex downstream processing, limited [...] Read more.

Microalgal biotechnology is gaining attention due to its potential to produce pigments, lipids, biofuels, and value-added products. However, challenges persist in terms of the economic viability of microalgal lipid production in photobioreactors due to slow growth rates, expensive media, complex downstream processing, limited product yields, and contamination risks. Recent studies suggest that co-cultivating microalgae with bacteria can enhance the profitability of microalgal bioprocesses. Immobilizing bacteria offers advantages such as protection against shear forces, the prevention of overgrowth, and continuous product secretion. Previous work has shown that biopolymeric immobilization of Paenibacillus polymyxa enhances 2,3-butanediol production. In this study, a novel co-fermentation process was developed by exploiting the chemical crosstalk between a freshwater microalga Scenedesmus obliquus, also known as Tetradesmus obliquus, and an immobilized plant-growth-promoting bacterium, Paenibacillus polymyxa. This co-cultivation resulted in increased metabolite production, with a 1.5-fold increase in the bacterial 2,3-butanediol concentration and a 3-fold increase in the microalgal growth rates compared to these values in free-cell co-cultivation. Moreover, the co-culture with the immobilized bacterium exhibited a 5-fold increase in the photosynthetic pigments and a 3-fold increase in the microalgal lipid concentration compared to these values in free-cell co-cultivation. A fixed bed photobioreactor was further constructed, and the co-cultivation bioprocess was implemented to improve the bacterial 2,3-butanediol and microalgal lipid production. In conclusion, this study provides conclusive evidence for the potential of co-cultivation and biopolymeric immobilization techniques to enhance 2,3-butanediol and lipid production. Full article

(This article belongs to the Special Issue The Application Potential of Microalgae in Green Biotechnology)

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Setup of the utilized self-constructed fixed bed photobioreactor (A) and a photo of the utilized self-constructed tubular fixed bed photobioreactor with the main light source of the tubular photobioreactor removed to visualize the medium (B). Fluidized bed photobioreactor can improve the product yields with proper mixing due to gas sparging and improved light transmission (C). Full article ">Figure 2
Maximum specific growth rates (μmax) determined in the cultivation of axenic microalgae in the PS medium with the addition of empty chitosan-coated carrageenan beads and the co-cultivation with immobilized bacteria. n = 5; mean ± SD. Different letters a, b, and c indicate a significant difference according to the one-way ANOVA F2,14 = 1501.51; p < 0.001 with Bonferroni’s post hoc test at p < 0.05. n = 5; mean <math display="inline"><semantics> <mrow> <mo>±</mo> </mrow> </semantics></math> SD; one-way ANOVA with Bonferroni’s post hoc test, p < 0.05 (A). Total chlorophyll contents determined in the cultivation of axenic microalgae in the PS medium with the addition of empty chitosan-coated carrageenan beads and the co-cultivation with immobilized bacteria. n = 5; mean ± SD. Different letters a, b, and c indicate a significant difference according to the one-way ANOVA F2,14 = 2894.11; p < 0.001 with Bonferroni’s post hoc test at p < 0.05 (B). Full article ">Figure 3
Determined 2,3-BDL concentrations of co-cultivation of free cells and chitosan-coated calcium alginate bead- and chitosan-coated carrageenan bead-immobilized bacteria. n = 5; mean ± SD. Different letters a and b indicate a significant difference according to one-way ANOVA F2,24 = 166.58; p < 0.001 with Bonferroni’s post hoc test at p < 0.05. Full article ">Figure 4
Co-cultivation of axenic S. obliquus with P. polymyxa immobilized in chitosan-coated carrageenan beads in PS medium using a self-constructed tubular fixed bed photobioreactor over 366 h; photos with the main light source of the tubular photobioreactor removed to visualize the medium. Full article ">Figure 5
Samples of S. obliquus in co-culture with chitosan-coated carrageenan P. polymyxa beads in PS medium in a photobioreactor for 0 h to 336 h. Full article ">

18 pages, 17133 KiB

Open AccessArticle

Influence of Inhibitors Generated in Lignocellulosic Hydrolysates from Group of Acids on the Growth of Strains TG1 and Tuner of Escherichia coli

by Suelen S. Gaspar, Júnia Alves-Ferreira, Patrícia Moniz, Talita Silva-Fernandes, Adriana I. R. Silvestre, Ivone Torrado, Gaetano R. Pesce, Florbela Carvalheiro, Luís C. Duarte and Maria C. Fernandes

Microorganisms 2025, 13(3), 605; https://doi.org/10.3390/microorganisms13030605 - 5 Mar 2025

Abstract

Concerns over fossil fuels are of increasing interest in biorefineries that utilize lignocellulosic residues. Besides sugars, inhibitors are formed during biomass pretreatment, including acetic acid (AI) and formic acid (FI), which can hinder microbial fermentation. The TG1 and Tuner strains of Escherichia coli [...] Read more.

Concerns over fossil fuels are of increasing interest in biorefineries that utilize lignocellulosic residues. Besides sugars, inhibitors are formed during biomass pretreatment, including acetic acid (AI) and formic acid (FI), which can hinder microbial fermentation. The TG1 and Tuner strains of Escherichia coli were subjected to various acid concentrations. Samples were taken during fermentation to monitor growth, sugar consumption, biomass yield, and product yield. With increasing AI, the TG1 strain maintained stable growth (0.102 1/h), while xylose consumption decreased, and product formation improved, making it better suited for high-acetic-acid industrial applications. In contrast, the Tuner strain performed better under low-inhibitor conditions but suffered metabolic inhibition at high AI levels, compensating by increasing lactic acid production—an adaptation absent in TG1. However, Tuner showed greater resistance to formic acid stress, sustaining higher growth and ethanol production, whereas TG1 experienced a greater metabolic decline but maintained stable acetic acid output. Both strains experienced inhibition in formic acid metabolism, but TG1 had a higher yield despite its lower overall robustness in formic acid conditions. The use of TG1 for value-added compounds such as ethanol or formic acid may help to avoid the use of chemicals that eliminate acetic acid. Tuner could be used for lactic acid production, especially in hydrolysates with under moderate concentration. Full article

(This article belongs to the Special Issue Microbial Advances in a Sustainable Environment: Biological Waste Treatment and Bioconversion Technology 2.0)

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18 pages, 5292 KiB

Open AccessArticle

In Vitro Transcribed Artificial Primary MicroRNA for the Inhibition of Hepatitis B Virus Gene Expression in Cultured Cells

by Creanne Shrilall, Patrick Arbuthnot and Abdullah Ely

Microorganisms 2025, 13(3), 604; https://doi.org/10.3390/microorganisms13030604 - 5 Mar 2025

Abstract

Available interventions for the management of chronic hepatitis B (hepB) exhibit limited efficacy and barriers to vaccination against the hepatitis B virus (HBV) have hampered prophylaxis programmes. Development of potent therapeutics capable of functional cure of chronic hepB thus remains a relevant medical [...] Read more.

Available interventions for the management of chronic hepatitis B (hepB) exhibit limited efficacy and barriers to vaccination against the hepatitis B virus (HBV) have hampered prophylaxis programmes. Development of potent therapeutics capable of functional cure of chronic hepB thus remains a relevant medical objective. RNA interference (RNAi) can be exploited to effect potent and specific silencing of target genes through the introduction of RNA sequences that mimic the natural activators of the pathway. To achieve a therapeutic effect, artificial primary microRNAs (pri-miRNAs) have been used extensively to target various viruses, including HBV. To date artificial pri-miRNAs have exclusively been produced from DNA expression cassettes. Although this achieves impressive silencing, eventual translation of this platform to the clinic is complicated by the requirement for viral vectors to deliver DNA. Consequently, clinical translation has been slow. Recently, the use of in vitro transcribed RNA, specifically to produce mRNA vaccines at industrial scale, has gained significant interest. We therefore sought to evaluate the feasibility of using in vitro transcribed artificial pri-miRNAs for the inhibition of HBV gene expression. Artificial HBV-targeting pri-miR-31 sequences, which are highly effective when expressed in cells from a DNA template, demonstrated modest silencing of viral replication when incorporated into mRNA that was transcribed in vitro. Off-target effects were also observed. Characterisation revealed that intracellular processing of the artificial pri-miRNAs was inefficient and non-specific effects were caused by stimulation of the interferon response. Nevertheless, optimised nuclear delivery of the artificial pri-miRNAs should improve their processing and achieve better anti-hepB efficacy. Full article

(This article belongs to the Special Issue Hepatitis B Virus Infection and Vaccine)

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Design and inhibitory capacity of artificial pri-miR-31 expression vectors. (A) Upper panel: Original CMV promoter-driven anti-HBV artificial pri-miR-31 vector. Bottom panel: Vector design for in vitro transcription of artificial pri-miR-31 sequences. The vector contains the T7 promoter to enable use of the T7 bacteriophage RNA polymerase. The 3′ end comprises a sequence encoding a 52 nucleotide stretch of adenosines, which makes up the polyA tail. RNAs are capped in a separate enzymatic reaction to add a 7-methylguanosine to the 5′ end. (B) HBsAg secretion from HepG2-hNTCP co-transfected with the pri-miR-31 expression vectors (CMV promoter-driven vectors, left and IVT vectors, right) and the HBV target plasmid, pCH-9/3091, were determined by ELISA. The graphs were plotted as the mean ± the standard error of the mean (SEM) (n = 3). The statistical significance was calculated using the Student’s paired two-tailed t-test (*** indicates p < 0.001 and ** indicates p < 0.01). Full article ">Figure 2
Inhibition of HBV gene expression by in vitro transcribed artificial pri-miR-31 sequences. (A) HepG2-hNTCP cells were co-transfected with the HBV target plasmid, pCH-9/3091, and an mCherry reporter plasmid. One day later the cells were transfected with the indicated in vitro transcribed sequences. Secreted HBsAg levels in culture supernatants at 24- and 48-h post-transfection were measured by ELISA and plotted as the mean ± standard error of the mean (SEM) (n = 3). The statistical significance was calculated using the Student’s paired two-tailed t-test (*** indicates p < 0.001; ** indicates p < 0.01; * indicates p < 0.05. (B) HepG2-hNTCP cells were co-transfected with the HBV target plasmid, psiCHECK-HBx, and an mCherry reporter plasmid. One day after the DNA transfection the cells were transfected with the indicated RNA sequences. Twenty-four and forty-eight hours after RNA transfection the cells were harvested and luciferase activity measured in cell lysates. Graphs show the mean ratios of Renilla luciferase to Firefly luciferase activities. Error bars represent the SEM. Statistical significance was calculated against the mock control using the Student’s paired two-tailed t-test (*** indicates p < 0.001; ** indicates p < 0.01; * indicates p < 0.05). Full article ">Figure 3
Assessing processing of the artificial pri-miR-31/5 sequence using Northern blot hybridisation. Total RNA was extracted from HEK293T cells that had been transfected with eGFP mRNA, in vitro transcribed pri-miR-31/5, or pCMV-pri-miR-31/5. The samples were separated on a 20% denaturing polyacrylamide gel and Northern blot analysis was performed using a radioactively labelled probe complementary to the expected guide sequence of pri-miR-31/5. The ladder was generated by radioactively labelling several oligonucleotides of different lengths and running them together alongside the RNA samples. The sizes indicated are approximations of the RNA sizes. Full article ">Figure 4
Assessing non-specific effects of in vitro transcribed artificial pri-miR-31 sequences. (A) HEK293T cells were transfected with the indicated RNA and their viability assessed by MTT assay 48-h later. The graph was plotted using the mean normalised to the mean of the untransfected cells. Error bars indicate the normalised SEM (n = 3). The statistical significance was calculated against the untransfected control using the Student’s paired two-tailed t-test (*** indicates p < 0.001; * indicates p < 0.05). (B) HEK293T cells were co-transfected with phRL-CMV, pUCAsc-ISRE×3 and the indicated in vitro transcribed RNA or poly I:C. Two days after transfection, Firefly and Renilla luciferase activity was measured using a dual-luciferase assay. The graph shows the mean ratios of Firefly to Renilla luciferase activity ± SEM (n = 3). Statistical significance was calculated against the mock control using the Student’s paired two-tailed t-test (* indicates p < 0.05). (C–E) Expression levels of the interferon stimulated genes IFN-β (C), OAS1 (D) and MxA (E) in HEK293T cells transfected with the indicated in vitro transcribed RNA were assessed using qRT-PCR. The graphs represent the relative levels of IFN-β, OAS1 and MxA to GAPDH mRNA. Each graph is normalised to its respective mock (mean ± SEM, n = 3). Statistical significance was calculated against the mock treated cells using the Student’s paired two-tailed t-test (*** indicates p < 0.001; ** indicates p < 0.01; * indicates p < 0.05). Full article ">Figure 4 Cont.
Assessing non-specific effects of in vitro transcribed artificial pri-miR-31 sequences. (A) HEK293T cells were transfected with the indicated RNA and their viability assessed by MTT assay 48-h later. The graph was plotted using the mean normalised to the mean of the untransfected cells. Error bars indicate the normalised SEM (n = 3). The statistical significance was calculated against the untransfected control using the Student’s paired two-tailed t-test (*** indicates p < 0.001; * indicates p < 0.05). (B) HEK293T cells were co-transfected with phRL-CMV, pUCAsc-ISRE×3 and the indicated in vitro transcribed RNA or poly I:C. Two days after transfection, Firefly and Renilla luciferase activity was measured using a dual-luciferase assay. The graph shows the mean ratios of Firefly to Renilla luciferase activity ± SEM (n = 3). Statistical significance was calculated against the mock control using the Student’s paired two-tailed t-test (* indicates p < 0.05). (C–E) Expression levels of the interferon stimulated genes IFN-β (C), OAS1 (D) and MxA (E) in HEK293T cells transfected with the indicated in vitro transcribed RNA were assessed using qRT-PCR. The graphs represent the relative levels of IFN-β, OAS1 and MxA to GAPDH mRNA. Each graph is normalised to its respective mock (mean ± SEM, n = 3). Statistical significance was calculated against the mock treated cells using the Student’s paired two-tailed t-test (*** indicates p < 0.001; ** indicates p < 0.01; * indicates p < 0.05). Full article ">

21 pages, 1957 KiB

Open AccessReview

A Pipeline for the Isolation and Cultivation of Microalgae and Cyanobacteria from Hypersaline Environments

by Petra Tavčar Verdev and Marko Dolinar

Microorganisms 2025, 13(3), 603; https://doi.org/10.3390/microorganisms13030603 - 5 Mar 2025

Abstract

Microorganisms in high-salinity environments play a critical role in biogeochemical cycles, primary production, and the biotechnological exploitation of extremozymes and bioactive compounds. The main challenges in current research include isolating and cultivating these microorganisms under laboratory conditions and understanding their complex adaptive mechanisms [...] Read more.

Microorganisms in high-salinity environments play a critical role in biogeochemical cycles, primary production, and the biotechnological exploitation of extremozymes and bioactive compounds. The main challenges in current research include isolating and cultivating these microorganisms under laboratory conditions and understanding their complex adaptive mechanisms to high salinity. Currently, universally recognized protocols for isolating microalgae and cyanobacteria from salt pans, salterns, and similar natural habitats are lacking. Establishing axenic laboratory cultures is essential for identifying new species thriving in high-salinity environments and for exploring the synthesis of high-value metabolites by these microorganisms ex situ. Our ongoing research primarily focuses on photosynthetic microorganisms with significant biotechnological potential, particularly for skincare applications. By integrating data from the existing literature with our empirical findings, we propose a standardized pipeline for the isolation and laboratory cultivation of microalgae and cyanobacteria originating from aqueous environments characterized by elevated salt concentrations, such as solar salterns. This approach will be particularly useful for researchers working with microorganisms adapted to hypersaline waters. Full article

(This article belongs to the Special Issue Photosynthetic Microorganisms: Culturing and Biotechnological Applications)

► Show Figures

Figure 1

Figure 1
Revival of a dry culture sample of green microalgae Dunaliella sp. in our laboratory. (A) Initial state of the sample, which was dried up by slow evaporation at room temperature. The culture was maintained in modified Johnson’s medium, supplemented with 170 g/L NaCl, prior to that. (B) The same sample one month after adding distilled water to the dried algae. (C) Microscopic view of the revived Dunaliella sp., showing two ovoid-shaped cells in the vegetative motile form. Scale bar corresponds to 10 µm. Microscopy was performed using a Primostar optical microscope with a 100× Pan-Achromat immersion objective (Zeiss, Jena, Germany). Full article ">Figure 2
Macroscopic and microscopic view of filamentous cyanobacterium, isolated from the Sečovlje salterns. (A) Macroscopic view showing multilayered sheet organization of filamentous cyanobacteria cultivated on a non-nutrient agarose plate topped with liquid nutrient medium. (B) Microscopic view of filament organization within the sheet. Scale bar corresponds to 20 µm. Microscopy was performed using a Primostar optical microscope with a 100× Pan-Achromat immersion objective (Zeiss, Germany). Full article ">Figure 3
Workflow for the isolation and cultivation of microalgae and cyanobacteria from environmental samples, including those obtained in hypersaline environments. The workflow consists of: (1) pre-sampling preparation of sampling equipment, labware, and reagents; (2) field sampling to collect various sample types, including water, microbial mats, and crystallized salt; (3) microscopic examination of samples to assess diversity and detect contaminants; (4) elimination of contaminants, especially grazers (if no grazers are observed, proceed directly to Step 5); (5) enrichment of environmental samples under controlled conditions; (6) laboratory cultivation using appropriate isolation and purification techniques; and (7) verification of culture axenicity and species identification. Full article ">

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