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The Real-Lab-Day active learning methodology is an innovative approach aimed at deepening undergraduate students' understanding of the scientific process by integrating theory with practical laboratory experience in microbiology. This study aimed to reassess the educational impact of Real-Lab-Day ten years after its initial implementation and to explore its potential for optimizing experimental microbiology protocols. Undergraduate students participated in authentic laboratory experiments under the supervision of graduate mentors. To assess the impact of the activity, formative questionnaires were administered to evaluate students' perceptions of learning, engagement, and motivation. In parallel, the methodology was employed to optimize a bacterial pathogenicity protocol using enteroinvasive Escherichia coli (EIEC) as a model organism. Our data revealed sustained student approval and confirmed the effectiveness of the methodology in consolidating theoretical knowledge, developing practical and cognitive skills, and fostering interest in microbiological research. Furthermore, Real-Lab-Day enabled the successful optimization of bacterial invasion and dissemination protocol. Overall, Real-Lab-Day emerges as a multifunctional pedagogical strategy that effectively connects teaching and research. It enhances student learning experiences while also contributing to the optimization and validation of experimental protocols for use in practical classes. The methodology aligns with contemporary higher education practices and holds strong potential for future educational research.

Soils are heterogeneous and dynamic systems characterized by complex physical, chemical, and biological interactions. Understanding these interactions is critical, as they influence plant productivity, global biogeochemical cycles, and ecosystem resilience. While ecologists have long studied soils in field, greenhouse, and laboratory settings, their complexity and heterogeneity make it challenging to pinpoint key properties driving biological processes and derive mechanistic insights. Advancements in synthetic biology, which seeks to engineer and control biological processes in soils, have increased the demand for standardized and controllable experimental platforms. These platforms, referred to here as 'synthetic soils', are systems designed to reproduce selected physicochemical characteristics of natural soils in a simplified and defined format, allowing scientists to systematically change soil physicochemical properties (i.e. texture, mineralogy, pH) to study how biological components (i.e. microbes, plants, soil fauna, etc.) respond to, modify, or interact within these controlled environments. This review explores existing synthetic soils, their advantages, limitations, and applications in ecology and synthetic biology, and discusses potential directions for their future development.

The lizard microbiome is a dynamic community that plays a crucial role in the health and survival of these animals. As global change poses significant threats to lizard populations around the world, understanding the interactions between lizards and their microbial communities is increasingly important. Here, we synthesize a rapidly growing body of research on the composition, diversity, transmission, and functional roles of lizard microbiomes. We discuss the implications of microbiome variation for lizard physiology, as well as the potential for microbiomes to inform conservation strategies for threatened species. Finally, we highlight priorities for future research, which include the need to quantify microbiome diversity and function across additional taxa, as lizards remain underrepresented in the microbiome literature. We also stress the importance of experimental and field research that can reveal the adaptive significance of lizard microbiomes in the face of environmental change. Our synthesis highlights the contributions of lizard microbiome science to the fields of ecology, evolution, and conservation biology and demonstrates how the microbial communities that live in and on lizards enhance our understanding of their biodiversity and inform efforts to protect vulnerable populations.

Salmonella is a major zoonotic bacterial pathogen with significant impacts on public health, food safety, and veterinary medicine. The genus Salmonella includes exactly two recognized species, S. enterica and S. bongori. Most routine molecular surveillance assays target conserved genus-level genes, such as invA, which do not distinguish Salmonella enterica and Salmonella bongori, potentially obscuring species-specific ecology and source attribution. Here, we present the first introduction of Multiple accessory-genes sequence typing (MAST), a pan-genome-guided framework for binary (two species) bacterial PCR target screening, primer design, and experimental validation, demonstrated here in Salmonella. Applying MAST to 2 236 high-quality Salmonella genomes, we identified sopD2 as a species-discriminative locus. sopD2-targeted primers with the highest MAST Diff score (=0.999) demonstrated high analytical specificity for Salmonella, with no cross-amplification observed against Escherichia coli, Streptococcus suis, or Pasteurella multocida, and enabled clear PCR differentiation of S. enterica from S. bongori across laboratory isolates. Taken together, our results position MAST as a broadly applicable and practical pipeline for species‑level PCR marker discovery across diverse bacterial taxa and validate sopD2 as a robust target for discriminating Salmonella species, enhancing the accuracy of molecular surveillance and the clarity of source attribution.

This work describes a laboratory activity designed to illustrate the phenomenon of bacterial Quorum Sensing (QS), a communication mechanism in bacterial communities. The activity focuses on the bioluminescence production regulated by QS of bacteria that live in symbiosis with cephalopods. This activity targets undergraduate students in biology, biochemistry, or other sciences and aims to promote their interest in microbiology and to help students to understand the role and mechanism of QS in microorganisms by means of a visual example of symbiotic interactions between bacteria and animals. At the same time, students are expected to develop lab skills in bacterial isolation, pure culture obtention and interpretation of microbiological results. The work also provides references and resources to help students understand the subject and teachers assess student learning.

Esca is one of the most damaging fungal diseases of grapevine and continues to defy Koch's postulates. Although Phaeomoniella chlamydospora, Phaeoacremonium minimum, and Fomitiporia mediterranea are consistently associated with wood necrosis in esca-symptomatic vines, they also occur in asymptomatic vines and even in apparently healthy wood tissues without visible necrosis, and single-species but also mixed-species inoculations rarely reproduce the characteristic foliar symptoms. We hypothesize that esca is best understood as a stress-mediated pathobiome disorder of the grapevine holobiont rather than a predictable outcome of specific fungal combinations, shifting focus from pathogen identity to holobiont functional state and environmental context. In this Review, we integrate evidence from community ecology, vascular biology, and multi-omics studies to link microbial community structure and activity with host hydraulics, defence, and environmental drivers. Metabarcoding and metatranscriptomics indicate that symptom expression correlates with functional reprogramming of trunk-inhabiting fungi more than their mere presence, while metabolomics and epigenomics reveal localized physiological disruption combined with systemic regulatory responses. Climatic and edaphic stresses, particularly drought, are strongly associated with holobiont destabilization and dysbiosis, altering symptom expression without necessarily modifying pathogen occurrence. We propose a temporal, multi-phase model integrating colonization history, microbiome restructuring, and host stress physiology through long-term feedbacks. This framework emerges through convergent multi-omics evidence and generates testable predictions for early detection, microbiome-informed biocontrol, and resilience-oriented vineyard management strategies.

Anoxic coastal sediments are significant natural sources of the potent greenhouse gas methane where, methylotrophic methanogenesis may predominate. However, the underlying methanogen community composition as well as the impact of salinity levels and vegetation on their diversity and potential activity remain poorly resolved. To assess methane production and characterize methanogen diversity underlying methanol and trimethylamine (TMA) degradation, we incubated anoxic sediments from three sites along the Medway Estuary (UK)-a high-salinity marine site and two brackish sites, one vegetated (saltmarsh with cordgrass) and one non-vegetated. Saltmarsh sediments exhibited the highest methane yields (46% from TMA, 27% from methanol), whereas we did not observe methanogenesis from methanol in the high-salinity marine samples. Methylotrophic methanogen genera Methanolobus, Methanomethylophilus, and Methanofastidiosum were abundant in all original sediments, where metabolic generalist Methanosarcinales were also present. Methanoculleus were the most abundant taxon (31.5%) in original brackish sediments, while they were only at 1% in saltmarsh sediments. Following the amendment with TMA or methanol, Methanolobus dominated all the sediments with methane production (80%-99.5% relative abundance), however Methanoculleus were still in high relative abundance in brackish incubations with methanol or TMA (4.6% and 10%, respectively). These findings indicate that both salinity and vegetation shape the community composition of methylotrophic methanogens in coastal sediments, yet key methylated substrates are primarily metabolized by Methanolobus, regardless of differences in salinity and vegetation.

In host-microbe interactions, host diet and environmental stress are key driving factors shaping the gut microbiota. Although previous studies have shown that hypoxia affects the structure and function of the gut microbiota in rodents, most have relied on 16S rRNA gene sequencing and lacked analysis of community assembly mechanisms, co-occurrence networks, and functional pathways. Here, we used metagenomic next-generation sequencing (mNGS) to examine the gut microbiota of rats exposed to hypobaric hypoxia (WH, simulated 6000 m altitude) compared to WL group (2100 m altitude). Hypoxia significantly altered β-diversity of gut microbiota, but did not affect its α-diversity. Community assembly was primarily governed by stochastic processes, with hypoxia stress reducing their impact. Microbial co-occurrence networks were dominated by positive correlations, although network resilience and stability declined under hypoxia. Helicobacter and Eubacterium were identified as high-abundance differentiating genera, and Akkermansia muciniphila was significantly enriched in WH group. Functional analysis revealed alterations in pathways related to protein synthesis and carbohydrate metabolism, suggesting that hypoxia may affect nutrient utilization by the host. Overall, these findings provide a comprehensive view of how hypoxic stress reshapes the gut microbiota of rats, offering new insights into microbial dynamics under environmental stress.

This study investigated the antibacterial and potentiating activity of ammonium polyoxomolybdate tetrahydrate (NHMO; (NH4)6Mo7O24·4H2O; 1235.86 g/mol), particularly in combination with ampicillin against Staphylococcus aureus and Escherichia coli strains. Spectroscopic characterization by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and microdilution tests with resazurin were used. NHMO showed no direct antibacterial activity, with a minimum inhibitory concentration (MIC) ≥1024 μg/mL (≈0.83 mM). However, when used at a subinhibitory concentration (MIC/8, 128 μg/mL; ≈0.10 mM) with antibiotics initially tested at 1024 μg/mL, NHMO reduced the MIC of ampicillin by approximately 50% for both bacterial strains. Sulbactam, a known β-lactamase inhibitor, showed superior results, confirming the presence of enzymatic resistance. NHMO also demonstrated a resistance-reversal effect in β-lactamase-producing S. aureus strains, lowering the MIC of ampicillin from 20.16 to 16 μg/mL in K4100 and from 322.54 to 256 μg/mL in K4414. Ciprofloxacin did not show relevant modulation, whereas gentamicin potentiation was observed against S. aureus 10. These findings indicate that NHMO can enhance the activity of antibiotics, particularly ampicillin and gentamicin, in resistant bacterial strains. Its action in β-lactamase-related assays suggests that it is a promising candidate in the search for alternatives to combat antimicrobial resistance.

The occurrence of drug-resistant strains exacerbates the difficulty of treating stomach cancer caused by Helicobacter pylori. DNA replication and cell division offer attractive targets for the development of antibacterial therapies. Both DNA replication and cell division in H. pylori start at one of the poles and terminate at the middle of the cell. The absence of a SlmA homolog and the presence of a unique Min CDE system may explain polar divisome assembly in Helicobacter. We previously observed the co-localization of replisome and divisome proteins; however, it is unclear if they interact directly. We report, for the first time, the interaction between two complexes in vitro and in vivo. These results highlight the mechanistic aspects of the unique polar co-assembly of the replisome/divisome complex in Helicobacter, through interactions among members of each component, which might be useful for identifying new targets.

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Mycoplasma pneumoniae (M. pneumoniae) is a major pathogen causing community-acquired pneumonia in children. Its pathogenic process relies on the adherence to and colonization of host respiratory epithelial cells. P1 protein is the primary adhesin of M. pneumoniae, directly mediating its binding to host cells. To explore the interaction mechanism between P1 recombinant protein and host cells, we conducted protein expression and purification, glutathione S-transferase (GST) pull-down assay, and transcriptome sequencing. The rP1-GST fusion protein was expressed under confirmed induction conditions (16 °C, 0.1 mM IPTG). GST pull-down assay identified 22 differentially expressed membrane proteins in the rP1-GST group, among which annexin A2 (ANXA2) and C-C chemokine receptor type 5 (CCR5) were significantly altered and interact with P1 adhesin. Both ANXA2 and CCR5 possessed multiple functions including protein binding, receptor activity and signal sensor activity. Transcriptome analysis indicated that differentially expressed genes from rP1-A549 cell interaction were significantly enriched in multiple Gene Ontology (GO) terms and KEGG pathways. These results suggest that ANXA2 and CCR5 may serve as potential binding partners of P1 adhesin. P1 adhesin may regulate host genes involved in mitochondria and energy metabolism. These findings provide clues for understanding the adhesion and pathogenesis of M. pneumoniae.

Microplastics (MP) are relevant contaminants in agroecosystems, influencing soil nutrient dynamics and soil-plant-microbial interactions. As agriculture shifts from conventional to biodegradable plastics, their impacts on different crop rhizosphere microbiomes considering both total (DNA-derived) and active (rRNA-derived) communities have not been clearly elaborated. We hypothesized that microbiome impacts would be distinct across different plants and polymer types. Maize and strawberry plants were cultivated with 1% MP by soil weight, including two conventional polymers (LDPE, PET) and one biodegradable polymer (PBAT). Strawberry plants increased biomass across all MP treatments, accompanied by greater soil nitrate depletion. MP-induced impacts on soil prokaryotic communities were mostly additive to plant effects, as determined by 16S rRNA amplicon sequence profiling. PBAT stimulated Cupriavidus spp. and members of Saccharimonadales, suggesting a selection of potential polymer-degraders and microbial interactions, independent of plant species and root proximity. In contrast, conventional MPs induced a less selective response with compositional shifts across a greater number of taxa. MP-induced changes were more apparent in rRNA- than DNA-derived profiles, suggesting a profound response of putative active taxa. Together, we demonstrate that plant species and MP type jointly modulate rhizosphere microbial community response to MP pollution, with direct implications for soil biogeochemistry, rhizosphere functioning, and crop performance.

Given the ability of microorganisms to develop drug resistance at a faster pace than the development of new drugs, it becomes urgent to seek alternatives capable of restoring the efficacy of antibiotics, especially against pathogens such as Staphylococcus aureus, known for its versatility in expressing resistance mechanisms. Synthetic chalcones have been extensively investigated as efflux pump inhibitors due to their structural flexibility and ease of chemical modification. Can cite studies that show chalcones derived from Croton anisodontus for their ability to inhibit the NorA and MepA efflux pumps in S. aureus strains (1199B and K2068) and studies chalcones act as inhibitors of the NorA efflux pump in whole cells and everted membrane vesicles of S. aureus. Among these mechanisms, the NorA and MepA efflux pumps stand out, as they are responsible for expelling antibiotics from the bacterial cell interior. In this context, the present study investigated the potential of the chalcone (E)-1-(3-aminophenyl)-3-(4-chlorophenyl)prop-2-en-1-one as a possible inhibitor of these efflux systems. Initial screening was conducted through in silico studies, followed by in vitro assays, determination of minimum inhibitory concentration (MIC), and fluorescence tests using ethidium bromide EtBr and SYTOX Green. Molecular docking results revealed that this chalcone interacts with amino acid residues described in the literature as essential for efflux pump activity (Glu222 and Phe303 in NorA; Tyr35 and Met172 in MepA). The binding distances and energies obtained were consistent with potential inhibitory activity, suggesting that chalcone may interfere with the functionality of these proteins. In vitro assays showed a significant reduction in the MIC of antibiotics such as norfloxacin and ciprofloxacin in resistant strains, indicating a potentiating effect. Furthermore, fluorescence tests demonstrated increased retention of ethidium bromide by up to 736.3% for NorA and 234.8% for MepA, supporting the inhibition of efflux pumps. Increased bacterial membrane permeability was also observed, evidenced by an increase in SYTOX Green fluorescence by 70-855%. Although the results demonstrate the potential of chalcone as a therapeutic adjuvant, the ADMET analyses revealed toxicological alerts that require further investigation. These findings suggest that rational structural modifications may be necessary to ensure its safety and efficacy in combating resistant bacterial infections.

Some Serratia marcescens (Sm) strains produce the red pigment prodigiosin, which exhibits immunosuppressive, antimicrobial, and antitumoral properties. Prodigiosin biosynthesis is controlled by a complex regulatory network that includes SlyA, a MarR-family transcriptional regulator. However, the molecular mechanism by which SlyA regulates prodigiosin production remains largely unknown. Here, we showed that methyl benzoate, paeonol, salicylic acid, and p-coumaric acid reduce pigment production in Sm. Given previous evidence that small aromatic molecules can allosterically regulate MarR-family proteins, we generated a homodimeric structural model of Sm SlyA. Molecular docking analyses suggested that these aromatic metabolites bind within the two cavities of the SlyA homodimer, and molecular dynamics simulations identified methyl benzoate as the most stable ligand. Consistently, in vitro assays showed that the binding of recombinant SlyA to the prodigiosin promoter was altered in the presence of methyl benzoate and phenolic metabolites. Finally, exogenous methyl benzoate, paeonol, and salicylic acid significantly reduced pigA-gfp transcription in Sm, and this effect became more pronounced upon expression of slyA from a multicopy plasmid. Together, these findings support a model in which SlyA functions as an allosterically regulated sensor, coupling the perception of aromatic carbonyl compounds to the control of prodigiosin biosynthesis in Sm.

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Bivalves bioaccumulate microorganisms and, therefore, serve as effective indicators of microbial activity in intertidal ecosystems. Monitoring bivalve-associated viruses can provide insight into circulating viral communities and their relationship with environmental change. The New Zealand cockle (Austrovenus stutchburyi) is culturally, ecologically, and economically important, yet little is known about the viruses it hosts, how these change over time, or whether cockles act as reservoirs for pathogenic viruses affecting bivalves or humans. We used a metatranscriptomic approach to characterise the cockle RNA virome over 12 months and assess whether environmental parameters influenced viral dynamics. Longitudinal sampling revealed a highly diverse RNA virome spanning 16 viral orders within the kingdom Orthornavirae. Phylogenetic analysis of RNA-dependent RNA polymerase sequences identified 358 viral transcripts representing 213 distinct viral species, of which 199 were likely novel, including several putative new virus families and three orders. No viruses known to be pathogenic to humans or bivalves were detected. While total virome composition showed no strong seasonal patterns, environmental parameters influenced virus abundance within specific viral orders, including the highly abundant Picornavirales, Wolframvirales, Bunyavirales, and Sobelivirales. Overall, New Zealand cockles harbour an exceptionally diverse RNA virome, with environmental drivers acting in a taxon-specific rather than community-wide manner.

The functioning of various aquatic ecosystems is greatly influenced by the composition of their microbial communities. However, the prokaryotic and eukaryotic organisms present in the microbiome remain to be characterized in the waters of various tropical islands. Here, we used DNA metabarcoding to assess differences in the richness and abundance of prokaryotic and eukaryotic microbial communities in coastal, mangrove, and urban surface waters in Guadeloupe (French West Indies). We found that turnover was an important driving force in these three compartments, and that the urban compartment was the most diverse. We identified 119 prokaryotic and 80 eukaryotic OTUs with differential abundance between these three compartments. Furthermore, functional predictions revealed the importance of photosynthetic organisms (including Bacillariophyceae, Chrysophyceae, Chlorophyceae, and Cyanobacteria) in the three compartments, and an enrichment of urban waters in chemoheterotrophic prokaryotes and eukaryotic consumers. Interestingly, we detected several putative harmful algal bloom taxa that had not yet been reported in Guadeloupe. By cataloging the taxa restricted to particular water bodies, this inventory will facilitate analyses of the long-term effects of urbanization and industrialization on the evolution of microbial assemblages in Guadeloupe.

Pathogenic bacteria generally resume growth upon removal of bacteriostatic antibiotics. However, it is poorly understood how pre-growth conditions influence: i) the lag time before growth resumption, and ii) induction of antibiotics tolerance upon removal of the antibiotics. To study these traits in Mycobacterium smegmatis, we used a stringent-response-inducing bacteriostatic inhibitor, serine hydroxamate (SHX), which mimics a growth-rate reducing antibiotic. We find that SHX-induced, growth-arrested cells display characteristics typical of persister cells i.e. low ATP levels, increased antibiotic tolerance, and delayed growth-recovery. Single-cell microscopy shows morphological changes in SHX-treated cells (elongation and polar swelling) and growth resumption upon SHX removal after a lag time. During this lag time, the cells displayed increased antibiotic survival, which remained evident after complete recovery from SHX stress. Varying the SHX dose, exposure time, and pre-exposure growth rate indicated that the duration of the lag phase depends on the SHX exposure time and the prior growth rate, not on the SHX dose. The lag phase could be shortened by over two-fold with the addition of amino acids, pyruvate, or spent medium, indicating that growth inhibition induces a relievable metabolic bottleneck. Our findings align with studies with other (evolutionarily distant) microorganisms, suggesting that mycobacteria may be limited by similar phenotypic trade-offs associated with adaptive responses to sudden growth rate reductions.