Bacteria

The increasing prevalence of antimicrobial resistance, especially in methicillin-resistant Staphylococcus aureus (MRSA), underscores the need for alternative therapies from natural sources. This study investigated the chemical composition, antibacterial activity, and gene expression modulation of Melaleuca cajuputi essential oils. Gas chromatography–mass spectrometry (GC-MS) identified 91 compounds, with naphthalene (23.90%), guaiol (12.92%), caryophyllene oxide (9.69%), D-limonene 98% (8.59%), and gamma terpinene (7.54%) among the most abundant. In Silico molecular docking against MRSA virulence proteins revealed that alloaromadendrene had the strongest binding to toxic shock syndrome toxin-1 (TSST-1) (−7.948 kcal/mol), suggesting high inhibitory potential, while cyclohexane showed weak binding with staphylococcal enterotoxin A (SEA) (−3.532 kcal/mol). Antibacterial assays demonstrated concentration-dependent inhibition, with the zones ranging from 6.33 ± 0.33 mm to 16.67 ± 0.88 mm. MIC and MBC values ranged from 1.56 to 12.5% and 3.13 to 25%, respectively, with most isolates showing bactericidal effects (MBC/MIC ≤ 2). Gene expression analysis of MRSA isolate 4 indicated that sea was moderately upregulated (FC = 1.44), while sec remained unchanged (FC = 1.02). In contrast, fnbA (FC = 0.72), seb (FC = 0.33), and mecA (FC = 0.23) genes were downregulated, and the tsst-1 gene (FC = 0.05) was nearly silent. These findings highlight M. cajuputi essential oils as a promising candidate with both antibacterial efficacy and regulatory effects on MRSA virulence genes. Full article

Hot springs represent unique geothermal ecosystems where extreme physicochemical conditions intersect with remarkable microbial diversity and metabolic innovation. These natural laboratories harbor specialized communities of thermophilic and hyperthermophilic microorganisms that have evolved exceptional adaptations to elevated temperatures, extreme pH, and high salinity. This review synthesizes current understanding of hot spring systems as multifunctional natural resources, examining their roles in fundamental microbiology, biotechnology, and sustainable development. We explore the ecological principles governing microbial community assembly, the taxonomic and functional diversity of prokaryotic and eukaryotic microorganisms, and the genomic mechanisms underlying thermophilic adaptation. Hot springs yield enzymes revolutionizing molecular biology and industrial catalysis, bioactive metabolites with pharmaceutical potential, and novel bioremediation capabilities including plastic degradation. Beyond biological significance, these systems contain valuable minerals and rare earth elements, supporting an emerging bioeconomy integrating wellness tourism, bioprospecting, and sustainable resource extraction. However, critical knowledge gaps remain regarding viral ecology, horizontal gene transfer, eukaryotic diversity, and climate change impacts. We emphasize that hot springs merit renewed interdisciplinary attention as model systems for understanding extremophile physiology, early life evolution, and the development of nature-based biotechnological solutions. Realizing their full potential requires balanced management strategies that preserve ecosystem integrity while enabling responsible utilization of these irreplaceable geobiological resources. Full article

Sequencing technologies have reshaped the study of the subgingival microbiome, but selecting the appropriate method remains challenging because of differences in resolution, cost, host DNA contamination, and computational complexity. This review compares 16S rRNA sequencing, full-length 16S, shotgun metagenomics, and metatranscriptomics with respect to taxonomic resolution, functional output, sample requirements, and analytical limitations. Key practical issues, including low microbial biomass, contamination control, and the choice of appropriate bioinformatic tools, are emphasized to help researchers avoid common pitfalls. A decision-making framework is provided to link study goals to suitable sequencing methods while outlining realistic budget and sample-handling constraints. The review concludes with recommendations for integrating sequencing with complementary techniques to improve the accuracy, reproducibility, and clinical relevance of periodontal microbiome studies. Full article

(1) Background: Carbapenem-resistant Escherichia coli (CREC) is widespread and resistant to almost all available antimicrobial agents. In this study, we aimed to assess the phenotypic and molecular characteristics of CREC isolated from retail meats in Hat Yai, Songkhla, Thailand. (2) Methods: A total of 155 retail meat samples were randomly collected, and 412 presumptive carbapenem-non-susceptible isolates were screened via culturing on imipenem-containing eosin methylene blue (EMB) agar. Susceptibility to imipenem and meropenem was tested using the disk diffusion method, and carbapenemase and virulence genes in CREC isolates were detected using PCR. Phylogenetic groups and genetic relatedness of carbapenemase-positive CREC isolates were analyzed using gene markers and BOX-PCR, respectively. (3) Results: The results revealed a high prevalence of presumptive carbapenem-non-susceptible E. coli (CNSEC) isolates in beef samples. Over 89% of the CNSEC isolates from all meat types were identified as CREC. Of these, only 4.8% of the isolates from beef samples were positive for the bla_NDM gene, and one was also positive for the bla_VIM gene. These isolates carried only the fimH gene as a virulence factor. The bla_NDM-positive CREC isolates were classified in phylogenetic Group D. (4) Conclusions: Identifying antimicrobial-resistant pathogens, particularly CREC, in food-producing animals is critical due to potential risks to public health. Full article

Klebsiella pneumoniae is a prominent pathogen implicated in a wide range of infections, including pneumonia, urinary tract infections, and septicemia. Its ability to acquire and disseminate antibiotic resistance, coupled with the rising prevalence of hypervirulent strains, represents a significant public health threat. Understanding the molecular basis of drug resistance can guide the design and development of effective treatment strategies. Antimicrobial resistance (AMR) in these bacteria is a complicated process and cannot be attributed to a single resistance mechanism. K. pneumoniae develops resistance to antibiotics through a variety of mechanisms, ranging from single molecular mechanisms to complex interactions, where molecular synergy exacerbates resistance. This review summarizes the current understanding of the molecular mechanisms that contribute to the drug resistance and virulence of this pathogen. Key antibiotic resistance mechanisms include drug inactivation via B-lactamases and carbapenemases, membrane remodeling, efflux pump systems, such as AcrAB-TolC and OqxAB, and biofilm formation facilitated by quorum sensing. Additionally, the role of ribosomal changes in resistance is highlighted. This review also examines the mechanisms of virulence, emphasizing fimbriae, iron acquisition systems, and immune evasion strategies. Understanding these mechanisms of drug resistance and virulence is crucial for remodeling existing antibiotics and developing new therapeutic strategies. Full article

Background: Multidrug-resistant (MDR) Gram-negative bacilli (GNBs) significantly compromise the effective management of urinary tract infections (UTIs) worldwide. As antimicrobial resistance varies across regions, locally tailored data are essential to guide empirical therapy. This study investigated the prevalence, determinants, and temporal dynamics of MDR GNBs in UTI patients from Central Portugal between 2018 and 2022. Methods: We conducted a retrospective observational study at a hospital center in Central Portugal, analyzing data from 2018 to 2022. Data from 5194 UTI patients with GNB-positive cultures were analyzed. Binary logistic regression was used to identify determinants of MDR GNBs, defined as resistance to ≥1 agent in ≥3 antibiotic classes. Results: The study population had a mean age of 64.5 ± 25.3 years, and females represented two-thirds of the sample (67.0%). The overall prevalence of MDR GNBs was 35.8%. Advanced age (≥75 years), male sex, and specific treatment contexts—particularly day treatment and laboratory-only cases—were independently associated with MDR. SBL-producing Enterobacterales and non-fermenting GNBs showed the highest risk levels. Conclusions: MDR GNBs are highly prevalent among UTI patients in Central Portugal, and their increasing trend—particularly in 2022—highlights an urgent need for strengthened surveillance and updated empirical treatment strategies. The observed temporal increase highlights the urgent need for strengthened regional surveillance and updated empirical treatment guidelines. Full article

The taxonomy of the genus Arcobacter has been subject to substantive turmoil in recent years following a proposal to subdivide the genus into six genera. This proposal has been challenged by a number of multidisciplinary studies employing phenotypic, genomic, and phylogenetic analyses. Following several discussions among members of the International Committee on Systematics of Prokaryotes (ICSP) subcommittee on the taxonomy of Campylobacter and related bacteria, this group now unanimously recommends the use of the genus term Arcobacter to refer to these species. Full article

Streptococcus uberis is a bovine mastitis pathogen with a demonstrated ability to form biofilms. However, the dynamics of this process remain poorly characterized. This study aimed to comprehensively characterize biofilm formation in four S. uberis strains that differed in their biofilm-forming capacity, from weak to strong producers, and in the presence of key virulence-associated genes, such as sua, hasA and hasC. To achieve this, we integrated structural, biochemical, physiological and transcriptional analyses using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), spectral flow cytometry and qRT-PCR. The multi-faceted analysis revealed a coordinated maturation peak at 48 h, characterized by a structured architecture with water channels, a distinct biochemical signature rich in polysaccharides and proteins, and a predominantly viable bacterial population. This peak coincided with a marked upregulation of key virulence-associated genes, with sua expression increasing 2.5-fold and hasA increasing 3-fold at 48 h. This mature biofilm conferred high tolerance to antibiotics, with eradication concentrations (>256 µg/mL) exceeding planktonic MICs, although tetracycline was notably effective. At 72 h, the biofilm entered a dispersion phase characterized by structural collapse and reduced viability. These findings establish S. uberis biofilm maturation as a highly coordinated process, providing new insights into the biofilm lifecycle of this important pathogen and identifying key temporal and molecular targets for future interventions. Full article

Globally, large quantities of animal waste and human sewage sludge are generated annually. Their application as soil amendments can enhance soil quality and support a circular economy. However, these wastes may harbour pathogenic bacteria, posing contamination risks to soil and water and potential transmission to animals and humans. This study investigated the survival of five bacterial pathogens during six months of storage in five types of organic waste and following their subsequent application to soil. During storage, T₉₀ values ranged as follows: Salmonella Typhimurium (2.3–17.7 days), Campylobacter jejuni (0 to 23.9 days), Escherichia coli O157:H7 (4.3 to 57.8 days), and Listeria monocytogenes (1.9 to 170.4 days). In soil, T₉₀ values were S. Typhimurium (4.2 to 17.4 days), C. jejuni (4.8 to 26.8 days), E. coli O157:H7 (4.3 to 52.9 days), and L. monocytogenes (2 to 83.7 days). Clostridium sporogenes remained stable throughout both experiments, preventing T₉₀ calculation. Contrary to our initial hypothesis that soil microbiota would accelerate pathogen decline, T₉₀ values were higher during storage in 11 cases and higher in soil in nine scenarios. These findings highlight the need for pre-treatment strategies for animal waste and biosolids before land spreading to consistently mitigate risks of pathogen transmission and environmental contamination. Full article

Perinatal depression (PND) is a severe mood disorder affecting mothers during pregnancy and postpartum, with implications for both maternal and neonatal health. Emerging evidence suggests that gut microbiota-derived metabolites play a critical role in neuroinflammation and neurotransmission. In this study, we employed an in silico approach to evaluate the pharmacokinetic and therapeutic potential of metabolites produced by Lactobacillus helveticus and Bifidobacterium longum in targeting key proteins implicated in PND, including BDNF, CCL2, TNF, IL17A, IL1B, CXCL8, IL6, IL10. The ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) profiles of selected microbial metabolites, including acetate, lactate, formate, folic acid, riboflavin, kynurenic acid, γ-aminobutyric acid, and vitamin B12 were assessed using computational tools to predict their bioavailability and safety. Enrichment analysis was performed to identify biological pathways and molecular mechanisms modulated by these metabolites, with a focus on neuroinflammation, stress response, and neurogenesis. Additionally, molecular docking studies were conducted to evaluate the binding affinities of these metabolites toward the selected PND-associated targets, providing insights into their potential as neuroactive agents. Our findings suggest that specific microbial metabolites exhibit favorable ADMET properties and strong binding interactions with key proteins implicated in PND pathophysiology. These results highlight the therapeutic potential of gut microbiota-derived metabolites in modulating neuroinflammatory and neuroendocrine pathways, paving the way for novel microbiome-based interventions for perinatal depression. Further experimental validation is warranted to confirm these computational predictions and explore the clinical relevance of these findings. Full article

This study focuses on discovering novel quorum-sensing inhibitors (QSIs) from endophytes of Coelothrix irregularis, aiming to develop new strategies against drug-resistant bacterial infections. From the endophytic bacterial strain Bacillus strain W10-B1, isolated from C. irregularis, twelve compounds were isolated and structurally identified. Subsequent screening against Serratia marcescens NJ01 revealed that compound (12), 3,3′-dibromo-4,4′-biphenyldiol, exhibited significant inhibitory activity against the quorum-sensing system of S. marcescens NJ01. It effectively suppressed biofilm formation, swimming, and swarming motilities of the bacterium. This work is the first to demonstrate that endophytes from C. irregularis are a novel source of potent QSIs, providing both material and theoretical foundations for combating pathogen virulence, drug resistance, and pathogenicity. Full article

Migratory birds have been implicated in the spread of diverse emerging infectious pathogens, including West Nile virus, Usutu virus, Avian influenza viruses, Salmonella, Campylobacter, antimicrobial-resistant (AMR) bacteria, and antibiotic resistance genes (ARGs). Beyond their roles as vectors and reservoirs, migratory birds are also susceptible hosts whose own health may be compromised by these infections, reflecting their dual position in the ecology of pathogens. As facilitators of pathogen transmission during their long-distance migrations, often spanning thousands of kilometres and connecting ecosystems across continents, these birds can easily cross-national borders and circumvent traditional biosecurity measures, thereby acting as primary or secondary vectors in the transmission of cross-species diseases among wildlife, livestock, and humans. Africa occupies a pivotal position in global migratory bird networks, yet comprehensive data on pathogen carriage remain limited. Gaps in knowledge of pathogen diversity constrain current surveillance systems, resulting in insufficient genomic monitoring of pathogen evolution and a weak integration of avian ecology with veterinary and human health. These limitations hinder early detection of novel pathogens and reduce the continent’s preparedness to manage outbreaks. Therefore, this review provides a holistic assessment of these challenges by consolidating existing knowledge concerning the pathogens transmitted by migratory birds in Africa, while recognizing the adverse effect of pathogens, which potentiates population decline, extinction, and ecological imbalance. It further advocates for the adoption of a comprehensive One Health-omics approach that not only strengthens surveillance and technological capacity but also prioritizes the protection of avian health as an integral component of ecosystem and public health. Full article

Extensively drug-resistant (XDR) bacteria pose a serious global public health threat due to their high levels of resistance to multiple classes of antibiotics. This study aimed to characterize bacterial isolates obtained from clinical samples, evaluate their antibiotic resistance patterns, and investigate the antimicrobial and anticancer potential of essential oils (EOs) and their nanoemulsions (NEs). A total of 175 bacterial isolates were collected from various clinical sources, identified, and subjected to antibiotic susceptibility testing using both conventional methods and the VITEK^® 2 system. Among these, nine isolates were identified as extensively drug-resistant. Among the tested EOs, carvacrol exhibited the strongest antibacterial activity, with minimum inhibitory concentrations (MICs) ranging from 14 to 35 µg/mL, compared to 8 to 19 µg/mL for meropenem. To enhance its stability and efficacy, carvacrol nanoemulsions (CANE) were prepared via ultrasonication and characterized using zeta potential measurements, which indicated a positive surface charge of +14.2 mV, while dynamic light scattering (DLS) analysis revealed a narrow size distribution with a mean hydrodynamic diameter of 411.3 nm. High-resolution transmission electron microscopy (HR-TEM) showed spherical droplets ranging from 18 to 144 nm in size, with an average diameter of 69 ± 28 nm. The nanoemulsion formulation significantly enhanced antibacterial activity, with MICs reduced to 11 ± 0.0–23 ± 0.21 µg/mL, compared to 14 ± 0.13–35 ± 0.11 µg/mL for pure carvacrol oil. Gas chromatography–mass spectrometry (GC–MS) analysis identified major active constituents, including thymol, methoxyphenyl, estragole, and D-limonene, which are likely contributors to the observed antimicrobial and anticancer effects. In addition, carvacrol nanoemulsions demonstrated potent cytotoxicity against multiple human cancer cell lines (HepG2, MCF-7, PC-3, and Caco-2) while showing minimal toxicity toward normal cells. Confocal microscopy further confirmed apoptosis induction in treated cancer cells, suggesting a mitochondria-mediated apoptotic pathway. In conclusion, this study highlights the strong therapeutic potential of essential oils—particularly carvacrol and its nanoemulsion formulation—as dual-action agents exhibiting broad-spectrum antibacterial activity against XDR pathogens and selective cytotoxicity against cancer cells. Full article

Extracellular vesicles, encompassing eukaryotic exosomes and bacterial outer membrane vesicles (OMVs), play multifaceted roles in mediating host–pathogen interactions. These nanoscale structures act as critical mediators of intercellular communication, transporting diverse bioactive cargo such as miRNAs, cytokines, proteins, and bacterial components. Exosomes contribute to host immunity by delivering antimicrobial agents and modulating inflammatory responses, but they can also be hijacked by pathogens to suppress defenses and promote persistent infection. OMVs, on the other hand, enable bacteria to disseminate virulence factors, deliver toxins directly into host cells, and modulate immune signaling. For example, exosomes from infected macrophages can stimulate dendritic cell activation and T-cell priming, whereas bacterial OMVs have been shown to suppress host immunity or trigger excessive inflammation depending on their molecular cargo. Importantly, OMVs facilitate horizontal gene transfer and nutrient exchange within microbial communities, thereby influencing microbiome composition and adaptation. Together, these complex dynamics position both exosomes and OMVs as central players in immunity and pathogenesis. This review synthesizes recent insights into how host- and pathogen-derived vesicles modulate infection biology and immune responses, while also exploring their potential as diagnostic biomarkers and therapeutic carriers, and discussing current limitations in their clinical translation. Full article

The aim of this paper is to provide an overview of the main trends and progress in the biostimulation approach, which represents a crucial component of the broader multi-factor bioremediation process. A comprehensive search was carried out in the Scopus database. The stimulating roles of individual and complex nutrient amendments are reviewed, with particular emphasis on plant extracts, molasses, and surfactants. Methodological approaches for optimising nutrient formulations and conditions to strengthen the biostimulation effect are analysed, taking into account microbial ecology and physiology. Aspects of interspecies microbial interactions, such as cross-feeding connections, are discussed. The roles of directed evolution, starvation, and statistical optimisation in enhancing microbial activity are also highlighted. Overall, substantial theoretical knowledge on this topic has been accumulated in the scientific literature. However, data from long-term field studies remain scarce. Looking forward, modern methodological approaches may bridge these knowledge gaps by enabling the prediction of microbial activity, interactions, and cross-feeding, supported by comprehensive monitoring. In particular, artificial intelligence tools for the statistical optimisation of biostimulation conditions are expected to significantly improve process performance. This review summarises recent scientific papers alongside findings from our own long-term studies. Full article

Toxin–antitoxin (TA) modules represent sophisticated regulatory networks that have evolved from simple plasmid maintenance factors into multifunctional genetic modules orchestrating bacterial stress responses, pathogenesis, and ecological adaptation. This review highlights a compelling correlation between the abundance of toxin–antitoxin (TA) modules and bacterial pathogenicity, as exemplified by Mycobacterium tuberculosis (M.tb), which encodes 118 TA loci—significantly more than the fewer than 10 found in closely related saprophytic species. The clinical significance of TA modules extends beyond traditional stress response roles to encompass antimicrobial persistence, where systems like VapBC and MazEF facilitate dormant subpopulations that survive antibiotic therapy while maintaining chronic infections. Recent discoveries have revealed TA modules as sophisticated bacterial defense mechanisms against bacteriophage infection, with DarTG and ToxIN systems representing novel antiviral immunity components that complement CRISPR-Cas and restriction–modification systems. The immunomodulatory capacity of TA modules demonstrates their role in host–pathogen interactions, where systems such as VapC12 in M.tb promote macrophage polarization toward permissive M2 phenotypes while inducing anti-inflammatory cytokine production. Large-scale genomic analyses reveal that TA modules function as drivers of horizontal gene transfer networks, with their signatures enabling accurate prediction of plasmid community membership and serving as determinants of microbial community structure. The biotechnological applications of TA modules have expanded to include genetic circuit stabilization, biocontainment device construction, and multi-species microbial community engineering, while therapeutic strategies focus on developing multi-target inhibitors against conserved TA protein families as promising approaches for combating drug-resistant bacterial infections. The evolutionary conservation of TA modules across diverse bacterial lineages underscores their fundamental importance as central organizing principles in bacterial adaptation strategies, where their multifunctional nature reflects complex selective pressures operating across environmental niches and host-associated ecosystems. This review provides an integrated perspective on TA modules as dynamic regulatory elements that support bacterial persistence, immune evasion, and ecological versatility, establishing them as genetic elements with truly “many faces and functions” in prokaryotic biology. Full article

Escherichia coli LS5218 is an attractive host for producing polyhydroxybutyrate. The strain, however, strongly requires heterologous gene expressions like phaC for efficient production. For enhancing the production, the whole gene expressions relating to end product-producing flow should be optimized so that not only heterologous induced-genes but also other relating genes are comprehensively analyzed on the transcription levels, resulting in normally time-consuming mutant-creation. Additionally, the explanation for each transcriptional relationship is likely to follow the relationships on known metabolic pathway map to limit the consideration. This study aimed to infer gene regulatory networks within glycolysis, a central metabolic pathway in LS5218, using machine learning-based causal discovery methods. To construct a directed acyclic graph representing the gene regulatory network, we employed the NOTEARS algorithm (Non-combinatorial Optimization via Trace Exponential and Augmented lagRangian for Structure learning). Using transcription data of 264 time-resolved sampling points, we inferred the gene regulatory network and identified several distal regulatory relationships. Notably, gapA, a key enzyme controlling the transition between the preparatory and rewarding phases in glycolysis, was found to influence pgi, the enzyme at the pathway’s entry point. These findings suggest that inferring such nonlocal regulatory interactions can provide valuable insights for guiding genetic engineering strategies. Full article

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by motor symptoms like tremor, rigidity, and bradykinesia. The WHO estimates that 10 million people currently have PD, with its prevalence expected to double to 20 million by 2050. Key risk factors include age, male sex, environmental contaminants, and family history. Emerging evidence links gut microbiota dysbiosis to PD, suggesting it contributes to neuroinflammation and disease progression, though the role of dietary interventions remains unclear. This study used computational simulations with genome-scale metabolic models (GEMs) to analyze how diet impacts the gut microbiota in PD patients. Fecal microbiota from PD patients and healthy controls were compared across three diets: high-fiber, Mediterranean, and vegan. Simulations revealed increased pro-inflammatory bacteria (e.g., Escherichia coli O157) in PD patients, likely due to reduced bacterial competition, alongside the decreased production of beneficial metabolites like butyrate, phenylalanine, and cysteine. The Mediterranean diet showed higher short-chain fatty acid production, potentially benefiting PD patients. These findings underscore the importance of dietary interventions in modulating the gut microbiome and suggest that targeted diets may complement PD therapies, improving patient outcomes. Full article

This report investigates the interaction between the metabolites of the highly virulent bacteria Salmonella enterica and RTGill-W1 cells, a cell line derived from rainbow trout gills. As a facultative intracellular pathogen, Salmonella enterica infects both animals and humans through many routes. Upon entering an organism it can cause severe infection and pathology, which is also influenced by the bacterial metabolites. Although no intracellular presence of the pathogen in the exposed cell line could be detected, a dose-dependent effect of the metabolites on the cell line was observed, as exposure to 5%, 10%, and 20% concentrations led to enhanced metabolic activity and increased cytoplasmic neutral lipid droplets accumulation, whereas the lower dosage of 2.5% induced a lower metabolic rate compared to control and no significant intracellular lipid accumulation. The combination of all of the metabolites might be speculated to have increased the metabolic rate and lipid droplet production at higher concentrations due to possessing a growth factor or an endocrine effect, or as a response to a toxin. This paper may be the first report investigating the effect of a complete bacterial metabolite mixture in cultured cells. Full article