Multidrug-resistant bacteria in biofilms are a growing public health threat, due to their resistance to conventional antibiotics. Phytochemicals are attractive candidates because of their structural diversity and ability to potentiate antimicrobial activity. This study investigated the antibiofilm and resistance-modifying effects of two monoterpenes, menthol and linalool, alone and in combination with ten antibiotics, against Escherichia coli and Staphylococcus epidermidis. Menthol exhibited MIC and MBC of 800 µg/mL against E. coli and the same MIC against S. epidermidis, while linalool showed MICs of 800 µg/mL and 400 µg/mL, respectively. Combination assays revealed enhanced activity of erythromycin with both monoterpenes against E. coli and of amoxicillin with menthol against S. epidermidis, although sessile cells were largely unaffected. When applied individually, both monoterpenes caused a 3-log reduction in culturable E. coli biofilm cells. The overall findings highlight the antibiofilm activity of linalool and, particularly, menthol, supporting their role as antibiotic adjuvants against biofilm-associated infections.

Delftia acidovorans, a Gram-negative bacterium commonly found in diverse environments, can occasionally cause infections in immunocompromised individuals. Despite its environmental prevalence and clinical relevance, there is a notable lack of studies on the cellular ultrastructure of D. acidovorans. Characterizing this aspect is essential for understanding the bacterium aggregation behavior, which significantly influences biofilm formation, environmental adaptability, and potential pathogenicity in clinical contexts. This study employs cryo-electron tomography to investigate the cellular ultrastructure of Delftia acidovorans. Our observations of D. acidovorans revealed a supercoiling pattern in flagellar filaments and diverse outer membrane projections. Our major finding was the observation of cytoplasmic membrane invaginations resembling mesosomes seen in Gram-positive bacteria, offering new insights into the cellular architecture and potential functions of these structures in Gram-negative bacteria. Together, these ultrastructural insights reveal adaptations potentially linked to environmental persistence and interspecies aggregation.

This chapter summarizes the current knowledge on the practical, methodological, and interpretative aspects of applying metaproteomics in water biotechnology. We outline the full metaproteomic workflow-from sampling and protein extraction to LC-MS/MS acquisition, database construction, quantitative analysis, and bioinformatic interpretation-and emphasize critical considerations specific to complex matrices such as EPS-rich biofilms, granular sludge, and low-biomass drinking water. Case studies illustrate how metaproteomics can clarify mechanisms of micropollutant degradation, nitrogen-transforming pathways, biofilm functional architecture, and microbial resilience under operational stress. Recent advances in data-independent acquisition, metagenome-informed databases, and integrative multi-omics are shown to substantially improve depth, reproducibility, and functional resolution. Finally, we discuss emerging applications in wastewater-based epidemiology, where metaproteomics complement nucleic-acid-based surveillance by enabling the detection of large biomolecule biomarkers of population health and industrial activity. Although metaproteomics is already being applied across a wide range of water cycle contexts and is producing promising, robust results, several challenges, including limitations in analytical chemistry, database completeness, and bioinformatics workflows, continue to hinder its broader implementation. Continued technical research and innovation are therefore essential to fully unlock its potential in water biotechnology.

Bacterial coaggregation is the specific recognition and adhesion of genetically distinct bacteria, and it is central to multispecies biofilm formation. Here, we present a protocol integrating visual, turbidimetry, epifluorescence microscopy, and thermodynamic analysis to study coaggregation. We describe procedures for performing coaggregation assays and analyzing complementary data from different assays. This integrated workflow provides a robust and multidimensional framework for investigating bacterial coaggregation in different contexts. For complete details on the use and execution of this protocol, please refer to Afonso et al.1,2.

Drinking water (DW) is paramount to human health, serving as a cornerstone of public health worldwide. However, DW is not a sterile product and can harbor a large diversity of microorganisms, including pathogens. This comprehensive review addresses the critical importance of DW for human health and the ongoing challenges posed by microbial pathogens and biofilms in water distribution systems. It further analyzes the growing challenges driven by contemporary factors such as antimicrobial resistance, biofilms, climate change, and micro-pollutants. Biofilms, in particular, remain underestimated in conventional water treatment processes despite their significant contribution to microbial contamination, the formation of disinfection by-products, and disinfectant resistance. Emerging challenges, such as climate change and micropollutants, have become significant concerns due to their profound impact on microbial communities and their role in shaping biofilm formation. This review highlights the key microbiological threats to DW distribution systems, focusing on the role of biofilms and the emerging challenges posed by climate change. It explores the factors promoting biofilm development, including water composition, pipe materials, and treatment strategies. It also explores the limitations of current water treatment strategies, which often fail to address biofilms effectively, and highlights the need to integrate microbiological considerations into water quality management. This review aims to underscore the urgent need to reassess water treatment and management practices to address current microbiological challenges and ensure the delivery of safe and sustainable DW.

Acinetobacter calcoaceticus, a ubiquitous Gram-negative bacterium, exhibits remarkable adaptability across diverse environments, including drinking water distribution systems (DWDS), where its biofilm-forming and coaggregation capabilities pose significant public health challenges. This study integrates physicochemical, structural, and proteomic analyses to elucidate the mechanisms underlying A. calcoaceticus aggregation and biofilm dynamics. Surface characterization through contact angle measurements, zeta potential, and co-adhesion energy assessments revealed a predominantly hydrophilic surface with strong electron donor properties and a highly negative charge, promoting intercellular adhesion. Transmission electron microscopy unveiled dense cellular aggregates with extracellular filamentous structures, indicative of enhanced cell-to-cell interactions and potential extracellular polymeric substance involvement. Proteomic profiling identified 2593 differentially expressed proteins between aggregation stages, highlighting metabolic shifts, stress response activation, and upregulation of biofilm-associated proteins, including chaperones and quorum-sensing regulators. Our multidisciplinary approach emphasizes the importance of surface characterization in understanding bacterial community and underscores the critical role of physicochemical properties and proteomic flexibility in A. calcoaceticus biofilm and aggregation ability.

AIM:Coaggregation, a highly specific cell-cell interaction mechanism, plays a pivotal role in multispecies biofilm formation. While it has been mostly studied in oral environments, its occurrence in aquatic systems is also acknowledged. Considering biofilm formation's economic and health-related implications in engineered water systems, it is crucial to understand its mechanisms. Here, we hypothesized that traceable differences at the proteome level might determine coaggregation ability. METHODS AND RESULTS:Two strains of Delftia acidovorans, isolated from drinking water were studied. First, in vitro motility assays indicated more swarming and twitching motility for the coaggregating strain (C+) than non-coaggregating strain (C-). By transmission electronic microscopy, we confirmed the presence of flagella for both strains. By proteomics, we detected a significantly higher expression of type IV pilus twitching motility proteins in C+, in line with the motility assays. Moreover, flagellum ring proteins were more abundant in C+, while those involved in the formation of the flagellar hook (FlE and FilG) were only detected in C-. All the results combined suggested structural and conformational differences between stains in their cell appendages. CONCLUSION:This study presents an alternative approach for identifying protein biomarkers to detect coaggregation abilities in uncharacterized strains.

Bacterial coaggregation is a critical process in multispecies biofilm formation, driven by specific molecular interactions that facilitate the adhesion and aggregation of bacterial cells. These interactions are essential for the development and persistence of complex microbial communities. This review provides a comprehensive analysis of the roles of the proteosurfaceome and exoproteome in bacterial coaggregation. The proteosurfaceome, comprising surface-bound molecules such as adhesins, drives species-specific interactions crucial for partner recognition and adhesion. In parallel, the exoproteome, particularly extracellular polymeric substances (EPS), enhances aggregate stability by reinforcing structural integrity and facilitating intercellular communication, although its direct role in coaggregation remains to be fully clarified. By integrating these perspectives, this review aims to elucidate how the proteosurfaceome and exoproteome influence bacterial coaggregation, offering insights into their combined impact on microbial community structure and function. Furthermore, we highlight existing knowledge gaps and propose directions for future research.

Physical exercise has positive effects on clinical outcomes of breast cancer survivors such as quality of life, fatigue, anxiety, depression, body mass index, and physical fitness. We aimed to study its impact on immune, inflammatory, cardiometabolic, and fatty acids (FA) biomarkers. An exploratory sub-analysis of the MAMA_MOVE Gaia After Treatment trial (NCT04024280, registered July 18, 2019) was performed. Blood sample collections occurred during the control phase and at eight weeks of the intervention phase. Samples were subjected to complete leukocyte counts, cytokine, and cardiometabolic marker evaluation using flow cytometry, enzyme-linked immunoassays, and gas chromatography. Ninety-three percent of the 15 participants had body mass index ≥ 25 kg/m2. We observed a decrease of the plasmatic saturated FA C20:0 [median difference − 0.08

Biofilms in drinking water (DW) systems persistently challenge traditional disinfection methods due to intricate microbial interactions, with coaggregation playing a crucial role in forming multispecies biofilms. This study examined the implications of coaggregation on tolerance towards sodium hypochlorite (NaOCl) disinfection. Dual-species biofilms were formed for seven days on polyvinyl chloride coupons, comprising a strain of the emerging pathogen Stenotrophomonas maltophilia and the coaggregating strain Delftia acidovorans 005 P. For comparison, dual-species biofilms were also formed with a non-coaggregation strain (D. acidovorans 009 P). The minimum bactericidal concentration (MBC) for each planktonic strain varied (D. acidovorans: 1 mg/L, S. maltophilia: 1.5 mg/L) below the safe DW treatment limits. However, high NaOCl doses (10 xMBC and 100 xMBC,) showed low efficacy against dual-species biofilms, indicating significant biofilm tolerance to disinfection. Membrane damage occurred at sub-MBC without culturability loss, underscoring biofilm resilience. The biofilm analysis revealed a complex interplay between the composition of extracellular polymeric substances and the architecture, which was influenced by the presence of the coaggregating strain. Overall, coaggregation significantly influenced biofilm formation and resilience, impacting NaOCl disinfection. These findings underscore the challenges of microbial interactions in biofilms, emphasizing the need for improved disinfection strategies to control biofilms in drinking water systems.

Bacterial coaggregation is a highly specific type of cell-cell interaction, well-documented among oral bacteria, and involves specific characteristics of the cell surface of the coaggregating strains. However, the understanding of the mechanisms promoting coaggregation in aquatic systems remains limited. This gap is critical to address, given the broad implications of coaggregation for multispecies biofilm formation, water quality, the performance of engineered systems, and diverse biotechnological applications. Therefore, this study aims to comprehensively characterize the cell surface of the coaggregating strain Delftia acidovorans 005P, isolated from drinking water, alongside a non-coaggregating strain, D. acidovorans 009P. By analyzing two strains of the same species, we aim to identify the factors contributing to the coaggregation ability of strain 005P. To achieve this, we employed a combination of physicochemical characterization, Fourier-transform infrared spectroscopy (FTIR), and advancing imaging techniques [transmission electron microscopy and cryo-electron tomography (cryo-ET)]. The coaggregating strain (005P) exhibited higher surface hydrophobicity, negative surface charge, and cell surface and co-adhesion energies than the non-coaggregating strain (009P). The chemical characterization of bacterial surfaces through FTIR revealed subtle differences, particularly in spectral regions linked to carbohydrates and phosphodiesters/amide III of proteins (860-930 cm-- 1 and 1212-1240 cm-- 1 , respectively). Cryo-ET highlighted significant differences in pili structures between the strains, such as variations in length, frequency, and arrangement. The pili in the 005P strain, identified as pili-like adhesins, serve as key mediators of coaggregation. By integrating physicochemical analyses and high-resolution imaging techniques, this study conclusively links the coaggregation ability of D. acidovorans 005P to its unique pili characteristics, emphasizing their crucial role in microbial coaggregation in aquatic environments.

Cognitive impairment and anxiety are common side effects of chemotherapy, particularly with the use of doxorubicin (DOX), known as “chemobrain”. This study aimed to examine the dose-dependent effects of DOX on cognitive decline, anxiety, and locomotor activity in healthy female Wistar rats. The rats were divided into groups receiving low (2 mg/kg), intermediate (4 mg/kg), and high (5 mg/kg) doses of DOX for four weeks, alongside a control group. Behavioral tests, including open field, elevated plus maze, and Y-maze tests, assessed anxiety, locomotion, and cognitive performance, while brain tissue analysis evaluated neuroinflammation using markers such as GFAP and Iba-1. The results showed that all doses of DOX induced anxiety-like behavior, reduced locomotion, and caused neuroinflammation in the hippocampus, with more severe effects at higher doses. Notably, high-dose DOX also caused short-term memory deficits. These findings highlight the dose-dependent nature of DOX’s impact on behavior and cognition, suggesting that DOX plays a key role in the development of cognitive symptoms during chemotherapy. Further research is needed to understand the mechanisms behind these effects and to explore potential interventions.

Coaggregation plays an important role in the development of multispecies biofilms in different environments, often serving as an active bridge between biofilm members and other organisms that, in their absence, would not integrate the sessile structure. The ability of bacteria to coaggregate has been reported for a limited number of species and strains. In this study, 38 bacterial strains isolated from drinking water (DW) were investigated for their ability to coaggregate, in a total of 115 pairs of combinations. Among these isolates, only Delftia acidovorans (strain 005P) showed coaggregating ability. Coaggregation inhibition studies have shown that the interactions mediating D. acidovorans 005P coaggregation were both polysaccharide-protein and protein-protein, depending on the interacting partner bacteria. Dual-species biofilms of D. acidovorans 005P and other DW bacteria were developed to understand the role of coaggregation on biofilm formation. Biofilm formation by Citrobacter freundii and Pseudomonas putida strains highly benefited from the presence of D. acidovorans 005P, apparently due to the production of extracellular molecules/public goods favouring microbial cooperation. This was the first time that the coaggregation capacity of D. acidovorans was demonstrated, highlighting its role in providing a metabolic opportunity for partner bacteria.

DNA double-strand breaks (DSBs) trigger specialized cellular mechanisms that collectively form the DNA damage response (DDR). In proliferating cells, the DDR serves the function of mending DNA breaks and satisfying the cell-cycle checkpoints. Distinct goals exist in differentiated cells that are postmitotic and do not face cell-cycle checkpoints. Nonetheless, the distinctive requirements and mechanistic details of the DDR in differentiated cells are still poorly understood. In this study, we set an in vitro differentiation model of human skeletal muscle myoblasts into multinucleated myotubes that allowed monitoring DDR dynamics during cell differentiation. Our results demonstrate that myotubes have a prolonged DDR, which is nonetheless competent to repair DSBs and render them significantly more resistant to cell death than their progenitors. Using live-cell microscopy and single-molecule kinetic measurements of transcriptional activity, we observed that myotubes respond to DNA damage by rapidly and transiently suppressing global gene expression and rewiring the epigenetic landscape of the damaged nucleus. Our findings provide novel insights into the DDR dynamics during cellular differentiation and shed light on the strategy employed by human skeletal muscle to preserve the integrity of the genetic information and sustain long-term organ function after DNA damage.

The treatment of bacterial infections has been troubled by the increased resistance to antibiotics, instigating the search for new antimicrobial therapies. Phytochemicals have demonstrated broad-spectrum and effective antibacterial effects as well as antibiotic resistance-modifying activity. In this study, perillyl alcohol and hydrocinnamic acid were characterized for their antimicrobial action against Escherichia coli. Furthermore, dual and triple combinations of these molecules with the antibiotics chloramphenicol and amoxicillin were investigated for the first time. Perillyl alcohol had a minimum inhibitory concentration (MIC) of 256 µg/mL and a minimum bactericidal concentration (MBC) of 512 µg/mL. Hydrocinnamic acid had a MIC of 2048 µg/mL and an MBC > 2048 µg/mL. Checkerboard and time-kill assays demonstrated synergism or additive effects for the dual combinations chloramphenicol/perillyl alcohol, chloramphenicol/hydrocinnamic acid, and amoxicillin/hydrocinnamic acid at low concentrations of both molecules. Combenefit analysis showed synergism for various concentrations of amoxicillin with each phytochemical. Combinations of chloramphenicol with perillyl alcohol and hydrocinnamic acid revealed synergism mainly at low concentrations of antibiotics (up to 2 μg/mL of chloramphenicol with perillyl alcohol; 0.5 μg/mL of chloramphenicol with hydrocinnamic acid). The results highlight the potential of combinatorial therapies for microbial growth control, where phytochemicals can play an important role as potentiators or resistance-modifying agents.

Aims: The use of phytochemicals to improve the effectiveness of antibiotics is a promising strategy for the development of novel antimicrobials. In this study, the antibiofilm activity of perillyl alcohol and hydrocinnamic acid, both phytochemicals present in several plants, and two antibiotics from different classes (amoxicillin and chloramphenicol) was tested, alone and in combination, against Escherichia coli. Methods and results: Each molecule was tested at the minimum inhibitory concentration (MIC), 5 x MIC, and 10 x MIC, and characterized concerning biomass removal, metabolic inactivation, and cellular culturability. The highest percentages of metabolic inactivation (88.5% for 10 x MIC) and biomass reduction (61.7% for 10 x MIC) were obtained with amoxicillin. Interestingly, for 5 x MIC and 10 x MIC, phytochemicals provided a total reduction of colony-forming units (CFUs). Dual and triple combinations of phytochemicals and antibiotics (at MIC and 5 x MIC) demonstrated high efficacy in metabolic inactivation, moderate efficacy in terms of biomass reduction, and total reduction of cellular culturability for 5 x MIC. Conclusions: The results demonstrated the antibiofilm potential of phytochemicals, highlighting the advantage of phytochemical/antibiotic combinations for biofilm control.

As in human medicine, in veterinary medicine, chronic wounds are often related to polymicrobial infections and the presence of a biofilm, which compromises the effectiveness of therapeutic approaches. In this study, a Lusitano mare presented a 21-day-old chronic wound that was only being treated with an antiseptic. A swab sample was collected, and three isolates of Staphylococcus aureus and one of Pseudomonas aeruginosa were isolated. S. aureus did not show resistance to a panel of antibiotics. However, the P. aeruginosa isolate showed a resistance profile to carbapenems and fluoroquinolones, which may suggest a cross-resistance between antiseptic and antibiotics, given that no antibiotic therapy was applied to the wound or the mare in the previous year. Further experiments were conducted to assess the ability of the isolates to form biofilms, and to ascertain their susceptibility to gentamicin. The results demonstrated that the isolates produced biofilms. Gentamicin at the minimum inhibitory concentration (MIC) and 10× MIC caused biofilm removal between 59.3% and 85.7%, with the highest removal percentage being obtained for the P. aeruginosa isolate (at 10× MIC concentration). This study reveals that an equine wound was colonized by antibiotic resistant bacteria, and that all the wound colonizers could form biofilms, demonstrating the relevance of an adequate diagnosis and treatment when there is a suspicion of a biofilm-infected wound. It also highlights the possibility of resistance transmission between animals, animals and humans, or animals and the environment.

Although bacterial coaggregation has already been identified as a key mechanism in the multispecies biofilms development in diverse environments, little is known about this highly specific type of cell-cell interaction in aquatic systems. In this study, a strain of Delftia acidovorans isolated from drinking water (DW) and previously described with coaggregation ability, was studied for its role in biofilm development. Single and dual-species biofilms, of D. acidovorans and the DW representative bacteria Citrobacter freundii and Pseudomonas putida, were grown in 96-well microtiter plates and characterized in terms of metabolic activity. In addition, to prove that D. acidovorans can facilitate the metabolic activity of other bacteria, single biofilms were formed in the presence of D. acidovorans cell-free supernatant (CFS). The metabolic activity was higher when comparing single with dual-species biofilms. Furthermore, in the presence of CFS, it was evident an increase in the metabolic activity of C. freundii and P. putida biofilms. These results suggest the production of extracellular metabolites by D. acidovorans, that possibly act as public goods. That said, the presence of coaggregating species, namely D. acidovorans, in biofilms appears to create a functional cooperative microbial community, providing a favorable metabolic opportunity for partner bacteria.

Doxorubicin (DOX) is commonly used in several chemotherapies to treat various cancers, but it is known to cause cardiotoxicity and cardiac symptoms. Autonomic dysfunction is thought to contribute to the cardiotoxic effects of DOX, but the specific dose required to disrupt homeostatic processes is still unclear and is influenced by numerous factors. This study aimed to investigate how the DOX dosage affects autonomic function and physiological parameters, to elucidate the neurocardiac mechanisms underlying the observed cardiovascular side effects. Wistar rats were treated with DOX for four weeks and divided into three dosing groups: DOX8 (2 mg/kg/week), DOX16 (4 mg/kg/week), and DOX20 (5 mg/kg/week). A control group received NaCl 0.9% saline (1 mL/kg/week). In an acute experiment, we recorded blood pressure (BP), electrocardiogram, heart rate (HR), and respiratory rate (RF). Baroreflex gain and chemoreflex sensitivity were calculated, and cardiac tissue was analyzed with picrosirius histochemistry to measure collagen content. Our results showed that the LF/HF ratio, indicative of autonomic activity, was altered along with hypotension and bradycardia at a cumulative DOX dose threshold of 16 mg/kg. We observed a positive correlation between DOX dose and BP, HR, urinary norepinephrine, LF/HF ratio, and fibrotic heart area. Lower LF/HF ratios were associated with high DOX doses, reflecting drug-induced impairment of autonomic control of HR. This study provides valuable insights into the dose-dependent effects of DOX on physiological parameters and the development of cardiovascular dysfunction. These findings are critical, which is important for optimizing the management and therapeutic strategies for patients undergoing DOX-based chemotherapy.