Osteomyelitis is a deep bone tissue infection caused by pathogenic microorganisms, with the primary pathogen being methicillin-resistant Staphylococcus aureus (MRSA). Due to the tendency of the infection site to form biofilms that shield drugs and immune cells to kill bacteria, combined with the severe local inflammatory response causing bone tissue destruction, the treatment of osteomyelitis poses a significant challenge. Herein, we developed a remotely controlled nanotherapeutic (TLBA) with immunomodulatory to treat MRSA-induced osteomyelitis. TLBA, combined with baicalin and gold nanorods, is positively charged to actively target and penetrate biofilms. Near-infrared light (808 nm) triggers spatiotemporal, controllable drug release, while bacteria are eliminated through synergistic interaction of non-antibiotic drugs and photothermal therapy, enhancing bactericidal efficiency and minimizing drug resistance. TLBA eliminated nearly 100 % of planktonic bacteria and dispersed 90 % of biofilms under NIR light stimulation. In MRSA-induced osteomyelitis rat models, laser irradiation raised the infection site temperature to 50 °C, effectively eradicating bacteria, promoting M2 macrophage transformation, inhibiting bone inflammation, curbing bone destruction, and fostering bone tissue repair. In summary, TLBA proposes a more comprehensive treatment strategy for the two characteristic pathological changes of bacterial infection and bone tissue damage in osteomyelitis.

Encapsulated in a self-produced negatively charged extracellular polymeric substance (EPS) matrix, the wound infected bacterial biofilms exhibit formidable resistance to conventional positively charged antibiotics and host's immune responses, which can undoubtedly lead to persistent infections and lethal complications. Nevertheless, developing efficacious strategies to root out stubborn biofilm and promote tissue regeneration still remains a challenge. To resolve this dilemma, a versatile vacancies-rich Z-scheme MoSSe Van der Waals heterojunction (MoSSe VdW HJ) is rationally fabricated as nanoplatform for hydrogen sulfide (H2S)-sensitized synergistic therapy of wound bacterial biofilm infection. The rich anion vacancies and Z-scheme heterostructure make the fabricated MoSSe VdW HJ can effectively augment H2S, localized hyperthermia, and reactive oxygen species production under the stimulation of biofilm microenvironments (BME) and irradiation of 808 nm near-infrared (NIR) light. Therefore, MoSSe VdW HJ is capable to integrate H2S gas, chemodynamic, photothermal, and photodynamic therapies to effectively destroy eDNA and polysaccharides in the EPS matrix, thereby breaching the biofilm barrier to eradicate bacteria and facilitate wound healing. The synergistic strategy exhibits superior anti-biofilm and wound repair effects both in vivo and in vitro, thus providing guideline for the development of BME and NIR light activated synergistic therapeutics to fight against refractory biofilm infections.

Biofilm infections represent the greatest risk associated with medical devices and implants, constituting 65 %-70 % of all device associated infections. Efforts to develop antimicrobial technologies for biomedical applications aim to reduce infection rates, antibiotic use, and the induction of antimicrobial resistance. However, standard laboratory test conditions often overestimate efficacy, highlighting the need for experimental designs that simulate real-world settings. To this end, we evaluated four commercially available antimicrobial materials containing silver (AG1, AG2, AG3) or zinc (ZN1) to assess their ability to mitigate microbial proliferation in for longer duration or multi-use medical devices. The materials' homogeneity and surface topography were characterized through Scanning Electron Microscopy (SEM) coupled with Energy Dispersive Spectroscopy (EDS) and Atomic Force Microscopy (AFM). Antimicrobial efficacy was tested using a modified ISO 22196 protocol under clinically relevant conditions and a dry contact test developed to mimic in-use conditions for many extracorporeal medical devices. Results revealed homogeneous elemental distributions in AG1, AG2, and ZN1, and heterogeneous clusters for AG3. Surface roughness was highest for AG2 (170.1 nm), followed by TPE control (155.3 nm), ZN1 (83.51 nm) and silicone control (66.74 nm). All test materials demonstrated antimicrobial efficacies against S. aureus and E. coli, but not against C. albicans. In the dry contact assay, only AG2 proved effective against E. coli, and P. aeruginosa, underlining the role of humidity in antimicrobial action. Results were further corroborated by measurement of ion release by the materials at various temperatures, revealing greater release at higher temperatures. These outcomes emphasize the importance of testing antimicrobial materials under in use conditions to minimize discrepancies between laboratory results and clinical outcomes. Our findings provide a valuable framework for testing and integrating these materials into next-generation multi-use medical devices.

We prepared a series of enones containing different substituents as potential antibiofilm molecules. The design considered the structural features previously found in N-acylhomoserine lactones, but it replaced the labile furanone with different imides portions. After evaluation, some of the analogs inhibited 50 % or more the formation of the biofilm from P. aeruginosa or A. baumannii; moreover, substituents attached at the phenyl ring, the size of the enone as well as the type of imide seemed relevant for the selectivity against the tested pathogens. In the end, we performed a molecular docking study using the crystallized LasR to describe the main interactions of the ligand-receptor complex and propose a plausible mechanism of action.

Vernal keratoconjunctivitis (VKC) is a chronic allergic ocular surface disease with seasonal recurrences and severe forms showing vision threatening complications. The purpose of the study is to understand the prevalence and diversity of biofilm-forming bacteria and antimicrobial resistance in VKC compared to healthy individuals (HC). For this, conjunctival swab samples were collected from VKC (n = 26) and HC (n = 23), of which culture positive samples were 77 % and 78.26 % respectively. The 16S rRNA gene sequencing revealed a significant increase in bacterial diversity in VKC compared to HC (p < 0.05), identifying 16 and 9 bacterial species, respectively. Staphylococcus epidermidis emerged as the predominant bacterium in both groups, with relative abundances of 52.8 % in HC and 30.2 % in VKC (p < 0.001). Biofilm formation was observed in 64.15 % of bacterial species in VKC and 31 % in HC (p < 0.001). Scanning electron microscopy analysis confirmed temporal biofilm formation by Staphylococcus spp. in both groups. Minimum inhibitory concentration testing showed that biofilm forming Staphylococcus spp. from VKC exhibited multidrug resistance (>2 antibiotics) more frequently than those from HC. Additionally, Staphylococcus spp. in VKC demonstrated higher resistance to fluoroquinolones compared to HC. These findings indicate a significantly greater prevalence of biofilm-forming and antimicrobial resistant Staphylococcus bacteria in VKC Patients compared with HC.

Pseudomonas aeruginosa chronic lung infection is the leading cause of death in the cystic fibrosis (CF) population. The high genome versatility of this microorganism allows it to adapt to the hostile CF lung where the same clone can persist for decades. Paranasal sinuses serve as a reservoir for bacterial adaptation before lung infection. Our study investigates biofilm compatibility among identical and different P. aeruginosa genotypes from sinus and lungs of CF patients. Strains were further characterized by whole genome sequencing and motility assays were performed.

Motility, gentamicin susceptibility and growth rates were assessed in four strains coming from three CF patients. The strains were subjected to whole genome sequencing with the Illumina MiSeq platform.Conjugation assays using the mini Tn7 transposon were performed in order to tag bacteria with the fluorescent proteins YFP (yellow) and CFP (cyan). Biofilm experiments were carried out in a flow cell system and images were acquired using a confocal laser microscope (CLSM) on days 3 and 5. Four experiments were performed: Experiment 1 with two clonal isolates from sinus and lungs from patient P01 (CF430-142, CF430-11621); experiments 2 (CF430-11621 + 75885-B) and 3 (CF430-11621 + 80271-B) with two lung isolates belonging to two different clones from different patients (P02, P03) and experiment 4 with one lung strain (CF430-11621) and P. aeruginosa PAO1 reference strain.

P. aeruginosa clonal isolates coming from paranasal sinuses and lungs from the same patient were able to form mixed biofilm. When different clones were employed no mixed biofilms were observed. Similar results were observed when combining the lung strain and the reference strain PAO1. Biofilms of both strains were observed in the flow-cell channels but no mixed biofilms of them were observed, with the exception of strain 75887-B which did not appear to form any biofilm when mixed with strain CF430-11621. All strains performed swarming while strains CF430-142 and 75887B lacked twitching motility. An aminoacidic change in SadB was observed in the strain 75887B.

Mixed biofilms were only observed when identical clones from the same patient were cultured together. Our experiments indicate that twitching motility does not significantly affect biofilm formation or architecture in our isolates.

Antibiotic resistance in foodborne bacteria poses a substantial global health challenge. Reports indicate that antibiotic overuse in middle-class and low-income countries is a significant factor in the ever-increasing resistance. Resistance mechanisms have developed through enzymatic hydrolysis, reduced membrane permeability, efflux pumps, and target site mutations. Preventive measures like proper hygiene and safe food preparation, vaccination, antibiotic stewardship and surveillance, implementing infection prevention and control (IPC) measures, good agricultural practices, and investigating novel approaches like CRISPR, NGS, nanotechnology, and bacteriophages may be employed to address this challenge. Naturally occurring preservatives (e.g., nisin) are alternatives to antibiotics for food preservation. Prebiotics, probiotics, nanobiotics, phage treatment, and antimicrobial peptides are also substitutes for antibiotics. Furthermore, plant-derived compounds, such as essential oils and plant extracts, are promising substitutes for antibiotics in animal production. This review focuses on the mechanisms of underlying antibiotic resistance in foodborne pathogens, necessary preventive measures, and the challenges associated.

Created using BioRender https://www.biorender.com/.

Antibiotic resistance in bacteria is a critical global health challenge, driven by molecular mechanisms such as genetic mutations, efflux pumps, enzymatic degradation of antibiotics, target site modifications, and biofilm formation. Horizontal gene transfer (HGT) further accelerates the spread of resistance genes across bacterial populations. These mechanisms contribute to the emergence of multidrug-resistant (MDR) strains, rendering conventional antibiotics ineffective. Recent advancements in CRISPR/Cas9-based genome editing offer innovative solutions to combat drug resistance. CRISPR/Cas9 enables precise targeting of resistance genes, facilitating their deletion or inactivation, and provides a potential method to eliminate resistance-carrying plasmids. Furthermore, phage-delivered CRISPR systems show promise in selectively killing resistant bacteria while leaving susceptible strains unaffected. Despite challenges such as efficient delivery, off-target effects, and potential bacterial resistance to CRISPR itself, ongoing research and technological innovations hold promise for using CRISPR-based antimicrobials to reverse bacterial drug resistance and develop more effective therapies. These abstract highlights the molecular mechanisms underlying bacterial drug resistance and explores how CRISPR/Cas9 technology could revolutionize treatment strategies against resistant pathogens.

Staphylococcus aureus (S. aureus) is a frequent culprit in implant-associated infections and employs many virulence factors to escape killing by the host immune system. The specific immune evasion strategies used by small aggregates of S. aureus on a surface, precursors to mature biofilm, are still relatively unknown. Time-lapse confocal microscopy was leveraged to quantify interactions between S. aureus aggregates and human neutrophils in vitro and identify specific mechanisms of resistance to neutrophil killing. Surface-associated wild-type S. aureus rapidly formed small biofilm aggregates when grown in human serum. Conversely, aggregation was inhibited when the SaeR/S two-component gene regulatory system was deleted. Wild-type aggregates began to show individual and population-level resistance to neutrophil killing upon reaching sizes of approximately 50 to 75 µm2, whereas Δsae clusters failed to reach these sizes and were readily cleared. Aggregation of Δsae strains was impaired by serum complement, and this inhibition required complement proteins C3 and factor B, but not C4 or C5, suggesting that this activity primarily occurs at the level of the alternative pathway. Several complement-inhibiting genes regulated by SaeR/S were identified that collectively facilitate biofilm aggregate formation in human, but not murine serum. Finally, aggregation of two related opportunistic pathogens, Staphylococcus epidermidis and Enterococcus faecalis, was inhibited by serum. These data demonstrate a function of serum complement, the ability to inhibit bacterial aggregation, that is potently blocked by S. aureus through the production of multiple complement-interfering proteins that are regulated by the SaeR/S system.

The emergence of bacterial ESKAPE pathogens presents a formidable challenge to global health, necessitating the development of innovative strategies for antibiotic discovery. Here, we leverage chemical synthetic lethality to locate therapeutic combinations of small molecules against multidrug-resistant Pseudomonas aeruginosa. Using a transposon screen, we identify PyrD as a target for sensitizing P. aeruginosa to subinhibitory doses of ceftazidime. High-throughput inhibitor screens identify two PyrD inhibitors, nordihydroguaiaretic acid (NDGA) and chlorhexidine (CHX), each of which does not significantly affect growth in isolation but exhibits chemical synthetic lethality when combined with low-dose ceftazidime. Downstream biochemical studies elucidate the mechanism of inhibition by NDGA and CHX. Remarkably, this combination is toxic to P. aeruginosa but leaves commensal bacteria, which are more susceptible to antibiotics, unscathed. Aside from advancing drug combinations that may be explored further in the future, our results offer a new approach for devising potent and specific drug combinations against recalcitrant pathogens.

The misuse and overuse of antibiotics have caused the emergence of antibiotic-resistant bacteria, making bacterial infections more challenging. The increasing prevalence of multidrug-resistant pathogens has driven researchers to explore novel therapeutic strategies. Phototherapy strategies that utilize photo-responsive biomaterials for their antibacterial properties have gained widespread attention due to their capability of precisely controlling bacterial inactivation with minimal side effects. Despite their potential, photodynamic therapies suffer from phototoxicity and low efficiency of photosensitizers, while photothermal therapy risks overheating, which may harm healthy tissues, thus restricting its broader application. Metal organic frameworks (MOFs) have unique physicochemical properties, which provide a promising way to deal with these challenges. MOFs can function as reservoirs, loading and releasing antibacterial agents, such as antibiotics or metal ions, upon light illumination by virtue of their metastable coordination bonds. Their porous structures enable controlled drug release and encapsulation of photosensitizers. Furthermore, MOFs' tunable composition and pore structure allow for the light-triggered generation of heat and reactive oxygen species, enhancing their antibacterial effectiveness. By doping MOFs with functional materials, it is possible to achieve multi-mode antibacterial effects. In this review, we will outline recent advancements of photo-responsive antibacterial MOFs, categorize their underlying mechanisms of action and highlight their prospects in addressing bacterial resistance.

Chromobacterium haemolyticum is an environmental bacterium that can cause severe and fatal opportunistic infections in humans and animals. Although C. haemolyticum is characterized by its strong β-hemolytic activity, the molecular basis of this phenotype has remained elusive over the more than 15 years since the species was first described. Herein, we report a family of cyclic lipodepsipeptides, the jagaricins, that are responsible for the potent hemolytic activity of C. haemolyticum. Comparative genomics of C. haemolyticum strains revealed a completely conserved gene locus (hml) encoding a nonribosomal peptide synthetase. Metabolic profiling of C. haemolyticum DSM 19808 identified a suite of cyclic lipodepsipeptides as the products, with the three main congeners (jagaricin A-C) being elucidated by a combination of tandem mass spectrometry, chemical derivatization, and nuclear magnetic resonance spectroscopy. Significantly, a C. haemolyticum hml deletion mutant is devoid of hemolytic activity. Moreover, purified jagaricins are hemolytic at low micromolar concentrations in an erythrocyte lysis assay. Further bioassays demonstrated that the cyclic lipodepsipeptides are crucial for the biofilm-forming and swarming behavior of C. haemolyticum. Matrix-assisted laser desorption ionization mass spectrometry imaging showed that primarily jagaricin B and C are involved in these processes in vitro. Our data shed light on the bioactivities of jagaricins, specialized metabolites that likely contribute to both successful niche colonization and the virulence potential of C. haemolyticum.IMPORTANCEDespite the rising incidence of Chromobacterium haemolyticum as a serious opportunistic pathogen, there is limited information on whether the competitive traits that ensure its survival in its freshwater niche also influence host infection. We reveal that C. haemolyticum produces specialized metabolites that not only cause its pronounced hemolytic phenotype but are also crucial for biofilm formation and swarming motility. These results exemplify a case of coincidental evolution, wherein the selective pressures encountered in a primary environmental niche drive the evolution of a trait impacting virulence. This knowledge provides a foundation for the development of antivirulence therapies against the emerging pathogen C. haemolyticum.

Soil salinization is a major constraint on agricultural productivity, particularly in arid and semi-arid regions where limited rainfall cannot wash salts from plant root zones. This leads to disruptions in water uptake, ion balance, photosynthesis, respiration, nutrient absorption, hormone regulation and rhizosphere microbiome disturbances in plants. Chemical and biological methods can help mitigate soil salinity, but biological approaches, like using halophytes and salt-tolerant microorganisms, are preferred for environmental sustainability. Halophytes, however, represent only about 1% of flora and are habitat specific, so halophilic plant growth-promoting (PGP) microbes have emerged as a key eco-friendly solution. Halophilic PGP bacteria have shown promise in remediating saline soils, enhancing fertility and boosting crop resilience by inducing salinity tolerance (IST) and promoting plant growth traits. In the era of modern agriculture where chemical inputs are at their peak of application rendering the soil infertile, halophilic PGP bacteria represent a promising, sustainable approach to support food security, aligning with Sustainable Development Goals for zero hunger.

Quorum sensing (QS) is widely utilized by both bacteria and fungi to mediate cell-cell communication. Previous studies have demonstrated that the indole derivative indole-3-ethanol (also known as tryptophol) controls morphogenesis as a QS molecule in fungi. However, whether this QS signal is involved in the modulation of biological functions in bacteria remains unknown. Here, we report that indole-3-ethanol controls the biological functions and pathogenicity of Enterobacter cloacae subsp. cloacae ATCC 13047. The biosynthesis of indole-3-ethanol is performed by YjgB (ECL_RS22935), an alcohol dehydrogenase. Deletion of yjgB results in impaired biological functions and virulence. Furthermore, we revealed that indole-3-ethanol from E. cloacae reduces the competitive fitness of Shigella sonnei by inhibiting its biofilm formation, extracellular polysaccharide synthesis, and virulence. Given that both E. cloacae and S. sonnei are common human intestinal microbes, our results highlight the critical roles of indole-3-ethanol in both intraspecies signaling and interspecies communication in bacteria.

Quorum sensing is a cell-cell communication mechanism widely employed by bacteria to control various biological functions and pathogenicity. In this study, we demonstrated that Enterobacter cloacae employs indole-3-ethanol as a quorum-sensing signal to control biological functions and virulence. We also revealed that indole-3-ethanol from E. cloacae effectively inhibits biofilm formation and virulence in Shigella sonnei. Our findings not only suggest the important role of indole-3-ethanol in the regulation of the pathogenicity of E. cloacae but also provide new insights into the development of indole-3-ethanol as an anti-virulence agent against S. sonnei.

Human-designed oligotrophic environments, such as cleanrooms, harbor unique microbial communities shaped by selective pressures like temperature, humidity, nutrient availability, cleaning reagents, and radiation. Maintaining the biological cleanliness of NASA's mission-associated cleanrooms, where spacecraft are assembled and tested, is critical for planetary protection. Even with stringent controls such as regulated airflow, temperature management, and rigorous cleaning, resilient microorganisms can persist in these environments, posing potential risks for space missions.

During the Phoenix spacecraft mission, genomes of 215 bacterial isolates were sequenced and based on overall genome-related indices, 53 strains belonging to 26 novel species were recognized. Metagenome mapping indicated less than 0.1% of the reads associated with novel species, suggesting their rarity. Genes responsible for biofilm formation, such as BolA (COG0271) and CvpA (COG1286), were predominantly found in proteobacterial members but were absent in other non-spore-forming and spore-forming species. YqgA (COG1811) was detected in most spore-forming members but was absent in Paenibacillus and non-spore-forming species. Cell fate regulators, COG1774 (YaaT), COG3679 (YlbF, YheA/YmcA), and COG4550 (YmcA, YheA/YmcA), controlling sporulation, competence, and biofilm development processes, were observed in all spore-formers but were missing in non-spore-forming species. COG analyses further revealed resistance-conferring proteins in all spore-formers (n = 13 species) and eight actinobacterial species, responsible for enhanced membrane transport and signaling under radiation (COG3253), transcription regulation under radiation stress (COG1108), and DNA repair and stress responses (COG2318). Additional functional analysis revealed that Agrococcus phoenicis, Microbacterium canaveralium, and Microbacterium jpeli contained biosynthetic gene clusters (BGCs) for ε-poly-L-lysine, beneficial in food preservation and biomedical applications. Two novel Sphingomonas species exhibited for zeaxanthin, an antioxidant beneficial for eye health. Paenibacillus canaveralius harbored genes for bacillibactin, crucial for iron acquisition. Georgenia phoenicis had BGCs for alkylresorcinols, compounds with antimicrobial and anticancer properties used in food preservation and pharmaceuticals.

Despite stringent decontamination and controlled environmental conditions, cleanrooms harbor unique bacterial species that form biofilms, resist various stressors, and produce valuable biotechnological compounds. The reduced microbial competition in these environments enhances the discovery of novel microbial diversity, contributing to the mitigation of microbial contamination and fostering biotechnological innovation. Video Abstract.

The problem of antimicrobial resistance (AMR) has caused global concern due to its great threat to human health. Evidences are emerging for a critical role of biofilms, one of the natural protective mechanisms developed by bacteria during growth, in resisting commonly used clinical antibiotics. Advances in nanomedicines with tunable physicochemical properties and unique anti-biofilm mechanisms provide opportunities for solving AMR risks more effectively. In this review, we summarize the five "A" stages (adhesion, amplification, alienation, aging and allocation) of biofilm formation and mechanisms through which they protect the internal bacteria. Aimed at the characteristics of biofilms, we emphasize the design "THAT" principles (targeting, hacking, adhering and transport) of nanomedicines in their interactions with biofilms and internal bacteria. Furthermore, recent progresses in multimodal antibacterial nanomedicines, including biofilms disruption and bactericidal activity, and the types of currently available antibiofilm nanomedicines contained organic and inorganic nanomedicines are outlined and highlighted their potential applications in the development of preclinical research. Last but not least, we offer a perspective for the effectiveness of nanomedicines designed to address AMR and challenges associated with their clinical translation.

Biofilms have extensive applications and important roles in biological processes. This study aimed to investigate the effect and mechanism of low-concentration sulfamethoxazole (SMX) on biofilm development in biofilters. The effects of various SMX concentrations (0, 100 ng/L, 1000 ng/L) on microbial development were compared. Compared with the control group without SMX, the start-up period of R2 and R3 filters with SMX added was decreased by 9 % and 21 %, respectively. Under antibiotic stimulation, reactive oxygen species (ROS) and bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) concentrations increased, aligning with changes in extracellular polymer content and biofilm formation. Microbial community results showed that the presence of SMX promoted the growth of some manganese-oxidizing bacteria (MnOB), such as Massilia, Pedomicrobium, Sphingopyxis, Pseudomonas, and Bacillus. Functional gene analysis further revealed higher expression levels of genes related to c-di-GMP transformation in the presence of SMX. These findings suggest that microbial communities can adapt to their environment by accelerating biofilm formation at lower antibiotic concentrations. The results of this study provide new insights into the impact of low-concentration antibiotics on biofilm development and offer a crucial reference for biofilter design and optimization.

Candida biofilm matrix components are delivered to the extracellular space by vesicles where they deposit and confer biofilm-associated drug resistance. Here, we present evidence that drugs designed to inhibit mammalian exosome production exhibit similar effects on C. albicans extracellular vesicles, ultimately eliminating biofilm matrix assembly. We find that vesicle reduction renders biofilm communities susceptible to the antifungal fluconazole. Our findings argue that vesicle trafficking pathways represent a promising target to optimize for recalcitrant fungal biofilms.

The development of personal protective equipment (PPE) is essential to control the spread of infectious diseases. However, traditional PPE has significant drawbacks, such as a lack of antibacterial capacity and nonreusability, which may result in direct or indirect secondary infections. Herein, we propose an octahedral (Fe-O6) Fe-gallate (Fe-GA) metal-organic framework nanozyme with light-enhanced oxidase-like (OXD-like) activity and extend the use onto nonwoven fabric (Fe-GA/NWF) by hot pressing. Specifically, visible-light-mediated carrier migration and the activity of the OXD-like species synergistically enhance the production of reactive oxygen species, achieving superior antibacterial effects without the addition of any chemical additives. In this case, Fe-GA/NWF spontaneously inactivates over 99.9% of real microbial aerosols and demonstrates excellent mechanical properties, reusability (15 cycles), and biocompatibility. Moreover, Fe-GA/NWF can be used as an ideal antibacterial platform for KN95 masks and protective clothing and can be extended to other substrates. This work provides a promising strategy to develop self-antibacterial PPE in complex environments.

Virulence factors of pathogens, such as toxin production and biofilm formation, often exhibit a public character, providing benefits to nearby non-producers. Consequently, anti-virulence drugs targeting these public traits may not select for resistance, as resistant mutants that resume production of the virulence factor share the benefits of their resistance with surrounding sensitive cells. In agreement with this, we show that even after long-term treatment with a 2-amino-imidazole (2-AI) biofilm inhibitor, Salmonella populations remained as susceptible to biofilm inhibition as the ancestral populations. Nonetheless, further genotypic and phenotypic analysis revealed that the Salmonella populations did adapt to the treatment and accumulated mutations in efflux pump regulators and alternative sigma factors. These mutations resulted in a reduced biofilm-forming capacity and increased efflux activity. Their selection was due to a growth delaying side effect of the biofilm inhibitor. Enhanced efflux activity helped overcome this growth delay, providing a fitness advantage over the ancestor. Finally, we demonstrate that chemical modification of the inhibitor enhances its specificity by partially alleviating the unintended growth delay while retaining the anti-biofilm activity, which in turn eliminated the selection pressure for increased efflux. Overall, our findings highlight that while unintended side effects can complicate anti-virulence strategies, adaptation to these effects does not necessarily restore the inhibited virulence trait. Moreover, chemical modification can mitigate these unintended side effects and enhance drug specificity.

Non-antibiotic treatments, such as carbon dots (C-dots), are gaining popularity in the search for effective alternatives. We aimed to evaluate the antibacterial properties of C-dots derived from Lactobacillus acidophilus (L-C-dots) alone and in combination against carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates.

Seventy clinical isolates of Klebsiella pneumoniae were tested for carbapenem resistance using a modified carbapenem inactivation assay. To determine the presence of fimH and mrkD genes, which are associated with biofilm formation, polymerase chain reaction (PCR) was conducted. L-C-dots were synthesized using a hydrothermal method and characterized. Furthermore, their antibacterial and anti-biofilm activities were evaluated against CRKP isolates. The combination of L-C-dots with the meropenem antibiotics were also tested using a checkerboard assay. Finally, the influence of L-C-dots on the expression levels of fimH and mrkD genes was examined using quantitative PCR (qPCR).

L-C-dots demonstrated significant antibacterial activity against CRKP isolates, with a minimum inhibitory concentration (MIC) of 50 mg/mL and a sub-MIC of 25 mg/mL. L-C-dots effectively inhibited biofilm formation at MIC and sub-MIC concentrations (P ˂ 0.05). Additionally, the L-C-dots had synergistic and additive interactions with the meropenem antibiotics against CRKP isolates, reducing the MIC of both agents. Furthermore, the L-C-dots decreased the expression of the fimH(p< 0.029) and mrkD (p < 0.015) genes.

The findings indicate that L-C-dots may serve as a promising new treatment option for CRKP infections. They show potential as a strong therapeutic choice, especially when used in combination with traditional antibiotics.

The transcriptional regulator FleQ contributes to Pseudomonas aeruginosa biofilm formation by activating the expression and biosynthesis of matrix exopolysaccharides in a manner dependent on c-di-GMP. However, little is known about the role of FleQ in the antibiotic tolerance phenotype of P. aeruginosa biofilms. Inactivation of fleQ impaired biofilm formation and rendered biofilms susceptible to tobramycin and norfloxacin. The phenotypes were similar to biofilms inactivated in sagS encoding the orphan sensor SagS that promotes the switch from planktonic to biofilm growth via BfiSR and antibiotic tolerance via BrlR. While FleQ was found to contribute to biofilm formation independently of SagS and BfiSR, FleQ instead converged with SagS-dependent regulation at the level of BrlR. This was supported by multicopy expression of sagS failing to restore biofilm antibiotic tolerance by ΔfleQ to wild-type levels (and vice versa) and by biofilms formed by the ΔfleQΔsagS double mutant being as susceptible as ΔfleQ and ΔsagS biofilms. Increased antibiotic susceptibility was independent of BrlR abundance or BrlR DNA binding but coincided with significantly reduced transcript abundance of the BrlR-activated mexCD-oprJ and PA1874-77, encoding an ABC transporter previously shown to contribute to the tolerance of biofilms to tobramycin and norfloxacin. FleQ- dependent regulation of gene expression was indirect. Co-immunoprecipitation and BACTH assays indicated FleQ to interact with SagS via its HisKA-Rec domain, likely suggesting FleQ and SagS to likely work in concert to enable biofilm antibiotic tolerance, by finetuning the expression of BrlR activated genes.IMPORTANCEIn P. aeruginosa, FleQ inversely regulates the expression of genes encoding flagella and biofilm matrix components, including exopolysaccharide (Pel, Psl) in a manner dependent on the levels of c-di-GMP. Our findings expand on the role of FleQ from regulating the transition to the biofilm mode of growth to FleQ contributing to the antimicrobial tolerance phenotype of biofilms, by FleQ affecting the expression of PA1874-77, a downstream target of the SagS-dependent transcriptional regulator BrlR. Importantly, our findings suggest FleQ works in concert with SagS, likely via FleQ-SagS protein-protein interactions, to enable the formation of inherently tolerant P. aeruginosa biofilms.

Among various treatments employed to solve the global problem of bacterial infection, photodynamic therapy (PDT) is recognized as a method with great potential to inactivate a wide range of bacteria without the development of drug resistance. However, many commonly used photosensitizers (PSs) have the disadvantages of poor water-solubility and potential toxicity, which limits their clinical application. Additionally, nitric oxide (NO) has unique advantages in antibacterial treatments due to its small molecular weight. Herein, protoporphyrin IX (PpIX), L-arginine (L-Arg), and glycol chitosan (GC) are used to construct a self-assembled cationic Arg-GC-PpIX nanoreactor for efficient bacterial inactivation under white light illumination. The Arg-GC-PpIX nanoreactor with excellent water dispersity and stability can rapidly bind to bacteria through electrostatic interaction and produce local singlet oxygen (1O2)/NO under light irradiation, leading to a high antibacterial efficiency toward both Gram-negative and Gram-positive bacteria. Besides, these NPs also possess a desirable antibiofilm ability. Finally, Arg-GC-PpIX@Gel which is obtained through loading Arg-GC-PpIX into the sodium alginate (SA)/Ca2+ hydrogel shows a satisfactory ability to promote infected wound healing when combined with white light irradiation. Therefore, the rationally designed Arg-GC-PpIX nanoreactor with light-triggered 1O2/NO release is a promising antibacterial agent for achieving effective PDT/NO gas therapy.

Mutanofactins are a family of natural products produced by Streptococcus mutans from the human oral microbiome. We report a unified approach to all mutanofactins by developing a total synthesis amenable to diversification. The key to success for the most complex members, mutanofactins 607 and 697, was an acyl ketene based strategy. Access to the family enabled comprehensive biological profiling, where we demonstrate that all mutanofactins are biofilm promoting in Streptococcus mutans. Experiments were extended to other inhabitants of the oral microbiome for the first time: Streptococcus gordonii and Streptococcus oralis, two early colonizers, were similarly affected with mutanofactins being biofilm promoting. Conversely, Veillonella dispar and Fusobacterium nucleatum showed little to no reaction to mutanofactins. Biophysical investigations based on quartz crystal microbalance with dissipation monitoring and atomic force microscopy reveal a previously unknown mucin-mutanofactin 697 interaction. Incubation of a mucin layer with mutanofactin 697 induces a morphology change within the mucin layer, which promotes bacterial adhesion and biofilm formation. This unique property of mutanofactin 697 might be key to early stages of biofilm formation in the human oral microbiome. Combined, an interdisciplinary approach consisting of total synthesis, microbiology and biophysical characterization provides insight into the roles of mutanofactins in the oral microbiome.

The microenvironment of Candida albicans biofilms create a hypoxic microenvironment, which exerts a profound influence on host immune responses during infection. Neutrophils are key defenders against C. albicans; however, the impact of biofilm-induced hypoxia on neutrophil function remains unclear.

We co-cultured human neutrophils in vitro with C. albicans biofilms at various stages of maturation, using both wild-type strains and extracellular matrix (ECM)-deficient mutants. Intracellular hypoxia was assessed using a fluorescent oxygen-sensitive probe. Neutrophil effector functions were evaluated by measuring caspase-3/7 activity, stabilization of hypoxia-inducible factor 1-alpha (HIF-1α), and accumulation of the anti-apoptotic Mcl-1 protein. Analyses included also quantification of reactive oxygen species (ROS) production, neutrophil extracellular trap (NET) formation, chemokine secretion (IL-8 and MIP-1β), and neutrophil elastase release. To assess the role of hypoxia signaling in neutrophil responses, cells were treated with the selective HIF-1α inhibitors LW6 and PX478.

Neutrophils infiltrating C. albicans wild-type biofilms experience progressive hypoxia, which intensifies with biofilm maturation. This hypoxia results from high fungal metabolic activity and extracellular matrix (ECM) production. Within the biofilm microenvironment, neutrophils exhibit increased stabilization of HIF-1α and Mcl-1, elevated secretion of MIP-1β, IL-8, and reduced caspase 3/7 activity, collectively suggesting a biofilm-induced pro-survival phenotype. Simultaneously, mature biofilms markedly suppress NET formation and ROS production while enhancing degranulation. Comparative analyses using mannan-deficient C. albicans mutants highlight the critical role of ECM composition in modulating hypoxia-driven immune responses. Pharmacological inhibition of HIF-1α with LW6 and PX478 partially restores NETosis and ROS production, underscoring the pivotal role of this protein in regulation of neutrophil function.

These findings provide novel insights into the impact of biofilm-induced hypoxia on neutrophil responses, identifying HIF-1α as a key regulator of immune adaptation in fungal biofilms. Targeting hypoxia pathways may offer new therapeutic strategies to modulate neutrophil responses and enhance host defenses against fungal infections.

The attachment of bacteria to solid surfaces has been studied primarily through the modes of pili or flagella tethering. We report on a common feature of tethering in pililess strains of three species of monotrichous bacteria-Vibrio alginolyticus, Pseudomonas aeruginosa, and Caulobacter crescentus-namely, that they may become tethered to the surface by their cell body rather than by a flagellum. These tethered bacteria rotate in alternating directions about a pivot point located under the cell body. Using high-intensity dark-field microscopy, we observed that, in most cases, the flagellum of a tethered Vibrio alginolyticus rotates together with the cell body. We name this distinct mode of attachment body tethering. Observing hundreds of rotating bacteria tethered to the surface, we find that body tethering is a more common mode of attachment than flagellum tethering for these three strains of bacteria. Our results confirm that body tethering is a key mechanism for the surface attachment of bacteria without pili. Recognizing body tethering as a robust mode of bacterial attachment to surfaces may have broad implications in the study of bacterial adhesion and biofilm formation.

Biofilms are highly organized, cooperating communities of microorganisms encased in a self-produced extracellular matrix, providing resilience against external stress such as antimicrobial agents and host defenses. A hallmark of biofilms is their phenotypic heterogeneity, which enhances the overall growth and survival of the community. In this study, we demonstrate that removing the dnaK and tig genes encoding the core molecular chaperones DnaK (Hsp70 homolog) and Trigger factor disrupted protein homeostasis in Bacillus subtilis and resulted in the formation of an extremely mucoid biofilm with aberrant architecture, compromised structural integrity, and altered phenotypic heterogeneity. These changes include a large reduction in the motile subpopulation and an overrepresentation of matrix producers and endospores. Overproduction of poly-γ-glutamic acid contributed crucially to the mucoid phenotype and aberrant biofilm architecture. Homeostasis impairment, triggered by elevated temperatures, in wild-type cells led to mucoid and aberrant biofilm phenotypes similar to those observed in strains lacking both dnaK and tig. Our findings show that disruption of protein homeostasis, whether due to the absence of molecular chaperones or because of environmental factors, severely changes biofilm features.

Pseudomonas aeruginosa biofilms, driven by extracellular polysaccharides (EPSs), exacerbate pathogenicity and drug resistance, posing critical threats to public health. While EPS biosynthesis pathways are central to biofilm formation, their distinct contributions and regulatory dynamics remain incompletely understood. Here, we systematically dissect the roles of three core EPS pathways-Psl, Pel, and alginate-in biofilm architecture and function using multi-omics approaches. Key findings reveal Psl as the dominant regulator of biofilm elasticity and thickness, with its deletion disrupting chemotaxis, quorum sensing, and 3',5'-Cyclic GMP (c-di-GMP)/amino acid metabolism. Pel redundantly enhances biofilm biomass, but elevates flagellar synthesis efficiency when Psl is absent. Alginate exhibited negligible transcriptional or metabolic influence on biofilms. These insights clarify hierarchical EPS contributions and highlight Psl as a priority target for therapeutic strategies to dismantle biofilm-mediated resistance.

Dental caries arise from intricate interactions among oral microorganisms, impacting ecological stability and disease progression. In this study, we aimed to investigate the microbial diversity and inter-kingdom interactions in severe early childhood caries (S-ECC) and assess the influence of environmental factors such as salivary pH and trace elements. We analyzed 61 children aged 3-4 years with complete deciduous dentition, evaluating salivary pH, buffering capacity, and trace elements (iron, fluoride). We examined the performance of 16S rRNA V1-V9 regions gene and internal transcribed spacer (ITS) primers for bacteria and fungi from plaque and saliva to characterize community compositions and diversity. Findings revealed significant shifts in bacterial diversity in S-ECC saliva samples, marked by decreased diversity and elevated abundance of cariogenic species, particularly Streptococcus mutans. Candida albicans was notably more prevalent in the S-ECC group, implicating its potential role in pathogenesis. Iron and fluoride concentrations showed no significant correlation with microbial community structure. Network analyses uncovered complex intra- and inter-kingdom interactions, underscoring cooperative and competitive dynamics. S-ECC children exhibited higher abundances of bacteria (Streptococcus mutans, Granulicatella, Actinomyces) and fungi (Candida albicans), with specific microbial taxa associated with reduced salivary pH.

This study illuminates the intricate relationship between bacteria and fungi within the oral microbial community of children, specifically highlighting differences between those with S-ECC and those without caries. Through an extensive analysis of the microbial composition in both saliva and dental plaque, we identified a significant increase in the abundance of specific bacterial taxa (e.g., S. mutans, Granulicatella, Actinomyces) and fungal species (e.g., C. albicans) in the oral cavities of children with S-ECC. This finding underscores the potential role of these microorganisms in the development of caries. Contrary to previous studies that emphasize the importance of iron and fluoride in oral health, our research found no significant correlation between the concentrations of these elements and the composition of oral microbial communities. This result challenges conventional understanding and opens new avenues for future research. Additionally, our findings revealed an association between Veillonella sp., Propionibacterium sp., and Candida sp. and reduced salivary pH. This provides novel insights into the relationship between the oral microenvironment and caries development. The implications of our findings are substantial for the development of prevention and intervention strategies targeting childhood caries. They also underscore the critical need for a deeper exploration of oral microbial interactions and their environmental influences.

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that plays a crucial role in regulating biofilm development, yet the role in Gram-positive bacteria remains elusive. Here, we demonstrated that dispersed cells from biofilms of Bacillus velezensis FZB42 exhibit a unique phenotype and gene expression compared to planktonic cells. Transcriptomic analysis revealed 1327 downregulated and 1298 upregulated genes, among which the c-di-GMP phosphodiesterase coding yuxH gene was remarkably upregulated. Deletion of the yuxH gene led to elevated c-di-GMP levels accompanied by reduced amounts of "actively dispersed cells" from the pellicle and the capacity of motility. Deletion of spoIIIJ, spo0J, and kinA resulted in increased c-di-GMP levels and reduced biofilm dispersal ability. Also, the level of c-di-GMP was increased when adding the cues of inhibition biofilm dispersal such as glucose and calcium ions. Collectively, these present findings suggest the c-di-GMP level is negatively correlated with biofilm dispersal in Bacillus velezensis FZB42, which sheds new light on biofilm regulation in Bacillus velezensis FZB42.

Endodontic infections are biofilm-mediated, demanding effective biofilm eradication from the root canal. Root canal complexities, coupled with bacterial biofilm resistance, pose challenges to thorough disinfection. Irrigation, particularly with sodium hypochlorite, is crucial in endodontics. Activation techniques, like sonic or ultrasonic oscillations, enhance irrigant penetration and biofilm disruption, improving decontamination and treatment outcomes.The aim of the present study was to evaluate the effectiveness of XP Finisher, EndoUltra, Eddy and Irriflex in the reduction of the multispecies endodontic biofilm formed by Enterococcus faecalis, Pseudomonas aeruginosa, Candida albicans and Proteus mirabilis.

A total of 44 single-rooted mandibular premolars were selected and divided into groups for investigation: Group A: Irriflex, Group B: XP Finisher, Group C: Eddy system, and Group D: EndoUltra system. Multispecies biofilms, comprising Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa, and Candida albicans, were cultured and inoculated into the pre-treated dentinal canals, which were then incubated for 16 days. Following this, the canals were subjected to the respective irrigation protocols. Bacterial counts were assessed using sterile paper points and culture techniques post-irrigation. Additionally, four non-inoculated root canals were used as negative controls for comparison.

EndoUltra achieved the highest reduction in Total Bacterial Count (TBC) with a median decrease of 75% (interquartile range [IQR]: 70-80%), significantly better than XP Finisher (p = 0.001) and Irriflex (p = 0.001). Eddy led to a reduction in Pseudomonas aeruginosa (PA) with a median decrease of 85% (IQR: 80-90%), significantly outperforming Irriflex (p = 0.001) and XP Finisher (p = 0.001). For Enterococcus faecalis (EF), EndoUltra had a median reduction of 70% (IQR: 65-75%), significantly better than Eddy (p = 0.01) and Irriflex (p = 0.001), while XP Finisher resulted in a reduction of 60% (IQR: 55-65%). EndoUltra showed the highest reduction in Proteus mirabilis (ProM) with 80% (IQR: 75-85%), significantly better than Irriflex (p = 0.001) and XP Finisher (p = 0.001), with Eddy also better than Irriflex (p = 0.009). EndoUltra reduced Candida albicans (CA) by 65% (IQR: 60-70%), significantly outperforming XP Finisher (p = 0.001) and Eddy (p = 0.001).

Within its limitations, this study identified EndoUltra as highly effective in reducing bacterial counts, indicating its potential utility in disinfecting root canals. These findings underscore the significance of such methods in enhancing treatment outcomes and addressing root canal infections.

Vibrio cholerae pathogens cause cholera, an acute diarrheal disease resulting in significant morbidity and mortality worldwide. Biofilms in vibrios enhance their survival in natural ecosystems and facilitate transmission during cholera outbreaks. Critical components of the biofilm matrix include the Vibrio polysaccharides produced by the vps-1 and vps-2 gene clusters and the biofilm matrix proteins encoded in the rbm gene cluster, together comprising the biofilm matrix cluster. However, the biofilm matrix clusters and their evolutionary patterns in other Vibrio species remain underexplored. In this study, we systematically investigated the distribution, diversity, and evolution of biofilm matrix clusters and proteins across the Vibrio genus. Our findings reveal that these gene clusters are sporadically distributed throughout the genus, even appearing in species phylogenetically distant from Vibrio cholerae. Evolutionary analysis of the major biofilm matrix proteins RbmC and Bap1 shows that they are structurally and sequentially related, having undergone structural domain and modular alterations. Additionally, a novel loop-less Bap1 variant was identified, predominantly represented in two phylogenetically distant V. cholerae subspecies clades that share specific gene groups associated with the presence or absence of the protein. Furthermore, our analysis revealed that rbmB, a gene involved in biofilm dispersal, shares a recent common ancestor with Vibriophage tail proteins, suggesting that phages may mimic host functions to evade biofilm-associated defenses. Our study offers a foundational understanding of the diversity and evolution of biofilm matrix clusters in vibrios, laying the groundwork for future biofilm engineering through genetic modification.

Biofilms help vibrios survive in nature and spread cholera. However, the genes that control biofilm formation in vibrios other than Vibrio cholerae are not well understood. In this study, we analyzed the biofilm matrix gene clusters and proteins across diverse Vibrio species to explore their patterns and evolution. We discovered that these genes are spread across different Vibrio species, including those not closely related to V. cholerae. We also found various forms of key biofilm proteins with different structures. Additionally, we identified genes involved in biofilm dispersal that are related to vibriophage genes, highlighting the role of phages in biofilm development. This study not only provides a foundational understanding of biofilm diversity and evolution in vibrios but also leads to new strategies for engineering biofilms through genetic modification, which is crucial for managing cholera outbreaks and improving the environmental resilience of these bacteria.

Bacterial infections remain a significant and escalating threat to global health, exacerbated by multidrug-resistant strains that undermine the efficacy of conventional antibiotics. This pressing issue underscores the urgent need for the development of new antimicrobial materials. Among these, molecular-based crystalline porous materials, such as metal-organic frameworks (MOFs), covalent organic frameworks (COFs), hydrogen-bonded organic frameworks (HOFs), and supramolecular assembly frameworks (SAFs), have emerged as a promising class of antibacterial agents. These materials exhibit well-defined crystallinity and tunable structures, offering exceptional versatility for antibacterial applications. Notably, their high surface area, adjustable pore size, and potential for functionalization enable efficient loading and controlled release of antibacterial agents, including metal ions and antibacterial molecules. This review provides a comprehensive analysis of recent advancements in this field, highlighting design strategies, structural diversity, antibacterial mechanisms, and applications. Finally, we discuss the current challenges and outline future opportunities for the practical development and deployment of antibacterial porous materials.

Colorectal cancer (CRC) is a prevalent global malignancy with complex etiologies, including microbiota alterations. This study investigates gut microbiota and biofilm-producing bacteria in 35 Thai CRC patients, analyzing paired normal and tumor biopsy samples. Bacterial DNA from the V3-V4 region of 16S rRNA was sequenced, and biofilms were visualized via scanning electron microscopy and fluorescence in situ hybridization (FISH). Results revealed Firmicutes as the dominant phylum, followed by Bacteroidota, Proteobacteria, and Fusobacteriota, with Fusobacteriota and Bacteroidota notably enriched in left-sided CRC. Key biofilm producers-Bacteroides fragilis, Fusobacterium nucleatum, and Pasteurella stomatis-showed significantly higher gene expression in tumor tissues. Dense biofilms and higher Fusobacterium abundance, localized within the crypts of Lieberkuhn, were observed in CRC tissues. These findings highlight CRC-associated microbiota alterations and pathogenic biofilm production, emphasizing a spatial relationship between tumor location and microbial distribution, with potential implications for understanding CRC pathogenesis and therapeutic targeting.

Pulmonary diseases, arising from infections caused by bacteria, fungi, and viruses, or stemming from underlying genetic factors are one of the leading causes of mortality in humans, accounting for millions of deaths every year. At the onset of pulmonary diseases, crucial roles are played by phagocytic immune cells, particularly tissue-resident macrophages, in regulating the immune response at the mucosal barrier. Recent strides have illuminated the pivotal role of host bioenergetics modulated by metabolites derived from both pathogens and hosts in influencing the pathophysiology of major organs. Their influence extends to processes such as the infiltration of immune cells, activation of macrophages, and the polarization phenomenon. Furthermore, host-derived metabolites, such as itaconate, contribute to the promotion of anti-inflammatory responses, thereby preventing immunopathology and facilitating the preservation of mucosal niches to thrive for the long-term. This review explores recent advancements in the field of immunometabolism, with a particular emphasis on the intricacies of disease progression in pulmonary infections caused by bacteria such as P. aeruginosa, M. tuberculosis and S. aureus and fungi like C. albicans.

Escherichia coli (E. coli) is one of the most common pathogens causing endometritis in dairy cows. The presence of genes encoding extended-spectrum β-lactamase (ESBL) and biofilm formation are important factors contributing to bacterial resistance, which poses a significant challenge to the treatment of endometritis in dairy cows. Essential oils containing linalool have been shown to improve the cure rate of bovine endometritis, but whether linalool can inhibit E. coli biofilm has not yet been reported. We proposed to ascertain the linalool implications on the development of E. coli biofilm and its extracellular polysaccharides, as well as to assess the impacts of linalool on E. coli in both planktonic and biofilm states. We discovered that the minimum biofilm inhibitory concentrations (MBICs) of linalool against E. coli were twice as high as the minimum inhibitory concentrations. Linalool exhibited a strong bactericidal effect on clinical E. coli strain producing ESBL and forming strong biofilm, regardless of whether they were in a planktonic or biofilm condition. Linalool suppressed the biofilm development in a way that was dependent on the dosage, with an MBIC 4 µL/mL. This was verified by the use of crystal violet test and scanning electron microscopy. Moreover, the CCK-8 assay and confocal laser scanning microscopy (CLSM) manifested significant reductions in live bacteria within the biofilm. The concentrations of extracellular polymeric compounds in the E. coli biofilm were also reduced. Furthermore, CLSM and RT-qPCR analysis confirmed that linalool (2 µL/mL) significantly suppressed exopolysaccharide (EPS) and the pgaABCD gene expression, regulating an essential exopolysaccharide expression in biofilm formation. These findings revealed that linalool effectively suppressed viable bacteria, EPS production, and E. coli biofilm formation, providing a theoretical foundation for alternative antibiotic therapy in endometritis in dairy cows and as a potential agent for preventing E. coli biofilm-related infections.

Probiotic strains with health-promoting activities have emerged as a promising strategy to prevent or treat different metabolic syndrome-related disturbances, including obesity or type 2 diabetes. In this work, we characterize the probiotic properties of a novel strain of Latilactobacillus sakei (L. sakei) CNTA 173, and we demonstrate its anti-obesity properties using the in vivo model Caenorhabditis elegans (C. elegans). This new strain exhibited sensitivity to the entire spectrum of antibiotics analysed, gastric and intestinal in vitro resistance, β-galactosidase activity, and the ability to form biofilm and to produce acetic acid in vitro. Cell culture analyses demonstrated that L. sakei CNTA 173 was able to reduce the adhesion to Caco-2 cells of the pathogenic E. coli O157:H7 and to exert immunomodulatory capacity in RAW 264.7 and HT-29 in vitro models. Furthermore, supplementation with L. sakei CNTA 173 counteracted the deleterious effects of glucose in C. elegans by significantly reducing fat accumulation, enhancing the oxidative stress response, and extending lifespan by directly regulating the carbohydrate and lipid metabolism-related genes acox-1, maoc-1, and daf-16. Our results unveil new strain-specific mechanisms of action by which L. sakei CNTA 173 exerts beneficial effects in vitro and in C. elegans, and suggest potential application of this novel probiotic strain in the prevention and treatment of metabolic syndrome-related disturbances.

A series of 1, 2, 4, 5-tetrasubstituted imidazole derivatives were synthesized and their antibiofilm potential against Candida albicans was evaluated in vitro. Two of the synthesized derivatives 5e (IC50 = 25 µg/mL) and 5m (IC50 = 6 µg/mL),displayed better antifungal and antibiofilm potential than the standard drug Fluconazole (IC50 = 40 µg/mL) against C. albicans. Based on the in vitro results, we escalated the real time polymerase chain reaction (RT-PCR) analysis to gain knowledge of the enzymes expressed in the generation and maintenance of biofilms and the mechanism of biofilm inhibition by the synthesized analogues. We then investigated the possible interactions of the synthesized compounds in inhibiting agglutinin-like proteins, namely Als3, Als4 and Als6 were prominently down-regulated using in-silico molecular docking analysis against the previously available crystal structure of Als3 and constructed structure of Als4 and Als6 using the SWISS-MODEL server. The stability and energy of the agglutinin-like proteins-ligand complexes were evaluated using molecular dynamics simulations (MDS). According to the 100 ns MDS, all the compounds remained stable, formed a maximum of 3, and on average 2 hydrogen bonds, and Gibb's free energy landscape analysis suggested greater affinity of the compounds 5e and 5m toward Als4 protein.

Antibiofilm treatment, particularly drug-containing wound healing dressings, does not typically penetrate the robust protective extracellular polymeric substance of biofilm and eradicate the bacteria. Here, a rational design of nitric oxide (NO) donor N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine (BNN6)-based injectable hydrogel, is reported in which the NO release can be triggered by a photothermal effect owing to semiconducting perylene diimide (PDI) J-aggregation fibers. The synthetic PDI derivatives self-assembling into 0D nanoparticles and then aggregating to 1D J fiber is accompanied by absorbance red-shifting from 700 to 790 nm and then to 852 nm. After encapsulating BNN6, a "sandwich roll" (SR) like structure is evenly crosslinked into an injectable hydrogel (SRH) exhibiting a high photothermal convenience efficiency of 72%, which enables the SRH to achieve highly efficient photocontrol NO release. The SRH shows excellent injectability, shape adaptability, and effective antibacterial efficacy over 99% to the E.coli and S. aureus. and remarkable in vivo antibiofilm efficiency of 99.58% by laser irradiation. Furthermore, the synergistic treatment displays the ability to eliminate inflammation, facilitate angiogenesis, and promote collagen deposition, thereby significantly stimulating the healing process of wounds. The semiconducting J-aggregation injectable hydrogel can be a versatile strategy for the treatment of biofilm.