Biofouling remains a significant challenge in water treatment fields, leading to a decline in the hydraulic performance, increased operational costs, and potential health risks. Previous biofouling control strategies primarily focused on the removal of particulates and microorganisms, often neglecting the role of extracellular proteins. Using a reclaimed water distribution system as an example, this study proposes a strategy to inhibit biofouling formation by utilizing urea, a reported protein denaturant with fertilizer functionality. Results indicated that urea significantly slowed the accumulation of biofouling, leading to a 16.4-49.4 % decrease in biofouling weight, an 18.6-55.3 % decrease in extracellular protein content, and a 25.9-45.3 % reduction in extracellular polymer substance (EPS) content. Urea mitigated biofouling through two mechanisms: (1) disrupting protein structures, which convert tightly bound EPS to loosely bound EPS, and (2) downregulating biofilm-forming signaling proteins, thereby inhibiting biofouling formation. In the process, proteins, polysaccharides, and microorganisms exhibited clear mutual promotion relationships. Additionally, urea weakened microbial symbiotic interactions by affecting protein signaling molecules, inhibiting microbial growth and polysaccharide metabolism. The research confirms that denaturing extracellular proteins to mitigate biofouling is a feasible and efficient approach. The findings aim to provide valuable insights for the development of sustainable and effective biofouling cleaning strategies.

Microbial fuel cells (MFCs) has attracted tremendous attention due to integrating clean energy generation and wastewater treatment. Electricigens are in charge of electron transfer and energy conversion, and therefore strain improvement is crucial for MFCs performance. Herein, the overexpression of sigma factor RpoF and the combined manipulation with other regulators (PmpR and RpoS) reinforced electricity generation of a model strain Pseudomonas aeruginosa PAO1. Next, RpoF was introduced into an isolate P. aeruginosa P2-A-12 with higher electroactivity, which not only yielded 3.2-fold increase in the maximal power density under non stress, but also generated 21.4 % improvement under 1.5 % NaCl. The comprehensive analysis at the levels of cells, metabolites and genes transcription ascertained its global regulatory mechanism, mainly including the enhanced biofilm formation by promoting cell attachment and cell-to-cell adhesion on the anode, more c-di-GMP and quorum sensing (QS) signal molecules accumulation, and the increase in phenazine-1-carboxamide (PCN), pyocyanin (PYO) and 1-hydroxyphenazine (1-OHPHZ) by controlling the expression levels of multiple genes involved in core biosynthesis and QS system. It was the first time to demonstrate the direct activation of RpoF on phzH responsible for PCN production and rhlR regulating N-butanoyl-HSL (C4-HSL) synthesis. Bioinformatic analysis indicated that the complex biological function of RpoF was closely linked with the conservation and diversity of sequences from various microorganisms. These findings strongly substantiated that RpoF acted as an efficient element to simultaneously optimize P. aeruginosa traits (such as electroactivity and stress tolerance) suitable for the practical MFCs, also broadened the theoretical recognition on its regulatory mechanism.

Synthesizing the microbial community with a high denitrifying capacity is the key for achieving efficient removal of nitrogen species in wastewater treatment plants. Here, we integrated the evolutionary top-down enrichment and bottom-up bioaugmentation to construct a high-efficiency Pseudomonas-recruited denitrifying consortium (PRDC). A PRDC with a high specific denitrification rate of 109.49 ± 10.58 mg N/(g MLVSS·h) was enriched after 181 days of microbiota construction with pre-inoculation of Pseudomonas strain onto carriers. The 16S rRNA gene sequencing analysis suggested that the pre-inoculated Pseudomonas was quickly washed out and replaced by dominant denitrifying genera, such as Halomonas and Thauera, under different hydraulic retention times (HRTs). The pre-inoculated Pseudomonas can facilitate PRDC by providing public goods, but compromising its nutrient requirements. The dominant community assembly processes switched from homogeneous selection to ecological drift and dispersal limitation under shortened HRT. Furthermore, a shortened HRT facilitated the colonization of new immigrants and intensified their competition with the pre-existing dominant denitrifiers. The PRDC carriers achieved a 1.65-fold enhancement in sludge denitrification and reduced the corresponding chemical oxygen demand consumption at a carrier filling ratio of 30%. Overall, our study developed a novel technique using Pseudomonas aeruginosa as a trigger to enrich high-efficiency denitrifying consortia for wastewater treatment.

Plastic pollution, particularly from polyethylene terephthalate (PET), has become a significant environmental concern, necessitating innovative and sustainable degradation strategies. The present study provides valuable perspectives on the genomic and functional characteristics of Brucella intermedia IITR130, a bacterium capable of degrading PET. Hybrid genome sequencing of IITR130 resulted in identification of two chromosomes combining 4.59 Mbp size. Genomic annotation revealed occurrence of key enzymes involved in the PET sheet biodegradation pathway, including hydrolases, ring hydroxylating dioxygenases, protocatechuate 3,4 dioxygenases, genes for metabolism of several other natural and synthetic plastic. A hydrolase gene Hy1 of 24 kDa, was identified, expressed, and characterized, demonstrating an optimal catalytic activity at 37 °C and pH 8.5. Scanning electron microscopy (SEM) and fourier-transform infrared spectroscopy (FTIR) confirmed substantial degradation of PET surfaces treated with Hy1 protein, resulted in surface erosion, crack formation, and functional group modifications in the range 2150-2550 cm⁻1 and 2950-3350 cm⁻1 suggestive of O=C=O stretching and O-H stretching respectively. Monomethyl terephthalate (MMT) and terephthalic acid (TPA) were identified as PET degradation metabolites formed by strain IITR130. Fluorescence quenching showed higher substrate affinity for bis(2-hydroxyethyl) terephthalate (BHET) (Kd = 148.2) than terephthalic acid (TPA) (Kd = 674). Moreover, phylogenetic analysis of Hy1 protein revealed that Hy1 containing conserved catalytic triad (Ser108, His188, Asp155) belonging to the family III of hydrolase enzyme sharing a clade with PET degrading hydrolase PETase from Ideonella sakaiensis. These results demonstrate the potential of B. intermedia IITR130 as an efficient biocatalyst for PET biodegradation which could be exploited appropriately for plastic waste management.

Pseudomonas aeruginosa is an opportunistic human pathogenic bacterium that is a common cause of both acute and chronic infections. Multidrug-resistant P. aeruginosa poses a significant challenge to antibiotics and therapeutic approaches due to its pathogenicity, virulence, and biofilm-forming ability mediated by quorum sensing. Understanding the pathogenic mechanisms is essential for developing potential drug targets. In this regard, strategies aimed at combating the targeted inhibition of virulence, quorum sensing pathways, secretion systems, biofilm-associated two-component systems, and signalling system regulators (such as c-di-GMP) associated with biofilm formation are critical. Several new antimicrobial agents have been developed using these strategies, including antimicrobial peptides, bacteriophages, nanoantibiotics, photodynamics, and natural products, which are considered promising therapeutic tools. In this review, we address the concept of biofilms, their regulation, and recent treatment strategies to target P. aeruginosa, a clinically significant pathogen known for biofilm formation.

Candida albicans stands as the foremost prevalent human commensal pathogen and a significant contributor to nosocomial fungal infections. In the metabolism of C. albicans, alcohol dehydrogenase 1 (Adh1) is one of the important enzymes that converts acetaldehyde produced by pyruvate decarboxylation into ethanol at the end of glycolysis. Leveraging the foundational processes of alcoholic fermentation, Adh1 plays an active role in multiple biological phenomena, including biofilm formation, interactions between different species, the development of drug resistance, and the potential initiation of gastrointestinal cancer. Additionally, Adh1 within C. albicans has demonstrated associations with regulating the cell cycle, stress responses, and various intracellular states. Furthermore, Adh1 is extracellularly localized on the cell wall surface, where it plays roles in processes such as tissue invasion and host immune responses. Drawing from an analysis of ADH1 gene structure, expression patterns, and fundamental functions, this review elucidates the intricate connections between Adh1 and various biological processes within C. albicans, underscoring its potential implications for the prevention, diagnosis, and treatment of candidiasis.

A major biofilm matrix determinant of Pseudomonas aeruginosa is the partially deacetylated α-1,4 linked N-acetylgalactosamine polymer, Pel. After synthesis and transport of the GalNAc polysaccharide across the inner membrane, PelA partially deacetylates and hydrolyzes Pel before its export out of the cell via PelB. While the Pel modification and export proteins are known to interact in the periplasm, it is unclear how the interaction of PelA and PelB coordinates these processes. To determine how PelA modifies the polymer, we determined its structure to 2.1 Å and found a unique arrangement of four distinct domains. We have shown previously that the hydrolase domain exhibits endo-α-1,4-N-acetylgalactosaminidase activity. Characterization of the deacetylase domain revealed that PelA is the founding member of a new carbohydrate esterase family, CE21. Further, we found that the PelAB interaction enhances the deacetylation of N-acetylgalactosamine oligosaccharides. Using the PelA structure in conjunction with AlphaFold2 modeling of the PelAB complex, we propose a model wherein PelB guides Pel to the deacetylase domain of PelA and subsequently to the porin domain of PelB for export. Perturbation or loss of the PelAB interaction would result in less efficient deacetylation and potentially increased Pel hydrolysis. In PelA homologs across many phyla, the predicted structure and active sites are conserved, suggesting a common modification mechanism in Gram-negative bacterial species containing a functional pel operon.

Pseudomonas aeruginosa is an opportunistic bacterium widely distributed in both natural and urban environments, playing a crucial role in global microbial ecology. This article reviews the interactive dynamics of P. aeruginosa across different ecosystems, highlighting its capacity for adaptation and resistance in response to environmental and therapeutic pressures. We analyze the mechanisms of antibiotic resistance, including the presence of resistance genes and efflux systems, which contribute to its persistence in both clinical and nonclinical settings. The interconnection between human, animal, and environmental health, within the context of the One Health concept, is discussed, emphasizing the importance of monitoring and sustainable management practices to mitigate the spread of resistance. Through a holistic approach, this work offers insights into the influence of P. aeruginosa on public health and biodiversity.

The reason why certain bacteria, e.g., Pseudomonas aeruginosa (PA), produce acetylated alginate (Alg) in their biofilms remains one of the most intriguing facts in microbiology. Being the main structural component of the secreted biofilm, like the one formed in the lungs of cystic fibrosis (CF) patients, Alg plays a crucial role in protecting the bacteria from environmental stress and potential threats. Nonetheless, to investigate the PA biofilm environment and its lack of susceptibility to antibiotic treatment, the currently developed in vitro biofilm models use native seaweed Alg, which is a non-acetylated Alg. The role of the acetyl side group on the backbone of bacterial Alg has never been elucidated, and the transposition of experimental results obtained from such systems to clinical conditions (e.g., to treat CF-infection) may be hazardous. We systematically investigated the influence of acetylation on the physico-chemical and mechanical properties of Alg in solution and Ca2+-crosslinked hydrogels. Furthermore, we assessed how the acetylation influenced the interaction of Alg with tobramycin, a common aminoglycoside antibiotic for PA. Our study revealed that the degree of acetylation directly impacts the viscosity and Young's Modulus of Alg in a pH-dependent manner. Acetylation increased the mesh size in biofilm-like Alg hydrogels, directly influencing antibiotic penetration. Our results provide essential insights to create more clinically relevant in vitro infection models to test the efficacy of new drugs or to better understand the 3D microenvironment of PA biofilms.

Antimicrobial resistance in Pseudomonas aeruginosa represents a major global challenge in public and veterinary health, particularly from a One Health perspective. This study aimed to investigate antimicrobial resistance, the presence of virulence genes, and the genetic diversity of P. aeruginosa isolates from diverse sources.

The study utilized antimicrobial susceptibility testing, genomic analysis for resistance and virulence genes, and multilocus sequence typing to characterize a total of 737 P. aeruginosa isolates that were collected from humans, domestic animals, and aquatic environments in Northern Portugal. Antimicrobial resistance profiles were analyzed, and genomic approaches were employed to detect resistance and virulence genes. The study found a high prevalence of multidrug-resistant isolates, including high-risk clones such as ST244 and ST446, particularly in hospital sources and wastewater treatment plants. Key genes associated with resistance and virulence, including efflux pumps (e.g. MexA and MexB) and secretion systems (T3SS and T6SS), were identified.

This work highlights the intricate dynamics of multidrug-resistant P. aeruginosa across interconnected ecosystems in Northern Portugal. It underscores the importance of genomic studies in revealing the mechanisms of resistance and virulence, contributing to the broader understanding of resistance dynamics and informing future mitigation strategies.

Bacterial biofilm infections are the root cause of persistent infections and the prevalence of resistance to specific or multiple antibiotics. Biofilms have unique features that provide a protective environment for bacteria under various stress conditions and contribute significantly to the pathogenesis of chronic infections. They cover bacterial cells with a self-produced extracellular polymeric matrix, effectively hiding the bacterial cells and their targets. Conventional therapies cannot effectively treat and control bacterial biofilm infections. Therefore, advanced therapeutic means like microneedles, targeted tissue therapy, phage therapy, nanodrug therapy, combination drug therapy, microbial therapy, and immune cell hijacking therapy are needed to tackle the complex issue. These advanced therapies have shown promising results not only in bacterial biofilm infections but also in diseases such as cancer and genetic disorders. Due to their unique features and mechanisms, they significantly contribute to preventing bacterial infections by disrupting biofilm. This article aims to serve as a comprehensive overview of the ongoing battle against biofilms with transformative therapies. This article compiles advancements in new therapies that have demonstrated effective roles in the disruption of bacterial biofilms. We also discuss the current developments and Food and Drug Administration-approved status of these therapies. Additionally, this article summarizes the limitations and future steps needed for these therapies in the field of bacterial biofilm prevention. Thus, these therapies represent the future of preventing bacterial biofilm infections and could be also effective in the reversal of resistance.

Antibiotic resistance and the persistence of sessile cells within biofilms complicate the eradication of biofilm-related infections using conventional antibiotics. This highlights the necessity for alternate therapy methods. The objective of this study was to investigate the biofilm destruction activity of α-tocopherol against Staphylococcus aureus, Proteus mirabilis, and Pseudomonas aeruginosa on polystyrene. α-Tocopherol showed significant biofilm destruction activity on the pre-formed biofilms of S. aureus (45%-46%), Pr. mirabilis (42%-54%), and Ps. aeruginosa (28%). Resazurin assay showed that α-tocopherol disrupted all bacterial biofilms without interfering with their cell viability. Scanning electron microscope images showed lower bacterial cell count and less compacted cell aggregates on polystyrene surfaces after treatment with α-tocopherol. This study demonstrated the biofilm destruction activity of α-tocopherol against S. aureus, Pr. mirabilis, and Ps. aeruginosa. α-Tocopherol could potentially be used to decrease biofilm-associated infections of these bacteria.

Pseudomonas aeruginosa, a common cause of morbidity in cystic fibrosis, chronically infects the patient's lungs by forming an alginate-containing biofilm. Alginate lyases are polysaccharide lyases that lyse alginate and are, therefore, potential biofilm-disruptive agents. However, cystic fibrosis sputum contains high levels of metals such as iron and zinc. The efficacy of alginate lyases under these conditions of elevated metal concentrations has not been categorically determined. Here, we have assessed the enzyme activity of two exolytic and five endolytic alginate lyases in the presence of metal ions (Fe2+, Zn2+, Mn2+, Mg2+, Ca2+, Ni2+, Cu2+) elevated in the cystic fibrosis lung milieu. Several of these alginate lyases exhibited increased activity in the presence of Ca2+, and the polysaccharide lyase family 7 members studied here exhibited decreased activity in the presence of Zn2+. The enzyme activity of the PL7 alginate lyases from Cellulophaga algicola (CaAly/CaFLDAly) and Vibrio sp. (VspAlyVI) was not affected in the presence of a mix of all the above-mentioned metal ions at the elevated concentrations found in the cystic fibrosis lung milieu. Specific alginate lyases might, therefore, retain the ability to degrade the alginate-containing Pseudomonas biofilm in the presence of metal ions elevated in the cystic fibrosis lung milieu.

Antimicrobial resistance, the biggest issue facing the global healthcare sector, quickly emerged and spread due to the frequent use of antibiotics in regular treatments. The investigation of polymer-based nanomaterials as possible antibiofilm treatment agents is prompted by the growing ineffectiveness of conventional therapeutic techniques against these resistant bacteria species. So far, several articles have been published on microbial biofilm eradication using various polymer-based nanomaterials due to their therapeutic efficacy and biocompatibility nature. Despite their potential, a comprehensive review of the chitosan-based hybrid nanomaterials to treat microbial biofilms and their infections is lacking. This review provides a comprehensive investigation of the current state of therapeutic efficacy, various nanoformulations, advantages, limitations, and regulations of chitosan-based hybrid nanomaterials for biofilm treatment. Special attention is given to the application of chitosan-based nanomaterials in wound care, urinary tract infections, and dental biofilms are discussed, highlighting their role in managing biofilm-associated complications. Researchers will be better able to comprehend and develop unique, marketable chitosan-based nanomaterials with increased activity to treat biofilm infections in near future with the aid of this review.

Cationic biocides (CBs), which include quaternary ammonium compounds (QACs), are employed to mitigate the spread of infectious bacteria, but resistance to such surface disinfectants is rising. CB exposure can have profound phenotypic implications that extend beyond allowing microorganisms to persist on surfaces. Pseudomonas aeruginosa is a deadly bacterial pathogen that is intrinsically tolerant to a wide variety of antimicrobials and is commonly spread in healthcare settings. In this study, we pursued resistance selection assays to the QAC benzalkonium chloride and quaternary phosphonium compound P6P-10,10 to assess the phenotypic effects of CB exposure in P. aeruginosa PAO1 and four genetically diverse, drug-resistant clinical isolates. In particular, we sought to examine how CB exposure affects defensive strategies and the virulence-associated "offensive" strategies in P. aeruginosa. We demonstrated that development of resistance to BAC is associated with increased production of virulence-associated pigments and alginate as well as pellicle formation. In an in vivo infection model, CB-resistant PAO1 exhibited a decreased level of virulence compared to wild type, potentially due to an observed fitness cost in these strains. Taken together, these results illustrate the significant consequence CB resistance exerts on the virulence-associated phenotypes of P. aeruginosa.

Bacteriophages, the natural predators of bacteria, are incredibly potent candidates to counteract antimicrobial resistance (AMR). However, the rapid development of phage-resistant mutants challenges the potential of phage therapy. Understanding the mechanisms of bacterial adaptations to phage predation is crucial for phage-based prognostic applications. Phage cocktails and combinatorial therapy, using optimized dosage patterns of antibiotics, can negate the development of phage-resistant mutations and prolong therapeutic efficacy. In this study, we describe the characterization of a novel bacteriophage and the physiology of phage-resistant mutant developed during infection. M12PA is a P. aeruginosa-infecting bacteriophage with Myoviridae morphology. We observed that prolonged exposure of P. aeruginosa to M12PA resulted in the selection of phage-resistant mutants. Among the resistant mutants, pyomelanin-producing mutants, named PA-M, were developed at a frequency of 1 in 16. Compared to the wild-type, we show that PA-M mutant is severely defective in virulence properties, with altered motility, biofilm formation, growth rate, and antibiotic resistance profile. The PA-M mutant exhibited reduced pathogenesis in an allantoic-infected chick embryo model system compared to the wild-type. Finally, we provide evidence that combinatory therapy, combining M12PA with antibiotics or other phages, significantly delayed the emergence of resistant mutants. In conclusion, our study highlights the potential of combinatory phage therapy to delay the development of phage-resistant mutants and enhance the efficacy of phage-based treatments against P. aeruginosa.

Bacterial cells often exist in the form of sessile aggregates known as biofilms, which are polymicrobial in nature and can produce slimy Extracellular Polymeric Substances (EPS). EPS is often referred to as a biofilm matrix and is a heterogeneous mixture of various biomolecules such as polysaccharides, proteins, and extracellular DNA/RNA (eDNA/RNA). In addition, bacteriophage (phage) was also found to be an integral component of the matrix and can serve as a protective barrier. In recent years, the roles of proteins, polysaccharides, and phages in the virulence of biofilms have been well studied. However, a mechanistic understanding of the release of such biomolecules and their interactions with antimicrobials requires a thorough review. Therefore, this article critically reviews the various mechanisms of release of matrix polymers. In addition, this article also provides a contemporary understanding of interactions between various biomolecules to protect biofilms against antimicrobials. In summary, this article will provide a thorough understanding of the functions of various biofilm matrix molecules.

Pseudomonas aeruginosa is well-known for its antimicrobial resistance and the ability to survive in harsh environmental conditions due to an abundance of resistance mechanisms, including the formation of biofilms and the production of exopolysaccharides. Exopolysaccharides are among the major components of the extracellular matrix in biofilms and aggregates of P. aeruginosa. Although their contribution to antibiotic resistance has been previously shown, their roles in resistance to oxidative stressors remain largely elusive. Here, we studied the function of the exopolysaccharides Psl and Pel in the resistance of P. aeruginosa to the commonly used disinfectants and strong oxidizing agents NaOCl and H2O2. We observed that the simultaneous inactivation of Psl and Pel in P. aeruginosa PAO1 mutant strain ∆pslA pelF resulted in a significant increase in susceptibility to both NaOCl and H2O2. Further analyses revealed that Pel is more important for oxidative stress resistance in P. aeruginosa and that the form of Pel (i.e., cell-associated or cell-free) did not affect NaOCl susceptibility. Additionally, we show that Psl/Pel-negative strains are protected against oxidative stress in co-culture biofilms with P. aeruginosa PAO1 WT. Taken together, our results demonstrate that the EPS matrix and, more specifically, Pel exhibit protective functions against oxidative stressors such as NaOCl and H2O2 in P. aeruginosa.

Biofilms are microbial communities of cells embedded in a self-produced polymeric matrix composed of polysaccharides, proteins, lipids, and extracellular DNA. Biofilm bacteria have been shown to possess unique characteristics, including increased stress resistance and higher antimicrobial tolerance, leading to failures in bacterial eradication during chronic infections or in technical settings, including drinking and wastewater industries. Previous studies have shown that in addition to conferring structure and stability to biofilms, the polysaccharides Psl and Pel are also involved in antibiotic resistance. This work provides evidence that these biofilm matrix components also contribute to the resistance of Pseudomonas aeruginosa to oxidative stressors including the widely used disinfectant NaOCl. Understanding the mechanisms by which bacteria escape antimicrobial agents, including strong oxidants, is urgently needed in the fight against antimicrobial resistance and will help in developing new strategies to eliminate resistant strains in any environmental, industrial, and clinical setting.

Reactive chlorine species (RCS) like sodium hypochlorite (NaOCl) are potent oxidizing agents and widely used biocides in surface disinfection, water treatment, and biofilm elimination. Moreover, RCS are also produced by the human immune system to kill invading pathogens. However, bacteria have developed mechanisms to survive the damage caused by RCS. Using the comprehensive Pseudomonas aeruginosa PA14 transposon mutant library in a genetic screen, we identified a total of 28 P. aeruginosa PA14 mutants whose biofilms showed increased susceptibility to NaOCl in comparison to PA14 WT biofilms. Of these, ten PA14 mutants with a disrupted apaH, PA0793, acsA, PA1506, PA1547, PA3728, yajC, queA, PA3869, or PA14_32840 gene presented a 4-fold increase in NaOCl susceptibility compared to wild-type biofilms. While none of these mutants showed a defect in biofilm formation or attenuated susceptibility of biofilms toward the oxidant hydrogen peroxide (H2O2), all but PA14_32840 also exhibited a 2-4-fold increase in susceptibility toward the antibiotic ciprofloxacin. Further analyses revealed attenuated levels of intracellular ROS and catalase activity only for the apaH and PA1547 mutant, providing insights into the oxidative stress response in P. aeruginosa biofilms. The findings of this paper highlight the complexity of biofilm resistance and the intricate interplay between different mechanisms to survive oxidative stress. Understanding resistance strategies adopted by biofilms is crucial for developing more effective ways to fight resistant bacteria, ultimately contributing to better management of bacterial growth and resistance in clinical and environmental settings.

Bacterial biofilm formation and attachment to hosts are mediated by carbohydrate-binding lectins, exopolysaccharides, and their interactions in the extracellular matrix (ECM). During tomato infection Ralstonia pseudosolanacearum (Rps) GMI1000 highly expresses three lectins: LecM, LecF, and LecX. The latter two are uncharacterized. We evaluated the roles in bacterial wilt disease of LecF, a fucose-binding lectin, LecX, a xylose-binding lectin, and the Rps exopolysaccharide EPS I. Interestingly, single and double lectin mutants attached to tomato roots better and formed more biofilm under static conditions in vitro. Consistent with this finding, static bacterial aggregation was suppressed by heterologous expression of lecFGMI1000 and lecXGMI1000 in other Ralstonia strains that naturally lack these lectins. Crude ECM from a ΔlecF/X double mutant was more adhesive than the wild-type ECM, and LecF and LecX increased Rps attachment to ECM. The enhanced adhesiveness of the ΔlecF/X ECM could explain the double mutant's hyper-attachment in static conditions. Unexpectedly, mutating lectins decreased Rps attachment and biofilm viscosity under shear stress, which this pathogen experiences in plant xylem. LecF, LecX, and EPS I were all essential for biofilm development in xylem fluid flowing through cellulose-coated microfluidic channels. These results suggest that under shear stress, LecF and LecX increase Rps attachment by interacting with the ECM and plant cell wall components like cellulose. In static conditions such as on root surfaces and in clogged xylem vessels, the same lectins suppress attachment to facilitate pathogen dispersal. Thus, Rps lectins have a dual biological function that depends on the physical environment.

Biofilm is a community of microorganisms attached to the substrate and plays a significant role in microbial pathogenesis and medical device-related infection. Pseudomonas aeruginosa (PA) is a highly infectious gram-negative opportunistic biofilm-forming bacterium with high antibiotic resistance. Several reports underscore the antimicrobial activity of natural and synthetic food coloring agents, including carmoisine, turmeric dye, red amaranth dye, and phloxine B. However, their ability to suppress the PA biofilm is not clearly understood. Carmoisine is a red-colored synthetic azo dye containing naphthalene subunits and sulfonic groups and is widely used as a food coloring agent. This study investigated the antibiofilm potential and possible mechanism of biofilm inhibition by carmoisine against PA. Computational studies through molecular docking revealed that carmoisine strongly binds to QS regulator LasR (-12.7) and relatively less strongly but significantly with WspR (-6.9). Further analysis of the docked LasR-carmoisine complex using 100 ns MD simulation (Desmond, Schrödinger) validated the bonding strength and stability. Crystal violet assay, triphenyl tetrazolium chloride salt assay, and confocal microscopic studies were adopted for biofilm quantification, and the results indicated the dose-dependent antibiofilm activity of carmoisine against PA. We hypothesise that the carmoisine-mediated reduction of biofilm in PA is due to its interaction with LasR and interference with the QS system. The computational and biochemical analysis of another compound, 1,2-naphthoquinone-4-sulphonic acid, reiterated the role of the naphthalene ring in biofilm inhibition. Hence, this work will pave the way for the future discovery of antibiofilm drugs based on naphthalene ring-based lead compounds.Communicated by Ramaswamy H. Sarma.

Cadmium (Cd) contamination poses a significant threat to agricultural soils and food safety, necessitating effective remediation strategies. Salix species, with their high coverage and Cd accumulating capacity, hold promise for remediation efforts. The rhizosphere microbiome is crucial for enhancing Cd accumulating capacity for Salix. However, the mechanisms by how Salix interacts with its rhizosphere microbiome to enhance Cd extraction remains poorly understood. In this study, we compared the remediation performance of two Salix ecotypes: 51-3 (High Cd-accumulating Ecotype, HAE) and P646 (Low Cd-accumulating Ecotype, LAE). HAE exhibited notable advantages over LAE, with 10.80 % higher plant height, 43.80 % higher biomass, 20.26 % higher Cd accumulation in aboveground tissues (93.09 μg on average), and a superior Cd translocation factor (1.97 on average). Analysis of the rhizosphere bacterial community via 16S rRNA amplicon sequencing revealed that HAE harbored a more diverse bacterial community with a distinct composition compared to LAE. Indicator analysis identified 84 genera specifically enriched in HAE, predominantly belonging to Proteobacteria, Actinobacteria, and Firmicutes, including beneficial microbes such as Streptomyces, Bacillus, and Pseudomonas. Network analysis further elucidated three taxa groups specifically recruited by HAE, which were highly correlated with functional genes that associated with biosynthesis of secondary metabolites, glycan biosynthesis and metabolism, and metabolism of cofactors and vitamins. These functions contribute to enhancing plant growth, Cd uptake, and resistance to Cd in Salix. Overall, our findings highlight the importance of the rhizosphere microbiome in facilitating Cd extraction and provide insights into microbiome-based strategies for sustainable agricultural practices.

The ability of bacteria to colonize diverse environmental niches is often linked to their competence in biofilm formation. It depends on the individual characteristics of a strain, the nature of the colonized surface (abiotic or biotic), or the availability of certain nutrients. Pseudomonas donghuensis P482 efficiently colonizes the rhizosphere of various plant hosts, but a connection between plant tissue colonization and the biofilm formation ability of this strain has not yet been established. We demonstrate here that the potential of P482 to form biofilms on abiotic surfaces and the structural characteristics of the biofilm are influenced by the carbon source available to the bacterium, with glycerol promoting the process. Also, the type of substratum, polystyrene or glass, impacts the ability of P482 to attach to the surface. Moreover, P482 mutants in genes associated with motility or chemotaxis, the synthesis of polysaccharides, and encoding proteases or regulatory factors, which affect biofilm formation on glass, were fully capable of colonizing the root tissue of both tomato and maize hosts. Investigating the role of cellular factors in biofilm formation using these plant-associated bacteria shows that the ability of bacteria to form biofilm on abiotic surfaces does not necessarily mirror its ability to colonize plant tissues. Our research provides a broader perspective on the adaptation of these bacteria to various environments.

Pseudomonas aeruginosa is an opportunistic pathogen causing acute and chronic infections, especially in immunocompromised patients. Its remarkable adaptability and resistance to various antimicrobial treatments make it difficult to eradicate. Its persistence is enabled by its ability to form a biofilm. Biofilm is a community of sessile micro-organisms in a self-produced extracellular matrix, which forms a scaffold facilitating cohesion, cell attachment, and micro- and macro-colony formation. This lifestyle provides protection against environmental stresses, the immune system, and antimicrobial treatments, and confers the capacity for colonization and long-term persistence, often characterizing chronic infections. In this review, we retrace the events of the life cycle of P. aeruginosa biofilm, from surface perception/contact to cell spreading. We focus on the importance of extracellular appendages, mechanical constraints, and the kinetics of matrix component production in each step of the biofilm life cycle.

Evaluate the effects of five disinfection methods on bacterial concentrations in hospital sink drains, focusing on three opportunistic pathogens (OPs): Serratia marcescens, Pseudomonas aeruginosa and Stenotrophomonas maltophilia.

Over two years, three sampling campaigns were conducted in a neonatal intensive care unit (NICU). Samples from 19 sink drains were taken at three time points: before, during, and after disinfection. Bacterial concentration was measured using culture-based and flow cytometry methods. High-throughput short sequence typing was performed to identify the three OPs and assess S. marcescens persistence after disinfection at the genotypic level.

This study was conducted in a pediatric hospitals NICU in Montréal, Canada, which is divided in an intensive and intermediate care side, with individual rooms equipped with a sink.

Five treatments were compared: self-disinfecting drains, chlorine disinfection, boiling water disinfection, hot tap water flushing, and steam disinfection.

This study highlights significant differences in the effectiveness of disinfection methods. Chlorine treatment proved ineffective in reducing bacterial concentration, including the three OPs. In contrast, all other drain interventions resulted in an immediate reduction in culturable bacteria (4-8 log) and intact cells (2-3 log). Thermal methods, particularly boiling water and steam treatments, exhibited superior effectiveness in reducing bacterial loads, including OPs. However, in drains with well-established bacterial biofilms, clonal strains of S. marcescens recolonized the drains after heat treatments.

Our study supports thermal disinfection (>80°C) for pathogen reduction in drains but highlights the need for additional trials and the implementation of specific measures to limit biofilm formation.

Antimicrobial resistance (AMR) is a silent critical issue that poses several challenges to health systems. While the discovery of novel antibiotics is currently stalled and prevalently focused on chemical variations of the scaffolds of available drugs, novel targets and innovative strategies are urgently needed to face this global threat. In this context, bacterial G-quadruplexes (G4s) are emerging as timely and profitable targets for the design and development of antimicrobial agents. Indeed, they are expressed in regulatory regions of bacterial genomes, and their modulation has been observed to provide antimicrobial effects with translational perspectives in the context of AMR. In this work, we review the current knowledge of bacterial G4s as well as their modulation by small molecules, including tools and techniques suitable for these investigations. Finally, we critically analyze the needs and future directions in the field, with a focus on the development of small molecules as bacterial G4s modulators endowed with remarkable drug-likeness.

The prevalence of carbapenem-resistant P. aeruginosa has dramatically increased over the last decade, and antibiotics alone are not enough to eradicate infections caused by this opportunistic pathogen. Phage therapy is a fresh treatment that can be administered under compassionate use, particularly against chronic cases. However, it is necessary to thoroughly characterize the virus before therapeutic application. Our work describes the discovery of the novel sequenced bacteriophage, vB_PaeP-F1Pa, containing an integrase, performs a phylogenetical analysis, describes its stability at a physiological pH and temperature, latent period (40 min), and burst size (394 ± 166 particles per bacterial cell), and demonstrates its ability to infect MDR and XDR P. aeruginosa strains. Moreover, this novel bacteriophage was able to inhibit the growth of bacteria inside preformed biofilms. The present study offers a road map to analyze essential areas for successful phage therapy against MDR and XDR P. aeruginosa infections, and shows that a phage containing an integrase is also able to show good in vitro results, indicating that it is very important to perform a genomic analysis before any clinical use, in order to prevent adverse effects in patients.

Polycyclic aromatic hydrocarbons (PAHs) are considered substances of potential human health hazards because of their resistance to biodegradation and carcinogenic index. Chrysene is a PAH with a high molecular weight (HMW) that poses challenges for its elimination from the environment. However, bacterial degradation is an effective, environmentally friendly, and cost-effective solution. In our study, we isolated a potential chrysene-degrading bacteria from crude oil-contaminated seawater (Bizerte, Tunisia). Based on 16SrRNA analysis, the isolate S5 was identified as Achromobacter aegrifaciens. Furthermore, the results revealed that A. aegrifaciens S5 produced a biofilm on polystyrene at 20 °C and 30 °C, as well as at the air-liquid (A-L) interface. Moreover, this isolate was able to swim and produce biosurfactants with an emulsification activity (E24%) over 53%. Chrysene biodegradation by isolate S5 was clearly assessed by an increase in the total viable count. Confirmation was obtained via gas chromatography-mass spectrometry (GC-MS) analyses. A. aegrifaciens S5 could use chrysene as its sole carbon and energy source, exhibiting an 86% degradation of chrysene on day 7. In addition, the bacterial counts reached their highest level, over 25 × 1020 CFU/mL, under the conditions of pH 7.0, a temperature of 30 °C, and a rotary speed of 120 rpm. Based on our findings, A. aegrifaciens S5 can be a potential candidate for bioremediation in HMW-PAH-contaminated environments.

Pseudomonas aeruginosa is an opportunistic pathogen found in a wide variety of environments, including soil, water, and habitats associated with animals, humans, and plants. From a One Health perspective, which recognizes the interconnectedness of human, animal, and environmental health, it is important to study the virulence characteristics and antibiotic susceptibility of environmental bacteria. In this study, we compared the virulence properties and the antibiotic resistance profiles of seven isolates collected from the Gulf of Mexico with those of seven clinical strains of P. aeruginosa. Our results indicate that the marine and clinical isolates tested exhibit similar virulence properties; they expressed different virulence factors and were able to kill Galleria mellonella larvae, an animal model commonly used to analyze the pathogenicity of many bacteria, including P. aeruginosa. In contrast, the clinical strains showed higher antibiotic resistance than the marine isolates. Consistently, the clinical strains exhibited a higher prevalence of class 1 integron, an indicator of anthropogenic impact, compared with the marine isolates. Thus, our results indicate that the P. aeruginosa marine strains analyzed in this study, isolated from the Gulf of Mexico, have similar virulence properties, but lower antibiotic resistance, than those from hospitals.

New antimicrobial molecules effective against Pseudomonas aeruginosa, known as an antibiotic-resistant "high-priority pathogen", are urgently required because of its ability to develop biofilms related to healthcare-acquired infections. In this study, for the first time, the anti-biofilm and anti-virulence activities of a polyphenolic extract of extra-virgin olive oil as well as purified oleocanthal and oleacein, toward P. aeruginosa clinical isolates were investigated. The main result of our study was the anti-virulence activity of the mixture of oleacein and oleocanthal toward multidrug-resistant and intermediately resistant strains of P. aeruginosa isolated from patients with ventilator-associated pneumonia or surgical site infection. Specifically, the mixture of oleacein (2.5 mM)/oleocanthal (2.5 mM) significantly inhibited biofilm formation, alginate and pyocyanin production, and motility in both P. aeruginosa strains (p < 0.05); scanning electron microscopy analysis further evidenced its ability to inhibit bacterial cell adhesion as well as the production of the extracellular matrix. In conclusion, our results suggest the potential application of the oleacein/oleocanthal mixture in the management of healthcare-associated P. aeruginosa infections, particularly in the era of increasing antimicrobial resistance.

The expanding urbanization of coastal areas has led to increased ocean sprawl, which has had both physical and chemical adverse effects on marine and coastal ecosystems. To maintain the health and functionality of these ecosystems, it is imperative to develop effective solutions. One such solution involves the use of biodegradable polymers as bioactive coatings to enhance the bioreceptivity of marine and coastal infrastructures. Our study aimed to explore two main objectives: (1) investigate PHA-degrading bacteria on polymer-coated surfaces and in surrounding seawater, and (2) comparing biofilm colonization between surfaces with and without the polymer coating. We applied poly(3-hydroxybutyrate) [P(3HB)) coatings on concrete surfaces at concentrations of 1% and 6% w/v, with varying numbers of coating cycles (1, 3, and 6). Our findings revealed that the addition of P(3HB) indeed promoted accelerated biofilm growth on the coated surfaces, resulting in an occupied area approximately 50% to 100% larger than that observed in the negative control. This indicates a remarkable enhancement, with the biofilm expanding at a rate roughly 1.5 to 2 times faster than the untreated surfaces. We observed noteworthy distinctions in biofilm growth patterns based on varying concentration and number of coating cycles. Interestingly, treatments with low concentration and high coating cycles exhibited comparable biofilm enhancements to those with high concentrations and low coating cycles. Further investigation into the bacterial communities responsible for the degradation of P(3HB) coatings identified mostly common and widespread strains but found no relation between the concentration and coating cycles. Nevertheless, this microbial degradation process was found to be highly efficient, manifesting noticeable effects within a single month. While these initial findings are promising, it's essential to conduct tests under natural conditions to validate the applicability of this approach. Nonetheless, our study represents a novel and bio-based ecological engineering strategy for enhancing the bioreceptivity of marine and coastal structures.

Bile microecology changes play an important role in the occurrence and development of choledocholithiasis. At present, there is no clear report on the difference of bile microecology between asymptomatic patients with gallbladder polyps and choledocholithiasis. This study compared bile microecology between gallbladder polyp patients and patients with choledocholithiasis to identify risk factors for primary choledocholithiasis. This study was conducted in 3 hospitals in different regions of China. Bile samples from 26 patients with gallbladder polyps and 31 patients with choledocholithiasis were collected by laparoscopic cholecystectomy and endoscopic retrograde choledocholithiasis cholangiography (ERCP), respectively. The collected samples were used for 16S ribosomal RNA sequencing and liquid chromatography mass spectrometry analysis. The α-diversity of bile microecological colonies was similar between gallbladder polyp and choledocholithiasis, but the β-diversity was different. Firmicutes, Proteobacteri, Bacteroidota and Actinobacteriota are the most common phyla in the gallbladder polyp group and choledocholithiasis group. However, compared with the gallbladder polyp patients, the abundance of Actinobacteriota has significantly lower in the choledocholithiasis group. At the genera level, the abundance of a variety of bacteria varies between the two groups, and Enterococcus was significantly elevated in choledocholithiasis group. In addition, bile biofilm formation-Pseudomonas aeruginosa was more metabolically active in the choledocholithiasis group, which was closely related to stone formation. The analysis of metabolites showed that a variety of metabolites decreased in the choledocholithiasis group, and the concentration of beta-muricholic acid decreased most significantly. For the first time, our study compared the bile of gallbladder polyp patients with patients with choledocholithiasis, and suggested that the change in the abundance of Actinobacteriota and Enterococcus were closely related to choledocholithiasis. The role of Pseudomonas aeruginosa biofilm in the formation of choledocholithiasis was discovered for the first time, and some prevention schemes for choledocholithiasis were discussed, which has important biological and medical significance.

The global demand for therapeutic prebiotics persuades the quest for novel exopolysaccharides that can retard the growth of pathobionts and healthcare-associated pathogens. In this regard, an exopolysaccharide (3.69 mg/mL) producing strain showing prebiotic and antibiofilm activity was isolated from indigenous pineapple pomace of Tripura and identified as Bacillus subtilis PR-C18. Zymogram analysis revealed EPS PR-C18 was synthesized by levansucrase (∼57 kDa) with a maximal activity of 4.62 U/mg. Chromatography techniques, FTIR, and NMR spectral data revealed the homopolymeric nature of purified EPS with a molecular weight of 3.40 × 104 Da. SEM and rheological study unveiled its microporous structure and shear-thinning effect. Furthermore, EPS PR-C18 showed remarkable emulsification, flocculation, water retention, water solubilization, and antioxidant activity. DSC-TGA data demonstrated its high thermostability and cytotoxicity analysis verified its nontoxic biocompatible nature. In addition, the antibiofilm activity of EPS PR-C18 was validated using molecular docking, molecular simulation, MM-GBSA and PCA studies, which exhibited its strong binding affinity (-20.79 kcal/moL) with PelD, a virulence factor from Pseudomonas aeruginosa. Together, these findings support the future exploitation of EPS PR-C18 as an additive or adjuvant in food and pharmaceutical sectors.

Biofilms are surface-associated communities of bacteria that grow in a self-produced matrix of polysaccharides, proteins, and extracellular DNA (eDNA). Sub-minimal inhibitory concentrations (sub-MIC) of antibiotics induce biofilm formation, potentially as a defensive response to antibiotic stress. However, the mechanisms behind sub-MIC antibiotic-induced biofilm formation are unclear. We show that treatment of Pseudomonas aeruginosa with multiple classes of sub-MIC antibiotics with distinct targets induces biofilm formation. Further, addition of exogenous eDNA or cell lysate failed to increase biofilm formation to the same extent as antibiotics, suggesting that the release of cellular contents by antibiotic-driven bacteriolysis is insufficient. Using a genetic screen for stimulation-deficient mutants, we identified the outer membrane porin OprF and the ECF sigma factor SigX as important. Similarly, loss of OmpA - the Escherichia coli OprF homolog - prevented sub-MIC antibiotic stimulation of E. coli biofilms. Our screen also identified the periplasmic disulfide bond-forming enzyme DsbA and a predicted cyclic-di-GMP phosphodiesterase encoded by PA2200 as essential for biofilm stimulation. The phosphodiesterase activity of PA2200 is likely controlled by a disulfide bond in its regulatory domain, and folding of OprF is influenced by disulfide bond formation, connecting the mutant phenotypes. Addition of reducing agent dithiothreitol prevented sub-MIC antibiotic biofilm stimulation. Finally, activation of a c-di-GMP-responsive promoter follows treatment with sub-MIC antibiotics in the wild-type but not an oprF mutant. Together, these results show that antibiotic-induced biofilm formation is likely driven by a signaling pathway that translates changes in periplasmic redox state into elevated biofilm formation through increases in c-di-GMP.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen causing chronic infections that are associated with the sessile/biofilm mode of growth rather than the free-living/planktonic mode of growth. The transcriptional regulator FleQ contributes to both modes of growth by functioning both as an activator and repressor and inversely regulating flagella genes associated with the planktonic mode of growth and genes contributing to the biofilm mode of growth. Here, we review findings that enhance our understanding of the molecular mechanism by which FleQ enables the transition between the two modes of growth. We also explore recent advances in the mechanism of action of FleQ to both activate and repress gene expression from a single promoter. Emphasis will be on the role of sigma factors, cyclic di-GMP, and the transcriptional regulator AmrZ in inversely regulating flagella and biofilm-associated genes and converting FleQ from a repressor to an activator.

One of the key mechanisms enabling bacterial cells to create biofilms and regulate crucial life functions in a global and highly synchronized way is a bacterial communication system called quorum sensing (QS). QS is a bacterial cell-to-cell communication process that depends on the bacterial population density and is mediated by small signalling molecules called autoinducers (AIs). In bacteria, QS controls the biofilm formation through the global regulation of gene expression involved in the extracellular polymeric matrix (EPS) synthesis, virulence factor production, stress tolerance and metabolic adaptation. Forming biofilm is one of the crucial mechanisms of bacterial antimicrobial resistance (AMR). A common feature of human pathogens is the ability to form biofilm, which poses a serious medical issue due to their high susceptibility to traditional antibiotics. Because QS is associated with virulence and biofilm formation, there is a belief that inhibition of QS activity called quorum quenching (QQ) may provide alternative therapeutic methods for treating microbial infections. This review summarises recent progress in biofilm research, focusing on the mechanisms by which biofilms, especially those formed by pathogenic bacteria, become resistant to antibiotic treatment. Subsequently, a potential alternative approach to QS inhibition highlighting innovative non-antibiotic strategies to control AMR and biofilm formation of pathogenic bacteria has been discussed.

The Pel exopolysaccharide is one of the most mechanistically conserved and phylogenetically diverse bacterial biofilm matrix determinants. Pel is a major contributor to the structural integrity of Pseudomonas aeruginosa biofilms, and its biosynthesis is regulated by the binding of cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP) to the PelD receptor. c-di-GMP is synthesized from two molecules of guanosine triphosphate (GTP) by diguanylate cyclases with GGDEF domains and degraded by phosphodiesterases with EAL or HD-GYP domains. As the P. aeruginosa genome encodes 43 c-di-GMP metabolic enzymes, one way signaling specificity can be achieved is through direct interaction between specific enzyme-receptor pairs. Here, we show that the inner membrane hybrid GGDEF-EAL enzyme, BifA, directly interacts with PelD via its cytoplasmic HAMP, GGDEF, and EAL domains. Despite having no catalytic function, the degenerate active site motif of the BifA GGDEF domain (GGDQF) has retained the ability to bind GTP with micromolar affinity. Mutations that abolish GTP binding result in increased biofilm formation but stable global c-di-GMP levels. Our data suggest that BifA forms a dimer in solution and that GTP binding induces conformational changes in dimeric BifA that enhance the BifA-PelD interaction and stimulate its phosphodiesterase activity, thus reducing c-di-GMP levels and downregulating Pel biosynthesis. Structural comparisons between the dimeric AlphaFold2 model of BifA and the structures of other hybrid GGDEF-EAL proteins suggest that the regulation of BifA by GTP may occur through a novel mechanism.IMPORTANCEc-di-GMP is the most common cyclic dinucleotide used by bacteria to regulate phenotypes such as motility, biofilm formation, virulence factor production, cell cycle progression, and cell differentiation. While the identification and initial characterization of c-di-GMP metabolic enzymes are well established, our understanding of how these enzymes are regulated to provide signaling specificity remains understudied. Here we demonstrate that the inactive GGDEF domain of BifA binds GTP and regulates the adjacent phosphodiesterase EAL domain, ultimately downregulating Pel-dependent P. aeruginosa biofilm formation through an interaction with PelD. This discovery adds to the growing body of literature regarding how hybrid GGDEF-EAL enzymes are regulated and provides additional precedence for studying how direct interactions between c-di-GMP metabolic enzymes and effectors result in signaling specificity.

Upon infection, mucosal tissues activate a brisk inflammatory response to clear the pathogen, i.e., resistance to disease. Resistance to disease is orchestrated by tissue-resident macrophages, which undergo profound metabolic reprogramming after sensing the pathogen. These metabolically activated macrophages release many inflammatory factors, which promote their bactericidal function. However, in immunocompetent individuals, pathogens like Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella evade this type of immunity, generating communities that thrive for the long term.

These organisms develop features that render them less susceptible to eradication, such as biofilms and increased tolerance to antibiotics. Furthermore, after antibiotic therapy withdrawal, "persister" cells rapidly upsurge, triggering inflammatory relapses that worsen host health. How these pathogens persisted in inflamed tissues replete with activated macrophages remains poorly understood.

In this review, we discuss recent findings indicating that the ability of P. aeruginosa, S. aureus, and Salmonella to evolve biofilms and antibiotic tolerance is promoted by the similar metabolic routes that regulate macrophage metabolic reprogramming.

Quorum sensing plays a major role in the expression of virulence and development of biofilm in the human pathogen Pseudomonas aeruginosa. Natural compounds are well-known for their antibacterial characteristics by blocking various metabolic pathways. The goal of this study is to find natural compounds that mimic AHL (Acyl homoserine lactone) and suppress virulence in P. aeruginosa, which is triggered by quorum sensing-dependent pathways as an alternative drug development strategy. To support this rationale, functional network analysis and in silico investigations were carried out to find natural AHL analogues, followed by molecular docking studies. Out of the 16 top-hit AHL analogues derived from phytochemicals, seven ligands were found to bind to the quorum sensing activator proteins. Cassialactone, an AHL analogue, exhibited the highest binding affinity for RhlI, RhlR, and PqsE of P. aeruginosa, with a docking score of -9.4, -8.9, and -8.7 kcal/mol, respectively. 2(5H)-Furanone, a well-known inhibitor, was also docked to compare the docking score and intermolecular interactions between the ligand and the target protein. Furthermore, molecular dynamics simulations and binding free energy calculations were performed to determine the stability of the docked complexes. Additionally, the ADME properties of the analogues were also analyzed to evaluate the pharmacological parameters. Functional network analysis further showed that the interconnectedness of proteins such as RhlI, RhlR, LasI, and PqsE with the virulence and biofilm phenotype of the pathogen could offer potential as a therapeutic target.Communicated by Ramaswamy H. Sarma.

The purpose of this study was to synthesize a nanoform of eugenol (an important phytochemical with various pharmacological potentials) and to investigate its antibiofilm efficacy on Pseudomonas aeruginosa biofilm.

Colloidal suspension of eugenol-nanoparticles (ENPs) was synthesized by the simple ultrasonic cavitation method through the emulsification of hydrophobic eugenol into hydrophilic gelatin. Thus, the nanonization process made water-insoluble eugenol into water-soluble nano-eugenol, making the nanoform bioavailable. The size of the ENPs was 20-30 nm, entrapment efficiency of eugenol within gelatin was 80%, and release of eugenol from the gelatin cap was slow and sustained over 5 days. Concerning the clinically relevant pathogen P. aeruginosa, ENPs had higher antibiofilm (for both formation and eradication) activities than free eugenol. Minimal biofilm inhibitory concentration and minimal biofilm eradication concentration of ENP on P. aeruginosa biofilm were 2.0 and 4.0 mM, respectively. In addition, the measurement of P. aeruginosa biofilm biomass, biofilm thickness, amount of biofilm extra-polymeric substance, cell surface hydrophobicity, cell swarming and twitching efficiencies, cellular morphology, and biofilm formation in catheter demonstrated that the antibiofilm efficacy of nano-eugenol was 30%-40% higher than that of bulk eugenol.

These results signify that future pharmacological and clinical studies are very much required to investigate whether ENPs can act as an effective drug against P. aeruginosa biofilm-mediated diseases. Thus, the problem of intrinsic antibiotic tolerance of biofilm-forming cells may be minimized by ENPs. Moreover, ENP may be used as a potential catheter-coating agent to inhibit pseudomonal colonization on catheter surfaces and, therefore, to reduce catheter-associated infections and complications.

Halophilic bacterial strains, designated SG2L-4T, SB1M4, and SB2L-5, were isolated from jeotgal, a traditional Korean fermented food. Cells are Gram-stain-negative, aerobic, non-motile, rod-shaped, catalase-positive, and oxidase-negative. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain SG2L-4T is closely related to Halomonas garicola KACC 18117T with a similarity of 96.2%. The complete genome sequence of strain SG2L-4T was 3,227,066 bp in size, with a genomic G + C content of 63.3 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain SG2L-4T and H. garicola KACC 18117T were 90.5 and 40.7%, respectively. The optimal growth conditions for strain SG2L-4T were temperatures between 30 and 37°C, a pH value of 7, and the presence of 10% (w/v) NaCl. The polar lipids identified included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unknown phospholipid, an unknown glycolipid, and an unknown polar lipid. The major cellular fatty acids were C16:0, summed features 8 (C18:1ω6c and/or C18:1ω7c), C19:0 cyclo ω8c, and summed features 3 (C16:1ω6c and/or C16:1ω7c). The predominant respiratory quinone was ubiquinone with nine isoprene units (Q-9). Based on the phenotypic, genotypic, and chemotaxonomic results, strain SG2L-4T represents a novel species within the genus Halomonas, for which the name Halomonas piscis sp. nov. is proposed. The type strain is SG2L-4T (=KCTC 92842T = JCM 35929T). Functional annotation of the genome of strain SG2L-4T confirmed the presence of exopolysaccharide synthesis protein (ExoD) and capsular polysaccharide-related genes. Strain SG2L-4T also exhibited positive results in Molisch's test, indicating the presence of extracellular carbohydrates and exopolysaccharides (EPS) production. These findings provide valuable insights into the EPS-producing capabilities of H. piscis sp. nov. isolated from jeotgal, contributing to understanding its potential roles in food and biotechnological applications.