Search Results (513)

RNA methyltransferases are key regulators of bacterial physiology, yet their specific roles in virulence remain poorly defined. In this study, we characterize PA3840, a putative RNA methyltransferase in Pseudomonas aeruginosa (P. aeruginosa). Deletion of PA3840 specifically impaired twitching motility without affecting bacterial growth, swimming, or swarming. Notably, PA3840 was found to suppress the expression of Type III Secretion System (T3SS) genes, thereby reducing cytotoxicity and host cell rounding. Consistent with these observations, PA3840 expression attenuated pro-inflammatory cytokine production in epithelial cells by inhibiting NF-κB activation. Mechanistic analysis revealed that PA3840 is translocated into host cells in a Type VI Secretion System (T6SS)-dependent manner. This translocation was reduced by hcp1 deletion and nearly abolished by a double deletion of pscF and hcp3, suggesting the involvement of multiple T6SS components and potential interplay with T3SS machinery. However, direct transfection of PA3840 into host cells failed to suppress cytokine expression, indicating that its immunomodulatory function is mediated by a bacterium-intrinsic mechanism rather than direct intracellular action. Collectively, these findings identify PA3840 as a translocated effector that modulates twitching motility and dampens host inflammation by repressing T3SS and NF-κB signaling, revealing a novel layer of post-transcriptional virulence regulation in P. aeruginosa. Full article

This work introduces an original sequential bio-inspired strategy combining lytic phage pretreatment with TiO₂–thyme essential oil (TEO) photocatalysis, achieving near-complete inhibition of both biofilm initiation and maturation. By simultaneously targeting planktonic cells, mature biofilms, and extracellular DNA (eDNA), this approach addresses key mechanisms involved in biofilm persistence. Pseudomonas aeruginosa ATCC 4114 was selected as the biological model due to its relevance in water distribution systems and its strong biofilm-forming ability. Experimental results showed that phage pretreatment alone inhibited biofilm formation by planktonic cells by up to 99.6% (inactivation rate constant k = 0.034 min⁻¹) and weakened bacterial attachment in mature biofilms by 89.06% (k = 0.011 min⁻¹). To further enhance photocatalytic efficacy, titanium dioxide (TiO₂) was combined with TEO at 0.05% (v/v) as a bio-inspired photosensitizer. UV–Vis spectroscopy confirmed TiO₂-TEO interactions that extended light absorption into the visible region (400–700 nm), thereby enhancing photocatalytic efficiency. This combination was designed to suppress residual biofilm development and disrupt extracellular DNA (eDNA), a critical component of biofilm structure and stability. The integrated approach involving phage pretreatment followed by TiO₂–TEO (0.05%) photocatalysis achieved 99.99% inhibition of both biofilm initiation and maturation phases, with significantly increased kinetic parameters (A = 2.62 for planktonic cells and A = 3.65 for sessile cells; k = 0.076 min⁻¹ and 0.063 min⁻¹, respectively; p < 0.01). This study provides novel insights into water disinfection strategies using photocatalytic treatment, emphasizing the importance of monitoring post-treatment bacterial virulence factor expression. Full article

Pseudomonas aeruginosa is a resilient Gram-negative pathogen frequently implicated in healthcare associated infections, particularly among immunocompromised individuals and those with chronic conditions such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), or cancer. It is well known for its high resistance to antibiotic treatment. This review briefly mentions P. aeruginosa’s resistance mechanisms, biofilm formation, and virulence factors, while primarily focusing on treatment challenges and recent advancements in therapeutic strategies aimed at overcoming resistance. Covered are novel non-antibiotic interventions such as quorum sensing inhibitors, quorum quenching agents, iron chelators, lectin and efflux pump inhibitors, as well as antimicrobial peptides and nanoparticles. Traditional medicine, phytochemicals, and probiotics are also evaluated. Additionally, this review explores the development of a viable vaccine, bacteriophage therapy, lactoferrin-hypothiocyanite combination, and topical use of electrochemical scaffolds. This review emphasizes the need for extensive safety studies and in vivo validation of these emerging non-antibiotic therapeutic strategies to determine their efficacy, pharmacological behavior, and clinical feasibility before they can be translated into practice. Many of these emerging treatments could play a vital role in future combination therapies by enhancing the efficacy of existing antibiotics and countering resistance and virulence mechanisms. Advancing these approaches from laboratory to clinical application remains a major challenge, making the development of approved therapies or vaccines a critical scientific and public health priority. Full article

Volatile organic compounds produced by microbes are increasingly recognized as modulators of microbial interactions and mediators of both intra- and inter-kingdom communication. This study explored the possible ecophysiological roles of nine amides from Streptomyces sp. NP10 in quorum sensing (QS) and biofilm formation in Pseudomonas aeruginosa PAO1. GC-MS profiling, synthesis, spectral validation, and co-injection experiments confirmed compound identities. Notably, N-(3-methyl-2-butenyl)acetamide is reported as a new natural product and N-(2-methylbutyl)acetamide as a new Streptomyces-produced metabolite. At subinhibitory concentrations (250 μg/mL), most of the amides enhanced P. aeruginosa biofilm formation, with N-(2-methylbutyl)acetamide, N-(3-methyl-2-butenyl)acetamide, and 2-phenylacetamide showing the strongest effects. Simultaneously, these compounds suppressed QS by reducing the production of N-acyl homoserine lactones (AHLs) and 2-alkyl-4-quinolones (AHQs). Aliphatic acetamides preferentially inhibited short-chain AHLs, while N-acetyltyramine and 2-phenylacetamide mainly affected quinolone signaling. These opposing effects on QS and biofilm are consistent with the involvement of alternative regulatory circuits. Motility assays showed biofilm stimulation was not correlated with altered swarming or twitching. Cross-species assays revealed limited QS inhibition, with only N-acetyltryptamine reducing violacein production in Chromobacterium violaceum CV026. Most of the amides were non-cytotoxic at 100 μM (10.5–20.2 μg/mL), except for 2-phenylacetamide. Overall, these amides likely serve as microbial signals influencing QS and biofilm formation, offering leads for anti-virulence strategies. Full article

Bacteriophages are powerful drivers of microbial evolution and are increasingly explored as alternatives to antibiotics against multidrug-resistant pathogens such as Pseudomonas aeruginosa. Here, we describe the isolation, phenotypic characterization and genomic, structural and evolutionary analysis of Pseudomonas phage Adele, a lytic myovirus representing a novel species within the genus Pakpunavirus (family Vandenendeviridae). Phage Adele exhibits a short latent period of 20 min, a burst size of 59 ± 11 virions per infected cell and a high virulence index, efficiently lysing non-O11 Pseudomonas aeruginosa strains and reducing biofilm biomass. In vivo, Adele confers marked protection in a Galleria mellonella infection model. Phylogenetic reconstruction, synteny analysis and structural modeling demonstrate the relatedness of Vandenendeviridae to phages of the Andersonviridae and Vequintavirinae clades, pointing to a stable, ancestral virion architecture that has undergone lineage-specific elaborations, including the duplication and divergence of tail tube proteins. The tail assembly chaperone gene employs a conserved −1 programmed ribosomal frameshift. Phage Adele encodes an elaborate set of metabolic reprogramming and anti-defense systems, reflecting extensive horizontal gene transfer. The combination of a conserved structural architecture and mosaic genome establishes Adele as an exemplary system for studying modular evolution in phages, alongside its demonstrated therapeutic efficacy. Full article

Background/Objectives: The emergence of hypervirulent Pseudomonas aeruginosa strains resistant to β-lactamase inhibitor antibiotics is a critical health problem as they impede the treatment of infections. The objective of this study was to determine the different molecular arrangements of the virulence genotype related to β-lactamase genotype and the resistance phenotype to a combination of β-lactam antibiotics and β-lactamase inhibitors, and the phylogroups in P. aeruginosa strains isolated from patients with healthcare-associated infections and community-acquired infections. Methods: P. aeruginosa, virulence genes, β-lactamase genes and phylogroups were identified using polymerase chain reaction. Resistance to β-lactam antibiotics and β-lactamase inhibitors was determined using the disk diffusion method. The MIC determination of ticarcillin/clavulanic acid and piperacillin/tazobactam was performed using the MIC test strip for antimicrobial susceptibility testing. Results: In total, 124 P. aeruginosa strains from patients with healthcare-associated (67/124) and community-acquired infections (57/124) were analyzed. Most strains from patients with healthcare-associated infections and community-acquired infections harbored genes for proteases (aprA), phospholipases (pIcH and pIcN), elastases (lasA and lasB), rhamnolipids (rhLA), quorum-sensing system (lasI and rhII), and β-lactamase (bla_oxa-4, bla_oxa-1, and bla_GES). In total, 100% (124/124) and 99.1% (123/124) of the strains isolated from patients with healthcare-associated and community-acquired infections were resistant to the β-lactamase inhibitor antibiotics, amoxicillin/clavulanic acid and ampicillin/sulbactam, respectively, while 54% (67/124) of the strains were resistant to piperacillin/tazobactam. Phylogroup 1 (22/124) was detected more frequently among the strains in relation to phylogroup 2 (8/12). Conclusions: We demonstrated different association profiles of virulence genotype related to the β-lactamase genotype, the β-lactamase inhibitor resistome, phylogroups, and clinical origin of the strains. Therefore, medical treatment regimens against infections caused by P. aeruginosa should be improved. Full article

Background/Objectives: Antibiotic-resistant strains have been reported in aquatic ecosystems, with varying prevalence and resistance patterns by region. In Tamaulipas, Mexico, little information has been generated on this topic, making it difficult to estimate their potential risk to environmental and human health. Therefore, the objective of this study was to evaluate the presence and virulence of antibiotic-resistant strains of Pseudomonas aeruginosa in environmental water from Tamaulipas, Mexico. Methods: One hundred water samples were collected from different water bodies in Tamaulipas to identify P. aeruginosa by PCR and MALDI-TOF, virulence gene detection, antimicrobial susceptibility testing, and detection class 1 integrons. Results: In this study, 109 P. aeruginosa strains were isolated. Eight virulence genes were identified in 47.7% to 80.7% of the strains, with the rhlAB gene being the most frequent. The strains showed resistance or intermedia resistance to 10 of the 16 antibiotics tested, in a range of resistance values 0.9–66.2%. In total, 100% (109/109) were susceptible to ceftazidime (CAZ), gentamicin (GM), amikacin (AN), netilmicin (NET), tobramycin (NN) and norfloxacin (NOR), and 65.7% were resistant to ticarcillin/clavulanic acid and 53.5% to ticarcillin; the resistance to the remaining antibiotics was between 19.4% and 0.9%. The class 1 integron was not identified in any of the strains analyzed. Conclusions:P. aeruginosa in environmental waters of Tamaulipas showed potential to cause infections and low rates of resistance to most of the antibiotics tested. However, 20% were resistant to one of the most common treatments, which could pose a risk to public health. Full article

Background/Objectives: Postbiotics derived from lactic acid bacteria are emerging as promising antimicrobial agents due to their antibacterial, antibiofilm, and immunomodulatory properties. Among their metabolites, lactic acid (LA) is thought to play a major role in antimicrobial activity. This study investigated the proteomic response of Pseudomonas aeruginosa and Staphylococcus aureus to Lacticaseibacillus rhamnosus cell-free supernatant (CFS) and compared it with that elicited by LA alone. Methods: Overnight bacterial cultures were exposed to sub-MIC LA or CFS (1:10 for P. aeruginosa and 1:8 for S. aureus; ~12.5–15.6 mM LA) for 6 h at 37 °C. Intracellular proteins were harvested and subsequently quantified and purified to be analysed by HPLC–MS/MS, for quantitative label-free proteomics. Results: Proteomic analysis revealed clear separation of treated samples from controls, with largely overlapping responses to CFS and LA. Hallmark acid-stress adaptations were observed, including urease-mediated pH buffering, confirming that part of the response was driven by mild organic acid. In P. aeruginosa, treatments suppressed virulence pathways (phenazines, T3SS), while shifting metabolism toward lactate utilisation and reinforcing the outer membrane (lipid A, polyamine). In S. aureus, decreased abundance of the SaeRS-regulated immune-evasion factor Sbi, together with changes in envelope, ROS and translation-related proteins, suggested a bacteriostatic-like state. S. aureus differences between CFS and LA were more pronounced; CFS uniquely increased cell-wall defences, oxidative stress (SodA, SodM) and chaperone expression (GroS, GrpE), suggesting stress beyond acidification alone. Conclusions: These findings shed light on the molecular mechanisms underlying bacterial adaptation to CFS and highlight their potential as a novel antimicrobial approach. Full article

Multi-drug-resistant Pseudomonas aeruginosa (P. aeruginosa) commonly causes infections that are difficult to treat, necessitating the development of new therapeutics. The search for more effective ways to combat the emergence of bacterial resistance has also led to research into phage-antibiotic synergy (PAS) as a potential therapeutic strategy. The aim of this study was to isolate and characterize virulent phages from water sources that are active against clinical carbapenem-resistant P. aeruginosa isolates, and to evaluate their in vivo efficacy using a Galleria mellonella larvae infection model. The biological and genomic characteristics of the isolated phages were determined using host range analysis, one-step growth curve analysis, transmission electron microscopy analysis and whole-genome sequencing. Two phages (vB_PaMB13 and vB_PaMB17) that demonstrated in vitro synergistic and bactericidal interactions with antipseudomonal antibiotics (tobramycin and ceftazidime) were selected for further investigation using the checkerboard method. The study revealed synergy between all phages and either antibiotic, tobramycin or ceftazidime, against P. aeruginosa. Similarly, the percentage survival rates increased in the in vivo model when both phages and antibiotics were used in combination. Overall, our study provides further support for the idea that phage-antibiotic synergy could be an effective strategy for improving treatment outcomes. Full article

Objectives: Pseudomonas aeruginosa is an opportunistic pathogen of high clinical relevance due to its ability to form biofilms, its inherent virulence regulated by quorum-sensing systems, and its multidrug resistance. In the present study, we evaluated the inhibitory potential of nine extracts from Inula species (chloroform and methanolic fractions, including a sesquiterpene lactone-enriched fraction) against biofilm formation and virulence-associated traits of P. aeruginosa PAO1 and three multidrug-resistant clinical isolates, as well as their cytotoxicity, biocompatibility, and ability to affect cytokine and nitric oxide production in infected skin explants. Methods: The following methods were applied: fractionation and extraction of plant extracts; cytotoxicity assessment on HFF cells; crystal violet assay for determining antibiofilm activity; fluorescence microscopy for evaluating biofilm viability; electron microscopy for assessing the 3D structure of biofilms and morphological alterations; inhibition assays of pyocyanin pigment, protease activity, bacterial motility, interleukin-17, and nitric oxide production; histological analysis of mouse skin explants. Results: Quantitative analyses of antibiofilm activity revealed that five of the tested extracts inhibited biofilm formation by more than 50%. Structural and functional analyses using confocal laser scanning microscopy and scanning electron microscopy demonstrated a substantial reduction in biofilm thickness, exfoliation of biofilm biomass, the presence of isolated bacterial clusters, metabolically inactive cell populations, and morphological abnormalities associated with cell elongation, invaginations, and polar deformations as a consequence of treatment. In addition, the plant extracts strongly affected virulence factors regulated by quorum sensing. The methanolic fractions from I. britannica and I. bifrons significantly suppressed pyocyanin synthesis. In contrast, the chloroform fractions from I. helenium and I. spiraeifolia produced the largest inhibition zones in assays for extracellular protease activity. Furthermore, all chloroform extracts suppressed bacterial motility, with the lowest swarming diameter observed for the chloroform and lactone-enriched fractions from I. britannica. The chloroform extracts of I. helenium and I. bifrons, methanolic extracts of I. britannica, and chloroform and methanolic extracts of I. spiraeifolia showed relatively low toxicity to normal diploid human fibroblasts. Methanolic and chloroform fractions from I. britannica disrupted biofilm integrity and reduced IL-17A and nitric oxide production in infected skin explants. Conclusions: All these findings indicate a possible synergistic action of the chemical constituents within the fractions on quorum-sensing regulation, biofilm formation, cellular viability, and modulation of host inflammatory responses. Full article

Background/Objectives: Biofilm-associated infections pose a major clinical challenge since bacteria within biofilms exhibit highly antibiotic tolerance. Pseudomonas aeruginosa forms persistent biofilms that cause chronic infections in vulnerable patients, including those with cystic fibrosis, burns, or medical implants. Such biofilm-associated chronic infections require prolonged treatments that promote antimicrobial resistance. To address this, recent strategies focus on enhancing biofilm dispersion. Methods: Thirty-six N-arylimino-1,2,3-dithiazoles were screened for their biofilm dispersal activity using a crystal violet assay. Their cytotoxicity was assessed on A549 and HaCat eukaryotic cells. Moreover, their influence on bacterial growth and virulence was investigated. Lastly, fluorescence anisotropy was used to measure membrane fluidity to obtain the first insights on the mechanism of action of these chemicals. Results: Our results showed that quinazoline-derivatives of imino-1,2,3-dithiazoles display biofilm dispersion activity. These compounds do not increase virulence through pyocyanin production, do not modify the growth kinetics of P. aeruginosa, and do not show cytotoxicity towards eucaryotic cells. Conclusions: These findings highlight the potential use of N-arylimino-1,2,3-dithiazole-derived compounds as safe and effective dispersal agents of P. aeruginosa biofilms. Full article

Pseudomonas aeruginosa is a Gram-negative, pathogenic, bacterium that produces biofilms comprising phenotypically distinct cell subpopulations. When separating and characterizing a single P. aeruginosa PA14 biofilm, three novel rugose small colony variants (RSCVs) (denoted RSCV_1, RSCV_2, and RSCV_3) were discovered. Characteristics of these stationary phase RSCVs differed between stationary phase wild-type (WT) PA14, between the PA14 biofilm subpopulations, and between the RSCVs themselves. The observed phenotypic changes in the RSCVs included differences in cellular morphology, exopolysaccharide production, biosynthesis of virulence factors, biofilm formation, and antibiotic tolerance. Stationary phase cell surface-associated molecules on the RSCVs were differently ionized as compared to WT PA14 using matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Many RNA transcripts were differentially expressed between the RSCVs and WT PA14 as well as between RSCV_1 and RSCV_3. DNA sequencing revealed single-nucleotide deletions and single-nucleotide polymorphisms (SNPs) among the RSCVs and between the RSCVs and WT PA14. The levels of the intracellular signaling molecule bis-(3′,5′)-cyclic-dimeric-guanosine monophosphate (cyclic-di-GMP) were higher in the RSCVs compared to WT PA14 and significantly lower in RSCV_3 as compared to both RSCV_1 and RSCV_2. The detected differences in the RSCVs have significant implications for biofilm production, antibiotic tolerance, and virulence. Full article

Background/Objectives: The rise in multidrug-resistant pathogens such as Pseudomonas aeruginosa (PA), coupled with declining antibiotic development, underscores the need for innovative therapeutic strategies. Repurposing approved drugs provides advantages of safety and rapid development. Since quorum sensing (QS) controls key virulence traits in PA, targeting this pathway represents a promising antivirulence approach. This study aimed to identify and repurpose existing drugs as QS inhibitors. Methods: An in silico docking screen of 3000 FDA-approved or clinically tested compounds was performed against the C4-HSL receptor RhlR. Seventeen candidates were tested in the laboratory strain PAO1 for lactone-dependent signaling inhibition. The most active compound, MK-8245, was further evaluated for effects on growth, cytotoxicity, lactone release, biofilm formation, pyocyanin, elastase, rhamnolipids, and swarming motility. Its activity was also assessed in 20 clinical PA isolates. Results: MK-8245 (40 µM) reduced QS-regulated gene expression by ~60% without affecting viability. In PAO1, it inhibited rhamnolipids (60%), pyocyanin (40%), elastase (25%), biofilm formation, and swarming motility (25%). MK-8245 also enhanced the efficacy of imipenem against biofilms. In clinical isolates, it consistently decreased lactone release (~60%), pyocyanin (~50%), rhamnolipids (~40%), biofilm formation (~30%), and swarming motility (~25%). Conclusions: MK-8245 emerges as a promising antivirulence candidate against P. aeruginosa. By disrupting QS signaling and impairing multiple virulence factors, it attenuates pathogenicity without bactericidal pressure. Its synergy with standard antibiotics and consistent activity in clinical isolates highlight its translational potential and warrant further preclinical evaluation. Full article

Multidrug resistance has also been accompanied by the prolonged use of antibiotics that makes complications in treatment. Biofilm in pathogenic bacteria is the most serious challenge linked with chronic illnesses and also contributes to virulence and drug resistance. Several bacterial pathogens employ the Quorum-sensing (QS) mechanism to coordinate their collective behaviors like bioluminescence, virulence, and biofilm formation. Therefore, agents that inhibit or interfere with bacterial QS and biofilm formation are emerging as a new class of next-generation antibacterial. Recently, nanoparticles have been employed to improve the efficacy of existing antibacterial agents. In the present study, gold nanocrystals were synthesized by using Koelreuteria paniculata (KP) leaf extract. Synthesized nanocrystals were characterized by a face-centered cubic structure of ~20 nm by XRD, FTIR, Zeta sizer, and TEM. Biogenic Gold nanocrystals (BGNCs) exhibited extended QS inhibition in bio-indicator strains Chromobacterium violaceum and Pseudomonas aeruginosa biosensor strains. BGNCs strongly suppressed QS-controlled violacein production in C. violaceum CV026, and elastase, protease, pyocyanin, alginate, and biofilm formation in P. aeruginosa (PA01). In addition, BGNCs notably suppressed the relative expression of PA01 quorum sensing, biofilm-forming, and virulence-regulating genes, as quantified by qRT-PCR. As a result of the broad-spectrum suppression of QS and biofilm by BGNCs, it is anticipated that these nontoxic bioactive nanocrystals can be employed as surface sterilization agents in nosocomial infections. Full article