Search Results (49)

Antimicrobial resistance (AMR) poses a global health threat, which is becoming more challenging due to the involvement of bacterial virulence mechanisms such as quorum sensing (QS) and biofilm formation. These systems regulate pathogenic traits and shield bacteria from conventional therapies. Phytocompounds offer promising antivirulence strategies by disrupting QS and biofilms without exerting selective pressure. In this study, emodin, a natural anthraquinone, was evaluated for its anti-QS and antibiofilm efficacy. Emodin inhibited violacein production by 63.86% in C. violaceum 12472. In P. aeruginosa PAO1, it suppressed pyocyanin (68.04%), pyoverdin (48.79%), exoprotease (58.55%), elastase (43.13%), alginate (74.12%), and rhamnolipids (56.37%). In S. marcescens MTCC 97, emodin reduced prodigiosin (55.94%), exoprotease (48.80%), motility (83.27%), and cell surface hydrophilicity (41.20%). Biofilm formation was inhibited by over 50% in all three bacteria, highlighting emodin’s potential as a broad-spectrum antibiofilm agent. Molecular docking analyses indicated that emodin exhibited affinity towards QS regulatory proteins CviR, LasR, and SmaR, implying a possible competitive interaction at their ligand-binding sites. Subsequent molecular dynamics simulations confirmed these observations by demonstrating structural stability in emodin-bound proteins. The collective insights from in vitro assays and computational studies underscore the potential of emodin in interfering with QS-mediated virulence expression and biofilm development. Such findings support the exploration of non-antibiotic QS inhibitors as therapeutic alternatives for managing bacterial infections and reducing dependence on traditional antimicrobial agents. Full article

Background/Objectives: The growing threat of antimicrobial resistance has intensified the search for alternative therapeutic approaches. Quorum sensing (QS) inhibition, which disrupts bacterial communication and virulence, represents a promising approach to mitigating infection. Given the complexity of the sponge holobiont, sponge-associated microorganisms may demonstrate QS inhibitory properties and serve as potential sources of novel anti-virulence agents. This study aimed to investigate the QS inhibitory potential of sponge-associated Bacillus species against Pseudomonas aeruginosa, a multidrug-resistant pathogen that relies on QS for virulence regulation. Methods: Ninety-eight bacterial isolates were obtained from seven intertidal South African sponges. Biosensor-based sandwich assays using Chromobacterium violaceum identified 15 isolates with putative QS inhibition (QSI) activity, including five classified as Bacillus species via 16S rRNA gene sequencing. Crude extracts from these isolates, cultivated in medium Mannitol (Mann) and medium 5294, were screened for their ability to inhibit QS-regulated virulence factors in P. aeruginosa. Results: Extracts, particularly from medium 5294, exhibited significant QSI activity without cytotoxic effects. The five most potent extracts, i.e., Bacillus mobilis SP2-AB7 (5294), Bacillus wiedmannii SP5-AB7 (Mann), B. mobilis SP2-AB7 (Mann), and Bacillus cereus SP1-AB4 (Mann and 5294), inhibited both Las- and Rhl-regulated virulence factors, including pyocyanin, pyoverdine, elastase, protease, rhamnolipid production, motility, and initial adhesion, achieving inhibition rates of up to 93% (p < 0.05). Molecular analysis confirmed the presence of the aiiA lactonase gene in key isolates, while GC-MS and FTIR profiling revealed medium-specific differences in metabolite production. Conclusions: Sponge-associated Bacillus species from KwaZulu-Natal exhibit robust QSI activity against P. aeruginosa, highlighting their potential as sources of alternative anti-virulence agents. Further characterization and in vivo validation are needed to assess their therapeutic application in combatting resistant infections. Full article

Pseudomonas aeruginosa is an opportunistic pathogen capable of forming antibiotic-resistant biofilms, contributing to persistent infections and treatment failure. Environmental factors such as osmolarity and nutrient availability are known to influence biofilm formation and virulence. In this study, we investigated the effects of NaCl depletion and glutamine supplementation on biofilm production in three P. aeruginosa strains: the laboratory strain ATCC 27853 and two clinical isolates with distinct antibiotic resistance profiles and phenazine production patterns (P. aeruginosa Pr, pyorubrin-producing, and P. aeruginosa Pc, pyocyanin-producing). Bacteria were cultured in standard Luria–Bertani (LB) medium, LB without NaCl, and LB in which yeast extract was replaced by glutamine. For each strain and condition, we assessed growth kinetics, phenazine production, and biofilm formation. Biofilm development was quantified via XTT assays and compared to secondary metabolite profiles. NaCl removal did not substantially affect growth, whereas glutamine supplementation reduced growth, especially in the laboratory strain. Both conditions modulated secondary metabolite production and biofilm formation in a strain-specific manner. In P. aeruginosa ATCC 27853, NaCl depletion significantly increased pyoverdine, pyocyanin, and QS gene expression, while biofilm formation showed significant differences only at 72 h; in contrast, glutamine supplementation affected only pyoverdine. A similar trend was observed in the clinical strain P. aeruginosa Pc, although NaCl depletion did not significantly impact pyoverdine production but already enhanced biofilm formation at 48 h. In P. aeruginosa Pr, only glutamine appeared to alter the considered parameters, increasing pyoverdine production while reducing pyocyanin and biofilm levels, although the absence of NaCl also negatively impacted biofilm formation. These findings highlight the impact of osmotic and nutritional signals on P. aeruginosa virulence traits. Full article

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium, responsible for several life-threatening infections due to its multiple virulence factors and problematic multi-drug resistance, hence the necessity to find alternatives such as competitive probiotics. Pediococcus pentosaceus MZF16 is an LAB strain, isolated from traditional dried meat “Ossban”, with high probiotic potential. Our study investigated the capacity of P. pentosaceus MZF16 to counteract P. aeruginosa H103 using several tests on intestinal cells (analysis of cytotoxicity, inflammation, adhesion/invasion) and on the in vivo Caenorhabditis elegans model. The effect of MZF16 on the quorum sensing of the pathogen was also examined. We found that P. pentosaceus MZF16 was able to reduce H103 cytotoxicity and inflammatory activity and prevented pathogen colonization and translocation across Caco-2/TC7 cells. MZF16 also exerted an anti-virulence effect by attenuating quorum-sensing (QS) molecules and pyoverdine production and extended C. elegans lifespan. The obtained results highlight the potential of P. pentosaceus MZF16 probiotic strain as an anti-Pseudomonas aeruginosa alternative and establish a basis for elucidating the mechanisms of P. pentosaceus MZF16 involved in countering P. aeruginosa virulence. Full article

Nonribosomal peptide synthetases (NRPS) are essential for the biosynthesis of therapeutically valuable molecules, including antibiotics, immunosuppressants, and anticancer agents. The assembly-line mechanism of NRPS offers significant potential for engineering novel natural products through reprogramming. However, the challenging purification of NRPS proteins has impeded the investigation of their assembly and catalytic mechanisms. In this study, we employed homologous recombination to insert a purification tag at the C-terminus of the NRPS gene within the chromosome. This genetic modification enabled efficient purification of NRPS proteins from the tagged mutant strain using a one-step affinity chromatography approach. Additionally, we discovered that MbtH-like proteins (MLPs) form stable complexes with all pyoverdine (PVD) NRPS subunits, allowing for the purification of the entire NRPS assembly line via tagged MLP. Negative stain electron microscopy analysis revealed that the purified PVD NRPS proteins exist as dynamically linear monomers. Our in-situ tag-based purification method enhances NRPS research in both biochemical and structural biology, providing a robust platform for further investigations into NRPS mechanisms and applications. Full article

Due to the emergence of drug resistance, many antimicrobial medications are becoming less effective, complicating the treatment of infections. Therefore, it is crucial to develop new active agents. This article aims to explore the ruthenium(IV) complexes with the following formulas: (Hdma)₂(HL)₂[Ru^IVCl₆]·2Cl·2H₂O (1), where Hdma is protonated dimethylamine and L is 2-hydroxymethylbenzimidazole, and [Ru^IVCl₄(AN)₂]·H₂O (2), where AN is acetonitrile. This paper delves into the physicochemical characteristics and crystal structures of these complexes, employing various techniques such as spectroscopy (IR, UV–Vis), electrochemistry (CV, DPV), and X-ray crystallography. Hirshfeld surface analysis was also performed to visualize intermolecular interactions. Furthermore, the potential antibiofilm activity of the complexes against Pseudomonas aeruginosa PAO1 was investigated and the effect of the compounds on the production of pyoverdine, one of the virulence factors of the Pseudomonas strain, was assessed. The results show that particularly complex 1 reduces biofilm formation and pyoverdine production. Additionally, the bioavailability of these complexes in biological systems (by fluorescence quenching of human serum albumin (HSA) and molecular docking studies) is discussed, assessing how their chemical properties influence their interactions with biological molecules and their potential therapeutic applications. Full article

As a result of drug resistance, many antimicrobial medicines become ineffective, making the infections more difficult to treat. Therefore, there is a need to develop new compounds with antibacterial activity. This role may be played, for example, by metal complexes with carboxylic acids. This study reports the formation and characterization of ruthenium complexes with pyridazine-3-carboxylic acid (pdz-3-COOH)—([(η⁶-p-cym)Ru^IICl(pdz-3-COO)] (1), [Ru^IIICl₂(pdz-3-COO)₂Na(H₂O)]_n(H₂O)_0.11 (2) and [Ru^IIICl₂(pdz-3-COO)₂Na(H₂O)₂]_n (3). The synthesized compounds were analyzed using various spectroscopic and electrochemical techniques, with structure confirmation via SC-XRD analysis. Experimental data showed the ligand binds to metal ions bidentately through the nitrogen donor of the pyridazine ring and one carboxylate oxygen. To visualize intermolecular interactions, Hirshfeld surface analysis and 2D fingerprint plots were conducted. Furthermore, the impact of ruthenium compounds (1 and 2) on the planktonic growth of selected bacterial strains and the formation of Pseudomonas aeruginosa PAO1 biofilm was examined. Both complexes demonstrated comparable anti-biofilm activity and outperformed the free ligand. The effect of the complexes on selected virulence factors of P. aeruginosa PAO1 was also investigated. Compounds 1 and 2 show high suppressive activity in pyoverdine production, indicating that the virulence of the strain has been reduced. This inhibitory effect is similar to the inhibitory effect of ciprofloxacin. Within this context, the complexes exhibit promising antibacterial activities. Importantly, the compounds showed no cytotoxic effects on normal CHO-K1 cells. Additionally, a molecular docking approach and fluorescence spectroscopy were used to determine the interactions of ruthenium complexes with human serum albumin. Full article

Pseudomonas aeruginosa strains with potential for degrading n-alkanes are frequently cultured from hydrocarbon-contaminated sites. The initial hydroxylation step of long-chain n-alkanes is mediated by the chromosomally encoded AlkB1 and AlkB2 alkane hydroxylases. The acquisition of an additional P. putida GPo1-like alkane hydroxylase gene cluster can extend the substrate range assimilated by P. aeruginosa to <C12 n-alkanes. Efficient niche colonization of hydrocarbon-contaminated sites is facilitated by avid iron-uptake systems, such as pyoverdine, and the production of several compounds with antimicrobial activities. A GPo1-like gene cluster can facilitate detoxification and solvent tolerance in P. aeruginosa. The overproduction of various multidrug efflux pumps, in particular, the MexAB-OprM system, can also contribute to solvent tolerance, which is often associated with reduced susceptibility or full resistance to certain clinically relevant antibiotics. These characteristics, together with the remarkable conservation of P. aeruginosa virulence determinants among human, animal, and environmental isolates, necessitate further studies from a One Health perspective into the acquired antibiotic resistance mechanisms of environmental P. aeruginosa strains and possible ways for their dissemination into the human population. Full article

Three new 24-membered macrolactines, amylomacrolactines A–C (1–3), along with two known compounds 4 and 5, were isolated from the Arctic bacteria Bacillus amyloliquefaciens SCSIO 41392. The configurations of 1–3 were assigned by a combination of coupling constants, NOESY, and analysis of MM2-optimized conformation, as well as by comparison with reports in the literature. Compounds 1 and 2 showed quorum sensing (QS) inhibitory activities against the Pseudomonas aeruginosa (P. aeruginosa) PQS system and suppressed PQS-regulated virulence factor pyocyanin synthesis. In addition, compounds 3–5 affected the production of another essential virulence factor, siderophore of pyoverdine (PVD), in P. aeruginosa. More importantly, compound 5 showed an anti-biofilm activity against P. aeruginosa. Altogether, the isolated compounds displayed multiple bacterial virulence inhibition activities, which is worthy of further exploration for novel analogues in antimicrobial drug development. Full article

Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are pathogens inhabiting the lungs of persons with cystic fibrosis (CF), or immune-compromised patients, causing or aggravating disease. We previously investigated their microbial interaction as well as susceptibility to anti-fungal drugs using RPMI medium (contains undetectable iron concentrations), as is standard for susceptibility testing. Here we investigated microbial interaction in synthetic sputum medium (SSPM), a complex mixture designed to mimic the milieu in CF lungs. SSPM contains Fe²⁺. Pa laboratory strain PA14 or PA14 siderophore mutant planktonic culture filtrate, prepared in RPMI or SSPM, were compared for inhibition of Af biofilm formation. SSPM enhanced bacterial and fungal growth and the production of the Pa molecules pyoverdine, phenazines, and rhamnolipids. Af was more susceptible to these molecules in SSPM (with the exception of pyoverdine). SSPM interfered with fungal susceptibility to pyoverdine. Studies with the mutant helped to reveal that the reduced anti-fungal activity of pyoverdine in SSPM appears to be compensated by higher production of other anti-fungal molecules, e.g., rhamnolipids, phenazines, and PQS, and higher Af sensitivity to these molecules. In summary, SSPM better defines Pa–Af intermicrobial competition in the milieu of CF lungs. Full article

Introduction: The emergence of colistin resistance threatens the treatment of Pseudomonas aeruginosa infections. Methods: In this study, in vitro development of colistin resistance was investigated using comparative phenotypic and proteomic analysis of P. aeruginosa ATCC 9027, its 14-day colistin sub-MIC exposed strain (Col-E1), and 10-day antibiotic-free cultured Col-E1 strain (Col-E2). Antibiotic susceptibility, morphology, virulence factors, and proteomic changes were assessed using disc-diffusion, agar-based, spectrophotometry, SEM, and iTRAQ-LC-MS/MS methods. Results: Colistin-exposed strains decreased susceptibility to colistin while remaining susceptible to other antibiotics. Col-E1 reduced the cell lengths by 17.67% and the colony size by 36.16% compared to the initial strain. The reduction remained in Col-E2. The pyocyanin production was reduced in Col-E1 (p=0.025, Tukey HSD) and increased again in Col-E2 (p=0.005, Tukey HSD). In contrast, no significant changes in elastase, protease, rhamnolipid, pyoverdine, and biofilm production were observed (p>0.05, Tukey HSD). In Col-E1, the proteome analysis showed 135 differentially expressed proteins (DEPs) of which 94 DEPs (69.23%) maintained their expression change in Col-E2. Among DEPs, 82 were involved in metabolism and protein synthesis. Some DEPs (6/135) played a role in stress response such as GrpE (fold change: 14.93) and Hmp (fold change: 12.08). In particular, membrane proteins like OprD, DdlB, and OprI showed significant colistin response with fold change of -8.47, 6.43 and 6.19, respectively. Conclusions: In summary, colistin response in P. aeruginosa seemed to affect morphology, production of pyocyanin, and proteins of metabolism, protein synthesis, stress response and membrane. Full article

Among sixty-eight pseudomonads, isolate QCS59 from the rhizosphere of H. schimperi was selected based on its siderophore level. Production was optimal in Kings B supplemented with 2% peptone and 0.5% fructose at pH 6.5 and 25 °C for 72 h. Additionally, the threshold potential of iron was found at a concentration of 10 µM. After purification, the acidified siderophore presented a maximum absorption peak of 360 nm, while the neutral form presented a maximum of 414 nm, confirming its pyoverdine (PVD) nature. Furthermore, a major peak appeared at a retention time (RT) of 27.5 min during RP-HPLC, confirming its homogeneity. Interestingly, it demonstrated effective antibacterial activity, especially against Escherichia coli ATCC 8739, with a minimum inhibitory concentration (MIC) of 6.3 µg/mL and a minimum bactericidal concentration (MBC) of 12.5 µg/mL. At ½ the MIC value, it inhibited 82.1% of well-established biofilms of Salmonella enterica. There was an increase in malondialdehyde (MDA) and antioxidative enzymes, especially catalase (CAT) in the treated bacteria because of the peroxidation of membrane lipids and oxidative stress, respectively. SEM proved cellular lysis and surface malformation in most of the treated bacteria. This study concludes that QCS59 siderophore is a promising antibacterial candidate for treating wastewater bacteria and skin pathogens. Full article

The application of synthetic iron chelates to overcome iron deficiency in crops is leading to a high impact on the environment, making it necessary to find more friendly fertilizers. A promising alternative is the application of biodegradable iron chelates, such as those based on siderophores. In the present work, seven bacterial strains of the genus Pseudomonas were selected for their ability to secrete pyoverdine, a siderophore with a high affinity for iron, which could be used as a biofertilizer. The concentration of siderophores secreted by each bacterium expressed as desferrioxamine B equivalents, and the pyoverdine concentration was determined. Their potential as Fe biofertilizers was determined based on their capacity to complex Fe, determining the maximum iron complexation capacity at alkaline pH and selecting the RMC4 strain. The biostimulant capacity of the RMC4 strain was evaluated through the secretion of organic acids such as the hormone Indol-3-acetic acid or glutamic acid, among others, in a kinetic assay. Finally, the genome of RMC4 was determined, and the strain was identified as Pseudomonas monsensis. The annotated genome was screened for genes and gene clusters implicated in biofertilization and plant growth promotion. Besides iron mobilization, genes related to phosphorus solubilization, production of phytohormones and biological control, among others, were observed, indicating the suitability of RMC4 as an inoculant. In conclusion, RMC4 and its siderophores are promising sources for Fe biofertilization in agriculture. Full article

Pseudomonas aeruginosa ST274 is an international epidemic high-risk clone, mostly associated with hospital settings and appears to colonize cystic fibrosis (CF) patients worldwide. To understand the relevant mechanisms for its success, the biological and genomic characteristics of 11 ST274-P. aeruginosa strains from clinical and non-clinical origins were analyzed. The extensively drug-resistant (XDR/DTR), the non-susceptible to at least one agent (modR), and the lasR-truncated (by ISPsp7) strains showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity and low motility. Furthermore, the XDR/DTR and modR strains presented low pigment production and biofilm formation, which were very high in the lasR-truncated strain. Their whole genome sequences were compared with other 14 ST274-P. aeruginosa genomes available in the NCBI database, and certain associations have been primarily detected: bla_OXA-486 and bla_PDC-24 genes, serotype O:3, exoS⁺/exoU⁻ genotype, group V of type IV pili, and pyoverdine locus class II. Other general molecular markers highlight the absence of vqsM and pldA/tleS genes and the presence of the same mutational pattern in genes involving two-component sensor-regulator systems PmrAB and CreBD, exotoxin A, quorum-sensing RhlI, beta-lactamase expression regulator AmpD, PBP1A, or FusA2 elongation factor G. The proportionated ST274-P. aeruginosa results could serve as the basis for more specific studies focused on better antibiotic stewardship and new therapeutic developments. Full article

Pseudomonas aeruginosa (PA), one of the ESKAPE pathogens, is an opportunistic Gram-negative bacterium responsible for nosocomial infections in humans but also for infections in patients affected by AIDS, cancer, or cystic fibrosis (CF). Treatment of PA infections in CF patients is a global healthcare problem due to the ability of PA to gain antibiotic tolerance through biofilm formation. Anti-virulence compounds represent a promising approach as adjuvant therapy, which could reduce or eliminate the pathogenicity of PA without impacting its growth. Pyocyanin is one of the virulence factors whose production is modulated by the Pseudomonas quinolone signal (PQS) through its receptor PqsR. Different PqsR modulators have been synthesized over the years, highlighting this new powerful therapeutic strategy. Based on the promising structure of quinazolin-4(3H)-one, we developed compounds 7a–d, 8a,b, 9, 10, and 11a–f able to reduce biofilm formation and the production of virulence factors (pyocyanin and pyoverdine) at 50 µM in two PA strains responsible for CF acute and chronic infections. The developed compounds did not reduce the cell viability of IB3-1 bronchial CF cells, and computational studies confirmed the potential ability of novel compounds to act as potential Pqs system modulators. Full article