NorA efflux pump mediates Staphylococcus aureus response to Pseudomonas aeruginosa pyocyanin toxicity.

Endogenous transporters protect Staphylococcus aureus against antibiotics and also contribute to bacterial defense from environmental toxins. We evaluated the effect of overexpression of four efflux pumps, NorA, NorB, NorC, and Tet38, on S. aureus survival following exposure to pyocyanin (PYO) of Pseudomonas aeruginosa, using a well diffusion assay. We measured the PYO-created inhibition zone and found that only an overexpression of NorA reduced S. aureus susceptibility to pyocyanin killing. The MICPYO of the NorA overexpressor increased threefold compared to that of wild-type RN6390 and was reduced 2.5-fold with reserpine, suggesting that increased NorA efflux caused PYO resistance. The PYO-created inhibition zone of a ΔnorA mutant was consistently larger than that of a plasmid-borne NorA overexpressor. PYO also produced a modest increase in norA expression (1.8-fold at 0.25 µg/mL PYO) that gradually decreased with increasing PYO concentrations. Well diffusion assays carried out using P. aeruginosa showed that ΔnorA mutant was less susceptible to killing by PYO-deficient mutants PA14phzM and PA14phzS than to killing by PA14. NorA overexpression led to reduced killing by all tested P. aeruginosa. We evaluated the NorA-PYO interaction using a collection of 22 clinical isolates from adult and pediatric cystic fibrosis (CF) patients, which included both S. aureus (CF-SA) and P. aeruginosa (CF-PA). We found that when isolated alone, CF-PA and CF-SA expressed varying levels of PYO and norA transcripts, but all four CF-PA/CF-SA pairs isolated concurrently from CF patients produced a low level of PYO and low norA transcript levels, respectively, suggesting a partial adaptation of the two bacteria in circumstances of persistent co-colonization.

Synthesis and Biological Evaluation of New Quinoline and Anthranilic Acid Derivatives as Potential Quorum Sensing Inhibitors.

Inhibiting quorum sensing (QS), a central communication system, is a promising strategy to combat bacterial pathogens without antibiotics. Here, we designed novel hybrid compounds targeting the PQS (Pseudomonas quinolone signal)-dependent quorum sensing (QS) of Pseudomonas aeruginosa that is one of the multidrug-resistant and highly virulent pathogens with urgent need of new antibacterial strategies. We synthesized 12 compounds using standard procedures to combine halogen-substituted anthranilic acids with 4-(2-aminoethyl/4-aminobuthyl)amino-7-chloroquinoline, linked via 1,3,4-oxadiazole. Their antibiofilm activities were first pre-screened using Gram-negative Chromobacterium violaceum-based reporter, which identified compounds 15-19 and 23 with the highest anti-QS and minimal bactericidal effects in a single experiment. These five compounds were then evaluated against P. aeruginosa PAO1 to assess their ability to prevent biofilm formation, eradicate pre-formed biofilms, and inhibit virulence using pyocyanin as a representative marker. Compound 15 displayed the most potent antibiofilm effect, reducing biofilm formation by nearly 50% and pre-formed biofilm masses by 25%. On the other hand, compound 23 exhibited the most significant antivirulence effect, reducing pyocyanin synthesis by over 70%. Thus, our study highlights the potential of 1,3,4-oxadiazoles 15 and 23 as promising scaffolds to combat P. aeruginosa. Additionally, interactive QS systems should be considered to achieve maximal anti-QS activity against this clinically relevant species.

Antibiofilm and antimicrobial activity of Lactobacillus cell free supernatant against Pseudomonas aeruginosa isolated from burn wounds.

The present study investigated the antimicrobial and anti-biofilm effects of indigenous Lactobacillus probiotic strains on Pseudomonas aeruginosa isolated from burn wound infection in laboratory conditions. The effect of 7 probiotic strains isolated from infant faeces on the pathogenicity factors of P. aeruginosa, including protease, elastase, antibiofilm and antipyocyanin was measured. Also, diffusion methods in the well and micro broth dilution were used to evaluate the antimicrobial activity of probiotics. All tests were performed in triplicate. A negative control and a positive control were used for each test. SPSS version 22 software was used for statistical analysis, and a p < 0.05 was considered statistically significant. A total of 30 clinical isolates of P. aeruginosa were isolated. The elastolytic activity of P. aeruginosa isolates decreased after adding Cell free supernatant (CFS) of each Lactobacillus. L1, L4, L5, and L6 strains had a 100% inhibitory effect on pathogen isolates. L3 and L7 strains had the lowest inhibitory effect. The inhibitory effect of CFS extracted from lactobacilli on protease production by P. aeruginosa. L1, L4, L5, and L6 strains had an inhibitory effect on all tested isolates. L2, L3, and L7 strains had a less inhibitory effect. L4 strain had the highest inhibitory effect on pyocyanin production by P. aeruginosa (50%), followed by L5 (43.3%), L1 (40%), and L6 (23.3%) strains. L3 and L7 strains had no inhibitory effect on the pyocyanin production of P. aeruginosa isolates. It was found that the CFS of 4 isolates (L1, L4, L5, and L6) was the most active extract and had a 100% inhibitory effect against biofilm formation of all P. aeruginosa strains. The L3 strain had the least inhibitory effect against the biofilm formation of pathogens. Overall, this study showed that probiotics could be promising alternatives to combat the pathogenicity of P. aeruginosa in burn wounds.

Real-time in-situ electrochemical monitoring of Pseudomonas aeruginosa biofilms grown on air-liquid interface and its antibiotic susceptibility using a novel dual-chamber microfluidic device.

Biofilms are communities of bacterial cells encased in a self-produced polymeric matrix that exhibit high tolerance toward environmental stress. Despite the plethora of research on biofilms, most P. aeruginosa biofilm models are cultured on a solid-liquid interface, and the longitudinal growth characteristics of P. aeruginosa biofilm are unclear. This study demonstrates the real-time and noninvasive monitoring of biofilm growth using a novel dual-chamber microfluidic device integrated with electrochemical detection capabilities to monitor pyocyanin (PYO). The growth of P. aeruginosa biofilms on the air-liquid interface (ALI) was monitored over 48 h, and its antibiotic susceptibility to 6 h exposure of 50, 400, and 1600 µg/ml of ciprofloxacin solutions was analyzed. The biofilm was treated directly on its surface and indirectly from the substratum by delivering the CIP solution to the top or bottom chamber of the microfluidic device. Results showed that P. aeruginosa biofilm developed on ALI produces PYO continuously, with the PYO production rate varying longitudinally and peak production observed between 24 and 30 h. In addition, this current study shows that the amount of PYO produced by the ALI biofilm is proportional to its viable cell numbers, which has not been previously demonstrated. Biofilm treated with ciprofloxacin solution above 400 µg/ml showed significant PYO reduction, with biofilms being killed more effectively when treatment was applied to their surfaces. The electrochemical measurement results have been verified with colony-forming unit count results, and the strong correlation between the PYO electrical signal and the viable cell number highlights the usefulness of this approach for fast and low-cost ALI biofilm study and antimicrobial tests.

Biosynthesis of Gold Nanoparticles and Its Effect against Pseudomonas aeruginosa.

Antimicrobial resistance has posed a serious health concern worldwide, which is mainly due to the excessive use of antibiotics. In this study, gold nanoparticles synthesized from the plant Tinospora cordifolia were used against multidrug-resistant Pseudomonas aeruginosa. The active components involved in the reduction and stabilization of gold nanoparticles were revealed by gas chromatography-mass spectrophotometry(GC-MS) of the stem extract of Tinospora cordifolia. Gold nanoparticles (TG-AuNPs) were effective against P. aeruginosa at different concentrations (50,100, and 150 µg/mL). TG-AuNPs effectively reduced the pyocyanin level by 63.1% in PAO1 and by 68.7% in clinical isolates at 150 µg/mL; similarly, swarming and swimming motilities decreased by 53.1% and 53.8% for PAO1 and 66.6% and 52.8% in clinical isolates, respectively. Biofilm production was also reduced, and at a maximum concentration of 150 µg/mL of TG-AuNPs a 59.09% reduction inPAO1 and 64.7% reduction in clinical isolates were observed. Lower concentrations of TG-AuNPs (100 and 50 µg/mL) also reduced the pyocyanin, biofilm, swarming, and swimming. Phenotypically, the downregulation of exopolysaccharide secretion from P. aeruginosa due to TG-AuNPs was observed on Congo red agar plates.

A purified and lyophilized Pseudomonas aeruginosa derived pyocyanin induces promising apoptotic and necrotic activities against MCF-7 human breast adenocarcinoma.

BACKGROUND: Pyocyanin, a specific extracellular secondary metabolite pigment produced by Pseudomonas aeruginosa, exhibits redox activity and has toxic effects on mammalian cells, making it a new and potent alternative for treating cancer. Breast cancer (BC) treatment is now defied by acquired and de novo resistance to chemotherapy, radiation, or targeted therapies. Therefore, the anticancer activity of purified and characterized pyocyanin was examined against BC in our study. RESULTS: The maximum production of pyocyanin (53 µg/ml) was achieved by incubation of the highest pyocyanin-producing P. aeruginosa strain (P32) in pH-adjusted peptone water supplemented with 3% cetrimide under shaking conditions at 37 °C for 3 days. The high purity of the extracted pyocyanin was proven by HPLC against standard pyocyanin. The stability of pyocyanin was affected by the solvent in which it was stored. Therefore, the purified pyocyanin extract was lyophilized to increase its shelf-life up to one year. Using the MTT assay, we reported, for the first time, the cytotoxic effect of pyocyanin against human breast adenocarcinoma (MCF-7) with IC50 = 15 µg/ml while it recorded a safe concentration against human peripheral blood mononuclear cells (PBMCs). The anticancer potential of pyocyanin against MCF-7 was associated with its apoptotic and necrotic activities which were confirmed qualitatively and quantitively using confocal laser scanning microscopy, inverted microscopy, and flow cytometry. Caspase-3 measurements, using real-time PCR and western blot, revealed that pyocyanin exerted its apoptotic activity against MCF-7 through caspase-3 activation. CONCLUSION: Our work demonstrated that pyocyanin may be an ideal anticancer candidate, specific to cancer cells, for treating MCF-7 by its necrotic and caspase-3-dependent apoptotic activities.

Virulence Induction in Pseudomonas aeruginosa under Inorganic Phosphate Limitation: a Proteomics Perspective.

Inorganic phosphate (Pi) is a central nutrient and signal molecule for bacteria. Pi limitation was shown to increase the virulence of several phylogenetically diverse pathogenic bacteria with different lifestyles. Hypophosphatemia enhances the risk of death in patients due to general bacteremia and was observed after surgical injury in humans. Phosphate therapy, or the reduction of bacterial virulence by the administration of Pi or phosphate-containing compounds, is a promising anti-infective therapy approach that will not cause cytotoxicity or the emergence of antibiotic-resistant strains. The proof of concept of phosphate therapy has been obtained using primarily Pseudomonas aeruginosa (PA). However, a detailed understanding of Pi-induced changes at protein levels is missing. Using pyocyanin production as proxy, we show that the Pi-mediated induction of virulence is a highly cooperative process that occurs between 0.2 to 0.6 mM Pi. We present a proteomics study of PA grown in minimal medium supplemented with either 0.2 mM or 1 mM Pi and rich medium. About half of the predicted PA proteins could be quantified. Among the 1,471 dysregulated proteins comparing growth in 0.2 mM to 1 mM Pi, 1,100 were depleted under Pi-deficient conditions. Most of these proteins are involved in general and energy metabolism, different biosynthetic and catabolic routes, or transport. Pi depletion caused accumulation of proteins that belong to all major families of virulence factors, including pyocyanin synthesis, secretion systems, quorum sensing, chemosensory signaling, and the secretion of proteases, phospholipases, and phosphatases, which correlated with an increase in exoenzyme production and antibacterial activity. IMPORTANCE Antibiotics are our main weapons to fight pathogenic bacteria, but the increase in antibiotic-resistant strains and their consequences represents a major global health challenge, revealing the necessity to develop alternative antimicrobial strategies that do not involve the bacterial killing or growth inhibition. P. aeruginosa has been placed second on the global priority list to guide research on the development of new antibiotics. One of the most promising alternative strategies is the phosphate therapy for which the proof of concept has been obtained for P. aeruginosa. This article reports the detailed changes at the protein levels comparing P. aeruginosa grown under Pi-abundant and Pi-depleted conditions. These data describe in detail the molecular mechanisms underlying phosphate therapy. Apart from Pi, several other phosphate-containing compounds have been used for phosphate therapy and this study will serve as a reference for comparative studies aimed at evaluating the effect of alternative compounds.

Evaluating the Antivirulence Effects of New Thiazolidinedione Compounds Against Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an opportunistic pathogen that causes several serious health problems and numerous forms of virulence. During the treatment of P. aeruginosa infections, the development of multidrug-resistant isolates creates significant clinical problems. Using antivirulence compounds to disrupt pathogenicity rather than killing the bacterium may be an interesting strategy to overcome this problem, because less harsh conditions will exist for the development of resistance. To reduce pathogenicity and biofilm formation, newly synthesized analogs of imidazolyl (8n) and previously synthesized analogs (8a-8m) with a similar backbone [the 5-(imidazolyl-methyl) thiazolidinediones] were tested against pyoverdine and pyocyanin production, protease activity, and biofilm formation. Compared to the positive control group, the best compounds reduced the production of pyoverdine (8n) by 89.57% and pyocyanin (8i) by 22.68%, and protease activity (8n) by 2.80% for PAO1 strain, at a concentration of 10 µM. Moreover, the biofilm formation assay showed a reduction of 87.94% (8i) for PAO1, as well as 30.53% (8d) and 44.65% (8m) for 1074 and 1707 strains, respectively. The compounds used in this study did not show any toxicity in the human dermal fibroblasts and 4T1 cells (viability higher than 90%). The in silico study of these compounds revealed that their antivirulence activity could be due to their interaction with the PqsR, PqsE, and LasR receptors.

Novel α-Amylase Inhibitor Hemi-Pyocyanin Produced by Microbial Conversion of Chitinous Discards.

α-Amylase inhibitors (aAIs) have been applied for the efficient management of type 2 diabetes. The aim of this study was to search for potential aAIs produced by microbial fermentation. Among various bacterial strains, Pseudomonas aeruginosa TUN03 was found to be a potential aAI-producing strain, and shrimp heads powder (SHP) was screened as the most suitable C/N source for fermentation. P. aeruginosa TUN03 exhibited the highest aAIs productivity (3100 U/mL) in the medium containing 1.5% SHP with an initial pH of 7-7.5, and fermentation was performed at 27.5 °C for two days. Further, aAI compounds were investigated for scaled-up production in a 14 L-bioreactor system. The results revealed a high yield (4200 U/mL) in a much shorter fermentation time (12 h) compared to fermentation in flasks. Bioactivity-guided purification resulted in the isolation of one major target compound, identified as hemi-pyocyanin (HPC) via gas chromatography-mass spectrometry and nuclear magnetic resonance. Its purity was analyzed by high-performance liquid chromatography. HPC demonstrated potent α-amylase inhibitory activity comparable to that of acarbose, a commercial antidiabetic drug. Notably, HPC was determined as a new aAI. The docking study indicated that HPC inhibits α-amylase by binding to amino acid Arg421 at the biding site on enzyme α-amylase with good binding energy (-9.3 kcal/mol) and creating two linkages of H-acceptors.

Evaluation of Blood-Brain-Barrier Permeability, Neurotoxicity, and Potential Cognitive Impairment by Pseudomonas aeruginosa's Virulence Factor Pyocyanin.

Pyocyanin (PCN) is a redox-active secondary metabolite produced by Pseudomonas aeruginosa as its primary virulence factor. Several studies have reported the cytotoxic potential of PCN and its role during infection establishment and progression. Considering its ability to diffuse through biological membranes, it is hypothesized that PCN can gain entry into the brain and induce oxidative stress across the blood-brain barrier (BBB), ultimately contributing towards reactive oxygen species (ROS) mediated neurodegeneration. Potential roles of PCN in the central nervous system (CNS) have never been evaluated, hence the study aimed to evaluate PCN's probable penetration into CNS through blood-brain barrier (BBB) using both in silico and in vivo (Balb/c mice) approaches and the impact of ROS generation via commonly used tests: Morris water maze test, novel object recognition, elevated plus maze test, and tail suspension test. Furthermore, evidence for ROS generation in the brain was assessed using glutathione S-transferase assay. PCN demonstrated BBB permeability albeit in minute quantities. A significant hike was observed in ROS generation (P < 0.0001) along with changes in behavior indicating PCN permeability across BBB and potentially affecting cognitive functions. This is the first study exploring the potential role of PCN in influencing the cognitive functions of test animals.