PAO1 Biofilm Research Articles

Dipeptides from Lactiplantibacillus plantarum limit Pseudomonas aeruginosa pathogenesis.

Lactobacilli have drawn a great deal of attention due to the heterogeneity of their cells, as well as their remarkable ability to adapt to a range of harsh conditions. One such adaptation response by Lactiplantibacillus plantarum was forming a V-shaped cell structure in low acidic pH conditions. This study aims to identify low pH induced-biofilm formation in L. plantarum and Limosilactobacillus reuteri and their antagonistic interactions with an opportunistic pathogen, Pseudomonas aeruginosa PAO1. Dual species bacterial interactions revealed a Lactobacilli biofilm-mediated suppression of PAO1 biofilms and pyocyanin pigment. Furthermore, filtrates from Lactobacilli grown at low acidic pH (pH 3.5) effectively arrest the pathogenesis of P. aeruginosa PAO1, including biofilm, swarming motility, and pyocyanin formation. Liquid chromatography-mass spectroscopy analysis revealed the presence of several Lactobacilli-secreted metabolites and dipeptides that exhibited strong LasR receptor antagonistic interactions in molecular docking analysis. Prolyl-tyrosine showed the best lasR antagonistic and PAO1 biofilm inhibitory activities of the identified dipeptides. Overall, the results will contribute to the development of anti-infective drugs against P. aeruginosa using Lactobacilli secreted dipeptides. Furthermore, the investigation identifies several dipeptides that can be extracted from V-shaped Lactobacilli for antimicrobial protein therapy against P. aeruginosa and other pathogens.

Antimicrobial and antibiofilm activity of human recombinant H1 histones against bacterial infections.

Histones possess significant antimicrobial potential, yet their activity against biofilms remains underexplored. Moreover, concerns regarding adverse effects limit their clinical implementation. We investigated the antibacterial efficacy of human recombinant histone H1 subtypes against Pseudomonas aeruginosa PAO1, both planktonic and in biofilms. After the in vitro tests, toxicity and efficacy were assessed in a P. aeruginosa PAO1 infection model using Galleria mellonella larvae. Histones were also evaluated in combination with ciprofloxacin (Cpx) and gentamicin (Gm). Our results demonstrate antimicrobial activity of all three histones against P. aeruginosa PAO1, with H1.0 and H1.4 showing efficacy at lower concentrations. The bactericidal effect was associated with a mechanism of membrane disruption. In vitro studies using static and dynamic models showed that H1.4 had antibiofilm potential by reducing cell biomass. Neither H1.0 nor H1.4 showed toxicity in G. mellonella larvae, and both increased larvae survival when infected with P. aeruginosa PAO1. Although in vitro synergism was observed between ciprofloxacin and H1.0, no improvement over the antibiotic alone was noted in vivo. Differences in antibacterial and antibiofilm activity were attributed to sequence and structural variations among histone subtypes. Moreover, the efficacy of H1.0 and H1.4 was influenced by the presence and strength of the extracellular matrix. These findings suggest histones hold promise for combating acute and chronic infections caused by pathogens such as P. aeruginosa.IMPORTANCEThe constant increase of multidrug-resistant bacteria is a critical global concern. The inefficacy of current therapies to treat bacterial infections is attributed to multiple mechanisms of resistance, including the capacity to form biofilms. Therefore, the identification of novel and safe therapeutic strategies is imperative. This study confirms the antimicrobial potential of three histone H1 subtypes against both Gram-negative and Gram-positive bacteria. Furthermore, histones H1.0 and H1.4 demonstrated in vivo efficacy without associated toxicity in an acute infection model of Pseudomonas aeruginosa PAO1 in Galleria mellonella larvae. The bactericidal effect of these proteins also resulted in biomass reduction of P. aeruginosa PAO1 biofilms. Given the clinical significance of this opportunistic pathogen, our research provides a comprehensive initial evaluation of the efficacy, toxicity, and mechanism of action of a potential new therapeutic approach against acute and chronic bacterial infections.

Antimicrobial and antibiotic-potentiating effect of calcium peroxide nanoparticles on oral bacterial biofilms

Bacterial biofilms represent a prominent biological barrier against physical and chemical attacks. Disturbing the anaerobic microenvironment within biofilms by co-delivery of oxygen appears as a promising strategy to enhance the activity of an antibiotic. Here, we report the effect of oxygen-producing calcium peroxide nanoparticles (CaO2 NP) in combination with tobramycin sulfate (Tob). On Pseudomonas aeruginosa PAO1 biofilms in vitro, the additive effect of CaO2 NP towards Tob activity enhanced biofilm eradication by 2 log compared to Tob alone. For natural biofilms grown in the oral cavity of human volunteers in situ, treatment by CaO2 NP alone slightly increased the fraction of dead bacteria from 44% in various controls, including Tob alone, to 57%. However, the combination of CaO2 NP with Tob further increased the fraction of dead bacteria to 69%. These data confirm the intrinsic antimicrobial and antibiotic-potentiating effect of CaO2 NP also in a clinically relevant setting.

Combinations of Bacteriophage Are Efficacious against Multidrug-Resistant Pseudomonas aeruginosa and Enhance Sensitivity to Carbapenem Antibiotics.

The Gram-negative ESKAPE bacterium Pseudomonas aeruginosa has become a pathogen of serious concern due its extensive multi-drug resistance (MDR) profile, widespread incidences of hospital-acquired infections throughout the United States, and high occurrence in wound infections suffered by warfighters serving abroad. Bacteriophage (phage) therapy has received renewed attention as an alternative therapeutic option against recalcitrant bacterial infections, both as multi-phage cocktails and in combination with antibiotics as synergistic pairings. Environmental screening and phage enrichment has yielded three lytic viruses capable of infecting the MDR P. aeruginosa strain PAO1. Co-administration of each phage with the carbapenem antibiotics ertapenem, imipenem, and meropenem generated enhanced overall killing of bacteria beyond either phage or drug treatments alone. A combination cocktail of all three phages was completely inhibitory to growth, even without antibiotics. The same 3× phage cocktail also disrupted PAO1 biofilms, reducing biomass by over 75% compared to untreated biofilms. Further, the phage cocktail demonstrated broad efficacy as well, capable of infecting 33 out of 100 diverse clinical isolate strains of P. aeruginosa. Together, these results indicate a promising approach for designing layered medical countermeasures to potentiate antibiotic activity and possibly overcome resistance against recalcitrant, MDR bacteria such as P. aeruginosa. Combination therapy, either by synergistic phage-antibiotic pairings, or by phage cocktails, presents a means of controlling mutations that can allow for bacteria to gain a competitive edge.

Edible Medicinal Guava Fruit (Psidium guajava L.) Are a Source of Anti-Biofilm Compounds against Pseudomonas aeruginosa.

Psidium guajava is one of the most common edible medicinal plants frequently used in Malagasy traditional medicine to treat gastrointestinal infections. In order to evaluate their probable antibacterial activities, three organic extracts (successive extractions by hexane, dichloromethane, and ethanol) of ripe guava fruits were assessed for their bactericidal and anti-virulence properties against P. aeruginosa PAO1. Although these three extracts have shown no direct antibacterial activity (MIC of 1000 µg/mL) and, at the non-bactericidal concentration of 100 µg/mL, no impact on the production of major P. aeruginosa PAO1 virulence factors (pyocyanin and rhamnolipids), the hexane and dichloromethane extracts showed significant anti-biofilm properties and the dichloromethane extract disrupted the P. aeruginosa PAO1 swarming motility. Bioguided fractionation of the dichloromethane extract led to the isolation and identification of lycopene and β-sitosterol-β-D-glucoside as major anti-biofilm compounds. Interestingly, both compounds disrupt P. aeruginosa PAO1 biofilm formation and maintenance with IC50 of 1383 µM and 131 µM, respectively. More interestingly, both compounds displayed a synergistic effect with tobramycin with a two-fold increase in its effectiveness in killing biofilm-encapsulated P. aeruginosa PAO1. The present study validates the traditional uses of this edible medicinal plant, indicating the therapeutic effectiveness of guava fruits plausibly through the presence of these tri- and tetraterpenoids, which deserve to be tested against pathogens generally implicated in diarrhea.

Efficacy and mechanisms of Pseudomonas aeruginosa PAO1 biofilm inactivation using high-power pulsed microwave

Foodborne pathogens and biofilm formation pose a critical threat to food safety. Pseudomonas aeruginosa is a pathogen of interest in the food industry. High-power pulsed microwave (HPPM) is a non-thermal technology with proven efficacy towards bacterial inactivation. This study investigated the inactivation mechanisms and performance of different HPPM treatments (50, 100, 150, 200, and 250 Hz for 3, 5, 7, 9, and 11 min, respectively) to identify the optimum conditions to inactivate P. aeruginosa PAO1 biofilms. The results showed significant cell inactivation at increasing frequency/time (p < 0.05). Hence, HPPM treatment (200 Hz, 9 min) was selected following its efficacy and limited thermal sublethal effects to elucidate further the inactivation mechanisms against P. aeruginosa and its biofilm-forming ability. Cell viability, scanning (SEM) and transmission electron microscopy (TEM) results demonstrated severe morphological disruptions, cell disintegration, and cell lysis in the treated cells. From the confocal laser scanning micrographs (CLSM), biofilm thickness was calculated, showing a significant reduction after treatment (17.56 vs. 42.16 μm in the treated and untreated groups, respectively), indicating that HPPM effectively compromised the integrity of the biofilm envelope. This study outlines useful insights into better understanding HPPM's efficacy on microbial inactivation and biofilm proliferation in the food industry.

BacA: a possible regulator that contributes to the biofilm formation of Pseudomonas aeruginosa.

Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for "Biofilm Associated Cluster." The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins.

The Potentiation Activity of Azithromycin in Combination with Colistin or Levofloxacin Against Pseudomonas aeruginosa Biofilm Infection.

Pseudomonas aeruginosa (PA) often displays drug resistance and biofilm-mediated adaptability. Here, we aimed to evaluate the antibiofilm efficacy of azithromycin-based combination regimens. Minimum inhibitory concentrations (MICs), minimal biofilm eradication concentrations (MBECs), and MBEC-combination of azithromycin, colistin, amikacin, and levofloxacin to bioluminescent strain PAO1 and carbapenem-resistant PAO1 (CRPAO1) were assessed. An animal biofilm infection model was established and detected using a live animal bio-photonic imaging system. In vitro, PAO1 and CRPAO1 were susceptible to colistin, amikacin, and levofloxacin, while they were unsusceptible to azithromycin. The combinations based on azithromycin have no synergistic effect on biofilm in vitro. In vivo, azithromycin plus colistin or levofloxacin could shorten the PAO1 biofilm eradication time, which totally eradicates the biofilm in all mice on the 8th or 6th day, while monotherapy only eradicate biofilm in 70% or 80% mice on the 8th day. For CRPAO1 biofilm, only azithromycin-colistin combination and colistin monotherapy eradicated the bacteria in 60% and 40% of mice at the 6th day. Azithromycin-based combinations containing levofloxacin or colistin had no synergistic effect in vitro, and they are promising for clinical applications due to the good synergistic activity against PAO1 biofilms in vivo.

In vitro inhibition of Pseudomonas aeruginosa PAO1 biofilm formation by DZ2002 through regulation of extracellular DNA and alginate production.

Pseudomonas aeruginosa (P. aeruginosa) is a common pathogen associated with biofilm infections, which can lead to persistent infections. Therefore, there is an urgent need to develop new anti-biofilm drugs. DZ2002 is a reversible inhibitor that targets S-adenosylhomocysteine hydrolase and possesses anti-inflammatory and immune-regulatory activities. However, its anti-biofilm activity has not been reported yet. Therefore, we investigated the effect of DZ2002 on P. aeruginosa PAO1 biofilm formation by crystal violet staining (CV), real-time quantitative polymerase chain reaction (RT-qPCR) and confocal laser scanning microscopy (CLSM). The results indicated that although DZ2002 didn't affect the growth of planktonic PAO1, it could significantly inhibit the formation of mature biofilms. During the inhibition of biofilm formation by DZ2002, there was a parallel decrease in the synthesis of alginate and the expression level of alginate genes, along with a weakening of swarming motility. However, these results were unrelated to the expression of lasI, lasR, rhII, rhIR. Additionally, we also found that after treatment with DZ2002, the biofilms and extracellular DNA content of PAO1 were significantly reduced. Molecular docking results further confirmed that DZ2002 had a strong binding affinity with the active site of S-adenosylhomocysteine hydrolase (SahH) of PAO1. In summary, our results indicated that DZ2002 may interact with SahH in PAO1, inhibiting the formation of mature biofilms by downregulating alginate synthesis, extracellular DNA production and swarming motility. These findings demonstrate the potential value of DZ2002 in treating biofilm infections associated with P. aeruginosa.

Identification of genes involved in enhanced membrane vesicle formation in Pseudomonas aeruginosa biofilms: surface sensing facilitates vesiculation.

Membrane vesicles (MVs) are small spherical structures (20-400 nm) produced by most bacteria and have important biological functions including toxin delivery, signal transfer, biofilm formation, and immunomodulation of the host. Although MV formation is enhanced in biofilms of a wide range of bacterial species, the underlying mechanisms are not fully understood. An opportunistic pathogen, Pseudomonas aeruginosa, causes chronic infections that can be difficult to treat due to biofilm formation. Since MVs are abundant in biofilms, can transport virulence factors to the host, and have inflammation-inducing functions, the mechanisms of enhanced MV formation in biofilms needs to be elucidated to effectively treat infections. In this study, we evaluated the characteristics of MVs in P. aeruginosa PAO1 biofilms, and identified factors that contribute to enhanced MV formation. Vesiculation was significantly enhanced in the static culture; MVs were connected to filamentous substances in the biofilm, and separation between the outer and inner membranes and curvature of the membrane were observed in biofilm cells. By screening a transposon mutant library (8,023 mutants) for alterations in MV formation in biofilms, 66 mutants were identified as low-vesiculation strains (2/3 decrease relative to wild type), whereas no mutant was obtained that produced more MVs (twofold increase). Some transposons were inserted into genes related to biofilm formation, including flagellar motility (flg, fli, and mot) and extracellular polysaccharide synthesis (psl). ΔpelAΔpslA, which does not synthesize the extracellular polysaccharides Pel and Psl, showed reduced MV production in biofilms but not in planktonic conditions, suggesting that enhanced vesiculation is closely related to the synthesis of biofilm matrices in P. aeruginosa. Additionally, we found that blebbing occurred during bacterial attachment. Our findings indicate that biofilm-related factors are closely involved in enhanced MV formation in biofilms and that surface sensing facilitates vesiculation. Furthermore, this work expands the understanding of the infection strategy in P. aeruginosa biofilms.

2065. Efficacy of D-peptide-conjugated Antisense Compounds Against Pseudomonas aeruginosa

Abstract Background Pseudomonas aeruginosa (PA) causes significant morbidity and mortality in immunocompromised hosts and those with cystic fibrosis. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are antisense compounds designed to target specific genes and prevent translation. PPMOs with L-isomer peptides (L-PPMOs) were previously shown to have in vitro and in vivo activity. However, L-PPMOs are vulnerable to degradation by host proteases. We hypothesize that PPMOs with a D-isomer peptide (D-PPMOs) may be a more stable and potent alternative. We aimed to characterize the activity of L- and D-isomers in PA strains using planktonic and biofilm assays as well as an infected mouse model. Methods All L- and D-PPMOs were synthesized by Sarepta Therapeutics, Inc. We tested L- and D-isomers of the (RXR)4XB (X for 6-aminohexanoic acid and B for β-Ala) and R6G peptide [(Arg)6-Gly]. PMOs for rpsJ (ribosomal protein) and acpP (fatty acid synthesis protein) were conjugated to the 3’ end of each peptide. Minimum inhibitory concentration (MIC) assays were performed in unique lab and clinical (including MDR) isolates using MOPS medium. Strain PAO1 biofilms were grown in MBEC plates for 24 hours, with PPMOs then dosed every 8 hours for 24 hours, followed by colony enumeration. For in vivo studies, mice were infected with strain PA103 intratracheally and given intranasal PPMO at 6 hours post-infection. Lung burden was determined at 24 hours post-infection. Results MIC values of D-R6G-RpsJ and D-R6G-AcpP were comparable to their L-isomers, but D-RXR-RpsJ showed an improved MIC90 of 0.5 μM compared to the L-RXR-RpsJ MIC90 of 16 μM (Table 1). Both isomers demonstrated reduction of 48-hour biofilm to various degrees. D-PPMO biofilm reduction ranged from 1.5 logs to &amp;gt; 3 logs (D-R6G-AcpP and D-RXR-RpsJ, respectively). L-PPMO biofilm reduction ranged from 2 logs to &amp;gt; 4 logs. D-PPMOs were superior to their L-PPMO counterparts in our delayed in vivo acute pneumonia model, with most D-PPMOs resulting in an additional 50% decrease in lung burden. Conclusion D-PPMOs show similar or improved activity in vitro and in vivo when compared to L-PPMOs at the same dosage. Future studies will investigate the pharmacology of D-PPMOs, as they may represent a more biologically robust alternative to L-PPMOs for PA infections. Disclosures David E. Greenberg, MD, University of Texas Southwestern Medical Center: Dr. Greenberg has numerous patents on PPMOs

Evaluating Antibiofilm, Cytotoxic and Apoptotic Activities of Scutellaria brevibracteata subsp. brevibracteata Essential Oil.

Essential oils have many important biological properties, including antibacterial and antibiofilm activities. These unique properties make, essential oils good alternatives to synthetic chemical drugs, which have many side effects. In this study, we aimed to determine the chemical composition and biological activity of the essential oil obtained from Scutellaria brevibracteata subsp. brevibracteata. Specifically, its antibiofilm activity against Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 29213 biofilms using XTT assay. Cytotoxic and apoptotic properties of the essential oil were investigated in human lung cancer cells (A540 and H1299) using MTT assay, Annexin V-FITC and propidium iodide staining and q-PCR. Thirty-two different compounds were identified from the essential oil, of which elemol (20.42 %), γ-eudesmol (20.12 %) and β-eudesmol (14.85 %) were the main components. The essential oil was more effective against P. aeruginosa PAO1 biofilm (79 %) than S. aureus ATCC 29213 biofilm (27 %). The specific activity of the essential oil against P. aeruginosa biofilm may be related to its high terpene contents. In addition, the essential oil showed high cytotoxic activity towards A549 (IC50 9.09 μg/ml) and H1299 (IC50 55.04 μg/ml) cell lines, inducing apoptosis in these cancer cells. These results demonstrate the antibiofilm and anticancer activities of S. brevibracteata subsp. brevibracteata essential oil.

Alkaline phosphatase LapA regulates quorum sensing-mediated virulence and biofilm formation in Pseudomonas aeruginosa PAO1 under phosphate depletion stress.

Among various environmental stresses, the depletion of inorganic phosphate, which is an important signaling molecule that modulates virulence production, is notable. In our previous study, we demonstrated that lapA, encoding alkaline phosphatase, plays a key role in Pseudomonas aeruginosa biofilm formation in porcine skin explants, an ex vivo chronic wound model. However, the mechanism by which lapA participates in the virulence and biofilm formation of P. aeruginosa remains unclear. In the present study, the lapA deletion mutant was constructed in P. aeruginosa PAO1, and the phenotypic characterization was assessed. Our data demonstrated that phosphate depletion stress significantly enhanced elastase activity, hemolysis, rhamnolipid production, swarming and swimming motilities, and 3-oxo-C12-homoserine lactone (HSL) production in P. aeruginosa. Moreover, phosphate starvation evidently induced the virulence of P. aeruginosa in Caenorhabditis elegans (C. elegans) through fast-kill and slow-kill infections. Deletion of the lapA gene led to broad phenotypic changes, including reduced elastase activity, swimming motility, C4-HSL, and 3-oxo-C12-HSL production, as well as increased chitinase activity and rhamnolipid production under phosphate depletion stress, which was unrelated to C4-HSL. Deletion of the lapA gene also significantly inhibited PAO1 biofilm formation in porcine skin explants by reducing the expression levels of las and rhl quorum sensing systems and extracellular polymeric substance synthesis. Finally, lapA deletion also reduced the virulence of PAO1 in C. elegans in fast-kill and slow-kill infection assays. Thus, the findings suggest that targeting lapA can help control biofilm formation and reduce virulence in nonhealing-infected wounds, where P. aeruginosa is a persistent problem. IMPORTANCE Our previous study demonstrated that the expression of lapA was induced under phosphate depletion conditions, but its roles in virulence and biofilm formation by Pseudomonas aeruginosa remain largely unknown. This study presents a systematic investigation of the roles of lapA in virulence induction and biofilm formation by constructing a lapA-deficient strain with P. aeruginosa PAO1. The results showed that deletion of the lapA gene evidently reduced elastase activity, swimming motility, C4-HSL, and 3-oxo-C12-HSL production, and increased rhamnolipid production under phosphate depletion stress. Moreover, lapA gene deletion inhibited PAO1 biofilm formation in porcine skin explants by reducing the expression levels of las and rhl quorum sensing systems and extracellular polymeric substance synthesis. Finally, lapA gene deletion also reduced the virulence of PAO1 in Caenorhabditis elegans in fast-kill and slow-kill infection assays. This study provides insights into the roles of lapA in modulating P. aeruginosa virulence and biofilm formation under phosphate depletion stress.

Comparison of Antibiofilm Activity of Pseudomonas aeruginosa Phages on Isolates from Wounds of Diabetic and Non-Diabetic Patients.

The persistence of organisms as biofilms and the increase in antimicrobial resistance has raised the need for alternative strategies. The study objective was to compare the ability of isolated bacteriophages to remove in vitro biofilms formed by Pseudomonas aeruginosa isolated from the environment with those isolated from diabetic and non-diabetic wounds. P. aeruginosa were isolated from clinical and environmental sites, and antimicrobial susceptibility was tested. Bacteriophages were isolated and characterized based on plaque morphology and host range. A reduction in the viable count assayed the lytic ability of candidate phages. The crystal violet method was used to determine the residual biofilm after 24 h of phage treatment on 72-h-old biofilms. The statistical significance of phage treatment was tested by one-way ANOVA. Of 35 clinical isolates, 17 showed resistance to 1 antibiotic at least, and 7 were multidrug resistant. Nineteen environmental isolates and 11 clinical isolates were drug-sensitive. Nine phages showed 91.2% host coverage, including multidrug-resistant isolates. Phages eradicated 85% of biofilms formed by environmental isolates compared to 58% of biofilms of diabetic isolates and 56% of biofilms of non-diabetic isolates. Clinical isolates are susceptible to phage infection in planktonic form. Biofilms of P. aeruginosa isolated from diabetic wounds and non-diabetic wounds resist removal by phages compared to biofilms formed by environmental isolates. All phages were efficient in dispersing PAO1 biofilms. However, there was a significant difference in their ability to disperse PAO1 biofilms across the different surfaces tested. Partial eradication of biofilm by phages can aid in complementing antibiotics that are unable to penetrate biofilms in a clinical set-up.

Cholinium-Based Ionic Liquids as Promising Antimicrobial Agents in Pharmaceutical Applications: Surface Activity, Antibacterial Activity and Ecotoxicological Profile.

Cholinium-based ionic liquids are compounds increasingly studied in pharmaceutics and biomedicine to enhance bioavailability in drug delivery systems and as bioactive ingredients in pharmaceutical formulations. However, their potential as antimicrobial agents has scarcely been investigated. Herein, we explored the antimicrobial activity of a series of surface-active cholinium-based ionic liquids (Chol-ILs). For this purpose, Chol-ILs with alkyl chains of 10-16 carbon atoms were synthesized and their self-assembly in aqueous medium was investigated. Subsequently, their antimicrobial activity against a panel of clinically relevant bacteria and their ability to eradicate MRSA and P. aeruginosa PAO1 biofilms was evaluated. Finally, we analyzed the ecotoxicological profile of Chol-ILs in terms of susceptibility to aerobic biodegradation and acute aquatic toxicity against D. magna and V. fisheri. Our results reveal that cholinium-based ILs with alkyl chain lengths ≥12 C show a broad spectrum of antibacterial activity. Their antimicrobial efficacy depends on their hydrophobicity, with the C14-C16 homologs being the most effective compounds. These ILs exhibit antimicrobial activity similar to that of imidazolium ILs and quaternary ammonium antiseptics. Moreover, the longer alkyl chain Chol-ILs are able to eradicate established biofilms at concentrations as low as 16-32 µg/mL. The biodegradation rate of cholinium-based ILs decreases with alkyl chain elongation. Our results reinforce the suitability of Chol-ILs as promising multifunctional compounds for application in pharmaceutical and biomedical formulation.

Design, Synthesis, and Biological Evaluation of Phenyloxadiazole Sulfoxide Derivatives as Potent Pseudomonas aeruginosa Biofilm Inhibitors.

With the development of antimicrobial agents, researchers have developed new strategies through key regulatory systems to block the expression of virulence genes without affecting bacterial growth. This strategy can minimize the selective pressure that leads to the emergence of resistance. Quorum sensing (QS) is an intercellular communication system that plays a key role in the regulation of bacterial virulence and biofilm formation. Studies have revealed that the QS system controls 4-6% of the total number of P. aeruginosa genes, and quorum sensing inhibitors (QSIs) could be a promising target for developing new prevention and treatment strategies against P. aeruginosa infection. In this study, four series of phenyloxadiazole and phenyltetrazole sulfoxide derivatives were synthesized and evaluated for their inhibitory effects on P. aeruginosa PAO1 biofilm formation. Our results showed that 5b had biofilm inhibitory activity and reduced the production of QS-regulated virulence factors in P. aeruginosa. In addition, silico molecular docking studies have shown that 5b binds to the P. aeruginosa QS receptor protein LasR through hydrogen bond interaction. Preliminary structure-activity relationship and docking studies show that 5b has broad application prospects as an anti-biofilm compound, and further research will be carried out in the future to solve the problem of microbial resistance.

Design, Synthesis and In Vitro Studies of 3-Amidocoumarins as Novel Antibiofilm Agents

Pseudomonas aeruginosa, a life-threatening bacteria listed as a priority pathogen by World Health Organization WHO, is known to cause severe nosocomial infections and fatality in immunocompromised individuals through its quorum sensing (QS) mediated biofilm formation. P. aeruginosa’s antibiotic-resistant biofilms are highly challenging to the existing antibiotic treatment options. There is an urgent clinical need to develop novel alternative therapeutic molecules such as antibiofilm and antiquorum sensing agents to counter the emergence of an unprecedented pace of antibiotic resistance of pathogens. In this context, a library of seventy 3-amidocoumarin derivatives was designed, and docking studies were performed against the P. aeruginosa LasR receptor using AutoDock 4.0. Based on docking results, a final series of sixteen 3-amidocoumarin derivatives (4a–p) were synthesized and evaluated for antibiofilm activity in vitro. Eight compounds significantly inhibited the formation of P. aeruginosa PAO1 biofilm. Compounds 4f, 4l and 4o showed maximum % inhibition in antibiotic-resistant P. aeruginosa PAO1 biofilm formation in the range of 80% to 86%. Further, the structure–activity relationship (SAR) studies revealed that the presence of electron-donating and bromo substituents at benzamido and coumarin moieties, respectively, effectively enhances the antibiofilm activity. In addition, the binding interactions between the synthesized compounds and active sites of the LasR QS receptor (Protein Data Bank Code: 2uv0) in P. aeruginosa were also investigated by molecular docking. The high binding affinities indicate that these compounds might be suitable for development into potent inhibitors of QS and biofilm disruptors.

Design, synthesis, and biological evaluation of benzoheterocyclic sulfoxide derivatives as quorum sensing inhibitors in Pseudomonas aeruginosa

Six series of benzoheterocyclic sulfoxide derivatives were designed and synthesised as Pseudomonas aeruginosa (P. aeruginosa) quorum sensing inhibitors in this paper. We experimentally demonstrated that 6b significantly inhibited the formation of P. aeruginosa PAO1 biofilm without affecting the growth. Further mechanistic studies showed that 6b affected the luminescence of quorum sensing reported strain PAO1-lasB-gfp and the production of P. aeruginosa PAO1 elastase virulence factor which was regulated by las system. These experimental results indicate that 6b acts as a quorum sensing inhibitor mainly through the las system. Furthermore, silico molecular docking studies demonstrated that 6b and the P. aeruginosa quorum sensing receptor LasR were molecularly bound via hydrogen bonding interactions. Preliminary structure-activity relationship and docking studies illustrated that 6b shows great promise as anti-biofilm compounds for further studies in order to solve the problem of microbial resistance in future.

The aggregate distribution of Pseudomonas aeruginosa on biochar facilitates quorum sensing and biofilm formation

Biochar when applied into soil, together with soil clay minerals, may provide habitats for soil microbes and shift soil microbial community structure. Although several mechanisms have been proposed to explain the effects of biochar on microbial community, the impact of biochar on quorum sensing (QS) and QS-regulated behavior is poorly understood. In this study, we compared the effects of biochar and three common soil minerals (i.e., montmorillonite, kaolinite, and goethite) on QS and biofilm formation. Pseudomonas aeruginosa PAO1 with complete QS systems was selected as a model organism. Our results showed that biochar and goethite effectively promoted microbial QS and biofilm formation, while montmorillonite and kaolinite posed no significant effect. Live/Dead staining, SEM and density-dependent QS activity indicated that biochar was beneficial to cell viability maintenance and cell aggregations, which improved the efficiency of intercellular communications through QS. QS mutant strain experiments confirmed that biochar enhanced PAO1 biofilm formation by promoting QS. Goethite promoted biofilm formation with a different mechanism that cell debris induced by iron ions and positive charge on goethite surface provided raw materials for bacterial biofilm formation. Our findings provide evidence that the presence of biochar can enhance QS and biofilm formation through a feedforward loop of the QS system. This contributes to better understand biochar-mediated microbial cell to cell communications through QS.

The Efficacy of Colistin Combined with Amikacin or Levofloxacin against Pseudomonas aeruginosa Biofilm Infection.

ABSTRACTPseudomonas aeruginosa (PA) biofilm infection is clinically prevalent and difficult to eradicate. In the present work, we aimed to evaluate the in vitro and in vivo efficacy of colistin (COL)-based combinations against PA biofilm. MICs and fractional inhibitory concentration indexes (FICIs) of four antibiotics (COL, amikacin, levofloxacin, and meropenem) to bioluminescent strain PAO1, carbapenem-resistant PAO1 (CRPAO1), and clinically isolated strains were assessed. Minimal biofilm eradication concentrations (MBECs) of monotherapy and combinations were examined by counting the live bacteria in biofilm, accompanied by visual confirmation using confocal laser-scanning microscopy. An animal biofilm infection model was established by implanting biofilm subcutaneously, and the therapeutic effect was evaluated according to the change in luminescence through a live animal bio-photonic imaging system. In vitro, even combined with 4 or 8 mg/L COL, meropenem needed to reach 128 or 256 mg/L to eradicate the biofilm. Moreover, 2 mg/L COL combined with 32 mg/L amikacin or 4-8 mg/L levofloxacin could kill the PAO1 and CRPAO1 in biofilm within 24 h. In vivo, COL combined with amikacin or levofloxacin could shorten the eradication time of biofilm than monotherapy. For PAO1 biofilm, combination therapy could eradicate the biofilm in all mice on the 5th day, whereas monotherapy only eradicated biofilms in almost half of the mice. For CRPAO1 biofilm, the biofilm eradication rate on the 6th day in the COL+ amikacin, amikacin, or COL alone regimen was 90%, 10%, or 40%, respectively. COL combined with levofloxacin did not show a better effect than each individual antibiotic. COL-based combinations containing levofloxacin or amikacin were promising choices for treating PA biofilm infection.IMPORTANCE Infections associated with PA biofilm formation are extremely challenging. When monotherapy fails to achieve optimal efficacy, combination therapy becomes the last option. After evaluating multiple drug combinations through a series of experiments in vitro and in vivo, we confirmed that colistin-based combinations containing levofloxacin or amikacin were promising choices for treating PA biofilm infection. The efficacy of these combinations derives from the different bactericidal mechanisms and the bacterial susceptibility to each antibiotic. This study provided a new regimen to solve the incurable problem of biofilm by using COL combined with other antibiotics.