Tackling Pseudomonas aeruginosa Virulence by Mulinane-Like Diterpenoids from Azorella atacamensis.

Pseudomonas aeruginosa is an important multidrug-resistant human pathogen by dint of its high intrinsic, acquired, and adaptive resistance mechanisms, causing great concern for immune-compromised individuals and public health. Additionally, P. aeruginosa resilience lies in the production of a myriad of virulence factors, which are known to be tightly regulated by the quorum sensing (QS) system. Anti-virulence therapy has been adopted as an innovative alternative approach to circumvent bacterial antibiotic resistance. Since plants are known repositories of natural phytochemicals, herein, we explored the anti-virulence potential of Azorella atacamensis, a medicinal plant from the Taira Atacama community (Calama, Chile), against P. aeruginosa. Interestingly, A. atacamensis extract (AaE) conferred a significant protection for human lung cells and Caenorhabditis elegans nematodes towards P. aeruginosa pathogenicity. The production of key virulence factors was decreased upon AaE exposure without affecting P. aeruginosa growth. In addition, AaE was able to decrease QS-molecules production. Furthermore, metabolite profiling of AaE and its derived fractions achieved by combination of a molecular network and in silico annotation allowed the putative identification of fourteen diterpenoids bearing a mulinane-like skeleton. Remarkably, this unique interesting group of diterpenoids seems to be responsible for the interference with virulence factors as well as on the perturbation of membrane homeostasis of P. aeruginosa. Hence, there was a significant increase in membrane stiffness, which appears to be modulated by the cell wall stress response ECFσ SigX, an extracytoplasmic function sigma factor involved in membrane homeostasis as well as P. aeruginosa virulence.

Phenotypic detection of quorum sensing inhibition in Pseudomonas aeruginosa pyoverdine and swarming by volatile organic products.

Aim: To determine phenotypically the anti quorum-sensing (QS) activity of 30 volatile organic products (VOPs) through the inhibition of swarming motility and pyoverdine production in Pseudomonas aeruginosa. Materials & methods: Twenty-four essential oils and six small volatile organic compounds randomly selected were screened for their anti-QS activity by violacein inhibition on Chromobacterium violaceum. The VOPs with positive results were subsequently evaluated for swarming motility and pyoverdine production on P. aeruginosa determining the colony diameter and fluorescence under UV light, respectively. Results: Fifty percent of VOPs tested showed strong violacein inhibition, 40% presented anti-swarming activity and 33% inhibited pyoverdine production. Conclusion: Our data demonstrate that VOPs have a great potential to inhibit virulence factors mediated by QS in P. aeruginosa.

Hierarchical Rose Petal Surfaces Delay the Early-Stage Bacterial Biofilm Growth.

A variety of natural surfaces exhibit antibacterial properties; as a result, significant efforts in the past decade have been dedicated toward fabrication of biomimetic surfaces that can help control biofilm growth. Examples of such surfaces include rose petals, which possess hierarchical structures like the micropapillae measuring tens of microns and nanofolds that range in the size of 700 ± 100 nm. We duplicated the natural structures on rose petal surfaces via a simple UV-curable nanocasting technique and tested the efficacy of these artificial surfaces in preventing biofilm growth using clinically relevant bacteria strains. The rose petal-structured surfaces exhibited hydrophobicity (contact angle (CA) ≈ 130.8° ± 4.3°) and high CA hysteresis (∼91.0° ± 4.9°). Water droplets on rose petal replicas evaporated following the constant contact line mode, indicating the likely coexistence of both Cassie and Wenzel states (Cassie-Baxter impregnating the wetting state). Fluorescence microscopy and image analysis revealed the significantly lower attachment of Staphylococcus epidermidis (86.1 ± 6.2% less) and Pseudomonas aeruginosa (85.9 ± 3.2% less) on the rose petal-structured surfaces, compared with flat surfaces over a period of 2 h. An extensive biofilm matrix was observed in biofilms formed by both species on flat surfaces after prolonged growth (several days), but was less apparent on rose petal-biomimetic surfaces. In addition, the biomass of S. epidermidis (63.2 ± 9.4% less) and P. aeruginosa (76.0 ± 10.0% less) biofilms were significantly reduced on the rose petal-structured surfaces, in comparison to the flat surfaces. By comparing P. aeruginosa growth on representative unitary nanopillars, we demonstrated that hierarchical structures are more effective in delaying biofilm growth. The mechanisms are two-fold: (1) the nanofolds across the hemispherical micropapillae restrict initial attachment of bacterial cells and delay the direct contact of cells via cell alignment and (2) the hemispherical micropapillae arrays isolate bacterial clusters and inhibit the formation of a fibrous network. The hierarchical features on rose petal surfaces may be useful for developing strategies to control biofilm formation in medical and industrial contexts.

[Sodium houttuyfonate inhibits virulence related motility of Pseudomonas aeruginosa].

Sodium houttuyfonate (SH) is a derivative of effective component of a Chinese material medica, Houttuynia cordata, which is applied in anti-infection of microorganism. But, the antimicrobial mechanisms of SH still remain unclear. Here, we firstly discovered that SH effectively inhibits the three types of virulence related motility of.Pseudomonas aeruginosa, i.e., swimming, twitching and swarming. The plate assay results showed that the inhibitory action of SH against swimming and twitching in 24 h and swarming in 48 h is dose-dependent; and bacteria nearly lost all of the motile activities under the concentration of 1 x minimum inhibitory concentration (MIC) (512 mg x L(-1) same as azithromycin positive group (1 x MIC, 16 mg x L(-1)). Furthermore, we found that the expression of structural gene flgB and pilG is down-regulated by SH, which implies that inhibitory mechanism of SH against motility of P. aeruginosa may be due to the inhibition of flagella and pili bioformation of P. aeruginosa by SR Therefore, our presented results firstly demonstrate that SH effectively inhibits the motility activities of P. aeruginosa, and suggest that SH could be a promising antipseudomonas agents in clinic.

A comparative study on edible Agaricus mushrooms as functional foods.

Agaricus bisporus is a cultivated mushroom; A. bitorquis, A. campestris and A. macrosporus are edible mushrooms growing wild in nature. A chemical characterization was carried out with samples that originated in Serbia. Antioxidant, antimicrobial and anti-quorum sensing properties of their methanolic and ethanolic extracts were assessed. A. campestris had the lowest caloric value and total sugar content and showed the highest concentration in organic and phenolic acids, as also in tocopherols (mainly γ-tocopherol). In general, the methanolic extracts showed higher antioxidant, but lower antibacterial and antifungal potential than ethanolic ones. Sub-inhibitory concentrations of the ethanolic extracts demonstrated reduction of virulence factors, AQ inhibition zones, twitching and swimming motility. The biofilm forming capability of P. aeruginosa PAO1 was also reduced in a concentration-dependent manner at sub-MIC values. The extracts of the tested Agaricus species are a promising source of antioxidant, antimicrobial and antiquorum sensing compounds.

At a supra-physiological concentration, human sexual hormones act as quorum-sensing inhibitors.

N-acylhomoserine lactone (AHL)-mediated quorum-sensing (QS) regulates virulence functions in plant and animal pathogens such as Agrobacterium tumefaciens and Pseudomonas aeruginosa. A chemolibrary of more than 3500 compounds was screened using two bacterial AHL-biosensors to identify QS-inhibitors (QSIs). The purity and structure of 15 QSIs selected through this screening were verified using HPLC MS/MS tools and their activity tested on the A. tumefaciens and P. aeruginosa bacterial models. The IC50 value of the identified QSIs ranged from 2.5 to 90 µg/ml, values that are in the same range as those reported for the previously identified QSI 4-nitropyridine-N-oxide (IC50 24 µg/ml). Under the tested culture conditions, most of the identified QSIs did not exhibit bacteriostatic or bactericidal activities. One third of the tested QSIs, including the plant compound hordenine and the human sexual hormone estrone, decreased the frequency of the QS-regulated horizontal transfer of the tumor-inducing (Ti) plasmid in A. tumefaciens. Hordenine, estrone as well as its structural relatives estriol and estradiol, also decreased AHL accumulation and the expression of six QS-regulated genes (lasI, lasR, lasB, rhlI, rhlR, and rhlA) in cultures of the opportunist pathogen P. aeruginosa. Moreover, the ectopic expression of the AHL-receptors RhlR and LasR of P. aeruginosa in E. coli showed that their gene-regulatory activity was affected by the QSIs. Finally, modeling of the structural interactions between the human hormones and AHL-receptors LasR of P. aeruginosa and TraR of A. tumefaciens confirmed the competitive binding capability of the human sexual hormones. This work indicates potential interferences between bacterial and eukaryotic hormonal communications.

Synthesis, characterization, and oil recovery application of biosurfactant produced by indigenous pseudomonas aeruginosa WJ-1 using waste vegetable oils.

A bacterial strain was isolated and cultured from the oil excavation areas in tropical zone in northern China. The biochemical characteristics and partial sequenced 16S rRNA gene of isolate, WJ-1, was identical to those of cultured representatives of the species Pseudomonas aeruginosa. This bacterium was able to produce a type of biosurfactant. Compositional analysis revealed that the extracted biosurfactant was composed of high percentage lipid (∼74%, w/w) and carbohydrate (∼20%, w/w) in addition to a minor fraction of protein (∼6%, w/w). The best production of 50.2 g/l was obtained when the cells were grown on minimal salt medium containing 6.0% (w/v) glucose and 0.75% (w/v) sodium nitrate supplemented with 0.1% (v/v) element solution at 37 °C and 180 rpm after 96 h. The optimum biosurfactant production pH value was found to be 6.0-8.0. The biosurfactant of WJ-1, with the critical micelle concentration of 0.014 g/L, could reduce surface tension to 24.5 mN/m and emulsified kerosene up to EI(24) ≈95. The results obtained from time course study indicated that the surface tension reduction and emulsification potential was increased in the same way to cell growth. However, maximum biosurfactant production occurred and established in the stationary growth phase (after 90 h). Thin layer chromatography, Fourier transform infrared spectrum, and mass spectrum analysis indicate the extracted biosurfactant was affiliated with rhamnolipid. The core holder flooding experiments demonstrated that the oil recovery efficiency of strain and its biosurfactant was 23.02% residual oil.

Effect of biosurfactant and fertilizer on biodegradation of crude oil by marine isolates of Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa.

This study was conducted to investigate the effects of fertilizers and biosurfactants on biodegradation of crude oil by three marine bacterial isolates; Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa. Five sets of experiments were carried out in shake flask and microcosm conditions with crude oil as follows: Set 1-only bacterial cells added (no fertilizer and biosurfactant), Set 2-with additional fertilizer only, Set 3-with additional biosurfactant only, Set 4-with added biosurfactant+fertilizer, Set 5-with no bacterial cells added (control), all the above experimental sets were incubated for 168 h. The biosurfactant+fertilizer added Set 4, resulted in maximum crude oil degradation within shake flask and microcosm conditions. Among the three bacterial isolates, P. aeruginosa and biosurfactant produced by this strain resulted in maximum crude oil degradation compared to the other two bacterial strains investigated. Interestingly, when biosurfactant and bacterial cells were used (Set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in Set 4 with added fertilizer+biosurfactant were only 4-5% higher degradation level in shake flask and 3.2-7% in microcosm experiments for all three bacterial strains used. It is concluded that, biosurfactants alone capable of promoting biodegradation to a large extent without added fertilizers, which will reduce the cost of bioremediation process and minimizes the dilution or wash away problems encountered when water soluble fertilizers used during bioremediation of aquatic environments.

Polycaprolactone-based fused deposition modeled mesh for delivery of antibacterial agents to infected wounds.

Infections represent a significant source of site morbidity following tissue trauma. Scarring and tissue adhesion remain the challenging issues yet to be solved. Prolonged inflammation and morphology of the re-epithelisated layer are important considerations. We hypothesized that the solution lies not only in the biochemistry of biomaterial but also the micro-architecture of the scaffold used as the matrix for wound healing. Targeted delivery of antibiotics may provide an efficacious means of infection control through adequate release. Here, we study the use of 3-dimensional polycaprolactone-tricalcium phosphate (PCL-TCP) mesh for the delivery of gentamicin sulphate (GS) fabricated using a solvent-free method. PCL-TCP meshes incorporated with varying loads of GS were evaluated in vitro for elution profile, antimicrobial efficacy and cytotoxicity. Results showed that PCL-TCP meshes incorporated with 15 wt% GS (PT15) efficiently eliminate bacteria within 2 h and demonstrate low cytotoxicity. Subsequently, PT15 meshes were evaluated using an infected full thickness wound mice model, and observed to eliminate bacteria in the wounds effectively. Additionally, mice from the PT15 treatment group (TG) showed no observable signs of overall infection through neutrophil count by day 7 and displayed efficient wound healing (94.2% wound area reduction) by day 14. Histology also showed significantly faster healing in TG through neo-collagen deposition and wound re-epithelisation. The meshes from TG were also observed to be expelled from wounds while gauze fibers from CG were integrated into wounds during healing.

Investigation of functional and morphological changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Origanum compactum essential oil.

AIMS: Evaluation of the cellular effects of Origanum compactum essential oil on Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. METHODS AND RESULTS: The damage induced by O. compactum essential oil on these two strains has been studied using different techniques: plate count, potassium leakage, flow cytometry (FC) and transmission electron microscopy (TEM). The results showed that oil treatment led to reduction of cells viability and dissipated potassium ion gradients. Flow cytometric analysis showed that oil treatment promoted the accumulation of bis-oxonol and the membrane-impermeable nucleic acid stain propidium iodide (PI), indicating the loss of membrane potential and permeability. The ability to reduce 5-cyano-2,3-ditolyl tetrazolium chloride was inhibited. Unlike in Ps. aeruginosa, membrane potential and membrane permeability in Staph. aureus cells were affected by oil concentration and contact time. Finally, TEM showed various structural effects. Mesosome-like structures were seen in oil-treated Staph. aureus cells whereas in Ps. aeruginosa, coagulated cytoplasmic material and liberation of membrane vesicles were observed, and intracellular material was seen in the surrounding environment. Both FC and TEM revealed that the effects in Ps. aeruginosa were greater than in Staph. aureus. CONCLUSIONS: Oregano essential oil induces membrane damage showed by the leakage of potassium and uptake of PI and bis-oxonol. Ultrastructural alterations and the loss of cell viability were observed. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mode of antibacterial effect of the oil studied is of a great interest in it further application as natural preservative in food or pharmaceutical industries.

Biosurfactant production by Pseudomonas aeruginosa grown in residual soybean oil.

Pseudomonas aeruginosa PACL strain, isolated from oil-contaminated soil taken from a lagoon, was used to investigate the efficiency and magnitude of biosurfactant production, using different waste frying soybean oils, by submerged fermentation in stirred tank reactors of 6 and 10 l capacities. A complete factorial experimental design was used, with the goal of optimizing the aeration rate (0.5, 1.0, and 1.5 vvm) and agitation speed (300, 550, and 800 rpm). Aeration was identified as the primary variable affecting the process, with a maximum rhamnose concentration occurring at an aeration rate of 0.5 vvm. At optimum levels, a maximum rhamnose concentration of 3.3 g/l, an emulsification index of 100%, and a minimum surface tension of 26.0 dynes/cm were achieved. Under these conditions, the biosurfactant production derived from using a mixture of waste frying soybean oil (WFSO) as a carbon source was compared to production when non-used soybean oil (NUSO), or waste soybean oils used to fry specific foods, were used. NUSO produced the highest level of rhamnolipids, although the waste soybean oils also resulted in biosurfactant production of 75-90% of the maximum value. Under ideal conditions, the kinetic behavior and the modeling of the rhamnose production, nutrient consumption, and cellular growth were established. The resulting model predicted data points that corresponded well to the empirical information.

A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol.

AIMS: The minimum inhibitory concentration (MIC) of oregano essential oil (OEO) and two of its principle components, i.e. thymol and carvacrol, against Pseudomonas aeruginosa and Staphylococcus aureus was assessed by using an innovative technique. The mechanism of action of the above substances was also investigated. METHODS AND RESULTS: The applied technique uses 100-well microtitre plate and collects turbidimetric growth data. To produce the inhibition profiles, a wide range of concentrations were tested for each of the three compounds, as well as for carvacrol-thymol mixtures. Following a specific mathematical analysis of the observed inhibition profiles from all compounds, it was suggested that mixtures of carvacrol and thymol gave an additive effect and that the overall inhibition by OEO can be attributed mainly to the additive antimicrobial action of these two compounds. Addition of low amounts of each additive: (a) increased permeability of cells to the nuclear stain EB, (b) dissipated pH gradients as indicated by the CFDA-SE fluorescent probe irrespective of glucose availability and (c) caused leakage of inorganic ions. CONCLUSION: Mixing carvacrol and thymol at proper amounts may exert the total inhibition that is evident by oregano essential oil. Such inhibition is due to damage in membrane integrity, which further affects pH homeostasis and equilibrium of inorganic ions. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of extent and mode of inhibition of specific compounds, which are present in plant extracts, may contribute to the successful application of such natural preservatives in foods, since certain combinations of carvacrol-thymol provide as high inhibition as oregano essential oil with a smaller flavour impact.

[Effect of carbon, nitrogen and phosphorus nutrition on the R, S, and M dissociants of Pseudomonas aeruginosa in mixed cultures]. / Vliianie uglerodnogo, azotnogo i fosfornogo pitaniia na rost R-, S- i M-dissotsiantov Pseudomonas Aeruginasa v smeshannykh kul'turakh.

The effect of glucose, nitrate, and phosphate on the stationary-phase growth characteristics of R, S, and M dissociants of the hydrocarbon-oxidizing strain P. aeruginosa K-2 was studied. The optimal concentrations of glucose and phosphate providing for at least 90% of the maximal culture density were found to be 2-7% glucose and 0.02-0.12% phosphate. The main factor that determined the proportion of dissociants in bacterial populations was the initial concentration of phosphate. The fraction of R dissociant in populations increased linearly with the concentration of glucose and varied nonlinearly with the concentration of phosphate in the growth medium. The fraction of M dissociant depended solely on the concentration of phosphate in a manner inverse to that typical of R dissociant. In glucose-deficient media containing sufficient amounts of phosphorus, S dissociant prevailed over R dissociant.

Influence of culture conditions on expression of the mucoid mode of growth of Pseudomonas aeruginosa.

Five strains of Pseudomonas aeruginosa that are routinely distinguished by diagnostic laboratories on the basis of their colony morphology on agar media were grown in different media to assess the effects of culture conditions on mucoid growth, which we define as the copious production of exopolysaccharide. On brain heart infusion agar, only two of these strains (mucoid and gelatinous) grew as slimy mucoid colonies. None of the five strains produced a mucoid pattern of growth in Mueller-Hinton broth, in which all grew as turbid, nonmucoid, homogeneous suspensions of bacterial cells. When Mueller-Hinton broth was supplemented with Mg2+, all of the strains produced some mucoid aggregated growth, but growth in a modified version of the chemically defined medium of Vogel and Bonner, with elevated levels of Mg2+ and gluconate, produced patently mucoid growth in all strains. This mucoid growth in a liquid medium takes the form of large, coherent, slimy aggregates within the medium and of a "collar" of adherent microcolonies at the air-medium interface. Direct observations by light and electron microscopy showed the submerged aggregates and the adherent microcolonies to be composed of bacterial cells enmeshed in a copious exopolysaccharide matrix. When agar was added to the supplemented medium of Vogel and Bonner, only the mucoid and gelatinous strains produced slimy mucoid colonies on its surface. We conclude that both chemical and physical factors affect exopolysaccharide production by these clinical strains of P. aeruginosa and that the colony morphology on a single agar medium is an insufficient criterion for the designation of a given isolate as being mucoid or nonmucoid.

Ultrastructural and chemical alteration of the cell envelope of Pseudomonas aeruginosa, associated with resistance to ethylenediaminetetraacetate resulting from growth in a Mg2+-deficient medium.

Cells of Pseudomonas aeruginosa became resistant to the lytic effect of ethylenediametetraacetate (EDTA) when grown in a Mg(2+)-deficient medium. To correlate ultrastructural changes in the cell wall associated with the shift to EDTA-resistance, a freeze-etch study was performed. Upon fracturing, the outer cell wall membrane split down the hydrophobic center to reveal the outer (concave) and inner (convex) layers. The concave cell wall layer of EDTA-sensitive cells grown in Mg(2+)-sufficient medium contained spherical units resting on an underlying smooth support layer. Upon EDTA treatment, approximately one-half of these spherical units were extracted. Cells grown in Mg(2+)-deficient medium were resistant to EDTA. The concave cell wall layer of EDTA-resistant cells had increased numbers of highly compacted spherical units, giving this layer a disorganized appearance. The highly compacted appearance of this layer was unaltered by EDTA treatment. Thus, growth in Mg(2+)-deficient medium resulted in cells which were resistant to EDTA and which possessed an ultrastructurally altered outer layer of the outer cell wall membrane. Cell envelopes from EDTA-resistant cells were found to possess 18% less phosphorus, 16.4% more total carbohydrate, and 13.3% more 2-keto-3-deoxyoctonate than cell envelopes from EDTA-sensitive cells. There were also qualitative, but not quantitative, differences in the protein content of cell envelopes from EDTA-resistant and EDTA-sensitive cells.

Inhibition of Pseudomonas aeruginosa by hyperbaric oxygen. II. Ultrastructural changes.

The ultrastructure of Pseudomonas aeruginosa cells treated with hyperbaric oxygen for 20 to 24 hr was examined by electron microscopy. A marked difference in the morphology of the nuclear area and cytoplasm of experimental cells was noted when compared to control cells grown under normobaric conditions. This difference was characterized by the absence of a definitive nuclear area and a reduced granularity of the cytoplasm.