Hydrophobic Chitosan Nanoparticles Loaded with Carvacrol against Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa infections have become more challenging to treat and eradicate due to their ability to form biofilms. This study aimed to produce hydrophobic nanoparticles by grafting 11-carbon and three-carbon alkyl chains to a chitosan polymer as a platform to carry and deliver carvacrol for improving its antibacterial and antibiofilm properties. Carvacrol-chitosan nanoparticles showed ζ potential values of 10.5-14.4 mV, a size of 140.3-166.6 nm, and an encapsulation efficiency of 25.1-68.8%. Hydrophobic nanoparticles reduced 46-53% of the biomass and viable cells (7-25%) within P. aeruginosa biofilms. Diffusion of nanoparticles through the bacterial biofilm showed a higher penetration of nanoparticles created with 11-carbon chain chitosan than those formulated with unmodified chitosan. The interaction of nanoparticles with a 50:50 w/w phospholipid mixture at the air-water interface was studied, and values suggested that viscoelasticity and fluidity properties were modified. The modified nanoparticles significantly reduced viable P. aeruginosa in biofilms (0.078-2.0 log CFU·cm-2) and swarming motility (40-60%). Furthermore, the formulated nanoparticles reduced the quorum sensing in Chromobacterium violaceum. This study revealed that modifying the chitosan polarity to synthesize more hydrophobic nanoparticles could be an effective treatment against P. aeruginosa biofilms to decrease its virulence and pathogenicity, mainly by increasing their ability to interact with the membrane phospholipids and penetrate preformed biofilms.

Zinc Exposure Promotes Commensal-to-Pathogen Transition in Pseudomonas aeruginosa Leading to Mucosal Inflammation and Illness in Mice.

The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) is associated gastrointestinal (GI) inflammation and illness; however, factors motivating commensal-to-pathogen transition are unclear. Excessive zinc intake from supplements is common in humans. Due to the fact that zinc exposure enhances P. aeruginosa colonization in vitro, we hypothesized zinc exposure broadly activates virulence mechanisms, leading to inflammation and illness. P. aeruginosa was treated with excess zinc and growth, expression and secretion of key virulence factors, and biofilm production were determined. Effects on invasion, barrier function, and cytotoxicity were evaluated in Caco-2 cells co-cultured with P. aeruginosa pre-treated with zinc. Effects on colonization, mucosal pathology, inflammation, and illness were evaluated in mice infected with P. aeruginosa pre-treated with zinc. We found the expression and secretion of key virulence factors involved in quorum sensing (QS), motility (type IV pili, flagella), biosurfactants (rhamnolipids), toxins (exotoxin A), zinc homeostasis (CzcR), and biofilm production, were all significantly increased. Zinc exposure significantly increased P. aeruginosa invasion, permeability and cytotoxicity in Caco-2 cells, and enhanced colonization, inflammation, mucosal damage, and illness in mice. Excess zinc exposure has broad effects on key virulence mechanisms promoting commensal-to-pathogen transition of P. aeruginosa and illness in mice, suggesting excess zinc intake may have adverse effects on GI health in humans.

Molecular Insights into Binding Mode and Interactions of Structure-Based Virtually Screened Inhibitors for Pseudomonas aeruginosa Multiple Virulence Factor Regulator (MvfR).

Multi-drug resistance (MDR) bacterial pathogens pose a threat to global health and warrant the discovery of new therapeutic molecules, particularly those that can neutralize their virulence and stop the evolution of new resistant mechanisms. The superbug nosocomial pathogen, Pseudomonas aeruginosa, uses a multiple virulence factor regulator (MvfR) to regulate the expression of multiple virulence proteins during acute and persistent infections. The present study targeted MvfR with the intention of designing novel anti-virulent compounds, which will function in two ways: first, they will block the virulence and pathogenesis P. aeruginosa by disrupting the quorum-sensing network of the bacteria, and second, they will stop the evolution of new resistant mechanisms. A structure-based virtual screening (SBVS) method was used to screen druglike compounds from the Asinex antibacterial library (~5968 molecules) and the comprehensive marine natural products database (CMNPD) (~32 thousand compounds), against the ligand-binding domain (LBD) of MvfR, to identify molecules that show high binding potential for the relevant pocket. In this way, two compounds were identified: Top-1 (4-((carbamoyloxy)methyl)-10,10-dihydroxy-2,6-diiminiodecahydropyrrolo[1,2-c]purin-9-yl sulfate) and Top-2 (10,10-dihydroxy-2,6-diiminio-4-(((sulfonatocarbamoyl)oxy)methyl)decahydropyrrolo[1,2-c]purin-9-yl sulfate), in contrast to the co-crystallized M64 control. Both of the screened leads were found to show deep pocket binding and interactions with several key residues through a network of hydrophobic and hydrophilic interactions. The docking results were validated by a long run of 200 ns of molecular dynamics simulation and MM-PB/GBSA binding free energies. All of these analyses confirmed the presence of strong complex formation and rigorous intermolecular interactions. An additional analysis of normal mode entropy and a WaterSwap assay were also performed to complement the aforementioned studies. Lastly, the compounds were found to show an acceptable range of pharmacokinetic properties, making both compounds potential candidates for further experimental studies to decipher their real biological potency.

Clinical and bacterial characteristics of Pseudomonas aeruginosa affecting the outcome of patients with bacteraemic pneumonia.

Few studies have assessed the clinical and bacterial characteristics of Pseudomonas aeruginosa (PA) bacteraemic pneumonia (BP) episodes. This study analysed all non-duplicate PA-BP episodes from a tertiary hospital in 2013-2017. Epidemiology, clinical data, antimicrobial therapy and outcomes were recorded. Whole-genome sequencing was performed on PA blood isolates. The impact on early and late overall mortality of host, antimicrobial treatment and pathogen factors was assessed by multivariate logistic regression analysis. Of 55 PA-BP episodes, 32 (58.2%) were caused by extensively drug-resistant (XDR) PA. ST175 (32.7%) and ST235 (25.5%) were the most frequent high-risk clones. ß-Lactamases/carbapenemases were detected in 29 isolates, including blaVIM-2 (27.2%) and blaGES type (25.5%) [blaGES-5 (20.0%), blaGES-1 (3.6%) and blaGES-20 (1.8%)]. The most prevalent O-antigen serotypes were O4 (34.5%) and O11 (30.9%). Overall, an extensive virulome was identified in all isolates. Early mortality (56.4%) was independently associated with severe neutropenia (aOR = 4.64, 95% CI 1.11-19.33; P = 0.035) and inappropriate empirical antimicrobial therapy (aOR = 5.71, 95% CI 1.41-22.98; P = 0.014). Additionally, late mortality (67.3%) was influenced by septic shock (aOR = 8.85, 95% CI 2.00-39.16; P = 0.004) and XDR phenotype (aOR = 5.46, 95% CI 1.25-23.85; P = 0.024). Moreover, specific genetic backgrounds [ST235, blaGES, gyrA (T83I), parC (S87L), exoU and O11 serotype] showed significant differences in patient outcomes. Our results confirm the high mortality associated with PA-BP. Besides relevant clinical characteristics and inappropriate empirical therapy, bacteria-specific genetics factors, such as XDR phenotype, adversely affect the outcome of PA-BP.

Antipathogenic Compounds That Are Effective at Very Low Concentrations and Have Both Antibiofilm and Antivirulence Effects against Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a human pathogen, causes both acute and chronic infections that are mediated by virulence factor production and biofilm formation. Since both characteristics of P. aeruginosa are regulated by quorum sensing (QS), we screened 126 synthetic chemicals for anti-QS activity and finally selected the compounds that have both antivirulence and antibiofilm activities. To efficiently screen the chemical library, the following reporter-based bioassay systems were used: the QS- or biofilm-specific promoter-lacZ fusions (lasIp- or PA1897p-lacZ for the QS activity and cdrAp-lacZ for measuring the intracellular c-di-GMP levels). We also measured the production of virulence factors and biofilm formation in P. aeruginosa. A small-animal infection model using mealworms was also used for virulence analysis. From this screening, MHY1383 and MHY1387 were found to have both antivirulence and antibiofilm activities in P. aeruginosa. Most importantly, MHY1383 and MHY1387 exhibited these activities at very low concentrations, showing a significant anti-QS effect at 100 pM and an antibiofilm effect at 1 to 10 pM. By treating P. aeruginosa with these compounds, the virulence factor production and biofilm formation of P. aeruginosa were significantly reduced. These compounds can be developed as promising antipathogenic and antibiofilm drugs that can be applied in situations where such compounds must be used in an extremely low concentration. Our findings also offer a significant advantage for developing therapeutic agents with few adverse side effects. IMPORTANCE Many antibiotics are increasingly losing their efficacy due to antibiotic resistance mediated by biofilm formation. In this study, we screened a synthetic chemical library and discovered several compounds that have both antivirulence and antibiofilm effects against Pseudomonas aeruginosa, a notorious human pathogen. Two of them had these effects at extremely low concentrations and are expected not to develop resistance, unlike conventional antibiotics, because they have no effect on the growth of bacteria. Our results strongly suggest that these compounds act on the target in a noncompetitive manner, indicating that they are distinct from other previously known quorum sensing inhibitors or biofilm inhibitors. Our findings offer a significant advantage for developing therapeutic agents with few adverse side effects.

Bioengineered phytomolecules-capped silver nanoparticles using Carissa carandas leaf extract to embed on to urinary catheter to combat UTI pathogens.

Rising incidents of urinary tract infections (UTIs) among catheterized patients is a noteworthy problem in clinic due to their colonization of uropathogens on abiotic surfaces. Herein, we have examined the surface modification of urinary catheter by embedding with eco-friendly synthesized phytomolecules-capped silver nanoparticles (AgNPs) to prevent the invasion and colonization of uropathogens. The preliminary confirmation of AgNPs production in the reaction mixture was witnessed by the colour change and surface resonance plasmon (SRP) band at 410nm by UV-visible spectroscopy. The morphology, size, crystalline nature, and elemental composition of attained AgNPs were further confirmed by the transmission electron microscopy (TEM), selected area electron diffraction (SAED), X-ray diffraction (XRD) technique, Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). The functional groups of AgNPs with stabilization/capped phytochemicals were detected by Fourier-transform infrared spectroscopy (FTIR). Further, antibiofilm activity of synthesized AgNPs against biofilm producers such as Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were determined by viability assays and micrographically. AgNPs coated and coating-free catheters performed to treat with bacterial pathogen to analyze the mat formation and disruption of biofilm formation. Synergistic effect of AgNPs with antibiotic reveals that it can enhance the activity of antibiotics, AgNPs coated catheter revealed that, it has potential antimicrobial activity and antibiofilm activity. In summary, C. carandas leaf extract mediated synthesized AgNPs will open a new avenue and a promising template to embed on urinary catheter to control clinical pathogens.

A scaffolded approach to unearth potential antibacterial components from epicarp of Malaysian Nephelium lappaceum L.

The emergence and spread of antimicrobial resistance have been of serious concern to human health and the management of bacterial infectious diseases. Effective treatment of these diseases requires the development of novel therapeutics, preferably free of side effects. In this regard, natural products are frequently conceived to be potential alternative sources for novel antibacterial compounds. Herein, we have evaluated the antibacterial activity of the epicarp extracts of the Malaysian cultivar of yellow rambutan fruit (Nephelium lappaceum L.) against six pathogens namely, Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella enterica. Among a series of solvent extracts, fractions of ethyl acetate and acetone have revealed significant activity towards all tested strains. Chemical profiling of these fractions, via HPLC, LC-MS and GC-MS, has generated a library of potentially bioactive compounds. Downstream virtual screening, pharmacological prediction, and receptor-ligand molecular dynamics simulation have eventually unveiled novel potential antibacterial compounds, which can be extracted for medicinal use. We report compounds like catechin, eplerenone and oritin-4-beta-ol to be computationally inhibiting the ATP-binding domain of the chaperone, DnaK of P. aeruginosa and MRSA. Thus, our work follows the objective to propose new antimicrobials capable of perforating the barrier of resistance posed by both the gram positives and the negatives.

Lemon Oils Attenuate the Pathogenicity of Pseudomonas aeruginosa by Quorum Sensing Inhibition.

The chemical composition of three Citrus limon oils: lemon essential oil (LEO), lemon terpenes (LT) and lemon essence (LE), and their influence in the virulence factors production and motility (swarming and swimming) of two Pseudomonas aeruginosa strains (ATCC 27853 and a multidrug-resistant HT5) were investigated. The main compound, limonene, was also tested in biological assays. Eighty-four compounds, accounting for a relative peak area of 99.23%, 98.58% and 99.64%, were identified by GC/MS. Limonene (59-60%), γ-terpinene (10-11%) and ß-pinene (7-15%) were the main compounds. All lemon oils inhibited specific biofilm production and bacterial metabolic activities into biofilm in a dose-dependent manner (20-65%, in the range of 0.1-4 mg mL-1) of both strains. Besides, all samples inhibited about 50% of the elastase activity at 0.1 mg mL-1. Pyocyanin biosynthesis decreases until 64% (0.1-4 mg mL-1) for both strains. Swarming motility of P. aeruginosa ATCC 27853 was completely inhibited by 2 mg mL-1 of lemon oils. Furthermore, a decrease (29-55%, 0.1-4 mg mL-1) in the synthesis of Quorum sensing (QS) signals was observed. The oils showed higher biological activities than limonene. Hence, their ability to control the biofilm of P. aeruginosa and reduce the production of virulence factors regulated by QS makes lemon oils good candidates to be applied as preservatives in the food processing industry.

Potential of Silver Nanoparticles in Overcoming the Intrinsic Resistance of Pseudomonas aeruginosa to Secondary Metabolites from Carnivorous Plants.

Carnivorous plants are exemplary natural sources of secondary metabolites with biological activity. However, the therapeutic antimicrobial potential of these compounds is limited due to intrinsic resistance of selected bacterial pathogens, among which Pseudomonas aeruginosa represents an extreme example. The objective of the study was to overcome the intrinsic resistance of P. aeruginosa by combining silver nanoparticles (AgNPs) with secondary metabolites from selected carnivorous plant species. We employed the broth microdilution method, the checkerboard titration technique and comprehensive phytochemical analyses to define interactions between nanoparticles and active compounds from carnivorous plants. It has been confirmed that P. aeruginosa is resistant to a broad range of secondary metabolites from carnivorous plants, i.e., naphthoquinones, flavonoids, phenolic acids (MBC = 512 µg mL-1) and only weakly sensitive to their mixtures, i.e., extracts and extracts' fractions. However, it was shown that the antimicrobial activity of extracts and fractions with a significant level of naphthoquinone (plumbagin) was significantly enhanced by AgNPs. Our studies clearly demonstrated a crucial role of naphthoquinones in AgNPs and extract interaction, as well as depicted the potential of AgNPs to restore the bactericidal activity of naphthoquinones towards P. aeruginosa. Our findings indicate the significant potential of nanoparticles to modulate the activity of selected secondary metabolites and revisit their antimicrobial potential towards human pathogenic bacteria.

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.

Pectin based banana peel extract as a stabilizing agent in zinc oxide nanoparticles synthesis.

Various amounts of banana peel extract were successfully used as a stabilizing agent in the co-precipitation of zinc oxide nanoparticles. The obtained materials were characterized by means of Fourier transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC), X-ray diffraction (XRD), photoluminescence spectroscopy (PL), scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDS), N2 physisorption, thermogravimetric analysis (TGA), and quadrupole mass spectroscopy (QMS). On account of using such a broad spectrum of analytic methods, a thorough description of the interactions between the organic ingredients of the extract and ZnO particles was presented. It was indicated that the banana peel extract is based on pectin. These carbohydrate macromolecules adsorb on ZnO surface due to presence of active carboxylic groups. By increasing the concentration of polysaccharides, pectin-pectin interactions were also observed. The amount of the extract used for the synthesis significantly influenced the crystalline structure of zinc oxide particles along with their size and morphology. The shape and size were varying from thin flakes (450 × 24 nm) when the smallest amount of the extract was used, through nanocones with pointed tips (210 × 120 nm) agglomerated in a flower-like structure, until cubic-shaped nanoparticles (20-40 nm) agglomerated in a pinecone-like structure (430 × 180 nm) when the biggest amount of the extract was applied. The obtained particles have displayed apromising antimicrobial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria, and fungus (Candida albicans). The highest activity was demonstrated against S. aureus pathogen.

Hypertonic glucose inhibits growth and attenuates virulence factors of multidrug-resistant Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is the most common Gram-negative pathogen responsible for chronic wound infections, such as diabetic foot infections, and further exacerbates the treatment options and cost of such conditions. Hypertonic glucose, a commonly used prolotherapy solution, can accelerate the proliferation of granulation tissue and improve microcirculation in wounds. However, the action of hypertonic glucose on bacterial pathogens that infect wounds is unclear. In this study, we investigated the inhibitory effects of hypertonic glucose on multidrug-resistant P. aeruginosa strains isolated from diabetic foot infections. Hypertonic glucose represents a novel approach to control chronic wound infections caused by P. aeruginosa. RESULTS: Four multidrug-resistant P. aeruginosa clinical strains isolated from diabetic foot ulcers from a tertiary hospital in China and the reference P. aeruginosa PAO1 strain were studied. Hypertonic glucose significantly inhibited the growth, biofilm formation, and swimming motility of P. aeruginosa clinical strains and PAO1. Furthermore, hypertonic glucose significantly reduced the production of pyocyanin and elastase virulence factors in P. aeruginosa. The expression of major quorum sensing genes (lasI, lasR, rhlI, and rhlR) in P. aeruginosa were all downregulated in response to hypertonic glucose treatment. In a Galleria mellonella larvae infection model, the administration of hypertonic glucose was shown to increase the survival rates of larvae infected by P. aeruginosa strains (3/5). CONCLUSIONS: Hypertonic glucose inhibited the growth, biofilm formation, and swimming motility of P. aeruginosa, as well as reduced the production of virulence factors and quorum sensing gene expression. Further studies that investigate hypertonic glucose therapy should be considered in treating chronic wound infections.

Calpurnia aurea (Aiton) Benth Extracts Reduce Quorum Sensing Controlled Virulence Factors in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is the causative agent of several life-threatening human infections. Like many other pathogens, P. aeruginosa exhibits quorum sensing (QS) controlled virulence factors such as biofilm during disease progression, complicating treatment with conventional antibiotics. Thus, impeding the pathogen's QS circuit appears as a promising alternative strategy to overcome pseudomonas infections. In the present study, Calpurnia aurea were evaluated for their antibacterial (minimum inhibitory concentrations (MIC)), anti-quorum sensing/antivirulence (AQS), and antibiofilm potential against P. aeruginosa. AQS and antivirulence (biofilm formation, swimming, and swarming motility) activities of plant extracts were evaluated against Chromobacterium violaceum and P. aeruginosa, respectively. The in vitro AQS potential of the individual compounds were validated using in silico molecular docking. Acetone and ethanolic extracts of C. aurea showed MIC at 1.56 mg/mL. The quantitative violacein inhibition (AQS) assay showed ethyl acetate extracts as the most potent at a concentration of 1 mg/mL. GCMS analysis of C. aurea revealed 17 compounds; four (pentadecanol, dimethyl terephthalate, terephthalic acid, and methyl mannose) showed potential AQS through molecular docking against the CviR protein of C. violaceum. Biofilm of P. aeruginosa was significantly inhibited by ≥60% using 1-mg/mL extract of C. aurea. Confocal laser scanning microscopy correlated the findings of crystal violet assay with the extracts significantly altering the swimming motility. C. aurea extracts reduced the virulence of pseudomonas, albeit in a strain- and extract-specific manner, showing their suitability for the identification of lead compounds with QS inhibitory potential for the control of P. aeruginosa infections.

Calycopterin, a major flavonoid from Marcetia latifolia, modulates virulence-related traits in Pseudomonas aeruginosa.

Although bacterial resistance is a worldwide growing concern, the development of bacteriostatic and bactericidal drugs has been decreasing in the last decade. Compounds that modulate the microorganism virulence, without killing it, have been considered promising alternatives to combat bacterial infections. However, most signaling pathways that regulate virulence are complex and not completely understood. The rich chemical diversity of natural products offers a good starting point to identify key compounds that shed some light on this matter. Therefore, we investigated the role of Marcetia latifolia ethanolic extract, as well as its major constituent, calycopterin (5,4'-dihydroxy-3,6,7,8-tetramethoxylflavone), in the regulation of virulence-related phenotypes of Pseudomonas aeruginosa. Our results show that calycopterin inhibits pyocyanin production (EC50 = 32 µM), reduces motility and increases biofilm formation in a dose-dependent manner. Such biological profile suggests that calycopterin modulates targets that may act upstream the quorum sensing regulators and points to its utility as a chemical probe to further investigate P. aeruginosa transition from planktonic to sessile lifestyle.

Musa acuminata and its bioactive metabolite 5-Hydroxymethylfurfural mitigates quorum sensing (las and rhl) mediated biofilm and virulence production of nosocomial pathogen Pseudomonas aeruginosa in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Musa acuminata, a tropical plant belongs to the family Musaceae. The fruit peels of this plant have been well documented for their therapeutic value in Asia and Africa. It has also been previously reported for numerous biological applications such as antimicrobial, antioxidant, itching, psoriasis and anti-diarrheal activities. Moreover, M. acuminata peels have been well known for its anti-healing and antiseptic properties and most commonly used for healing wounds and heat burns in South Asian and African traditional medicines. AIM OF THE STUDY: To evaluate the QS-mediated antibiofilm and antivirulence potential of M. acuminata, and its bioactive metabolites 5-Hydroxymethylfurfural (5HMF) against Pseudomonas aeruginosa. MATERIALS AND METHODS: The M. acuminata peel methanol extract (MAM) was evaluated for its antibiofilm potential against P. aeruginosa with increasing concentration. Besides, biofilm related phenomenon's such as total biofilm proteins, microcolony formation exopolysaccharides (EPS) and cell surface hydrophobicity (CSH) productions were also examined to support the antibiofilm potential of MAM. Further, MAM was evaluated for its antivirulence efficacy against P. aeruginosa by assessing the protease, LasA protease, LasB elastase, pyocyanin, alginate and rhamnolipid productions at 400 µg ml-1 concentration. Transcriptional analysis of QS regulated virulence genes expression level was also done by real-time PCR analysis. Then, the MAM was subjected to column chromatography for further fractions and the bioactive compounds present in MAM were identified by gas chromatograph-mass spectrometry analysis. Further, the major compounds such as 5-hydroxymethylfurfural, vaccenic acid and pentanoic acid identified from active fraction of MAM were evaluated for their antibiofilm and antivirulence potential against P. aeruginosa. RESULTS: MAM significantly inhibited the biofilm formation in P. aeruginosa at 400 µg ml-1 concentration which also inhibited the production of biofilm proteins, biofilm adherence, EPS and CSH productions to the level of 79%, 82% and 77% respectively. Further, the antivirulence potential was confirmed through numerous virulence inhibition assays. The MAM at 400 µg ml-1 concentration inhibited the QS-mediated virulence production such as protease, LasA protease, LasB elastase, pyocyanin, alginate and rhamnolipid productions to the level of 77%, 75%, 68%, 80%, 78% and 69% respectively. Moreover, the results of qPCR analysis confirmed the downregulation of QS regulated virulence genes expression upon treatment with MAM. The chromatographic analysis revealed the presence of 5-Hydroxymethylfurfural (5HMF), vaccenic acid and pentanoic acid in MAM and the potential bioactive compounds with antibiofilm and antivirulence was identified as 5-hydroxymethylfurfural, without exerting any growth inhibition in P. aeruginosa. CONCLUSION: This study investigated the ideal antibiofilm and antivirulence potential of MAM and its bioactive compound 5HMF, and confirms the ethnopharmacological value of these peels against P. aeruginosa infections.

The Development of an Antimicrobial Contact Lens - From the Laboratory to the Clinic.

Contact lens wear is generally safe and provides excellent vision. However, contact lens wear is often associated with the risk of developing ocular surface infection and inflammation, and in severe cases, the infection can result in loss of vision. Antimicrobial peptide-coated contact lenses have been made to help reduce the incidence of infection and inflammation. This paper reviews the research progress from conception, through the laboratory and preclinical tests to the latest information on clinical testing of an antimicrobial contact lens. We provide insights into the pathways followed and pitfalls that have been encountered. The journey has not always been linear or smooth, but has resulted in some of the first published clinical testing of antimicrobial peptide-coated contact lenses in humans. We hope this may help lead to the development and commercialisation of antimicrobial contact lenses in the future.

A compound isolated from Alpinia officinarum Hance. inhibits swarming motility of Pseudomonas aeruginosa and down regulates virulence genes.

AIM: The study was aimed at purifying the active principle from Alpinia officinarum rhizomes responsible for inhibition of swarming motility of Pseudomonas aeruginosa and analysing the mechanism of action. METHODS AND RESULTS: The active compound from methanol extract of A. officinarum was purified by silica gel column chromatography followed by elution from Amberlite resin. The compound 1-(3,5-dihydroxyphenyl)-2-(methylamino)ethan-1-one, inhibited swarming motility at 12·5 µg ml-1 . This inhibition was independent of rhamnolipid production. Real-time PCR analysis showed significant down-regulation of virulence-associated genes including T3SS exoS, exoT and flagella master regulator fleQ. CONCLUSIONS: The compound from A. officinarum inhibited swarming motility and significantly down-regulated the expression of type III secretory system effector genes exoS and exoT and flagellar master regulator fleQ genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The study identifies a potent swarming inhibitory compound from the common medicinal plant A. officinarum and reinstates the potential of plant-derived compounds in tackling virulence properties of pathogenic bacteria.

A Population Pharmacokinetic Model-Guided Evaluation of Ceftolozane-Tazobactam Dosing in Critically Ill Patients Undergoing Continuous Venovenous Hemodiafiltration.

The aim of this work was to describe optimized dosing regimens of ceftolozane-tazobactam for critically ill patients receiving continuous venovenous hemodiafiltration (CVVHDF). We conducted a prospective observational pharmacokinetic study in adult critically ill patients with clinical indications for ceftolozane-tazobactam and CVVHDF. Unbound drug concentrations were measured from serial prefilter blood, postfilter blood, and ultrafiltrate samples by a chromatographic assay. Population pharmacokinetic modeling and dosing simulations were performed using Pmetrics. A four-compartment pharmacokinetic model adequately described the data from six patients. The mean (± standard deviation [SD]) extraction ratios for ceftolozane and tazobactam were 0.76 ± 0.08 and 0.73 ± 0.1, respectively. The mean ± SD sieving coefficients were 0.94 ± 0.24 and 1.08 ± 0.30, respectively. Model-estimated CVVHDF clearance rates were 2.7 ± 0.8 and 3.0 ± 0.6 liters/h, respectively. Residual non-CVVHDF clearance rates were 0.6 ± 0.5 and 3.3 ± 0.9 liters/h, respectively. In the initial 24 h, doses as low as 0.75 g every 8 h enabled cumulative fractional response of ≥85% for empirical coverage against Pseudomonas aeruginosa, considering a 40% fT>MIC (percentage of time the free drug concentration was above the MIC) target. For 100% fT>MIC, doses of at least 1.5 g every 8 h were required. The median (interquartile range) steady-state trough ceftolozane concentrations for simulated regimens of 1.5 g and 3.0 g every 8 h were 28 (21 to 42) and 56 (42 to 84) mg/liter, respectively. The corresponding tazobactam concentrations were 6.1 (5.5 to 6.7) and 12.1 (11.0 to 13.4) mg/liter, respectively. We suggest a front-loaded regimen with a single 3.0-g loading dose followed by 0.75 g every 8 h for critically ill patients undergoing CVVHDF with study blood and dialysate flow rates.

Zinc oxide nanoparticles inhibits quorum sensing and virulence in Pseudomonas aeruginosa.

BACKGROUND: Quorum sensing inhibitionis an advanced strategy that aims to interfere with bacterial cell-to-cell communication systems (quorum sensing), which regulate virulence factors production in Pseudomonas aeruginosa, in order to overcome the globalcrisis of antimicrobial resistance. OBJECTIVES: Study the potential quorum sensing inhibitory effect of Zinc oxide (ZnO)nanoparticlesin Pseudomonas aeruginosa and the impact on production of virulence factors. METHODS: Quorum sensing inhibitory effect of ZnO was evaluated by assessing its ability to reducePseudomonas aeruginosa virulence factors production; rhamnolipids, pyocyanin, pyoverdin, hemolysins, elastase and proteases. Furthermore, qRT-PCR was performed to determine ZnO inhibitory effect onQS-regulatory geneslasI, lasR, rhlI, rhlR, pqsA and pqsR that control virulence factors secretion. Moreover, mice survival test was conducted to investigate the influence of ZnO on Pseudomonas aeruginosa-induced mortality in vivo. RESULTS: ZnO revealed a statistically significant reduction in the production of QS-controlled virulence factors rhamnolipids, pyocyanin, pyoverdin, hemolysins, elastase and proteases. Furthermore, ZnO exhibited a significant decrease in the relative expression of QS-regulatory geneslasI, lasR, rhlI, rhlR, pqsA and pqsR. Additionally, ZnO significantly reduced the pathogenesis of Pseudomonas aeruginosa in vivo. CONCLUSION: ZnO nanoparticles can be used as a quorum sensing inhibitor in Pseudomonas aeruginosa infections either as an adjuvant or alternative to conventional antimicrobials.