Quenching of quorum sensing in multi-drug resistant Pseudomonas aeruginosa: insights on halo-bacterial metabolites and gamma irradiation as channels inhibitors.

BACKGROUND: Anti-virulence therapy is a promising strategy to treat multi-drug resistant (MDR) pathogens. Pseudomonas aeruginosa is a potent opportunistic pathogen because of an array of virulence factors that are regulated by quorum sensing systems. METHODS: The virulence features of four multi-drug resistant P. aeruginosa strains were investigated upon exposure to the sub-lethal dose of gamma rays (1 kGy), and sub-inhibitory concentrations of bioactive metabolites recovered from local halophilic strains in comparison to control. Then, the gene expression of AHL-mediated quorum sensing systems (las/rhl) was quantitatively determined in treated and untreated groups by real-time PCR. RESULTS: The bioactive metabolites recovered from halophilic strains previously isolated from saline ecosystems were identified as Halomonas cupida (Halo-Rt1), H. elongate (Halo-Rt2), Vigibacillus natechei (Halo-Rt3), Sediminibacillus terrae (Halo-Rt4) and H. almeriensis (Halo-Rt5). Results revealed that both gamma irradiation and bioactive metabolites significantly reduced the virulence factors of the tested MDR strains. The bioactive metabolites showed a maximum efficiency for inhibiting biofilm formation and rhamnolipids production whereas the gamma irradiation succeeded in decreasing other virulence factors to lower levels in comparison to control. Quantitative-PCR results showed that AHL-mediated quorum sensing systems (las/rhl) in P. aeruginosa strains were downregulated either by halo-bacterial metabolites or gamma irradiation in all treatments except the upregulation of both lasI internal gene and rhlR intact gene in P. aeruginosa NCR-RT3 and both rhlI internal gene and rhlR intact gene in P. aeruginosa U3 by nearly two folds or more upon exposure to gamma irradiation. The most potent result was observed in the expression of lasI internal gene that was downregulated by more than ninety folds in P. aeruginosa NCR-RT2 after treatment with metabolites of S. terrae (Halo-Rt4). Analyzing metabolites recovered from H. cupida (Halo-Rt1) and H. elongate (Halo-Rt2) using LC-ESI-MS/MS revealed many chemical compounds that have quorum quenching properties including glabrol, 5,8-dimethoxyquinoline-2-carbaldehyde, linoleoyl ethanolamide, agelasine, penigequinolones derivatives, berberine, tetracosanoic acid, and liquidambaric lactone in the former halophile and phloretin, lycoctonine, fucoxanthin, and crassicauline A in the latter one. CONCLUSION: QS inhibitors can significantly reduce the pathogenicity of MDR P. aeruginosa strains; and thus can be an effective and successful strategy for treating antibiotic resistant traits.

FBPP: software to design PCR primers and probes for nucleic acid base detection of foodborne pathogens.

Foodborne pathogens can be found in various foods, and it is important to detect foodborne pathogens to provide a safe food supply and to prevent foodborne diseases. The nucleic acid base detection method is one of the most rapid and widely used methods in the detection of foodborne pathogens; it depends on hybridizing the target nucleic acid sequence to a synthetic oligonucleotide (probes or primers) that is complementary to the target sequence. Designing primers and probes for this method is a preliminary and critical step. However, new bioinformatics tools are needed to automate, specific and improve the design sets to be used in the nucleic acid‒base method. Thus, we developed foodborne pathogen primer probe design (FBPP), an open-source, user-friendly graphical interface Python-based application supported by the SQL database for foodborne pathogen virulence factors, for (i) designing primers/probes for detection purposes, (ii) PCR and gel electrophoresis photo simulation, and (iii) checking the specificity of primers/probes.

Evaluation of antibacterial activity induced by Staphylococcus aureus and Ent A in the hemolymph of Spodoptera littoralis.

The problem of antibiotic resistance considers one of the most dangerous challenges facing the medical field. So, it is necessary to find substitutions to conventional antibiotics. Antimicrobial peptides (AMPs) are a bio-functional derivative that have been observed as one of the important solutions to such upcoming crisis. Owing to their role as the first line of defense against bacteria, fungi, and viruses. This study was conducted to induce the immune response of Spodoptera littoralis larvae by inoculation of sub lethal doses of Staphylococcus aureus and its enterotoxin. Since Staphylococcal enterotoxin A (SEA) considers the major causative agents of Staphylococcal food poisoning, our study oriented to purify and characterize this toxin to provoke its role in yielding AMPs with broad spectrum antimicrobial activity. A great fluctuation was recorded in the biochemical properties of immunized hemolymph not only in the total protein content but also protein banding pattern. Protein bands of ∼22 kDa (attacin-like) and ∼15 kDa (lysozyme-like) were found to be common between the AMPs induced as a result of both treatments. While protein bands of molecular weight ∼70 kDa (phenoloxidase-like) and ∼14 kDa (gloverin-like) were found specific for SEA treatment. Chromatographic analysis using HPLC for the induced AMPs showed different types of amino acids appeared with differences in their quantities and velocities. These peptides exhibited noticeable antimicrobial activity against certain Gram-positive and Gram-negative bacteria. In conclusion, the antimicrobial potential of the antimicrobial peptides (AMP) induced in the larval hemolymph of S. littoralis will be a promising molecule for the development of new therapeutic alternatives.

Biodegradation of 3-chlorobenzoic acid with electron shuttle systems: pathways and molecular identification.

A synergy of biodegradation and electron shuttle systems is a promising strategy for eliminating pollutants including chlorinated aromatic compounds. The present work studies the degradation products of 3-chlorobenzoic acid by Pseudomonas putida in the presence of an electron shuttle system (ESS) composed of citrate and pyruvate as electron donors and the pollutant as an electron acceptor. Chromatographic results showed different pathways involved in the biodegradation process under the influence of electron shuttle systems. These routes depend on oxidation and reduction reactions for output byproducts to be easily mineralized by the bacterium under investigation. A nucleotide sequence with about 380 bp of a ton B gene was detected in P. putida and it resembles Escherichia coli Ton B. The relatedness tree of the selected gene reveals a high similarity and is comparable to P. aeruginosa (100%) and the highest variation with that of P. citronellolis (21.99%). Accordingly, in the presence of electron shuttle systems, the genes responsible for bacterial influx were activated to ease the biodegradation process. In an application model, the remediated-water samples were handled by two recycling processes using Scenedesmus obliquus and Trigonella foenum-graecum to evaluate the efficiency of this non-conventional treatment. In conclusion, this strategy succeeded in remediating the polluted water with chlorinated aromatic compounds for further applications.

Radiation-induced synthesis of tween 80 stabilized silver nanoparticles for antibacterial applications.

Recently, Silver nanoparticles (AgNPs) have become widely applied nanomaterial in human contacting areas such as cosmetics, food and medicine due to their antibacterial property. On the other hand, surfactants are essential ingredient of several industrial and consumer formulations. Based on these important applications, the current research was aimed to carry out the synthesis and characterization of Tween 80 capped silver nanoparticles (T80-AgNPs) using gamma radiation reduction method. Characterization of T80-AgNPs was occurred by using UV-Vis, XRD, FTIR and TEM techniques. UV-Visible spectra showed surface plasmon resonance (SPR) peak in the range of 420 nm signifying the synthesis of colloidal AgNPs. TEM confirmed the formation of spherical and uniformly distributed AgNPs with average size of 18 nm. XRD analysis illustrated the formation of pure crystalline AgNPs. The FTIR analysis provides evidence for the stabilization of AgNPs by Tween 80. The synthesized T80-AgNPs were evaluated for antibacterial activity against both Escherichia coli (E. coli) as gram negative (G -ve) bacteria and Staphylococcus aureus (S. aureus) as gram positive (G + ve) bacteria and anti-biofilm activity to P. aeruginosa. The results show that T80-AgNPs exhibits excellent antibacterial and antibiofilm activities.

Construction, Expression and Evaluation of Recombinant VP2 Protein for serotype-independent Detection of FMDV Seropositive Animals in Egypt.

Foot-and-mouth disease virus (FMDV) is one of the most devastating viral pathogens of cloven-hoofed animals. The detection of antibodies (Ab) against FMDV structural proteins (SP) using virus neutralization test (VNT) and liquid-phase blocking ELISA (LPBE) is the standard procedure in use for monitoring seroconversion in animals post vaccination, the prevalence of infection-surveillance, proving clinical cases and seronegative status of FMDV-free/naïve-animals prior transportation. However, due to variations within SP of FMDV serotypes, each serotype-specific Ab should be detected separately which is laborious and time-consuming. Accordingly, it is crucial to develop a sensitive, rapid, and accurate test capable of detecting FMDV-specific Ab, regardless its serotype. This study describes the heterologous expression of VP2 protein in E. coli, and its evaluation as a capture antigen in a simple indirect ELISA for serotype-independent detection of anti-FMDV Ab. Sequence analysis revealed that the VP2-coding sequence is considerably conserved among FMDV serotypes. The recombinant VP2 (rVP2), a 22 kDa polypeptide, was purified to near homogeneity by affinity chromatography under native conditions. Immunoreactivity of the rVP2 was confirmed by using a panel of positive sera including sera from animals vaccinated with the local trivalent vaccine and guinea pig FMDV antiserum, which is routinely used as tracing/detecting Ab in LPBE testing. The results obtained from the VP2-based ELISA were comparable to those determined by VNT and LPBE standard diagnostic assays. Specificity and sensitivity of rVP2 in capturing anti-FMDV Ab were 98.3% and 100%, respectively. The developed VP2-ELISA is proved reliable and time-efficient assay for detection of FMDV seropositive animals, regardless the FMDV serotype that can be implemented in a combination with VNT and/or LPBE for rapid diagnosis of an ongoing FMDV infection.