الفهرس 
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Abstract
With increasing in the antibiotic resistance problem around the world, many pharmacological and pharmacognostical studies were designed to identify new drugs or discover new structures for the development of novel therapeutic agents; a great interest has been encouraged in the seeking of new anti‑virulence drugs.
Pseudomonas aeruginosa is a gram- negative bacillus found widely in nature and in the environment. Pseudomonas aeruginosa possesses many mechanisms for antibiotic resistance and a variety of virulence factors that collectively account for the broad-spectrum of infections it causes and the increasingly challenging treatment for the resulting antimicrobial resistance. P. aeruginosa gene expression responds to environmental conditions with discrete patterns typical of environmental isolates: motility, piliation, and expression of numerous exoproducts.
Quorum sensing (QS) is a bacterial intercellular communication system, which depends on bacterial cell population density and controls the pathogenesis of many organisms through regulating gene expression, including virulence determinants. QS has become an important attractive
Summary
target for the development of novel anti‑infective agents that do not rely on using antibiotics.
Forty clinical isolates belonging to Pseudomonas aeruginosa were collected from Demerdash Public Hospital. Their clinical origin was wounds 54%, urine 15%, burn 10%, blood 2%, sputum 15%, and shunt 4%. Three of them showed antibiotic resistivity comparable to kindly provided strain( Pseudomonas aeruginosa U3 strain) for seven antibiotics including amikacin (30µg), amoxicillin-clavulanic acid (30µg), cefepime (30µg), ceftazidime (30µg), ciprofloxacin (5µg), Co- gentamycin (10µg), meropenem (30µg), trimoxazole (25µg), and tobramycin (10µg). The prospective anti-virulence agents in this work were recovered from five halophilic bacterial strains (Halomonas cupida Halo-Rt1, H. elongate Halo-Rt2, Vigibacillus natechei Halo-Rt3, Sediminibacillus terrae Halo- Rt4, and H. almeriensis Halo-Rt5). Additionally, the partially purified metabolites recovered from the selected halophiles showed antagonistic activity against one of selected MDR P. aeruginosa strains (under investigation) at least.
The virulence features of P. aeruginosa including biofilm formation, rhamnolipid, pyocyanin, pyoverdin production, protease, hemolysin activity, and three types of motility behavior were studied both in presence and absence recovered
Summary
of metabolites at the sub-inhibitory concentration. Results revealed that the metabolites recovered from H. elongate Halo- Rt2, S. terrae Halo-Rt4, and H. almeriensis Halo-Rt5 decreased the rhamnolipid production by P. aeruginosa U3, P. aeruginosa NCR-RT2, and P. aeruginosa NCR-RT3; respectively by 100%. The proteolytic activity of P. aeruginosa NCR-RT3 strains declined by less than 10% after treatment with the metabolites recovered from H. cupida Halo-Rt1, H. elongate Halo-Rt2, and H. almeriensis Halo-Rt5; respectively. Interestingly, the metabolites from H. elongate Halo-Rt2 decreased the pyocyanin production in P. aeruginosa NCR- RT2 by 100% whereas the metabolites from H. cupida Halo- Rt1 enhanced the production of the same pigment in P. aeruginosa NCR-RT3 by 50%. The decrease in the other treatments was nearly 40% or more.
Regarding biofilm formation, the most efficient treatment was accomplished by metabolites recovered from S. terrae Halo-Rt4 with a decrease in the formation reached 70% for P. aeruginosa NCR-RT2. The pyoverdin pigment production in P. aeruginosa NCR-RT3 declined by half (or more) when treated with metabolites purified from H. cupida Halo-Rt1, and H. almeriensis Halo-Rt5. The decrease in hemolysin activity of the selected P. aeruginosa strains was in the range between 50% and 75% following the exposure to sub-inhibitory
Summary
concentrations of halo-bacterial metabolites in comparison to the control. All the three types of motilities (swimming, swarming, and twitching) decreased in the selected P. aeruginosa strains after treatment with bioactive metabolites recovered from tested halophiles strains.
The QS inhibitors that exist in the metabolites of H. cupida (Halo-Rt1) and H. elongate (Halo-Rt2) were identified using LC-ESI-MS/MS. Glabrol, 5,8-dimethoxyquinoline-2- carbaldehyde, agelasine, linoleoyl ethanolamide, penigequinolones derivatives, berberine, tetracosanoic acid, and liquidambaric lactone were recovered from the first strain whereas phloretin, lycoctonine, fucoxanthin, and crassicauline A were present in the second one.
In an individual experiment, the virulence features of four multi-drug resistant P. aeruginosa strains were investigated when exposed to the sub-lethal dose of gamma rays (1kGy). Gamma rays succeeded in completely inhibiting pyocyanin and pyoverdin pigment production as well as hemolysin activity for all P. aeruginosa strains. For most treatments, the 1.0 kGy dose decreased proteolytic activity and biofilm formation by 70% and 60% or more; respectively. The positive effect of gamma rays on rhamnolipids production was observed after exposing
Summary
P. aeruginosa NCR-RT2 and P. aeruginosa NCR-RT3 to the sub-lethal dose.
Quantitative-PCR results proved a decline in the expression of HALs-dependent QS genes (LasR internal, LasI internal, rhlR intact, and rhlI internal) in the four tested MDR
P. aeruginosa upon stressed with the sub-lethal dose of gamma radiation and the sub-inhibitory concentration of halo-bacterial metabolites in comparison with the control (Untreated group). The main results obtained could be summarized as follows:
• In P. aeruginosa U3, the metabolites recovered from H. elongate Halo-Rt2 decreased the expression of all AHL- dependent QS genes under investigation at a higher level than those extracted from H. cupida Halo-Rt1. The expression of the LasR internal gene was decreased by more than 90% in cells irradiated with 1kGy.
• In P. aeruginosa NCR-RT1, metabolites of H. cupida Halo-Rt1 and V. natechei Halo-Rt3 decline the LasI internal gene by nearly twenty-fold. Gamma rays at 1kGy succeeded to decrease the expression of all AHL-dependent QS genes, especially the “rhlR intact” gene.
• In P. aeruginosa NCR-RT2, metabolites from H. elongate Halo-Rt2 and S. terrae Halo-Rt4 showed the greatest reduction in the expression of “lasI internal” gene (ninety folds
Summary
or more). The expression of “rhlI” declined by thirty-fold when cells were stressed by gamma rays at a sub-lethal dose (1kGy).
• In P. aeruginosa NCR-RT3, the expression of “rhlI internal” gene was downregulated by more than ten folds after treatment with halophilic metabolites of H. almeriensis Halo- Rt5 and H. cupida Halo-Rt1 whereas irradiating cells with 1.0 kGy upregulate the expression of the “rhlR intact” gene and the “lasI internal” gene by two folds.
In conclusion, the expression of AHL-mediated QS genes responsible for the production of virulence factors could be explained by the crosslinked network of QS; this means the inhibition in any channel is compensated by activating another but not in the same degree i.e. complete blocking isn’t easy but an efficient inhibition is very possible. Therefore, targeting the QS system to treat bacterial infections provides a new direction for effectively slowing down the development of bacterial resistance against antibiotics and paving the way for describing quorum-quenching drugs with antibiotics as a successful treatment in MDRP infection.