الفهرس 
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Abstract
Paeruginosa is an opportunistic Gram-negative bacterium that contributes significantly to HAIs. It has been reported by the CDC as a major lead causative agent of pneumonia, the third cause of urinary tract infection, eighth commonly isolated microbe from bloodstream infections. Additionally, it is responsible for fatal infections in cystic fibrosis and immunocompromised patients (Flockton et al., 2019).
The success of P. aeruginosa, as an opportunistic pathogen, is partially attributed to the ability of whole bacterial populations of this bacterium to coordinate their activity using cell-to-cell communication, mediated by QS signal molecules (Cao et al., 2014). QS regulates over 10% of the P. aeruginosa genome including swarming motility, biofilm formation and antimicrobial resistance, as well as the production of virulence determinants such as elastases, pyocyanin, cyanide and exotoxins (Moradali et al.,2017).
Two dominating QS systems are employed by P. aeruginosa: las (LasR-LasI) and rhl (RhlR-RhlI). The synthase of LasI catalyzes the synthesis of N- (3-oxododecanoyl) homoserine lactone, RhlI catalyzes the synthesis of N-butyryl-homoserine lactone, which induces their respective cognate transcriptional regulators LasR and RhlR, responsible for the activation of numerous QS-controlled genes (Kostylev et al., 2019).
The rise of drug-resistant P. aeruginosa and the delay in introducing newer drugs threaten human well-being. As a better drug strategy, it has been suggested to focus on QS-regulated virulence factors instead of growth-related features (Pang et al., 2019). Among the most accepted materials is chitosan, a polysaccharide polymer composed of N-acetyl-D-glucosamine and D-glucosamine units connected by β-1,4-glycosidic linkages. It is derived from partial or total deacetylation of chitin (Kong et al., 2010). It possesses unique combination of properties mainly excellent biocompatibility, enzymatic biodegradability, ability to metal complexation, and nontoxicity (Vilar Junior et al., 2016; Ma, Garrido-Maestu and Jeong, 2017). It has a good antimicrobial action against a wide scope of microorganisms such as Staphylococcus aureus and Escherichia coli; also, it has an antifungal effect (Vilar Junior et al., 2016; Ma, Garrido-Maestu and Jeong, 2017).
Advances in nanotechnology have enabled the formulation of chitosan polymers as nanoparticles, which are more effective antimicrobial agents than chitosan itself, in addition to other benefits, such as better penetration of biofilm and higher solubility (Chandrasekaran, Kim and Chun, 2020). This study aimed to assess the inhibitory impact of CS-NPs on virulence characters of P. aeruginosa regulated by QS as motility and biofilm formation and the expression of LasI and RhlI genes in an attempt to find new alternatives to the existing antibiotic therapy for treating resistant infections.
The present study was conducted from October 2020 till February 2021 on 30 P. aeruginosa isolates obtained from inpatient and outpatient clinical samples submitted to the central microbiology laboratory at Ain Shams University Hospital. The Research Ethics Committee approved the study of Faculty of Medicine Ain Shams University (No. FMASU M D 94/2020).
The isolates included sputum, blood, urine, and swabs from surgical and burned wounds, collected under complete aseptic precautions, identified by conventional microbiologic methods (Tille, 2017). All P. aeruginosa isolates were stored at −80°C in nutrient broth with 20% (vol/vol) glycerol.
The antibiotic sensitivity pattern of isolated strains showed that they were highly resistant to cefepime, ceftazidime, and gentamycin with rates of 96%, 90% and 87%, respectively. About 80% (24/30) of the isolates were MDR.
All 30 P. aeruginosa isolates exhibited both swimming and swarming motility. The inhibitory activity of CS-NPs on the motility of clinical isolates was assessed at the sub-MICs (it differed among the strains, but it ranged from 5mg/ml to 10mg/ml). CS-NPs reduced the swimming and swarming motility of all bacterial isolates. The test differed from the control with a P-value of 0.0001. The mean (+ SD) diameter of swimming motility was decreased from 3.93 (±1.5) to 1.63 (±1.02) cm, and the mean diameter of the swarming motility was reduced from 3.5 (±1.6) to 1.9 (±1.07) cm.
All 30 P. aeruginosa isolates showed biofilm production by microtiter plate assay. About 83% (25/30) of the isolates were moderate biofilm producers (+2), and 17% (5/30) were weak biofilm producers (+1). CS-NPs with the sub-MICs significantly reduced the biofilm formation by P. aeruginosa isolates (p-value < 0.01) as 100% of isolates became a non-biofilm producer (0). The mean (+SD) of OD was decreased from 0.667 (±0.096) to 0.099 (±0.045). The mean percentage rate of biofilm inhibition was 84.95% (±6.18).
CS-NPs inhibited virulence activity on the genotypic level as well. At the sub-MIC of CS-NPs, the median of Las I cycle threshold (CT) was increased from 12.5 (±3.3) to 23 (±5.6), and the median CT of RhlI was increased from 12.22 (±2) to 21.8 (±4.5). The levels of LasI, and RhlI expression were significantly lowered compared with untreated cultures. The median fold decrease of LasI, and RhlI expression in selected most virulent 15 isolates was 90 and 100, respectively.