الفهرس 
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Abstract
Infections in burn patients are one of the main factors causing increased morbidity and mortality. Pseudomonas aeruginosa (P. aeruginosa) is an aerobic opportunistic pathogen which produces many enzymes and extracellular molecules. These share in the biofilm formation, causing P. aeruginosa to be one of the highly resistant pathogens that monotherapy cannot treat it.
Azithromycin alone doesn’t have antipseudomonal activity, but if it has been combined with Ciprofloxacin, it gives synergistic results in highly resistant microbial infections. Also, it has been documented that it can disrupt P. aeruginosa biofilms.
Since there is a limited antimicrobial pipeline discovery, more effective forms of already existing antimicrobial agents could be a good alternative to fight the worldwide antimicrobial resistance threat.
Chitosan [poly B-(1–4)-2-amino-2-deoxy-d-glucose], a poly cationic biopolymer, has high antimicrobial activity. This natural polysaccharide is non-toxic, biodegradable, low price, high biocompatible and non-antigenic. It has been widely used in studies for the delivery of drugs, vaccines and biological materials.
The current study aimed to assess the effect of Ciprofloxacin and Azithromycin on chitosan nanoparticles separately and in combination versus free forms in vitro on planktonic and biofilm forming P. aeruginosa isolates and in vivo on P. aeruginosa infected burn lesions in mice with burn as well as to detect the presence of some biofilm-specific antibiotic resistance genes. Fifty clinical isolates of P. aeruginosa collected from burn samples submitted to AMUH Diagnostic Medical Microbiology laboratory, throughout a period starting from June 2018 through April 2019.
The antimicrobial susceptibility of all P. aeruginosa isolates was determined by the Bauer-Kirby disk diffusion technique according to CLSI guidelines (2020). Six isolates (12%) were MDR, 37 isolates (74%) were XDR and 2 isolates (4%) were PDR. Isolates showed 96% susceptibility to Colistin followed by 32% susceptibility to Imipenem.
All the isolates were found to be biofilm producers, 39 isolates (78%) were strong biofilm producers, 10 (20%) were moderate biofilm producers, and one (2%) was weak biofilm producer. All the isolates (100%) were positive for ndvB and tssC1 genes by PCR.
The antibacterial activity of Ciprofloxacin was compared with that of Ciprofloxacin Chitosan NPs against all P. aeruginosa isolates. The MIC values were decreased significantly by 45.62% when challenged with Ciprofloxacin chitosan NPs (32µg/ml versus 16 µg/ml). The MIC values of Ciprofloxacin decreased by 38.50 % when Azithromycin was added to Ciprofloxacin in equal concentrations (32µg/ml versus 16 µg/ml). Furthermore, MIC values decreased by 68.37% when challenged with Ciprofloxacin Azithromycin Chitosan NPs (32 µg/ml versus 8 µg/ml).
Testing the in vitro effect of studied antibiotics on all P. aeruginosa producing biofilm using MTP method, showed that the MBEC was ≤ 1024 µg/ml in 54% of isolates for free Ciprofloxacin, 92% for free Ciprofloxacin-Azithromycin, 98% for Ciprofloxacin Chitosan NPs & 98 % for Ciprofloxacin-Azithromycin Chitosan NPs.
The in vivo study results showed that the administered P. aeruginosa inoculum resulted in 100% mortality in the untreated mice group by day 7 of the study. However, on treating with Ciprofloxacin Chitosan NPs (subgroup IIa) and Azithromycin Chitosan NPs (subgroup IIb) showed 20% and 50% mortality respectively by day 7. On the contrary, 100% survival rate was observed in combination therapy groups Ciprofloxacin-Azithromycin free and Chitosan NPs form till day 10 of the experiment. The positive control group showed the maximum bacterial load on day 6 with 1.7x109 cfu/ml. Following treatment with Ciprofloxacin Chitosan NPs (subgroup IIa) 5 logs reduction in skin bacterial load was observed in day 6 in comparison with positive control group. However, maximum reduction was observed In combination therapy groups Ciprofloxacin-Azithromycin free form and Chitosan NPs form with a decrease of 9 logs in day 6. The percentage of wound contraction was compared in all groups with respect to the original wound size. A significantly higher percentage of wound contraction was observed in groups treated with free Ciprofloxacin-Azithromycin (74.50 ± 4.38) % and Ciprofloxacin Azithromycin Chitosan NPs (78.0 ± 3.50) % (P <0.001) by day 10 post infection. However, the wound area contracted on monotherapy of Ciprofloxacin Chitosan NPs (subgroup IIa) by (43.75 ± 5.18)% and Azithromycin Chitosan NPs (subgroup IIb) by (30.0 ± 5.0)%.