الفهرس 
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Abstract
Pseudomonas aeruginosa is a good example of ubiquitous opportunistic environmental pathogens; found in soil, water, feed and farm equipment, causing serious problems in poultry farms resulting in significant economic losses due to high mortalities in all ages.
In the present study, a total of 220 samples collected from kidney samples from broiler chickens were studied applying conventional and advanced techniques for identification of Pseudomonas spp. with focusing on P. aeruginosa.
The results of bacteriological examination revealed isolation of 34 Pseudomonas spp. with a prevalence rate of 15.5%. Identification of Pseudomonas isolates with traditional methods; including morphology, cultural characters and biochemical tests, as well as Vitek2 compact system for identification using Vitek2 ID-GN kits revealed identification of 4 different Pseudomonas species. P. aeruginosa was the most prevalent isolates (n=22; 10%), followed by P. putida (n=8; 3.6%), and finally both of P. fragi and P. lundensis (n=2; 0.9% for each).
In the current study, all the recovered P. aeruginosa isolates (n=22) were tested in-vitro for their antibiotic sensitivity to 12 different antibiotics. All isolates were completely resistant to cephalexin, apramycin, kanamycin, doxycycline HCl and chloramphenicol. Moreover, most of isolates were resistant to the other tested antibiotics; cefotaxime-sodium, enrofloxacin, ceftazidime, aztreonam, amoxicillin, amoxicillin+ clavulanic acid and cefepime. All P. aeruginosa isolates were recorded as MDR.
In the present work, P. aeruginosa isolates showed growing resistance pattern against 1st and 3rd generations of cephalosporins and extended to the 4th generation. Therefore, ESβLs producing P. aeruginosa isolates were phenotypically detected using modified CLSI ESβLs confirmatory test. The results showed that out of 22 of P. aeruginosa isolates, 15 isolates (68.2%) were confirmed as ESβLs producers.
In the present work, biofilm producing P. aeruginosa isolates were phenotypically detected by cultivation on YESCA CR agar plates. The results showed that out of 22 of P. aeruginosa isolates, 14 isolates (63.6%) were phenotypically biofilm producer.
In this work, PCR was conducted on 6 MDR P. aeruginosa isolates to detect 3 resistance-associated genes including genes encoding resistance to β-lactams (blaTEM), tetracycline (tetA(A) and aminoglycosides (aadB). The results showed that blaTEM gene was found in 4/6 isolates (66.7%) and all positive isolates were ESβLs producers. Meanwhile both tetA(A) and aadB genes were detected in 3/6 isolates (50% for each). Results revealed the existence of blaTEM in isolates confirmed phenotypically as ESβLs producers only (n=4; 66.7%) while others non-ESβLs producers were negative for blaTEM gene.
PCR was applied also on the same 6 MDR P. aeruginosa isolates to detect 3 virulence-associated genes (exoS, filiC and toxA). The results showed that exoS gene was found in all tested P. aeruginosa isolates (100%) and filiC gene was detected in 3/6 isolates (50%) meanwhile toxA gene was not found at any isolate
In the present study, cinnamon, oregano and thyme EOs were tested for their antimicrobial activities against all P. aeruginosa isolates (n=22) at concentrations of 0.1, 0.01 and 0.005% for each EO. The results showed that cinnamon was the most effective EO followed by oregano and finally thyme. Both cinnamon and oregano at concentration of 0.1% completely inhibited the growth of examined P. aeruginosa isolates (100%) while thyme inhibited only the growth of 11 P. aeruginosa isolates (50%). Meanwhile, at 0.01% concentration, cinnamon inhibited only 3 isolates (13.6%) while oregano and thyme had no antibacterial effect. On the other hand, at the concentration of 0.005% all the tested EOs had no antibacterial on the tested isolates but showed little and retarded growth.
In the present study, the effects of the three EOs on phenotypic characters of 10 selected P. aeruginosa isolates had been evaluated. The unaffected isolates with cinnamon, oregano and thyme EOs at the concentration of 0.01% were further investigated for their phenotypic proprieties including biofilm production, motility and exopigments production.
Regarding biofilm formation, the results showed that biofilm production was inhibited in 80% of the tested P. aeruginosa isolates treated with the sub-inhibitory concentration of cinnamon and oregano meanwhile thyme inhibited biofilm formation in 40% only. Concerning the effect of EOs on the motility, the results showed that cinnamon, oregano and thyme inhibited the motility of 60% the tested P. aeruginosa isolates meanwhile thyme had no effect. Regarding the effect of EOs on the production of exopigments, all exopigments the selected were strong producer for the exopigments. The results showed that cinnamon, oregano inhibited the exopigments production in 50% of tested P. aeruginosa isolates meanwhile thyme had no effect.
In the current study, the qRT-PCR was applied on 2 MDR P. aeruginosa isolates; those unaffected with cinnamon treatment at concentration of 0.01% and harbored blaTEM, filiC, and exoS genes, for detection of possible fold changes of such genes expression after treatment with cinnamon. The 16S rDNA gene was used as a housekeeping gene for gene expression. The results indicated that the gene expression for all tested genes after treatment with cinnamon oil had decreased.