التوصيف المظهري والجزيئي لبعض عوامل الضراوة في بكتريا Pseudomonas aeruginosa المعزولة من مرضى إصابات الاذن في محافظة كربلاء المقدسة

Phenotypic and molecular characterization of some virulence factors in Pseudomonas aeruginosa isolated from ear infection patients in sacred Kerbala province

Summary
Ear infections are a common clinical issue worldwide and the leading cause of hearing loss in the world.110 swabs were collected from patients with ear infections at the Ear, Nose, and Throat practice at Imam Hussein Medical City and private clinics in different areas of the Holy Governorate of Karbala, their ages ranged from 1-75 year for the period from August 2023 to October 2023.
The results revealed that 97 (88.18%) isolates showed microbial growth, of which 85 (87.63%) isolates showed bacterial growth and 12 (12.37%) isolates showed fungal growth. The fungal isolates were Candida spp. and Aspergillus spp. with 10 (83.33%) and 2 (16.67%) isolates, respectively.
Of the total bacterial isolates, which showed Gram-negative bacteria were more common than Gram-positive bacteria with 67 (78.82%) isolates including 35 (41.18%) isolates belonging to the genus Pseudomonas spp. .Moreover, the other genera belonging to Gram-negative isolates included Escherichia spp., Klebsiella spp., Proteus spp. and Burkholderia spp. with 11 (12.94%) ,10 (11.76%) ,7(8.24%) and 2 (%2.35) isolates respectively. Bordetella spp. and Stenotrophomtrty23fgvonas spp. with one isolate (1.18%) for each bacterial genus. Gram-positive bacterial isolates included 18 (21.18%) isolates belonging to the genus Staphylococcus spp.
This study identified three bacterial species in the Vitek 2 compact system, namely Pseudomonas aeruginosa، Pseudomonas putida و Pseudomonas fluorescens with 30 (35.29%), 3 (3.53%) and 2 (2.35%) isolates, respectively. Polymerase chain reaction (PCR) results using the 16s rRNA gene confirmed that all P. aeruginosa isolates carried this gene.The study highlighted that P. aeruginosa is the common bacterial species among the microorganisms causing ear infections. The Vitek 2 compact system was used to determine the sensitivity of the P. aeruginosa isolates 12 antibiotics, 96.67% of the isolates were resistant to both Ticarcillin and Ticarcillin/Clavulanic acid. In addition, 93.33% of them were resistant to Piperacillin, Ceftazidime and Cefepime, 90% of these isolates showed resistance to Gentamicin and Imipenem . 86.67% of the bacterial isolates were resistant to both Tobramycin,Ciprofloxacin and Meropenem. Moreover, the bacteria showed resistance (80 and 20%) to Amikacin and Colistin respectively.
Some virulence factors of P. aeruginosa isolates were examined, the results indicated that 24 (80%) of the isolates produced Pyocyanin, 23 (76.67%) isolates produced protease and 20 (66.67%) isolates produced hemolysin. The study also revealed that each of the tested P. aeruginosa isolates had the ability to produce biofilms according to both qualitative and quantitative methods. In the qualitative detection, 6 (20%) isolates were identified as strong biofilm producers, 8 (26.67%) as moderate producers and 16 (53.33%) as weak producers. The quantitative assay results showed that 12 (40%) isolates were strong biofilm producers, 15 (50%) isolates were moderate producers, and 3 (10%) isolates were weak producers. In addition to the phenotypic examination of biofilm production, molecular detection using polymerase chain reaction (PCR) revealed that 7 (23.33%) and 15 (50%) isolates carried the psIA and psID biofilm formation genes respectively.
On the other hand, molecular detection showed that all 30 (100%) isolates of P. aeruginosa contain both ndvB and tssC1 genes that are involved in biofilm-specific antibiotic resistance. The development of biofilms is closely related to the activity of efflux pumps. The detection of efflux pumps in 30 bacterial isolates of P. aeruginosa was performed using the Ethidium Bromide-Agar Cartwheel method as a guide for phenotypic detection. The results revealed that 23 (76.67%) bacterial isolates were positive for phenotypic detection. This was based on the lowest concentration at which the isolates did not show fluorescence under Ultraviolet (UV) light source. These results were confirmed on the molecular level using PCR detections. As 17 (56.67%) of the P. aeruginosa isolates carried the mexB gene, the gene encodes to one of the three proteins that form mexAB-oprM system. The latter belongs to the family of resistance-nodulation divisions (RND).
Molecular modeling results indicated that the predicted structure of adenylate kinase (Adk) in P.aeruginosa is divided into three functional subdomains namely the core subdomain, the AMP-binding subdomain (AMPbd), and the ATP-binding subdomain (LID). After that, the model was vertified by MolProbity version 4.4 with high rate of confidence.