Pseudomonas Aeroginosa
Conditions
Brief summary
1. Detection of expression level of mexB efflux pump gene in multidrug resistant clinical isolates of Pseudomonas aeruginosa by real time PCR(QRT-PCR). 2. Phenotypic detection of efflux pump activity in multidrug resistant clinical isolates of Pseudomonas aeruginosa. 3. Phenotypic detection of biofilm formation and its degree (strong-moderate-weak) in multidrug resistant clinical isolates of Pseudomonas aeruginosa. 4. Relationship between expression level of mexB efflux pump gene and degree of biofilm formation in multidrug resistant clinical isolates of Pseudomonas aeruginosa.
Detailed description
Pseudomonas aeruginosa is an opportunistic gram-negative pathogen, causing life threatening nosocomial infections including respiratory system, urinary system and skin wounds (1). P. aeruginosa exhibits resistance to a range of antibiotic classes, including aminoglycosides, carbapenems, beta-lactams, quinolones, and cephalosporins (2). Frequent occurrence of drug resistance is due to many virulence factors in P. aeruginosa such as flagella, pili, lipopolysaccharides, secreted enzymes like DNase and lipase, toxins, and pigments as pyocyanin which play crucial roles in tissue damage and immune suppression. Multiple resistant mechanisms are developed by P. aeruginosa (3). Two prominent mechanisms, biofilm formation and efflux pump activity among other mechanisms, play an important role in persistence and antibiotic resistance (4). Biofilm formation produced by P. aeruginosa is a complex phenomenon that promotes antibiotic resistance and shields the pathogen from the host immune system. This results in severe clinical outcomes in critically ill patients (5). There are about 12 resistance-nodulation-division (RND) families of efflux pumps in P. aeruginosa. The mexAB-oprM multidrug efflux pump system of P. aeruginosa is involved in resistance (6). Efflux pumps play a role in biofilm formation by influencing Physical-chemical interactions, mobility, gene regulation, quorum sensing (QS), extracellular polymeric substances (EPS), and toxic compound extrusion (6)(7). It has been demonstrated that MexAB-OprM plays a role in the resistance of aztreonam, gentamicin, tetracycline and tobramycin in biofilm structures of P. aeruginosa (8). MexB is the most critical and specific gene for efflux activity within the MexAB-OprM system and contributes to antibiotic resistance in Pseudomonas aeruginosa (9).
Interventions
GENETICReal time PCR
Clinical isolates of Pseudomonas aeruginosa will undergo RNA extraction followed by cDNA (complementary DNA) synthesis. Quantitative real-time PCR will be performed using specific primers for the mexB efflux pump gene, and SYBR Green will be used to quantify gene expression. Housekeeping genes (e.g., rpoD or 16S rRNA) will serve as internal controls. The procedure allows precise quantification of mexB expression levels in each isolate.
Sponsors
Assiut University
Study design
Observational model
COHORT
Time perspective
CROSS_SECTIONAL
Eligibility
Sex/Gender
ALL
Healthy volunteers
No
Inclusion criteria
1. Clinical isolates confirmed as Pseudomonas aeruginosa by microbiological testing. 2. Isolates collected from different clinical specimens (e.g., wound swabs, sputum, urine, blood, and catheters).
Exclusion criteria
1. Bacterial species other than Pseudomonas aeruginosa. 2. Repeated clinical isolates from the same patient. 3. Repeated clinical specimens from different sources of the same patient.
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Detection of mexB efflux pump gene expression level in multidrug resistant clinical isolates of Pseudomonas aeruginosa | one year | molecular detection of mexB efflux pump gene expression level by real time PCR (QRT-PCR) |
Secondary
| Measure | Time frame | Description |
|---|---|---|
| Detection of efflux pump activity in multidrug resistant clinical isolates of Pseudomonas aeruginosa | one year | Phenotypic detection of efflux pump activity by MIC (minimal inhibitory concentration) testing with efflux pump inhibitors (EPIs) using Phenylalanine-arginine β-naphthylamide (PAβN)in P. aeruginosa |
| Detection of degree of biofilm formation (strong-moderate-weak) in multidrug resistant clinical isolates of Pseudomonas aeruginosa | one year | Phenotypic detection of biofilm formation using microtiter plate assay and assess the degree of biofilm (strong-moderate-weak) |
Contacts
CONTACTRehab AM Omran
STUDY_DIRECTORAssiut University
Assiut University
Outcome results
None listed