Molecular Detection Of Efflux Pump and Virulence Factors Genes in Pseudomonas Aeruginosa

Study Overview
=================
Brief Summary
-----------------
Pseudomonas aeruginosa (PA) is a ubiquitous aerobic, non-fermentative Gram-negative rod that is widely associated with nosocomial pneumonia and can lead to severe illness with poor outcomes, particularly in critically ill people due to the ability of some strains to cause lung epithelial injury and spread into the circulation. 2 In the intensive care unit, PA infection is ranked among the top five causes of the bloodstream, pulmonary, surgical site, urinary tract, and soft tissue infections.

Detailed Description
-----------------
The pathogenesis of PA infections is multifactorial, and it is frequently complicated by the bacteria's intrinsic resistance to some antimicrobial agents such as sulfonamides, tetracyclines, and trimethoprim, as well as its ability to acquire or quickly develop resistance to major classes of antibiotics such as aminoglycosides, quinolones, B-lactams, and polymyxins (Bassetti et al., 2018). The efflux systems, which mediate the expulsion of antibiotics out of the cell shortly after entry, the production of enzymes to inactivate antibiotics, and the decrease in permeability across the cell wall are some mechanisms used by PA to develop antimicrobial resistance (Meletis & Bagkeri, 2013). PA possesses a large number of virulence factors that play a significant role in pathogenesis and the determination of infection severity. These virulence factors act alone or in synergy with each other to cause tissue damage, necrosis, and cell death. Among the virulence factors of PA, the major determinants of virulence are the type III secretion system (T3SS) and quorum sensing (cell-to-cell signaling system). The T3SS is a needle-like complex, also known as the injectisome, that enables a bacterium to deliver different effector proteins such as ExoS, ExoT, ExoU, and ExoY across the membrane into a host cell, altering host cell functions and increasing bacterial survival rates ( Horna G and, Ruiz J, 2021). In this study, we aimed to evaluate the prevalence of antibiotic resistance caused by the presence of Efflux genes and some virulence factors in Pseudomonas aeruginosa from clinical isolates.

Official Title
-----------------
Molecular Detection Of Efflux Pump and Virulence Factors Genes in Pseudomonas Aeruginosa

Conditions
-----------------
Pseudomonas Aeruginosa

Intervention / Treatment
-----------------
* Diagnostic Test: culture on cetrimide agar
* Diagnostic Test: Staining with Gram stain
* Diagnostic Test: Antibiotic sensitivity testing
* Diagnostic Test: Molecular detection to efflux genes and some virulence genes


Participation Criteria
=================
Eligibility Criteria
-----------------
Inclusion Criteria: All patients suffering from infections that can be caused by pseudomonas aeruginosa Exclusion Criteria: Samples diagnosed to have organisms other than pseudomonas aeruginosa.

Ages Eligible for Study
-----------------
Minimum Age: 4 Weeks
Maximum Age: 80 Years

Sexes Eligible for Study
-----------------
All

Accepts Healthy Volunteers
-----------------
No

Study Plan
=================
How is the study designed?
-----------------

Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Patients with pseudomonas aeruginosa infections<br>All patients suffer from infections that can be caused by pseudomonas aeruginosa. Clinical Data will be obtained as: Data about clinical manifestations including fever, expectoration, pus from wounds, urinary symptoms, symptoms of upper respiratory tract infections, and symptoms of otitis externa. Samples will be cultured on cetrimide agar. Antibiotic sensitivity testing will be done by disc diffusion method according to CLSI. Molecular detection to efflux genes and some virulence genes by PCR. | Diagnostic Test: culture on cetrimide agar<br>* Samples will be inoculated on cetrimide agar using the plating out technique.<br>Diagnostic Test: Staining with Gram stain<br>* colonies on cetrimide agar will be spread on glass slide and stained by gram stain<br>Diagnostic Test: Antibiotic sensitivity testing<br>* Antibiotic sensitivity testing will be done by disc diffusion method according to CLSI<br>Diagnostic Test: Molecular detection to efflux genes and some virulence genes<br>* Molecular detection to efflux genes and some virulence genes by conventional PCR<br>|
| Patients with infections other than pseudomonas aeruginosa<br>Data about clinical manifestations including fever, expectoration, pus from wounds, urinary symptoms, symptoms of upper respiratory tract infections, and symptoms of otitis externa. Samples will be cultured on different culture media and automated identification by Vitek system. | Diagnostic Test: culture on cetrimide agar<br>* Samples will be inoculated on cetrimide agar using the plating out technique.<br>Diagnostic Test: Staining with Gram stain<br>* colonies on cetrimide agar will be spread on glass slide and stained by gram stain<br>Diagnostic Test: Antibiotic sensitivity testing<br>* Antibiotic sensitivity testing will be done by disc diffusion method according to CLSI<br>|

What is the study measuring?
-----------------
Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Isolation and identification of pseudomonas aeruginosa using culture and automated system techniques | identification of pseudomonas aeruginosa in different clinical samples collected from Sohag University hospital using different laboratory techniques as culture on citramide agar, Staining with Gram, biochemical reactions such as Oxidase test, sugar fermentation test, and automated identification using vitek2 system | 1 December 2022 to 1 February 2023 | 
| Identification of recent antibiotic sensitivity pattern using Modified Kerby -Disc Diffusion method | Determination of recent antibiotic sensitivity pattern using different antibiotics by disc diffusion method by spreading the inoculum in pitry dish containing Muller Hinton Agar, then different discs containing antibiotics are placed at a distance of 1.5 cm, then incubated at 37 co for 24 hrs. The diameter of the zone of inhibition is measured to determine MIC for each antibiotic according to the guidelines of CSLI 2022. | 1 December 2022 to 1 February 2023 | 
| Molecular Identification of some virulence factors and efflux genes using PCR | Molecular detection of some virulence factors and efflux genes using specific primers by conventional PCR. primers of the following genes will be used as exoS,exoU, toxA, mex A, mex B. Extraction of DNA will be done first, followed by amplification technique using the thermal cycler. Detection of amplified DNA will be done using Agrose gel electrophoresis stained with ethidium bromide. | 1 February 2023 to 30 March 2023 | 

 Terms related to the study
=================
Keywords Provided by Centre Hospitalier Valida
-----------------
Efflux genes, Pseudomonas , virulence, PCR