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

Recruiting

• Conditions: Pseudomonas Aeruginosa
• Interventions: 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

• Registration Number: NCT05642767
• Lead Sponsor: Sohag University

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.

Basic Information
|
Recruitment & Eligibility
|
Study & Design
|
Trial Locations

Study Timeline

• Study First Submitted: 2022-11-19
• Study First Submitted QC: 2022-11-29
• Study Start: 2022-12-01
• Study First Posted: 2022-12-08
• Primary Completion: 2023-03-30
• Status Verified: 2023-04
• Last Update Submitted: 2023-04-11
• Last Update Posted: 2023-04-12
• Study Completion: 2023-05-01

Recruitment & Eligibility

• Status: RECRUITING
• Sex: All
• Target Recruitment: 75

Inclusion Criteria

• All patients suffering from infections that can be caused by pseudomonas aeruginosa

Read More

Exclusion Criteria

• Samples diagnosed to have organisms other than pseudomonas aeruginosa.

Read More

Study & Design

• Study Type: OBSERVATIONAL
• Study Design: Not specified

Arm && Interventions

Group	Intervention	Description
Patients with pseudomonas aeruginosa infections	Staining with Gram stain	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.
Patients with infections other than pseudomonas aeruginosa	culture on cetrimide agar	* 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.
Patients with pseudomonas aeruginosa infections	culture on cetrimide agar	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.
Patients with pseudomonas aeruginosa infections	Antibiotic sensitivity testing	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.
Patients with pseudomonas aeruginosa infections	Molecular detection to efflux genes and some virulence genes	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.
Patients with infections other than pseudomonas aeruginosa	Staining with Gram stain	* 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.
Patients with infections other than pseudomonas aeruginosa	Antibiotic sensitivity testing	* 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.

Primary Outcome Measures

Name	Time	Method
Identification of recent antibiotic sensitivity pattern using Modified Kerby -Disc Diffusion method	1 December 2022 to 1 February 2023	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.
Molecular Identification of some virulence factors and efflux genes using PCR	1 February 2023 to 30 March 2023	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.
Isolation and identification of pseudomonas aeruginosa using culture and automated system techniques	1 December 2022 to 1 February 2023	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

Trial Locations

Locations (1)

Sohag University; 🇪🇬 Sohag, Egypt