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

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.

Study Overview

• Status: Recruiting
• 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

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.

• Study Type: Observational
• Enrollment (Anticipated): 75

Contacts and Locations

• Study Contact: Name: Noha S Shafik, lecturer; Phone Number: +20 01067261504; Email: Nohasaber@med.sohag.edu.eg
• Study Contact Backup: Name: Nesma A Mohammed, lecturer; Phone Number: +20 01006780725; Email: Nesmaaateef@med.sohag.edu.eg

Study Locations

• Egypt
  • Sohag, Egypt
    • Recruiting
    • Sohag University
    • Contact: Noha S Shafik, Lecturer; Phone Number: 01067261504; Email: nohasaber@med.sohag.edu.eg
    • Contact: Nesma A Mohamed, Lecturer

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 4 weeks to 80 years (Child, Adult, Older Adult)
• Accepts Healthy Volunteers: No

• Sampling Method: Non-Probability Sample

Study Population

This study will be carried out on all patients suffering from infections that can be caused by pseudomonas aeruginosa.

Samples (pus, urine, blood, sputum, ear discharge) will be collected from different departments at sohag university hospital.

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.

Description

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.

Study Plan

How is the study designed?

Design Details

• Observational Models: Case-Control
• Time Perspectives: Cross-Sectional

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Patients with pseudomonas aeruginosa infections; 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; Samples will be inoculated on cetrimide agar using the plating out technique.; Diagnostic test: Staining with Gram stain; colonies on cetrimide agar will be spread on glass slide and stained by gram stain; Diagnostic test: Antibiotic sensitivity testing; Antibiotic sensitivity testing will be done by disc diffusion method according to CLSI; Diagnostic test: Molecular detection to efflux genes and some virulence genes; Molecular detection to efflux genes and some virulence genes by conventional PCR
Patients with infections other than pseudomonas aeruginosa; 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; Samples will be inoculated on cetrimide agar using the plating out technique.; Diagnostic test: Staining with Gram stain; colonies on cetrimide agar will be spread on glass slide and stained by gram stain; Diagnostic test: Antibiotic sensitivity testing; Antibiotic sensitivity testing will be done by disc diffusion method according to CLSI

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

Collaborators and Investigators

• Sponsor: Sohag University
• Investigators: Study Chair: Mohamed H Alrawy, Faculty of Medicine, Sohag University; Study Chair: Ebtisam M Gad, Faculty of Medicine, Sohag University

Publications and helpful links

Helpful Links

• Related Info

Study record dates

Study Major Dates

• Study Start (Actual): December 1, 2022
• Primary Completion (Actual): March 30, 2023
• Study Completion (Anticipated): May 1, 2023

Study Registration Dates

• First Submitted: November 19, 2022
• First Submitted That Met QC Criteria: November 29, 2022
• First Posted (Actual): December 8, 2022

Study Record Updates

• Last Update Posted (Actual): April 12, 2023
• Last Update Submitted That Met QC Criteria: April 11, 2023
• Last Verified: April 1, 2023

More Information

Terms related to this study

• Keywords: Efflux genes, Pseudomonas , virulence, PCR
• Additional Relevant MeSH Terms: Infections; Gram-Negative Bacterial Infections; Bacterial Infections; Bacterial Infections and Mycoses; Pseudomonas Infections; Anti-Infective Agents, Local; Anti-Infective Agents; Anti-Bacterial Agents; Cetrimonium
• Other Study ID Numbers: Soh-Med-22-11-18

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No