Phenotypic and Genotypic Detection of Biofilm Formation and Efflux Pump Activity in Multi-drug Resistant Klebsiella Pneumoniae (Klebsiella)

1. Sample: urine, pus and sputum samples.
2. Culture: the samples will be inoculated on the MacConkey agar and subcultured on eosine methylene blue agar.

3. Identification of isolates will be done by:

   • Colony morphology.
   • Gram staining.
   • Biochemical reactions: including Indole, Methyl red, Voges-Proskauer, and Citrate utilization (IMViC) tests.
4. Antimicrobial susceptibility testing of the isolates will be performed using the Kirby-Bauer method (disc diffusion method) using Muller Hinton agar according to the Clinical and Laboratory Standards Institute (CLSI 2025) guidelines for selected groups of antibiotics.
5. Phenotypic detection of biofilm formation by the microtitre plate method.
6. Phenotypic detection of efflux pump activity by ethidium bromide-agar cartwheel method.
7. Detection of biofilm genes (fimH-1, mrkA, and mrkD) by conventional PCR.
8. Detection of efflux pump genes (acrAB, tolC, and mdtk) by conventional PCR.

Study Overview

• Status: Not yet recruiting
• Conditions: Multidrug Resistant Klebsiella Pneumoniae Infection
• Intervention / Treatment: Genetic: Genotypic Detection of Biofilm Formation and Efflux Pump Activity.

Detailed Description

K. pneumoniae is a member of the Gram negative Enterobacteriaceae family. It is a rod-shaped, capsulated, non-motile, non-spore-forming bacteria characterized by a unique mucoid look on agar plates.

Klebsiella pneumoniae is an opportunistic pathogen that is responsible for a wide range of community-acquired and healthcare-associated infections including urinary tract infections, wound infection, bacteremia, and pneumonia

1. Sample: urine, pus and sputum samples.
2. Culture: the samples will be inoculated on the MacConkey agar and subcultured on eosine methylene blue agar.

3. Identification of isolates will be done by:

   • Colony morphology.
   • Gram staining.
   • Biochemical reactions: including Indole, Methyl red, Voges-Proskauer, and Citrate utilization (IMViC) tests.
4. Antimicrobial susceptibility testing of the isolates will be performed using the Kirby-Bauer method (disc diffusion method) using Muller Hinton agar according to the Clinical and Laboratory Standards Institute (CLSI 2025) guidelines for selected groups of antibiotics.
5. Phenotypic detection of biofilm formation by the microtitre plate method.
6. Phenotypic detection of efflux pump activity by ethidium bromide-agar cartwheel method.
7. Detection of biofilm genes (fimH-1, mrkA, and mrkD) by conventional PCR.
8. Detection of efflux pump genes (acrAB, tolC, and mdtk) by conventional PCR.

• Study Type: Observational
• Enrollment (Estimated): 100

Contacts and Locations

• Study Contact: Name: Ghofran Mahrous Mohammed; Phone Number: 01154027711; Email: ghofran.azam898@gmail.com

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child; Adult; Older Adult
• Accepts Healthy Volunteers: Yes

• Sampling Method: Non-Probability Sample

Study Population

All selected patients to complete history all samples transported to medical microbiology department.

Description

Inclusion Criteria:1-Age and sex range: any age and any sex. 2-Clinical setting: hospital-acquired infections. 3-Single pathogen focus. 4-MDR K. pneumoniae which develop resistance to at least one agent from three or more antibiotic classes.

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Exclusion Criteria:1-Recent antibiotics : Use within 48-72 hours before enrollment.

2-Mixed infections.

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Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Group 1: MDR Klebsiella pneumoniae isolates.	Genetic: Genotypic Detection of Biofilm Formation and Efflux Pump Activity.; Phenotypic detection of biofilm formation by the microtitre plate method.; Phenotypic detection of efflux pump activity by ethidium bromide-agar cartwheel method.; Detection of biofilm genes (fimH-1, mrkA, and mrkD) by conventional PCR.; Detection of efflux pump genes (acrAB, tolC, and mdtk) by conventional PCR.
group 2: Non-MDR Klebsiella pneumoniae isolates.	Genetic: Genotypic Detection of Biofilm Formation and Efflux Pump Activity.; Phenotypic detection of biofilm formation by the microtitre plate method.; Phenotypic detection of efflux pump activity by ethidium bromide-agar cartwheel method.; Detection of biofilm genes (fimH-1, mrkA, and mrkD) by conventional PCR.; Detection of efflux pump genes (acrAB, tolC, and mdtk) by conventional PCR.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Time Frame
Prevelace of biofilm formation genes in MDR Klebsiella pneumonea isolates (%)	6 months

Secondary Outcome Measures

Outcome Measure	Time Frame
Prevelace of efflux pump genes in MDR Klebsiella pneumonea isolates (%)	6 months

Collaborators and Investigators

• Sponsor: Sohag University

Study record dates

Study Major Dates

• Study Start (Estimated): April 1, 2026
• Primary Completion (Estimated): August 1, 2026
• Study Completion (Estimated): February 1, 2027

Study Registration Dates

• First Submitted: March 31, 2026
• First Submitted That Met QC Criteria: April 16, 2026
• First Posted (Actual): April 21, 2026

Study Record Updates

• Last Update Posted (Actual): April 21, 2026
• Last Update Submitted That Met QC Criteria: April 16, 2026
• Last Verified: April 1, 2026

More Information

Terms related to this study

• Other Study ID Numbers: Soh-Med--25-12-4MS

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
• product manufactured in and exported from the U.S.: Yes