JPSIM Journal of Pakistan Society of Internal Medicine J Pak Soc Intern Med 2709-8729 2710-0804 Pakistan Society of Internal Medicine 10.70302/jpsim.v6i2.2535 jpsim.v6i2.2535 Original Research Antibiotic Susceptibility Profiling, Molecular Characterization, and16S rRNA-Based Phylogenetic Analysis of Pseudomonas Aeruginosa Isolates from Musculoskeletal Infections Batool Wajeeha Muddassir Maria Iqbal Muhammad Junaid Ghaffar Abdul Ali Muhammad Qasim Naqvi Syed Zeeshan Haider zeeshan.haider@imbb.uol.edu.pk * Institute of Molecular Biology and Biotechnology (IMBB), The University of Lahore, Pakistan M Islam Medical College, Gujranwala, Pakistan Sahiwal Medical College, Sahiwal, Pakistan Gulab Devi Educational Complex, Lahore, Pakistan *Correspondence: Syed Zeeshan Haider Naqvi (zeeshan.haider@imbb.uol.edu.pk) 05 06 2025 2025 6 2 185 190 09 01 2025 25 02 2025 28 04 2025 Copyright © 2025 The Author(s) 2025 The Author(s) https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective

The primary goal of this research was to describe antibiotic resistance and identification of virulence genes in Pseudomonas aeruginosa.

 Methodology

The study was cross-sectional, featuring a randomized selection of samples. Conducted from January to December 2023, the study encompassed a sample size of 320 including blood, pus, and wound swabs [Male:169(52.8%), Female 151(47.1%)]. Age-based categories were ascertained, G1:13-20 years, G2:21-40 years, G3:41-60 years, G4:61-80 years. Identification tests for Pseudomonas aeruginosa included API20NE (bioMérieux®, France). Antimicrobial susceptibility testing was following the CLSI,2020 for the Disc Diffusion Test.DNA extraction and purification were carried out through a Genome Jet DNA Purification kit and 16S rRNA primers for the identification of resistance genes exoA and oprL. Phylogenetic and taxonomic identification was conducted via ribosomal RNA sequencing of PCR-amplified products(BIO-RAD T100TM Thermocycler) and sequencing with an automated sequencer (Illumina MiniSeqTM). Phylogenetic tree was constructed by MEGA11.p-value

 Results

Prevalence of Pseudomonas aeruginosa was 22%. Positivity was recorded from pus (152/320:47.5%),blood(124/320-38.75%) and wound swabs(44/320-1 3.75%).G4 showed maximum positive isolates(91.25%).Maximum resistance was exhibited against Meropenem(76%) and Imipenem(70%).PCR identified the presence of resistance genes,exoA gene(125 bp), and oprL(105 bp). The phylogenetic tree was constructed through ribosomal RNA.The gene bank accession number for 16S rRNA gene of P. aeruginosa is PQ269824.

 Conclusion

Antimicrobial susceptibility testing helps improve the treatment alternatives for stubborn strains. The presence of resistance genes highlights how research at the molecular level enhances treatment approaches for managing multi-drug resistant pathogens.

 Keywords Pseudomonas aeruginosa antibiotic susceptibility musculoskeletal infections phylogenetic tree. Ethics Statement

Ethics committee: Bioethics and Biosafety Committee, Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistan. Approval number: IMBB/BBBC/23/910, dated March 15, 2023.

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