Abstract
<jats:p>Background Pseudomonas aeruginosa is a major opportunistic pathogen known for its genetic diversity and adaptability. This study analyzed the phylogenetic diversity of P. aeruginosa clinical isolates based on 16S rRNA gene sequences and investigated potential correlations between genetic variation and the anatomical site of infection. Methods Twenty clinical P. aeruginosa isolates were collected from otitis media, high vaginal swabs, and diarrheal and non-diarrheal stool. Genomic DNA was extracted, and a ~698 bp fragment of the 16S rRNA gene was amplified by PCR and sequenced. Phylogenetic analysis was conducted using the UPGMA method in MEGA11. Sequence homology, evolutionary distances, and single nucleotide polymorphism frequencies were calculated and compared across groups. Results All isolates were confirmed as P. aeruginosa, showing extremely high sequence identity, from 99.54% to 100%, to the reference strain. The phylogenetic tree showed tight clustering with very short branch lengths, indicating minimal evolutionary divergence. Isolates from otitis media and high vaginal swabs showed a higher average number of mutations, 2.33 and 2.00 mutations per isolate, compared to isolates from diarrheal and non-diarrheal stool, 0.43 and 0.17. Conclusion The 16S rRNA gene is an excellent marker for species confirmation but has limited discriminatory power for epidemiological typing of P. aeruginosa. The observed variations in mutation frequency suggest that different clinical niches may impose distinct selective pressures. The findings indicate that 16S rRNA sequencing alone is insufficient for epidemiological differentiation and that higher-resolution genomic tools are required for further investigation of genetic diversity among isolates further.</jats:p>