16S Ribosomal RNA Gene Sequencing of Acinetobacter baumannii
Three clinical isolates presumptively identified as Acinetobacter baumannii underwent molecular confirmation and phylogenetic analysis using 16S ribosomal RNA (16S rRNA) gene sequencing. Genomic DNA from overnight cultures grown in Luria-Bertani broth was extracted using commercial DNA extraction kits, and its quality was checked using spectrophotometry. The 16S rRNA gene was amplified by polymerase chain reaction (PCR) using universal bacterial primers 27F/1492R and produced approximately 1,500 base pair long PCR products, as visualized by agarose gel electrophoresis. Purified PCR products were sequenced bidirectionally using Sanger sequencing and aligned with reference sequences using MEGA software version 11. BLAST analysis against the 16S rRNA gene sequence database of the National Centre for Biotechnology Information (NCBI) showed that three sequence variants (AB-1 to AB-3) of 1,428-1,429 bp each showed at least 99% sequence identity with characterized A. baumannii reference strains and were thus confirmed as species-level identifications. Coverage analysis showed 95% uniformity of alignments between all three isolates (1,413-1,414 bp matches from 1,428-1,429 bp total length). Phylogenetic reconstruction placed all three clinical isolates together in the principal A. baumannii clade, phylogenetically separated from other related Acinetobacter species. The constant amplification of appropriately sized PCR products, high degree of sequence homology to reference organisms, and monophyletic clustering within established A. baumannii phylogenetic lineages provide robust molecular confirmation of the identity of the species. These 16S rRNA sequences are molecular markers for differentiation between strains and provide a genetic basis for downstream genomic epidemiology and virulence characterization of extensively drug-resistant (XDR) clinical isolates.