DIVERSITY AND PHYLOGENETIC CHARACTERIZATION OF RALSTONIA-ASSOCIATED 16S RRNA SEQUENCES DETECTED IN MYOCARDIAL INFARCTION BLOOD SAMPLES FROM PAKISTAN
Keywords:
myocardial infarction; 16S rRNA; Sanger sequencing; Ralstonia; molecular diversity; phylogenetic analysis; BLASTn; bacterial DNA; PakistanAbstract
Background: Bacterial DNA has been found in cardiovascular tissue and blood in several molecular studies, but little is known about the diversity of these sequences in patients with myocardial infarction (MI), and almost nothing has been reported from Pakistan. Broad-range 16S rRNA gene sequencing offers a practical way to characterize bacterial DNA detected by PCR and to place it within a phylogenetic framework.
Methods: Bacterial 16S rRNA PCR was carried out on peripheral blood from 50 clinically confirmed MI patients in Lahore, Pakistan. Fourteen samples gave usable Nested-PCR products (V3–V4 region), and the ten best amplicons were sequenced by the Sanger method. Sequences were cleaned in BioEdit and compared against GenBank with BLASTn. Alignment was done with MUSCLE in MEGA 12, and pairwise identity, ambiguous nucleotide positions and Maximum Composite Likelihood distances were calculated. A Neighbor-Joining tree with 500 bootstrap replicates was built from the ten study sequences and 20 Ralstonia reference sequences.
Results: The cleaned sequences were 391–420 bp long (4,083 bp in total). BLASTn placed all ten closest to Ralstonia or uncultured Ralstonia-associated sequences, with 95.82–99.75% identity and 99–100% query coverage. Identity among the study sequences ranged from 96.21% to 100.00%. Fourteen ambiguous positions (0.343% of analyzed bases) occurred in nine of the ten sequences. In the phylogenetic analysis the study sequences fell within the wider Ralstonia-associated group.
Conclusions: The ten sequences are highly similar to one another and are most closely related to the genus Ralstonia. The partial 16S rRNA fragments analyzed here may not provide sufficient resolution for definitive species-level identification, and detection of bacterial DNA does not establish bacterial viability or a causal role in MI. These sequences provide an initial molecular dataset of Ralstonia-associated 16S rRNA sequences detected in MI blood samples from Pakistan.
