16S RRNA GENE SEQUENCING FOR IDENTIFICATION AND PHYLOGENETIC ANALYSIS OF ENVIRONMENTAL BACTERIAL ISOLATES
Keywords:
16S rRNA gene sequencing, Urban microbiome, Environmental bacteria, Bacterial taxonomy, Phylogenetic analysis, Microbial diversity.Abstract
Background
Urban environments harbor diverse bacterial communities across water, soil, and air-deposition niches, yet systematic phylogenetic surveys from South Asian megacities remain scarce. Whether niche type predicts taxonomic composition and phylogenetic clustering in Lahore's cultivable aerobic bacteria remains unclear. This study addresses this knowledge gap.
Research Design and Methods
This cross-sectional, culture-based environmental survey enrolled 300 cultivable aerobic bacterial isolates at 15 geographically distributed sites in Lahore, Pakistan, from defined sampling dates. Sites comprised five surface-water, five soil, and five air-deposition locations representing residential, commercial, industrial, peri-urban, and green-space zones. Isolates underwent 16S rRNA gene sequencing (≥400 bp, ≥90% query coverage), MALDI-TOF MS, and VITEK 2 identification. Primary outcome was phylum-level taxonomic composition by niche. Phylogenetic trees were constructed using maximum-likelihood methods. Alpha-diversity and beta-diversity were assessed at site level.
Results
Of 300 isolates sequenced, 104 were from water, 97 from soil, and 99 from air-deposition. Mean 16S identity was 98.90%±0.80%. Phylum distribution differed significantly by niche (χ²=156.77, df=6, p<0.001): Pseudomonadota dominated water (76.0%), while Actinomycetota and Bacillota prevailed in soil (41.2%, 38.1%) and air-deposition (46.5%, 40.4%). Eighteen genera were recovered; several were niche-restricted. Alpha-diversity did not differ between niches (p=0.113–0.357). No adverse events applicable.
Conclusions
Among Lahore's urban isolates, niche type was strongly associated with taxonomic composition, with distinct phylum-level distributions across water, soil, and air-deposition surfaces. However, culture-based methods limit generalizability to total community diversity, and site-level sample size (n=15) constrains beta-diversity precision. These findings support niche-specific environmental monitoring. Further metagenomic research is recommended.
