Abstract
16S ribosomal RNA (16S rRNA) gene sequences used to study bacterial phylogeny and taxonomy have been by far the most common housekeeping genetic marker utilized for identification and ancestor determination. This study aimed to investigate, for the first time, the relationship between Klebsiella spp. isolated from clinical and environmental samples in Iraq.
 Fifty Klebsiella spp. isolates were isolated from clinical and environmental sources. Twenty-five isolates were collected from a fresh vegetable (Apium graveolens) and 25 from clinical samples (sputum, wound swab, urine). Enteric bacteria were isolated on selective and differential media and identified by an automatic identification system, vitek-2. The total DNA was extracted and PCR amplified for selected isolates. The 16S rRNA gene was amplified by using the universal primer 27F (5'- AGAGTTTGATCCTGGCTCAG- 3') and 1492R (5'- GGTTACCTTGTTACGACTT- 3’). The 16SrRNA gene sequence was analysed among some local isolates, and the results were compared with the standard data of similar registered strains in NCBI. The most common species of Klebsiella was Klebsiella pneumoniae pneumoniae (Kpp), followed by Klebsiella pneumoniae ozaenae (Kpo) and Klebsiella oxytoca (Ko). The results of the identification of species and sub species by using the biochemical test (vitek-2) were more precise than those obtained by the use of the universal primer.Phylogenetic tree strategies have clearly indicated a relatively close similarity amongst all analysed Klebsiella isolates and revealed the intra-species genetic distance between the individual isolates of the Klebsiella spp. In conclusion, our results revealed the main advantage of using universal primers for the identification of Klebsiella spp. and their root from nature.
Highlights
16S rRNA gene sequences used to study bacterial phylogeny and taxonomy have been, for several reasons, by far the most commonly used housekeeping genetic marker. These reasons include the following; the 16SrRNA sequence is present in almost all bacteria, often existing as a multigene family, or operons; the function of the 16S rRNA gene has not changed over time, suggesting that random sequence changes are a more accurate measure of time; and the 16S rRNA gene (1,500 bp) is large enough for informatics purposes [1].Phylogenetic markers include the presence of specific protein coding or structural genes, the combinations of such genes and their variants, and insertion and repeat elements
Environment (Env.), Clinical (Clin.) Total DNA of bacterial isolates was extracted by using the G- spin DNA Extraction Kit
Phylogenetic tree design A specific comprehensive bacterial tree was constructed, in which the observed bacterial variants were compared with their neighboring homologous sequences using the NCBI-BLASTn server [9]
Summary
16S rRNA gene sequences used to study bacterial phylogeny and taxonomy have been, for several reasons, by far the most commonly used housekeeping genetic marker These reasons include the following; the 16SrRNA sequence is present in almost all bacteria, often existing as a multigene family, or operons; the function of the 16S rRNA gene has not changed over time, suggesting that random sequence changes are a more accurate measure of time (evolution); and the 16S rRNA gene (1,500 bp) is large enough for informatics purposes [1].Phylogenetic markers include the presence of specific protein coding or structural genes, the combinations of such genes and their variants, and insertion and repeat elements. The aim of this study was to identify Klebsiella spp. isolated from different sources by using 16S gene sequencing and to detect the relationship between clinical and environmental samples.
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