1. Introduction Introduction Discussion Materials and Methods
Identification of E. coli through analysis of 16S rRNA sequencing, isolated from municipal water supply systems and household drinking water
Jannatul Ferdous1, 2, Ridwan Bin Rashid1*, Suhella Tulsiani2,3, Sabera Saima1, Peter Kjaer Mackie Jensen2,3, Anowara Begum1
1 Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh 2Section for Global Health, Institute of Public Health, University of Copenhagen, Denmark Copenhagen Centre for Disaster Research, University of Copenhagen, Denmark 1014
Introduction
Sequence analysis revealed that E. coli isolates from this study 2P-64, M-223-1, M-212-1, M-225-1, M , 3P-28, M were grouped in the same cluster with E. coli HUSEC2011 from the database as its closest relative in the Neighbor Joining tree. Five of the isolates are from water supply system and the other two are from household drinking water. This study demonstrates that Escherichia coli, which live in the intestines of warm blooded animals as commensals, might have considerable genetic relatedness to pathogenic strains.
The presence of fecal coliforms such as thermotolerant E. coli is widely accepted as indicator for fecal contamination. Water samples collected from a low income area Arichpur, located northwest of Dhaka in the Tongi sub-district of Bangladesh was used for the isolation of Escherichia coli. The aim of this study was to analyze the sequence of the 16S rRNA gene of strains isolated in this study and to compare them with the sequences of bacteria available in NCBI GenBank database. Using bioinformatics tools such as ClustalX2, MEGA 5.05(1).
3. Sequence Analysis
Chromatograms were imported into SEQMAN in the package DNAStar (Lasergene), assembled into contigs, edited, and exported as FASTA files. To identify the similarity of sequences all the sequences were submitted to online BLAST program of the National Center for Biotechnology Information website .
Figure 2- BLAST search in NCBI
Nine bacterial 16SrRNA gene sequences from this study and 28 bacterial 16SrRNA gene sequences from the NCBI database were aligned Multiple sequence alignment was performed using ClustalX2 Multiple Alignment version
Figure 3- Multiple alignment of E. coli 16S rRNA gene region using software Clustal X2.0.
It can be inferred from the result that most strains from the municipal water supply were clustered together which means they were related. One of the isolate form the household was clustered together with the from the municipal water supply. Thus some isolates might find its way to the household. In general we can asses the genetic relatedness of the circulating strains in a region so conclude if new strains are appearing or any evolution is taking place in prevalent strains. Gene profile for common species specific genes, genotyping techniques such as multi locus sequence typing and pulsed field gel electrophoresis could be used to provide details on genetic background and phylogenetic relationship of the isolates under study.
Discussion
Materials and Methods
1. Bacterial culture and DNA Extraction
Presumptive E. coli colonies were randomly chosen and identified using conventional methods based on morphological, biochemical, and cultural properties. DNA extraction was done by the boiling template method (2)
2. 16S rRNA PCR,
2. Purification of PCR products and Sequencing
Purification of PCR products was carried out by PureLink™ Quick Gel Extraction and PCR Purification Combo Kit (life technology, USA). Sequencing was conducted using BigDye Terminator v3.1 sequencing kit (Applied Biosystems, USA) Sequences were retrieved by ABI3730XL (Applied Biosystems, USA) system.
Figure 1- Chromatogram of sequence.
Introduction
The ribosomal operon size, nucleotide sequences, and secondary structures of the three rRNA genes are conserved within a bacterial species . Since 16SrRNA is the most conserved of these three rRNAs, it has been proposed as an ‘‘evolutionary clock’’,which has led to the reconstruction of the tree of life
This study was supported by funds from the project entitled “Combating Cholera Caused by Climate Change in Bangladesh, C5” of Danish International Development Agency. We thank International Centre for Diarrheal Disease (icddr, b) Bangladesh for providing the reference bacterial strains
Acknowledgement
Results
Figure 4- Phylogenetic tree was constructed using the MEGA version 5.05 software package. Genetic distances were calculated using the Tamura Nei method. The dendogram was constructed using neighbor-joining tree method.
1) Tamura, K., Dudley, J., Nei, M., & Kumar, S. (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:
2) Medici, D., Croci, L., Delibato, E., Di Pasquale, S., Filetici, E., & Toti, L. (2003). Evaluation of DNA extraction methods for use in combination with SYBR green I real-time PCR to detect Salmonella enterica serotype enteritidis in poultry. Appl Environ Microbiol.69 (6):
3) Hniman, A., Prasertsan, P., & Sompong, O. (2011). Community analysis of thermophilic hydrogen-producing consortia enriched from Thailand hot spring with mixed xylose and glucose. International Journal of Hydrogen Energy, 36(21),
Reference
Primer name
Sequence Tm
Amplicon size
Reference
27f
5'-GAG TTT GAT CCT GGC TCA G-3'
56.7C
1490
(3)
1492r
5'-GAA AGG AGG TGA TCC AGC C-3'
58.8 C
Contact details:
Jannatul Ferdous-
Ridwan Bin Rashid-