Need for Standardization in Drinking Water Analyses

Transcription

1 30/ Need for Standardization in Drinking Water Analyses Identification of bacteria using 16S rrna Amplicon Sequencing Jakob Brandt PhD student Per Halkjær Nielsen & Mads Albertsen CENTER FOR MICROBIAL COMMUNITIES

2 Bacteria are inherently present in drinking water Access to safe drinking water extremely important Drinking water contains between bacteria cells/ml Microorganisms are associated with both harmful and beneficial effects relating to drinking water

3 Drinking water may harbour pathogens Legionella Legionella, Clostridium, Cholera, etc mill. cases of gastrointestinal illnesses annually in the US. Biofilm

4 Bacteria can impact the water infrastructure Microbiologically induced corrosion Water leaks in the drinking water distribution system Water loss of 35-45% in some areas of Canada due to leaky pipes

5 Bacteria in drinking water as a resource Removal of contamination such as pesticides

6 Bacteria are important, but how do we identify them?

7 How do we identify the bacteria? Culture-based methods Few bacteria are cultivable in drinking water (approximately 0.25%) Slow and labor intensive Only four parameters regularly tested DNA Sequencing (16S rrna amplicon) Detects the full spectrum of bacteria present No culturing needed No microscopy needed Fast and cheap (in regard to other methods) Detection limit

8 16S rrna amplicon sequencing (Bacteria have genes) DNA Gene Genetic material 16S rrna Amplicon sequencing 16S rrna Can be used as a fingerprint. Unique for different bacteria.

9 Results from 16S rrna amplicon sequencing List of which bacteria detected in the sample and their abundance Name % Accumulibacter Nitrospira Tetrasphaera Chloroflexi Microthrix Thiothrix Nitrotoga Streptococcus Competibacter Defluviicoccus Brachythrix Gordonia B others

10 What is hindering the use of 16S rrna amplicon sequencing?...

11 No standard method for the lab work Reference Sampling method Sampling volume [L] 1 Liu et al Filtering (0.2 mm pore-size) Extraction method 100 FastDNA SPIN Kit Primer target region V4 2 Belila et al Filtering (0.2 mm pore-size) 3 FastDNA SPIN Kit for Soil V4-5 3 Bautista-de los Santos et al Filtering (0.2 mm pore-size) Phenolchloroform method V4 Test effect of extraction procedures 4 Gomez-Alvarez et al Filtering (0.2 mm pore-size) 5 Roeselers et al Filtering (0.2 mm pore-size) 5 UltraClean Soil DNA Kit 1 Lysis buffer and beat beating V1-3 V5-6 Test commonly used primers 6 El-Chakhtoura et al Filtering (0.2 mm pore-size) 2 FastDNA SPIN Kit 7 Shaw et al Centrifugation 0.05 UltraClean Soil DNA Kit V3-4 V3 Estimate detection limit 8 Prest et al Filtering (0.2 mm pore-size) 9 Holinger et al Filtering (0.2 mm pore-size) 2 FastDNA SPIN Kit 1.5 Lysis buffer and bead beating V3-4 V Huang et al Water purifiers 1000 FastDNA Soil Kit V3-4

12 DNA extraction procedure impact the observed community FastDNA SPIN Kit for Soil vs. PowerWater DNA Isolation Kit FastDNA: 855 different species PowerWater: 2037 different species

13 How low can you go? Estimating the detection limit Dilution series of water only containing E. coli cells 900 ml 900 ml 900 ml 900 ml 900 ml 900 ml Normal drinking water range Decreasing E. coli concentrations [cells/ml]

14 How low can you go? Estimating the detection limit

15 Summary 16S rrna amplicon sequencing as a supplement to current methods Standardization of the method is needed Cheap, fast and high throughput Complete overview of the bacterial community structure offers appealing possibilities: Detection of pathogens Control/prevent microbiologically induced corrosion Source tracking Removal of contamination Rapid development in DNA-sequencing technologies (!)

16 Summary 16S rrna amplicon sequencing as a supplement to current methods Standardization of the method is needed Cheap, fast and high throughput Complete overview of the bacterial community structure offers appealing possibilities: Detection of pathogens Control/prevent microbiologically induced corrosion Source tracking Removal of contamination Rapid development in DNA-sequencing technologies (!)

17 One picture is worth a thousand words

18 One picture is worth a thousand words

19 Thanks Postdoc, Mads Albertsen Group leader of Albertsen Lab Professor, Per Halkjær Nielsen Head of Center for Microbial Communities

20 Thanks Postdoc, Mads Albertsen Environmental Group leader of Albertsen Lab Biotechnology group AAU Professor, Per Halkjær Nielsen Head of Center for Microbial Communities