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Development of an Escherichia coli K12-specific quantitative polymerase chain reaction assay and DNA isolation suited to biofilms associated with iron drinking water pipe corrosion products
Citation:
LU, J., T. L. GERKE, H. Y. BUSE, AND N. ASHBOLT. Development of an Escherichia coli K12-specific quantitative polymerase chain reaction assay and DNA isolation suited to biofilms associated with iron drinking water pipe corrosion products. JOURNAL OF WATER AND HEALTH. IWA Publishing, London, Uk, 12(4):763-771, (2014).
Impact/Purpose:
The primary objective of the biofilm Legionella study is to determine if there is any correlation between the ability of Legionella isolates to invade and replicate within amoebal hosts and their in vivo virulence utilizing a murine model of acute bacterial pneumonia (6-8 week old A/j mice). If such a correlation is demonstrated, then a second objective will be development of an assay for the rapid identification of potentially pathogenic environmental isolates of Legionella (and latter, other intracellular bacterial pathogens) via co-culturing with an “indicator” amoebal host rather than infecting mice. Subsequent research will then focus on better understanding the stability and ecology of Legionella spp. in biofilms and the effect(s) of key stressors to select for virulent biotypes. For the development of a biofilm-like sampling device, the initial objective is to evaluate representative mineral iron oxyhydroxides (alpha- and gamma-FeOOH) and an iron oxide (Fe3O4) for their ability to sorb pathogen surrogates (MS2, E. coli and Saccharomyces cerevisiae representing viral, bacterial and parasitic protozoan pathogens respectively). Then, to evaluate these mineral phases on coated glass beads and glass wool by pilot plant distribution system challenges (1 h intrusion events for the same three surrogates & followed over one month) and at full-scale under normal operation of Cincinnati’s distribution system.
Description:
Escherichia coli is one of the most commonly used fecal indicator organisms for drinking water and groundwater systems. In order to understand various biogeochemical and biophysical factors affecting its interactions with biofilms, E. coli K12 was chosen as a model organism. A TaqMan qPCR assay (115 bp) specific to E. coli K12 combined with an ABI internal control was developed based on sequence data encoding the rfb gene cluster. The specificity of the assay was confirmed using the DNA from three E. coli K12 stains (ATCC W3110, MG1655 and DH1), 38 common bacterial strains representative of 23 genera and 24 non-K12 E. coli isolates from different samples including drinking water (DW) biofilms and source waters. Assay sensitivity was also evaluated by examining DNA extracts from DW biofilm samples within various matrices, using different purification methods and at different densities. The E. coli K12 assay was tested with biofilms spiked with the K12 strain within a small laboratory (CDC) biofilm reactor with PVC and copper coupons and in a 15.2 cm diameter pipe loop system with biofilm coupons consisting of magnetite-coated and uncoated quartz. The results indicate that the new assay provides a specific, sensitive and robust tool for the improved detection and quantification of E. coli K12 within pipe biofilms and is particularly well suited for studying enteric bacterial interactions within biofilms.
