Background: Water and High Purity Water (HPW) distribution systems can be contaminated with human
pathogenic microorganisms. This biocontamination may pose a risk to human health as HPW is commonly used in
the industrial, pharmaceutical and clinical sectors. Currently, routine microbiological testing of HPW is performed
using slow and labour intensive traditional microbiological based techniques. There is a need to develop a rapid
culture independent methodology to quantitatively detect and identify biocontamination associated with HPW.
Results: A novel internally controlled 5-plex real-time PCR Nucleic Acid Diagnostics assay (NAD), was designed and
optimised in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines,
to rapidly detect, identify and quantify the human pathogenic bacteria Stenotrophomonas maltophilia, Burkholderia
species, Pseudomonas aeruginosa and Serratia marcescens which are commonly associated with the biocontamination of
water and water distribution systems. The specificity of the 5-plex assay was tested against genomic DNA isolated from a
panel of 95 microorganisms with no cross reactivity observed. The analytical sensitivities of the S. maltophilia, B. cepacia, P.
aeruginosa and the S. marcescens assays are 8.5, 5.7, 3.2 and 7.4 genome equivalents respectively.
Subsequently, an analysis of HPW supplied by a Millipore Elix 35 water purification unit performed using standard
microbiological methods revealed high levels of naturally occurring microbiological contamination. Five litre water
samples from this HPW delivery system were also filtered and genomic DNA was purified directly from these filters. These
DNA samples were then tested using the developed multiplex real-time PCR NAD assay and despite the high background
microbiological contamination observed, both S. maltophilia and Burkholderia species were quantitatively detected and
identified. At both sampling points the levels of both S. maltophilia and Burkholderia species present was above the
threshold of 10 cfu/100 ml recommended by both EU and US guidelines.
Conclusions: The novel culture independent methodology described in this study allows for rapid (<5 h), quantitative
detection and identification of these four human pathogens from biocontaminated water and HPW distribution systems.
We propose that the described NAD assay and associated methodology could be applied to routine testing of water and
HPW distribution systems to assure microbiological safety and high water quality standards.