Development of novel culture independent nucleic acid based diagnostics technologies for the detection and identification of microbial contaminants in building water distribution and premise plumbing systems
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Water is a vital component of the industrial, pharmaceutical and healthcare sectors. In industrial and pharmaceutical sectors water is a commonly used raw material, solvent and ingredient. While in healthcare facilities water is used for cleaning, bathing, sanitation, mixing or diluting drug solutions and rinsing medical devices. Building water distribution and premise plumbing systems can become contaminated with microorganisms. Currently microbial analysis of building water distribution and premise plumbing systems is routinely performed by culture incubation and can take 5-7 days to achieve a definitive result. The use of Nucleic Acid Diagnostics (NAD) technologies for the detection of biocontamination in building water distribution and premise plumbing systems has the potentially to quantitatively detect and identify contaminating microorganisms and also significantly reduce the turnaround time to result. In the industrial and pharmaceutical sectors a shorter time to result utilizing NAD technologies could potentially reduce manufacturing costs by enabling raw materials and end products to be released more quickly. It can also shorten the manufacturing production cycle and reduce inventory requirements. These rapid and specific NAD detection methods can provide the means to identify, contain and recover from a contamination event quickly which can result in significant cost savings for industry. Additionally, in healthcare settings such as hospitals, doctor’s clinics, nursing homes, clinical diagnostics testing laboratories and dental facilities contamination of building water distribution and premise plumbing systems can result in an increased risk of healthcare associated infections (HCAI’s). The use of NAD technologies for the rapid and early detection and identification of contamination in healthcare building water distribution and premise plumbing systems can help to decrease the risks of HCAI’s. This in turn can reduce the cost impact to the healthcare system and shorten the average healthcare stay for patients. The overall aim of this study was to design, develop, optimise and validate robust, internally controlled, NAD based methodologies for the simultaneous quantitative detection and identification of the most common microorganisms typically associated with the contamination of building water distribution and premise plumbing systems. The designed NAD assays were developed in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. In the first study, an internally controlled multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify Pseudomonas aeruginosa and Burkholderia species. These microorganisms are commonly associated with contamination of building water distribution and premise plumbing systems. These microorganisms are pathogenic and can cause infection in humans especially in immuno-compromised patients. In the second study, two additional targets were incorporated into the previous triplex assay for the detection of Serratia marcescens and Stenotrophomonas maltophilia resulting in an internally controlled 5-plex assay. The specificity of these multiplex real-time PCR assays was optimised and validated against genomic DNA from a panel of 95 isolates. Analytical sensitivities of less than 10 genome equivalents were determined for each diagnostics assay developed in multiplex format. Additionally in studies one and two a methodology was developed for the recovery and quantitative analysis of microbial nucleic acid from water. In the first study this methodology was demonstrated using sterile High Purity Water (HPW) spiked with bacterial cells. The microbial cells were trapped on a filter, suspended in PBS followed by mild sonication to detach cells from filter before chemical cell lysis and column purification of DNA. This DNA was then used as a template in the developed multiplex real-time PCR. This developed methodology detected a spiked bacterial load of 1.06 x 102 cfu / 100 ml for P. aeruginosa and 2.66 x 102 cfu / 100 ml for Burkholderia cepacia. In study two this methodology was slightly altered to increase the sensitivity. Water was filtered to trap microbial cells which were then subjected to direct chemical lysis on the filter followed by column purification of DNA. Subsequently an analysis of HPW supplied by a Millipore Elix 35 water purification unit was performed using traditional culture and using the developed culture independent methodology. Both of these analyses revealed the presence of S. maltophilia and Burkholderia species in the HPW. The quantitative analysis using the developed culture independent methodology combining filtration, nucleic acid purification and multiplex real-time PCR NAD assay identified the levels of S. maltophilia and Burkholderia species present in the HPW was above the threshold of 10 cfu / 100 ml recommended by both EU and US pharmacopoeial guidelines. In the third study additional biomarkers were developed for the detection of three other microorganisms associated with contamination of building water distribution and premise plumbing systems namely Legionella species, Staphyloccus aureus and Enterobacter aerogenes. All of the developed assays were modified for compatibility with Luminex magnetic bead suspension array technologies. An 8-plex internally controlled PCR assay was developed for the detection of each of these seven human pathogens. There is a need to develop rapid NAD technologies for the microbial analysis of building water distribution and premise plumbing systems to provide appropriate alternatives to the traditional culture based methodologies currently in use. The methodology developed in this study is rapid specific, sensitive and quantitative with a turnaround time to results of < 5 h. We propose that this methodology could be applied to the residential, industrial, pharmaceutical and healthcare sectors to assure the safety and quality of building water distribution and premise plumbing systems.