Abstract
Pseudomonas aeruginosa contaminations in tap water systems have caused severe health problems in both hospital and household settings. To ensure fast and reliable detection, culture-independent methods are recommendable. However, the typically low cell number in water samples requires sample enrichment prior to analysis. Therefore, we developed and optimized an adsorption elution method using monolithic adsorption filtration and subsequent centrifugal ultrafiltration that can be combined with culture-independent detection methods. The principle of adsorption of Pseudomonas aeruginosa by hydrophobic and ionic interactions was studied in modified epoxy-based monoliths. Optimized conditions (5-L initial sample volume at pH 3 filtered for 30 min through hydrolyzed monoliths (MAF-OH) and eluted with beef extract glycine buffer at pH 9.5) achieved a recovery of 67.1 ± 1.2% and a concentration factor of 103. For the first time, we therefore present a culture-independent approach for rapid enrichment and subsequent molecular biological quantification of P. aeruginosa by qPCR from tap water samples by monolithic adsorption filtration. The total enrichment and quantification process takes 4 h. This work further stresses the versatility of the monolithic adsorption filtration and its possibilities as a concentration tool for culture-independent analytics of pathogenic bacteria in the environment.Graphical abstract
Highlights
Providing safe drinking water — or lowering risk for workers in contact with technical water — nowadays requires very sensitive analytical techniques to detect and rapidly quantify pathogenic bacteria
The possibility to process high volumes enables high concentration factors that are crucial for a rapid detection of low cell numbers in environmental samples. While this adsorption elution process is commonly used for the enrichment of viruses, it was first applied to the enrichment of Salmonella sp. and Escherichia coli as early as 1976 using DEAE cellulose columns [12] and adapted for monolithic adsorption filters (MAFs) for Escherichia coli [10] and Legionella sp. [9]
Primer sequences for regA detection in P. aeruginosa are shown in Table 4 and additional information about specificity of the primer pair including cross-reactivity experiments, qPCR procedure, and calibration experiments can be found in the ESM
Summary
Providing safe drinking water — or lowering risk for workers in contact with technical water — nowadays requires very sensitive analytical techniques to detect and rapidly quantify pathogenic bacteria. It is necessary to concentrate bacteria from large water sample volumes prior to subsequent analysis by cultivation-independent methods. Traditional sterile filtration has its disadvantages as the filters tend to clog quickly due to matrix components when large water sample. One of the applications of an organic monolith is the monolithic adsorption filtration which has already been applied efficiently in concentrating different bacteria and viruses from water [8, 9]. An epoxy-based monolith with free epoxy groups on the filter surface can be synthesized within 1 h with a defined pore size of 21 μm [10]. The production costs of the unfunctionalized monolithic adsorption filters (MAFs) are below 1 € per single use MAF. The free epoxide groups allow functionalization for desired adsorption properties in an easy
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