Macroporous epoxy-based monoliths for rapid quantification of Pseudomonas aeruginosa by adsorption elution method optimized for qPCR

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

Detection of Pseudomonas aeruginosa Serogroup G Using Real-Time PCR for Novel Target Genes Identified Through Comparative Genomics

“…In a previous study, PCR amplification of wbpP had a limit of ≥10 3 CFU/mL, and ihfB had a limit of ≥10 5 CFU/mL for P. aeruginosa serogroup G (Wu et al, 2016). Therefore, our real-time PCR approach is 1-3 orders of magnitude more sensitive than the previously reported method, indicating that we successfully detected P. aeruginosa serogroup G. These values are comparable to those obtained using most PCR methods applied to detect microorganisms in water without prior enrichment (Goepfert et al, 2020). This method has a good consistency for the detection of P. aeruginosa serogroup G without interference from non-target bacteria and actual samples.…”

Polymer monoliths for the concentration of viruses from environmental waters: A review

“…The first MAF was designed to capture Eschericha coli, and the dynamic binding of the bacterial cells was independent of the flow rate in a range between 1 and 10 mL/min, indicating that mass transfer did not limit the adsorption [60]. After this pioneering work, MAF has been used to retain bacterial Adapted from [60] with permission cells [60][61][62][63], bacteriophages indicators for virus contamination [64], and model human adenoviruses (HAdV) [65] and murine noroviruses (MNV) [66]. MAF has been used as a secondary concentration step after the primary crossflow ultrafiltration (CUF) [64,66] or as the primary concentration before centrifugal ultrafiltration (CeUF) [61,62], aiming at least 10 4 -fold enrichment factors, implying in processing sample volumes as large as 93 m 3 [67].…”

“…After this pioneering work, MAF has been used to retain bacterial Adapted from [60] with permission cells [60][61][62][63], bacteriophages indicators for virus contamination [64], and model human adenoviruses (HAdV) [65] and murine noroviruses (MNV) [66]. MAF has been used as a secondary concentration step after the primary crossflow ultrafiltration (CUF) [64,66] or as the primary concentration before centrifugal ultrafiltration (CeUF) [61,62], aiming at least 10 4 -fold enrichment factors, implying in processing sample volumes as large as 93 m 3 [67]. Pei et al published the first work describing MAF to concentrate viruses in a hydroxylated monolith [66].…”

Schnelle Anreicherung und kulturunabhängige Detektion von Pseudomonas aeruginosa in Trinkwasser

“…Chem. 2021, 413, 999-1007 [3] Raysyan, A., Galvidis, I. A., Schneider, R. J., Eremin, S. A., Burkin, M. A., Development of a latex particles-based lateral flow immunoassay for group determination of macrolide antibiotics in breast milk, J. Pharm.…”

“…In dieser Studie präsentieren wir die Ergebnisse der Quantifizierung von P. aeruginosa aus aufgestockten Trinkwasserproben mittels einer kulturunabhängigen Schnellmethode. Diese wurden durch die Kopplung der monolithischen Adsorptionsfiltration mit zentrifugaler Ultrafiltration und anschließender Quantifizierung der DNA mittels qPCR untersucht [3].…”