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

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.

Isothermal haRPA detection of blaCTX-M in bacterial isolates from water samples and comparison with qPCR.

Antibiotic resistant bacteria complicate infection treatment worldwide. Rapid and inexpensive detection of the current occurrence of antibiotic resistant bacteria in surface and irrigation water as well as treated wastewater is essential to minimize exposure and further spread. To reduce cost and analysis time compared to current qPCR (quantitative polymerase chain reaction), isothermal nucleic acid amplification tests are promising bioanalytical methods which can be integrated in simplified molecular biological detection systems. This study establishes heterogeneous asymmetric recombinase polymerase amplification (haRPA) for the detection of antibiotic resistance genes in water. After DNA extraction of bacteria cultivated from water, the target DNA for blaCTX-M cluster 1 was amplified at 39 °C for 40 min on a microfluidic DNA chip. The amplified DNA on each spot was quantified by a flow-based chemiluminescence reaction. Even though slightly less sensitive than conventional qPCR, the haRPA method was successful in identifying the blaCTX-M cluster 1 in bacterial isolates with a limit of detection of 0.013 ng µL-1. In a proof-of-principle study, 37 bacterial isolates from environmental water samples were classified according to blaCTX-M cluster 1 occurrence and gave 100% agreement in cross-reference with PCR. Importantly, haRPA allows for a quick in-field monitoring at low incubation temperatures and by an easy visual readout. This study paves the path to establish haRPA as a quick on-site monitoring option for antibiotic resistance gene occurrence without the need for a thermal cycling device or long data processing.

Reaction kinetic studies for comparison of mutagenic potency between butadiene monoxide and glycidamide.

DNA adducts can be formed from covalent binding of electrophilic reactive compounds to the nucleophilic N- and O-atoms of the biomolecule. The O-sites on DNA, with nucleophilic strength (n) of ca. 2, is recognized as a critical site for mutagenicity. Characterization of the reactivity of electrophilic compounds at the O-sites can be used to predict their mutagenic potency in relative terms. In the present study, reaction kinetic experiments were performed for butadiene monoxide (BM) in accordance with the Swain-Scott relation using model nucleophiles representing N- and O-sites on DNA, and earlier for glycidamide (GA) using a similar approach. The epoxide from the kinetic experiments was trapped by cob(I)alamin, resulting in formation of an alkylcobalamin which was analyzed by liquid chromatography tandem mass spectrometry. The Swain-Scott relationship was used to determine selectivity constant (s) of BM and GA as 0.86 and 1.0, respectively. The rate constant for the reaction at n of 2 was extrapolated to 0.023 and 0.038 M-1 h-1 for BM and GA, respectively, implying a higher mutagenic potency per dose unit of GA compared to BM. The reaction kinetic parameters associated with mutagenic potency were also estimated by a density functional theory approach, which were in accordance to the experimental determined values. These types of reaction kinetic measures could be useful in development of a chemical reactivity based prediction tool that could aid in reduction of animal experiments in cancer risk assessment procedures for relative mutagenicity.