Comparative analysis of wastewater sample processing methods for antimicrobial resistance surveillance.

Area of Science:

• Environmental microbiology
• Molecular biology
• Public health surveillance

Background:

• Antimicrobial resistance (AMR) gene monitoring in wastewater is vital for public health.
• Standardized methods are needed for efficient wastewater surveillance (WS) of AMR.
• Various sample processing techniques can impact microbial diversity and gene recovery.

Purpose of the Study:

• To evaluate the impact of different sample processing techniques on microbial diversity and antimicrobial resistance gene (ARG) recovery from wastewater.
• To compare DNA extraction kits and quantification instruments for wastewater analysis.
• To establish an optimized protocol for AMR monitoring in wastewater.

Main Methods:

• Analysis of 38 wastewater subsamples from three treatment plants.
• Comparison of centrifugation volumes (50, 250 mL), speeds (3,400 × g vs 15,000 × g), and concentration methods (pellet vs. filter).
• Evaluation of two DNA extraction kits (DNeasy PowerSoil Pro, DNeasy PowerLyzer) and two quantification instruments (NanoDrop One, Qubit 4).
• 16S rRNA gene sequencing and quantitative PCR (qPCR) for ARG detection.

Main Results:

• Higher centrifugation speeds and larger sample volumes improved DNA yield.
• Pellet concentration yielded higher ARG recovery (1.32 times) than filters.
• DNA quantification methods showed strong positive correlations with each other and qPCR data.
• The DNeasy PowerSoil Pro Kit demonstrated effective ARG recovery.

Conclusions:

• Optimized sample processing, including high-speed centrifugation (15,000 × g) and pellet concentration, enhances ARG recovery from wastewater.
• The DNeasy PowerSoil Pro Kit is suitable for AMR monitoring.
• This optimized approach supports standardized wastewater surveillance for AMR, aiding public health protection.