Biology subjects

Manaia, C.

Publications and source records attributed to Manaia, C..

2 recordsLinked to original sources

bioRxiv2023

Influence of DNA extraction methods on microbiome and resistome analysis in activated sludge

Amplicon sequencing, metagenomics, and quantitative polymerase chain reaction (qPCR) are commonly used techniques to analyse microorganisms and antibiotic resistance genes (ARGs) in activated sludge from wastewater treatment plants (WWTPs). However, the lack of workflow harmonisation poses challenges in comparing measurements across studies and research groups. To address this issue, we examined the impact of DNA extraction procedures on 16S rRNA gene amplicon sequencing, shotgun metagenomics, and qPCR analyses of activated sludge by combining two widely used DNA extraction kits (PowerSoil and FastDNA) and two commonly employed disruption instruments (bead-beater and vortex) through a 2x2 factorial experimental design involving four groups of three analysts performing DNA extractions in triplicates. Our findings revealed significant differences in DNA yield, purity, and reproducibility of amplicon sequencing profiles among the extraction kits. Operator variability also influenced the results. We compared microbiome profiles obtained by amplicon sequencing and metagenomics and observed that bead-beating introduced more variability among triplicates compared to vortexing. The combinations of extraction kits and disruption instruments impacted the relative abundances of specific phyla such as Actinobacteriota, Bacteroidota, and Nitrospirota. For resistome analysis, we employed metagenomics for high-resolution profiling and qPCR for high-sensitivity detection of ARGs. The compositions and diversities of resistome datasets were not significantly affected by the choice of extraction kits and disruption instruments. Although using the same method is ideal for accurate comparisons, our results suggest that acceptable reproducibility can still be achieved when using different methods. This finding encourages the implementation of ARG monitoring in wastewater treatment processes. However, it is important to consider biases introduced by DNA extraction workflows when designing analytical studies, interpreting their results, and comparing their findings. Striving for more harmonised molecular workflows is crucial in the field of wastewater microbiology and engineering. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/546617v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@caa36eorg.highwire.dtl.DTLVardef@1afabfdorg.highwire.dtl.DTLVardef@448b9aorg.highwire.dtl.DTLVardef@296404_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

molecular biology↗

bioRxiv2022

Microbiome and resistome dynamics along a sewage-effluent-reservoir continuum underline the role of natural attenuation in effluent receiving reservoirs

This study assessed temporal dynamics of total and antibiotic resistant fecal bacterial indicators and antibiotic resistance genes (ARG) along a sewage-effluent-reservoir continuum, in an experimental system consisting of a sewage-fed membrane-aerated bioreactor (MABR) whose effluent fed a 4500 L polypropylene basin that mimicked an effluent storage reservoir. We applied a multidisciplinary approach that coupled physicochemical analyses, cultivation of total and cefotaxime-resistant E. coli, microbiome (bacterial and eukaryotic) analysis and qPCR/ddPCR quantification of selected ARGs. Total and cefotaxime-resistant E. coli loads dropped by approximately 1.5 log units in both the MABR and the reservoir, but the relative reduction (normalized to 16S rRNA genes) in both E. coli and ARGs was higher in the reservoir. Reservoir microbiomes were significantly different from those in the MABR, and were characterized by temporal shifts and periodic algal (Chlorophyta) blooms that were coupled to oxygen and pH fluctuations. Collectively, the data indicates that the decrease in E. coli and ARGs in the MABR was primarily facilitated by sludge removal, whereas in the reservoir, it was predominantly associated with microbial community shifts. The study highlights the capacity of ecological interactions in mitigating antibiotic resistance in both engineered and natural ecosystems. ImportanceAntibiotic resistance is considered one of the most significant public health predicaments of the 21st century, and there is growing evidence that anthropogenically impacted environments such as those receiving raw and treated sewage can contribute to this phenomenon. In this study, we evaluated the dynamics of total and antibiotic resistant fecal pathogen indicators and antibiotic resistance genes along a sewage-treated wastewater-effluent reservoir continuum, concurrent to evaluation of microbial community composition and physicochemical parameters. Results indicate that both the treatment bioreactor and the effluent storage reservoir removed resistant bacteria and antibiotic resistance encoding genes. However, in the reactor removal was primarily linked to physical processes, whereas in the storage reservoir it appeared to be strongly facilitated by ecological interactions. The study highlights the capacity of aquatic ecosystems to alleviate antibiotic resistance, and suggests that ecological processes in aquatic ecosystems can be harnessed to mitigate antibiotic resistance.

microbiology↗