High-throughput single-cell method reveals novel species and genetic diversity

Activated sludge is essential for removing contaminants from wastewater, and understanding the diverse microbial communities it harbors has been a significant task. Traditional methods fail to capture the full spectrum of microbial interactions. Due to these limitations, researchers recognized the need for single-cell level investigations to gain deeper insights into microbial heterogeneity.

Led by the University of Hong Kong and published in Environmental Science and Ecotechnology on September 19, 2024, the study applied single-cell sequencing to samples from a Hong Kong wastewater plant. This method overcame the constraints of traditional metagenomics, revealing the intricate genetic networks within microbial communities and offering new perspectives on antibiotic resistance gene transfer.

The research team sequenced 15,110 individual microbial cells, classifying them into 2,454 single-amplified genome bins, and discovered that 27.5% represent previously unclassified species. The identification of 1,137 ARGs, along with numerous plasmids and phages, underscored the frequent horizontal gene transfer between species.

Plasmids were found to be instrumental in spreading ARGs, raising concerns over the potential spread of antibiotic resistance. The findings highlight the need for closer monitoring of wastewater systems to prevent public health risks.

This study marks the first application of high-throughput single-cell sequencing to analyze the activated sludge (AS) microbiome. The analysis detected 1,137 antibiotic resistance genes, 10,450 plasmid fragments, and 1,343 phage contigs. The shared relationships for plasmid (12,819) and phage (184) hosts indicate a high frequency of horizontal gene transfer. In addition, a set of 1,529 metagenome-assembled genomes was used to further classify 98 previously unclassified SAG bins.

Prof. Tong Zhang, the senior researcher, states, “Our research highlights the power of single-cell sequencing in revealing the hidden dynamics within wastewater treatment. These findings on antibiotic resistance gene transfer pave the way for improved monitoring and mitigation strategies, which are crucial for safeguarding public health under a One Health approach.”

The research has wide-reaching implications, particularly in enhancing wastewater treatment processes and curbing the spread of antibiotic resistance. The ability to track ARGs at a single-cell level could influence future environmental policies and public health measures, while also opening new avenues for applications in other ecosystems, such as soil and the human gut.