Metagenomic sequencing enables cultivation-free analysis of microbial communities by capturing the total DNA content of environmental samples. In wastewater, this approach provides a powerful tool for monitoring pathogen spread and antimicrobial resistance genes relevant to public health. Here, we compared metagenomic data with genomes from cultured isolates of carbapenemase-producing Klebsiella pneumoniae (CPKP), a pathogen of global health concern according to the WHO.

A total of 12 metagenomic samples from two wastewater treatment plants in Germany were sequenced with Illumina and 56 CPKP isolates from the same samples with Illumina and Oxford Nanopore Technologies (ONT). Hybrid assemblies of the isolate samples yielded high-quality genome reconstructions, which were then analyzed alongside metagenomes using comprehensive antimicrobial resistance gene screening methods.

Genomic analysis of the isolates revealed diverse sequence types and resistance patterns. Although ONT data improved assembly contiguity, it failed to capture several small plasmids and antimicrobial resistance genes, indicating that a combined sequencing approach might be advantageous for a comprehensive genomic characterization. In contrast, metagenomic sequencing results revealed only 0.03% CPKP reads, which were insufficient for genome recovery. In addition, approximately 50% of metagenomic reads remained unclassified, which indicates high dataset complexity. While cultivation-based sequencing enabled detailed genomic profiling, metagenomics provided broader insights into microbial community composition and the resistome, including resident environmental species.

This study highlights the complementary strengths and limitations of cultivation-based and metagenomic approaches for pathogen and antimicrobial resistance gene surveillance in wastewater.

Co-authors: Vladimir Bajic, Katharina Werner, Mustafa Helal, Vitor C. Piro, Maximilian Driller, Silver A. Wolf, Christian Blumenscheit, Birgit Walther, Martin Hölzer