Strain-level characterization of bacterial pathogens using metagenomic sequencing for patients with pneumonia.

BACKGROUND

Metagenomic next-generation sequencing (mNGS) has emerged as an indispensable diagnostic tool for infectious diseases. The disparity in virulence and antimicrobial resistance among strains of the same species requires mNGS to reach strain-level resolution.

METHODS

To compare the subtyping performance between mNGS and culture, thirty bronchoalveolar lavage fluid (BALF) or blood specimens positive by culture were sequenced using the Illumina NextSeq platform, while whole genome sequencing (WGS) was conducted on the corresponding cultured colonies. Additionally, thirty BALF specimens underwent both mNGS and Oxford nanopore technology (ONT)-based metagenomic third-generation sequencing (mTGS) to compare the subtyping efficacy of the two platforms. To characterize the strain-level composition of pneumonia pathogen Acinetobacter baumannii and Klebsiella pneumoniae, 185 BALF specimens from three hospitals were analyzed by mNGS.

RESULTS

WGS of the cultured colonies yielded identical subtyping results to mNGS at the level of clonal complex (CC). Although mNGS and mTGS predicted largely consistent primary CCs, mTGS demonstrated less accuracy and precision in CC identification. Co-infections at the CC level were detected in 5.40% of A. baumannii-positive and 19.55% of K. pneumoniae-positive BALF specimens. CC composition differed markedly according to bacterial load and between primary and secondary CCs in co-infection specimens. Antimicrobial resistance profiles remained constant for patients with single-infection but varied for those with co-infection. Spatial and temporal consistency of CC composition was observed within individual patients.

CONCLUSIONS

The heterogeneity in virulence and antimicrobial resistance among CCs, together with the prevalence of strain-level co-infections, highlights the need to extend pathogen identification to the strain level. Under current technical conditions, mNGS is a more suitable subtyping tool compared to culture and mTGS.