Nanopore R10.4 metagenomic detection of bla_CTX-M/bla_DHA antimicrobial resistance genes and their genetic environments in stool.

Abstract

The increasing prevalence of gut colonization with CTX-M extended-spectrum β-lactamase- and/or DHA plasmid-mediated AmpC-producing Escherichia coli is a concern. Here, we evaluate Nanopore-shotgun metagenomic sequencing (Nanopore-SMS) latest V14 chemistry to detect bla_CTX-M and bla_DHA genes from healthy stools. We test 25 paired samples characterized with culture-based methods (native and pre-enriched). Antimicrobial resistant genes (ARGs) are detected from reads and meta-assembled genomes (MAGs) to determine their associated genetic environments (AGEs). Sensitivity and specificity of native Nanopore-SMS are 61.1% and 100%, compared to 81.5% and 75% for pre-enriched Nanopore-SMS, respectively. Native Nanopore-SMS identifies only one sample with an AGE, whereas pre-enriched Nanopore-SMS recognizes 9/18 plasmids and 5/9 E. coli chromosomes. Pre-enriched Nanopore-SMS identifies more ARGs than native Nanopore-SMS (p < 0.001). Notably, bla_CTX-Ms and bla_DHAs AGEs (plasmid and chromosomes) are identified within 1 hour of sequencing. Furthermore, microbiota analyses show that pre-enriched Nanopore-SMS results in more E. coli classified reads (47% vs. 3.1%), higher differential abundance (5.69 log₂ fold) and lower Shannon diversity index (p < 0.0001). Nanopore-SMS has the potential to be used for intestinal colonization screening. However, sample pre-enrichment is necessary to increase sensitivity. Further computational improvements are needed to reduce the turnaround time for clinical applications.