Single-molecule nanopore sequencing reveals extreme target copy number heterogeneity in arylomycin-resistant mutants

Abstract

Tandem gene amplification is a frequent and dynamic source of antibiotic resistance in bacteria. Ongoing expansions and contractions of repeat arrays during population growth are expected to manifest as cell-to-cell differences in copy number (CN). As a result, a clonal bacterial culture could comprise subpopulations of cells with different levels of antibiotic sensitivity that result from variable gene dosage. Despite the high potential for misclassification of heterogenous cell populations as either antibiotic-susceptible or fully resistant in clinical settings, and the concomitant risk of inappropriate treatment, CN distribution among cells has defied analysis. Here, we use the MinION single-molecule nanopore sequencer to uncover CN heterogeneity in clonal populations ofEscherichia coliandAcinetobacter baumanniigrown from single cells isolated while selecting for resistance to an optimized arylomycin, a member of a recently discovered class of Gram-negative antibiotic. We found that gene amplification of the arylomycin target, bacterial type I signal peptidase LepB, is a mechanism of unstable arylomycin resistance and demonstrate inE. colithat amplification instability is independent of RecA. This instability drives the emergence of a nonuniform distribution oflepBCN among cells with a range of 1 to at least 50 copies oflepBidentified in a single clonal population. In sum, this remarkable heterogeneity, and the evolutionary plasticity it fuels, illustrates how gene amplification can enable bacterial populations to respond rapidly to novel antibiotics. This study establishes a rationale for further nanopore-sequencing studies of heterogeneous cell populations to uncover CN variability at single-molecule resolution.

Plasmids Shape the Current Prevalence of tmexCD1-toprJ1 among Klebsiella pneumoniae in Food Production Chains

“…The mean copy numbers of tmexCD1-toprJ1 were lower in MinION data than in Illumina data ( Fig. 5c ), which is consistent with a similar study ( 37 ). Amplification losses likely occurred during enrichment culture with no selection pressure.…”

Co‐evolutionary adaptations of Acinetobacter baumannii and a clinical carbapenemase‐encoding plasmid during carbapenem exposure

“…Gene duplication is a major source of evolutionary innovation (Toll-Riera et al, 2016). Our results emphasized the importance of gene instability in rapidly tuning levels of gene expression, as noted in other studies (Girgis et al, 2021;Tomanek et al, 2020). We noted that plasmid fitness and host fitness are not necessarily aligned.…”

Antibiotic Heteroresistance in Klebsiella pneumoniae

“…The most common mechanisms underlying heteroresistance in Gram-negative bacteria are spontaneous tandem gene amplification [ 22 , 31 , 32 , 33 ]. For example, amplification of the prmD gene was observed in colistin-heteroresistant Salmonella typhimurium isolates [ 13 ].…”

“…Other studies have demonstrated that the unstable tobramycin resistance of Acinetobacter baumannii emerged due to the extensive RecA-dependent amplification of the aadB gene encoding an aminoglycoside adenylyltransferase [ 32 ]. Amplification of the lepB gene encoding the target of arylomicin, signal peptidase LepB, was recently detected in unstable arylomicin-heteroresistant E. coli subpopulations [ 33 ]. The number of lepB copies in different subpopulations was remarkably variable, from 1 to 50 copies.…”