Single nucleotide polymorphisms (SNPs) in dacB/ampD alter population plasticity of gene amplification based heteroresistance

Introduction Heteroresistance (HR) describes the ability of a subpopulation to grow in the presence of inhibitory antibiotic concentrations. We found HR to ceftazidime (CAZ) in a clinical Enterobacter cloacae complex (ECC) strain (IMT49658).

Material & Methods We performed extensive phenotypic (population analysis profiles, stability analysis of resistance, ScanLag) and molecular microbiological techniques (qRT-PCR, whole genome sequencing, raw read analysis) in order to show the plasticity and mechanism of HR in this ECC strain. We re-investigated the genome and phenotype of IMT 49658 after long-term evolution in 32 µg/ml CAZ and after exposure to 128 µg/ml CAZ in plates.

Goals By revealing mechanism and evolution of HR to conventional resistance, we aim for a better detection and treatment of bacterial heteroresistant infections.

Results WGS detected a plasmidal gene amplification with β-lactamase ampC blaDHA-1. qRT-PCR showed a high genomic copy number of blaDHA‑1 in resistant subpopulations, decreasing when they reverted to susceptibility. Gene amplifications varied in single cells of one colony (raw read analysis). Resistant subpopulations showed heterogeneous lag times in ScanLag. After evolving ECC for 21 days in CAZ, we discovered a SNP in dacB and ampD, encoding for a stop codon. These mutants displayed low amplification levels but resistance in disk diffusion and homogenous lag times. The dacB mutant was more frequent than the ampD mutant on plates with 128 µg/ml CAZ.

Summary Long-term evolution in antibiotic niches drives the emergence of new resistant mutants, balancing the fitness costs of e.g., gene amplifications. Comprehension of the transition from HR to resistance is inevitable for successful treatment of bacterial infections.