Loss and resurgence of multi-drug-resistance during laboratory evolution of a carbapenem-resistant isolate of Escherichia coli

A clinical Escherichia coli isolate (CDC AR Isolate Bank #0118) was found to be resistant to all beta-lactam antibiotics, including DOR, IPM, MER, and ERT, aztreonam-avibactam excluded. Genes associated with these resistance traits included bla NDM-1 , bla CMY-6 , bla OXA-2 , bla TEM-1A , and OmpF . Isolate 0118 was subjected to continual antibiotic-free broth culture for 294 days, with approximately 1 × 10 7 colony-forming units transferred at 24-h intervals. Following a 64-fold reduction in DOR resistance by day 287, a sub-inhibitory concentration of DOR was introduced during days 294–360 to introduce selective pressure. During this interval, DOR resistance increased approximately 250-fold, exceeding the initial level observed. Concurrently, loss of the plasmid-borne bla NDM-1 gene was detected by Day 140. bla CMY-6 was lost by day 319, while bla OXA-2 and bla TEM-1A remained. Following growth in sub-inhibitory DOR levels, changes in MICs to non-beta-lactam antibiotics were also observed, and will be the subject of future investigations. Whole-genome sequencing identified structural mutations and large genomic deletions that coincided with these phenotypic changes, including loss of envelope- and stress-associated genes ( nlpD , rpoS , mutS ) and truncations in genes involved in membrane and peptidoglycan biosynthesis (murein transglycosylase; UDP-galactose LPS α1,2-galactosyltransferase). RNAseq analysis revealed convergent transcriptomic signatures including downregulation of DNA repair genes mutS and mutY , and repression of the global regulator csrA. Differential gene expression of chromosomally encoded genes involved in outer membrane remodeling, efflux pump activation, and cell wall precursor restructuring was also found.