Shared and Unique Evolutionary Trajectories to Ciprofloxacin Resistance in Gram-Negative Bacterial Pathogens

Abstract

The challenge of spreading antibiotic resistance calls for systematic efforts to develop more “irresistible” drugs based on a deeper understanding of dynamics and mechanisms of antibiotic resistance acquisition. To address this challenge, we have established a comparative resistomics approach which combines experimental evolution in a continuous-culturing device, the morbidostat, with ultradeep sequencing of evolving microbial populations to identify evolutionary trajectories (mutations and genome rearrangements) leading to antibiotic resistance over a range of target pathogens.

A broad-spectrum synthetic antibiotic that does not evoke bacterial resistance

“…Therefore, E. coli mutL (JW4128) and wild-type A. baumannii ATCC 17978 were assessed for COE R mutants since both strains possess an inherently higher frequency of mutation (∼10-100-fold), resulting from deficiencies in either methyl-directed mismatch repair or the intrinsic DNA damage-inducible response, respectively. 45 , 46 Only low-level COE R mutants (2-4 × MIC) were recovered from either of these hypermutable strains, which is in marked contrast with the high-level CIP R mutants (128 × MIC) observed for A. baumannii 35 ( Supplementary Fig. S1b ; Supplementary Table S4c–e ).…”

“…Morbidostat . Although high-level CIP R mutants of wild-type E. coli (BW25113) were obtained after morbidostat culture for >3 days (64-128 × MIC), 35 no COE R mutants were observed under these conditions. Therefore, E. coli mutL (JW4128) and wild-type A. baumannii ATCC 17978 were assessed for COE R mutants since both strains possess an inherently higher frequency of mutation (∼10-100-fold), resulting from deficiencies in either methyl-directed mismatch repair or the intrinsic DNA damage-inducible response, respectively.…”

“…These data suggest that the COE either targets membrane-associated functions encoded by essential genes or resistance results from alteration of membrane biochemical dynamics that inhibit essential membrane protein function(s). Furthermore, no mutations were observed that affect any of the numerous efflux pumps, which were readily obtained using the same approach for ciprofloxacin resistance, 35 suggesting that COE2-2hexyl is not an efflux pump substrate or effector. MDR pathogens whose resistance is driven primarily by pre-existing efflux upregulation would thus remain susceptible to COEs.…”

“…The morbidostat measures the growth rates of evolving microbial populations and automatically adjusts drug concentrations to maintain a constant drug-induced inhibition. 34 , 35 The hardware, software components and experimental methods and work-flow were as described. 34 Briefly, bacterial populations were placed under increasing selective drug pressure in parallel reactors.…”

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

“…Thus, we hypothesized that plasmid–host adaptations between the clinically relevant plasmid pAZJ221 and its A. baumannii host would occur in the presence of a carbapenem. To elucidate the mechanisms of compensatory adaptations, the widely available and well‐characterized laboratory strain A. baumannii ATCC 17978 was used as a host (Ridenhour et al, 2017 ; Santos‐Lopez et al, 2019 ; Zlamal et al, 2021 ). We subjected A. baumannii ATCC 17978 containing pAZJ221 (ATCC 17978/pAZJ221 hereafter) to experimental evolution at a sub‐inhibitory concentration of imipenem for nearly 400 generations and applied population‐wide whole‐genome sequencing (WGS) to elucidate the dynamics of genomic adaptations.…”