Low-Level Cefepime Exposure Induces High-Level Resistance in Environmental Bacteria: Molecular Mechanism and Evolutionary Dynamics

Abstract

Antibiotics exert selective pressures on clinically relevant antibiotic resistance. It is critical to understand how antibiotic resistance evolves in environmental microbes exposed to subinhibitory concentrations of antibiotics and whether evolutionary dynamics and emergence of resistance are predictable. In this study, Comamonas testosteroni isolated from wastewater activated sludge were subcultured in a medium containing 10 ng/mL cefepime for 40 days (∼300 generations). Stepwise mutations were accumulated, leading to an ultimate 200-fold increase in the minimum inhibitory concentration (MIC) of cefepime. Early stage mutation in DNA polymerase-encoding gene dnaE2 played an important role in antibiotic resistance evolution. Diverse resistance mechanisms were employed and validated experimentally, including increased efflux, biofilm formation, reduced antibiotic uptake, and drug inactivation. The cefepime minimal selective concentrations (MSCs) and relative fitness of susceptible, intermediate, and resistant mutants were determined. Agent-based modeling of the modified Moran process enabled simulations of resistance evolution and predictions of the emergence time and frequency of resistant mutants. The unraveled cefepime resistance mechanisms could be employed by broader bacteria, and the newly developed model is applicable to the predictions of general resistance evolution. The improved knowledge facilitates the assessment, prediction, and mitigation of antibiotic resistance progression in antibiotic-polluted environments.

High-Throughput Evolution Unravels Landscapes of High-Level Antibiotic Resistance Induced by Low-Level Antibiotic Exposure

“…1a ), where resistance to AZI (78.9%) and TOB (18.8%) were the most prevalent. A similar evolution of AZI-resistant strains was recently reported for Comamonas testosteroni 10 . Only two evolved populations stressed by marbofloxacin and ceftriaxone displayed significantly decreased IC 90 values to at least one antibiotic.…”

“…Whole-genome sequencing was performed for the WT strain E. coli QSHQ1 as well as the 96 evolved populations, following the procedures described previously 10 . Briefly, the WT strain was sequenced using PacBio RS combined with Illumina sequencing (NovaSeq6000).…”

“…Antibiotics are increasingly released into the environment via wastewater discharge and disposal of solid waste, where 40-90% are excreted as parent compounds 6 . Environmental bacteria exposed to these antibiotics at low-level, sub-minimal inhibitory concentrations (sub-MIC) can evolve antibiotic resistance by accumulating diverse low-frequency mutants, which are different from those evolved at clinical concentrations [7][8][9][10][11] . The environmental research community essentially needs to elucidate the contribution of mutation-driven resistance evolution in the environment by crafting analytical measures to pinpoint the responsible selective forces (i.e., antibiotics) driving AMR development 12 .…”

Lipid Metabolites as Potential Regulators of the Antibiotic Resistome in Tetramorium caespitum

“…51 For example, low-level cefepime exposure increased the abundance of ARGs that encoded for multiple antibiotic resistance with different mechanisms such as cellular protection and efflux pump. 52 Therefore, the upregulation of ROS might be a response to the increase of ARG with a mechanism of antibiotic deactivation. Accordingly, a higher relative abundance of cuminaldehyde in the adult gut increased ARG abundance, and gut metabolites further developed the resistance.…”

“…Notably, cuminaldehyde not only inhibited bacteria but also led to an increase of ROS content, which may promote the enrichment of ARGs via cell mutation and by ARG transformation . For example, low-level cefepime exposure increased the abundance of ARGs that encoded for multiple antibiotic resistance with different mechanisms such as cellular protection and efflux pump . Therefore, the upregulation of ROS might be a response to the increase of ARG with a mechanism of antibiotic deactivation.…”