Progressive sub-MIC Exposure of Klebsiella pneumoniae 43816 to Cephalothin Induces the Evolution of beta-lactam Resistance without Acquisition of beta-lactamase Genes

• Published in: bioRxiv
• Main Authors: Anderson, Jasmine R, Lam, Nghi B, Jackson, Jazmyne L, Dorenkott, Sean M, Taylor Ticer, Maldosevic, Emir, Velez, Amanda, Camden, Megan R, Ellis, Terri N
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 04.12.2022
• Subjects: Antibiotic resistance; Antibiotics; Bacteria; Cell culture; Cephalothin; DNA-directed RNA polymerase; Evolutionary genetics; Genomes; Klebsiella pneumoniae; Lipopolysaccharides; Minimum inhibitory concentration; Mutation; Phenotypes; RNA polymerase; RpoB protein; Whole genome sequencing; β Lactamase; β-Lactam antibiotics
• DOI: 10.1101/2021.11.19.469034

Bacterial exposure to antibiotic concentrations below the minimum inhibitory concentration (MIC) may result in a selection window allowing for the rapid evolution of resistance. These sub-MIC concentrations are commonly found in the greater environment. This study aimed to evaluate the adaptive genetic changes in Klebsiella pneumoniae 43816 after prolonged but increasing sub-MIC levels of the common antibiotic cephalothin over a fourteen-day period. Over the course of the experiment, antibiotic concentrations increased from 0.5 μg/mL to 7.5 μg/mL. At the end of this extended exposure, the final adapted bacterial culture exhibited clinical resistance to both cephalothin and tetracycline, altered cellular and colony morphology, and a highly mucoid phenotype. Cephalothin resistance exceeded 125 μg/mL without the acquisition of beta-lactamase genes. Whole genome sequencing identified a series of genetic changes that could be mapped over the fourteen-day exposure period to the onset of antibiotic resistance. Specifically, mutations in the rpoB subunit of RNA Polymerase, the tetR/acrR regulator, and the wcaJ sugar transferase each fix at specific timepoints in the exposure regimen where the MIC susceptibility dramatically increases. These mutations indicate that alterations in the secretion of colanic acid and attachment of colonic acid to LPS, may contribute to the resistant phenotype. These data demonstrate that very low, sub-MIC concentrations of antibiotics can have dramatic impacts on the bacterial evolution of resistance. Additionally, this study demonstrates that beta-lactam resistance can be achieved through sequential accumulation of specific mutations without the acquisition of a beta-lactamase gene.Competing Interest StatementThe authors have declared no competing interest.Footnotes* This revision addressed reviewer concerns. Major additions include analysis of wcaJ knockout strain and expanded analysis of multiple generations of cephalosporin antibiotics.