Detection and Investigation of Eagle Effect Resistance to Vancomycin in Clostridium difficile With an ATP-Bioluminescence Assay

Abstract

Vancomycin was bactericidal against <i>Clostridium difficile</i> at eightfold the minimum inhibitory concentration (MIC) using a traditional minimum bactericidal concentration (MBC) assay. However, at higher concentrations up to 64 × MIC, vancomycin displayed a paradoxical "more-drug-kills-less" Eagle effect against <i>C. difficile</i>. To overcome challenges associated with performing the labor-intensive agar-based MBC method under anaerobic growth conditions, we investigated an alternative more convenient ATP-bioluminescence assay to assess the Eagle effect in <i>C. difficile</i>. The commercial BacTiter-Glo^TM assay is a homogenous method to determine bacterial viability based on quantification of bacterial ATP as a marker for metabolic activity. The ATP-bioluminescence assay was advantageous over the traditional MBC-type assay in detecting the Eagle effect because it reduced assay time and was simple to perform; measurement of viability could be performed in less than 10 min outside of the anaerobic chamber. Using this method, we found <i>C. difficile</i> survived clinically relevant, high concentrations of vancomycin (up to 2048 μg/mL). In contrast, <i>C. difficile</i> did not survive high concentrations of metronidazole or fidaxomicin. The Eagle effect was also detected for telavancin, but not for teicoplanin, dalbavancin, oritavancin, or ramoplanin. All four pathogenic strains of <i>C. difficile</i> tested consistently displayed Eagle effect resistance to vancomycin, but not metronidazole or fidaxomicin. These results suggest that Eagle effect resistance to vancomycin in <i>C. difficile</i> could be more prevalent than previously appreciated, with potential clinical implications. The ATP-Bioluminescence assay can thus be used as an alternative to the agar-based MBC assay to characterize the Eagle effect against a variety of antibiotics, at a wide-range of concentrations, with much greater throughput. This may facilitate improved understanding of Eagle effect resistance and promote further research to understand potential clinical relevance.