Abstract
Vancomycin was bactericidal against Clostridium difficile 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 C. difficile. 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 C. difficile. The commercial BacTiter-GloTM 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 C. difficile survived clinically relevant, high concentrations of vancomycin (up to 2048 μg/mL). In contrast, C. difficile 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 C. difficile tested consistently displayed Eagle effect resistance to vancomycin, but not metronidazole or fidaxomicin. These results suggest that Eagle effect resistance to vancomycin in C. difficile 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.
Highlights
Clostridium difficile is a spore forming, anaerobic bacteria that causes colon inflammation and diarrhea, which in severe cases can be life threatening
Vancomycin was bactericidal at 8 μg/mL, 8 × minimum inhibitory concentration (MIC) (1 μg/mL), but with higher concentrations of antibiotic (16–64 × MIC) more bacteria survived (n = 6)
Given that the Eagle effect was detected for vancomycin, one of the main therapeutic treatments of C. difficile, but not for metronidazole or fidaxomicin, we examined other glycopeptide antibiotics for the Eagle effect in C. difficile strain 630
Summary
Clostridium difficile is a spore forming, anaerobic bacteria that causes colon inflammation and diarrhea, which in severe cases can be life threatening. C. difficile infections (CDIs) are usually treated orally with metronidazole, vancomycin or fidaxomicin antibiotics; in recent years fecal transplant has become an important alternative investigational biotherapeutic to treat severe recurrent disease (Jarrad et al, 2015). Metronidazole is only recommended to treat mild to moderate cases, while vancomycin and fidaxomicin have improved cure rates relative to metronidazole for more severe infections (Crook et al, 2012; Surawicz et al, 2013). Metronidazole is nearly completely systemically absorbed following oral dosing, resulting in relatively low concentrations of antibiotic at the desired location of the site of infection in the colon (Bolton and Culshaw, 1986). Vancomycin and fidaxomicin have minimal systemic absorption, resulting in high concentrations in the colon (>1000 μg/g) (Sears et al, 2012). The capacity of fidaxomicin to inhibit spore formation (Babakhani et al, 2012) and the enhanced selectivity of fidaxomicin for C. difficile over beneficial gut microbiota compared to vancomycin (Louie et al, 2012) are thought to contribute to this outcome
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