Abstract
The rapid rise of antimicrobial resistance (AMR) poses a global threat to human health. Key to combating antimicrobial resistance will be to increase our understanding of how resistance develops. Conventional antimicrobial susceptibility assays determine the minimum inhibitory concentration (MIC) of an antimicrobial to a bacterial infection to classify clinically-relevant resistance. However, this is problematic because it means we only identify resistance once it has appeared, not as it has developed. Antimicrobial tolerance is the ability of an organism to persist in the presence of antimicrobials and has been shown to precede the development of resistance. The Cook laboratory have previously shown that enterococci, opportunistic bacterial pathogens, have extraordinary tolerance to a wide range of antimicrobials, including the recently discovered teixobactin. Teixobactin is a new class of antimicrobial that targets bacterial cell wall biosynthesis and has proven efficacy against multidrug-resistant pathogens such as vancomycin-resistant enterococci. There are no reported mechanisms of teixobactin resistance, however, we have previously generated teixobactin-tolerant mutants using serial passaging. Tolerance is identified as a change in the minimum bactericidal concentration (MBC) compared to resistance which is a change in MIC. Whole-genome sequencing of these mutants revealed single nucleotide polymorphisms responsible for this phenotype, mapping to the hprK and mvaD genes. I investigated the respective roles of hprK and mvaD in teixobactin tolerance using the Enterococcus faecalis mutant A8 which showed a 4-fold increase in MBC (128 μg/mL) compared to wild-type (WT) (16-32 μg/mL). Through genetic complementation, it was found that both mutations are needed for a teixobactin tolerant phenotype, as restoration of one gene (64 μg/mL) failed to reduce teixobactin tolerance to wild-type levels (16-32 μg/mL). A key aim of the study was to investigate the role of HPrK in teixobactin tolerance in E. faecalis. HPrK alters the phosphorylation status of (HPr), a key regulator of carbon metabolism and inhibitor of the cell wall stress response regulator CroR. To identify a link between carbon metabolism and teixobactin susceptibility, the MIC and MBC of E. faecalis WT and the A8 mutant were determined on different carbon sources. Biolog plates identified teixobactin susceptibility profiles of numerous carbon sources, and a subset were selected for MBC characterisation. Overall, no significant differences in teixobactin susceptibility on different carbon sources were determined. Phosphorylation of HPr is essential for HPr-mediated repression of CroR. To investigate the putative role of HPrK in HPr-mediated CroR inhibition MICs and MBCs were carried out on an E. faecalis croRS deletion strain (croRS). The ΔcroRS strain had an 8-fold reduction in MBC (2 μg/mL) compared to the WT (16-32 μg/mL); a finding that was also observed for other cell wall antibiotics, including vancomycin and bacitracin. This suggests that HprK may play an important role in regulating HPr-mediated repression of CroR thereby influencing intrinsic antimicrobial tolerance in E. faecalis. Further work is needed to fully elucidate the role of HPrK, and to identify which genes within the CroRS regulon are responsible for conferring this tolerance.