Methods: 3 sets of isogenic paired strains consisting of an index-WT and persistent-FKS mutant (S663P), two un-paired FKS mutant strains (S663F and S629P), and a WT reference strain (CBS138) were included. Growth kinetics were measured over 24 h in 96-well microplate containing liquid RPMI. After overnight growth in RPMI and staining with a chitin specific fluorescent marker, morphology and chitin were assessed at the single-cell level utilizing high-content imaging technique. Virulence was evaluated in Galleria mellonella larvae by injecting 107 cells/larvae. Mortality was assessed daily for 5 d.
Results: Significant differences in growth kinetics, frequency of morphologic phenotypes within the cell populations (non-budding, single-bud, multiple-buds), and virulence were observed between strains obtained from different patients (p<0.05 for each). However no difference was observed between paired index-WT and persistent-FKS S663P mutants. Compared to index-WT and the CBS138 reference strain, FKS mutant isolates (S663P, S629P, and S663F) had significantly elevated cell wall chitin content (p<0.05). Neither chitin content, the presence of an FKS mutation, nor in-vitro growth characteristics were found to be associated with virulence. Virulence was strongly correlated with the frequency of multi-bud cells within the population however, with 5-d post-injection survival rates of 4% vs. 28% for high-frequency (>12% multi-bud cells) and low frequency strains, respectively (p<0.001).
Conclusion: Acquired FKS-mediated echinocandin resistance induced significant alterations in cell wall chitin content but was not observed to attenuate fitness or virulence. Virulence was highly associated with the frequency of cells with distinct morphology.
N. D. Beyda, Astellas: Grant Investigator and Scientific Advisor , Consulting fee and Research grant .