Perseverance: a mechanism of proliferation in fungistatic drugs

Alex Rosenberg 1 Iuliana Ene 2 Arnaldo Colombo 3 Richard Bennett 2 Judith Berman 1
1Molecular Cell Biology and Biotechnology, Tel Aviv University, Tel Aviv, Israel
2Molecular Microbiology and Immunology, Brown University, Rhode Island, Providence, USA
3Medicine, Federal University of São Paulo, São Paulo, Brazil

Candida albicans infections obey the 90-60 rule: susceptible isolates respond to therapy 90% of the time; resistant isolates respond 60% of the time. Thus, susceptibility cannot account for all therapeutic responses. Indeed, in disk diffusion assays with azoles, C.albicans sub-populations often grow within the zone of inhibition (ZOI) via a non-genetic mechanism that we term perseverance, which provides a quantitative measure of trailing growth or tolerance. Here, we quantified growth parameters that affect the ability of subpopulations of cells to grow, albeit slowly, at supra-MIC drug concentrations for a broad range of clinical isolates using diskImageR (Gerstein AC et al, Microbiology 2016), an image analysis pipeline that resistance as the radius of the ZOI (RAD) and perseverance as the Fraction of Growth (FoG), the degree to which a subpopulation of cells grows within the ZOI. RAD corresponds to MIC, and as expected, RAD is heritable, concentration-dependent and time-independent. FoG is due to non-genetic heterogeneity in the population, is independent of drug concentration and of RAD and the degree of heterogeneity is heritable for a given strain. Interestingly, C.albicans strains with higher FoG levels exhibit shorter lag phase length than those with lower FoG levels, a feature very different from tolerance in bacteria. The size of the subpopulation of cells that form colonies on supraMIC drug concentration correlated with FoG levels, while colony growth rates did not. Importantly, several adjuvant drugs, including inhibitors of Hsp90 and calcineurin, eliminate perseverance without altering the MIC. We also find that perseverance and resistance are sensitive to mutations in different drug resistance and stress response pathways, underscoring the distinct nature of the two phenomena. Importantly, clinical persistence was associated with higher FoG levels, suggesting that quantitative measurements of perseverance may provide important prognostic information for clinical considerations.