diskImageR: quantification of resistance and tolerance to antimicrobial drugs using disk diffusion assays

Abstract

Microbial pathogens represent an increasing threat to human health. Although many infections can be successfully treated and cleared, drug resistance is a widespread problem. The existence of subpopulations of 'tolerant' cells (where a fraction of the population is able to grow above the population resistance level) may increase the rate of treatment failure; yet, existing methods to measure subpopulation effects are cumbersome. Here we describe diskImageR, a computational pipeline that analyses photographs of disk diffusion assays to determine the degree of drug susceptibility [the radius of inhibition, (RAD)], and two aspects of subpopulation growth [the fraction of growth (FoG) within the zone of inhibition, (ZOI), and the rate of change in growth from no drug to inhibitory drug concentrations, (SLOPE)]. diskImageR was used to examine the response of the human fungal pathogen Candida albicans to the antifungal drug fluconazole across different strain backgrounds and growth conditions. Disk diffusion assays performed under Clinical and Laboratory Standards Institute (CLSI) conditions led to more susceptibility and less tolerance than assays performed using rich medium conditions. We also used diskImageR to quantify the effects of three drugs in combination with fluconazole, finding that all three combinations affected tolerance, with the effect of one drug (doxycycline) being very strain dependent. The three drugs had different effects on susceptibility, with doxycycline generally having no effect, chloroquine generally increasing susceptibility and pyrvinium pamoate generally reducing susceptibility. The ability to simultaneously quantitate different aspects of microbial drug responses will facilitate the study of mechanisms of subpopulation responses in the presence of antimicrobial drugs.

Clearing the FoG: Antifungal tolerance is a subpopulation effect that is distinct from resistance and is associated with persistent candidemia

“…We quantified drug responses in C. albicans isolates from different genetic backgrounds and types of infections using diskImageR, a quantitative analysis tool that measures RAD, the radius of the zone of inhibition, an indicator of susceptibility that relates to MIC 69,70 , and FoG, the fraction of growth within the zone of inhibition, relative to the maximum possible growth 65 (Fig. 1a).…”

“…Here, we measured susceptibility and tolerance in a broad range of clinical isolates spanning the major C. albicans clades 62-64 and asked how these parameters influence fungal infection outcomes. We analyzed disk diffusion assays using diskImageR , and quantified the radius (RAD) of the zone of inhibition, a parameter that relates to the MIC 65 , and the fraction of growth (FoG) within the zone of inhibition, a parameter that measures tolerance 19,38,50,66-68 . We found that a range of adjuvant drugs, used in combination with FLC, increased drug cidality by reducing FoG and not MIC and were effective both in vitro and in vivo at inhibiting strains with high (and not low) FoG levels.…”

Pseudouridine synthase 7 impacts Candida albicans rRNA processing and morphological plasticity

“…Fifteen microlitres of 1‐mM fluconazole or 20 μl of 1‐mM caspofungin was added to a sterile paper disc at the centre of each plate. Assays were repeated in triplicate, and disk image R was used to analyse the data as described in Gerstein, Rosenberg, Hecht, and Berman ().…”

Candida albicansgenetic background influences mean and heterogeneity of drug responses and genome stability during evolution to fluconazole

“…Antifungal drug tolerance, a property distinct from drug resistance (2,3) , is the ability of some cells in a strain to grow slowly in the presence of a drug at concentrations above the MIC. In tolerant strains, the subpopulation of cells that grow (generally from 10-90% of cells, depending on the strain) is usually evident when growth is assessed after 48 h or longer in the drug (reviewed in 4) .…”

“…A mechanistic link between drug resistance and specific aneuploidies exists in multiple pathogenic fungal species (e.g., C. albicans, 22, 25-28 ; Cryptococcus neoformans , 29, 30 ; suggested in Candida auris, 31,32) . Because fluconazole tolerance and drug resistance are distinct and tolerance is sensitive to inhibitors that do not affect resistance (2,3) , it follows that antifungal tolerance likely evolves via a different subset of genes than resistance, although it is also possible that there is overlap between genes involved in tolerance and resistance.…”