Identification of Clinical Isolates of Candida albicans with Increased Fitness in Colonization of the Murine Gut

Abstract

The commensal and opportunistic pathogen Candida albicans is an important cause of fungal diseases in humans, with the gastrointestinal tract being an important reservoir for its infections. The study of the mechanisms promoting the C. albicans commensal state has attracted considerable attention over the last few years, and several studies have focused on the identification of the intestinal human mycobiota and the characterization of Candida genes involved in its establishment as a commensal. In this work, we have barcoded 114 clinical C. albicans isolates to identify strains with an enhanced fitness in a murine gastrointestinal commensalism model. The 114 barcoded clinical isolates were pooled in four groups of 28 to 30 strains that were inoculated by gavage in mice previously treated with antibacterial therapy. Eight strains that either exhibited higher colonization load and/or remained in the gut after antibiotic removal were selected. The phenotypic analysis of these strains compared to an RFP-tagged SC5314 wild type strain did not reveal any specific trait associated with its increased colonization; all strains were able to filament and six of the eight strains displayed invasive growth on Spider medium. Analysis of one of these strains, CaORAL3, revealed that although mice required previous bacterial microbiota reduction with antibiotics to be able to be colonized, removal of this procedure could take place the same day (or even before) Candida inoculation. This strain was able to colonize the intestine of mice already colonized with Candida without antibiotic treatment in co-housing experiments. CaORAL3 was also able to be established as a commensal in mice previously colonized by another (CaHG43) or the same (CaORAL3) C. albicans strain. Therefore, we have identified C. albicans isolates that display higher colonization load than the standard strain SC5314 which will surely facilitate the analysis of the factors that regulate fungal colonization.

Keywords: Candida albicans; adaptation; commensalism; fitness; murine gut; mycobiota.

Figure 1

Assay to identify C. albicans clinical isolates with increased ability to colonize the murine gut. (A) Schematic representation of the assay. (B) Fungal colonization load of C. albicans strains in pool. Inoculum A, B, C and D correspond to the four pools of C. albicans barcoded strains (supplementary file S1). Each C. albicans strains pool was inoculated intragastrically in 3 mice with a partially depleted microbiota. Antibiotic treatment was maintained for 21 days after C. albicans gavage then, it was removed. Stools were taken at different time points and processed for CFUs counting. Data are represented and expressed as log CFUs per stool gram versus time (days). Each replicate is shown and each line represents the mean of the data.

Figure 2

Competitive colonization assays. CaORAL3 and CaHG43 strains were inoculated in a 1:1 ratio with SC5314-RFP in 3 mice by gavage. The time course of fungal colonization load was followed by CFUs counting of stools in SD-chloramphenicol medium. Antibiotic treatment was removed at day 17. Figure S1 shows the colonization load for the other six selected C. albicans strains in competition with the SC5314-RFP.

Figure 3

Ability to perform the yeast-to hypha transition in liquid and on solid media. (A) The strains indicated in the upper row were grown in YPD overnight at 37 °C (time 0) and refreshed in pre-warmed serum. Samples were taken 3 h later and photographed under the microscope. (B) In total 50 to 100 cells from cultures in the stationary phase were spread on Spider medium and incubated for 5 days at 37 °C.

Figure 4

Effect of the antibiotic treatment on CaORAL3 colonization. (A) A suspension of CaORAL3 cells was inoculated in non-antibiotic treated mice. (B) CaORAL3 cells were inoculated in Ab-treated mice 4 days prior plus 9 days after C. albicans inoculation. Colonization rate was followed in time and expressed as Log CFUs per g of stools. (C) Effect of nystatin and fluconazole administration over CaORAL3 colonization rate.

Figure 5

Determination of the minimum antibiotic regimen to allow gut colonization (A) Schematic representation of the assay, three groups of mice were treated with antibiotics for 10 days, 10⁷ CaORAL3 cells were inoculated 3 days before (B), just after (J) or 7 days after (A) the 10-days antibiotic treatment. Stool samples were taken at different time and processed for CFUs count. Data are shown as Log CFU/g of feces (B). Arrows indicate C. albicans inoculation for each group.

Figure 6

Influence of the microbiota in mice stably colonized with CaORAL3 strain and without antibiotic treatment for 30 days. (A) Feces from naïve mice was inoculated in F group and colonization load was followed in time. Group C was followed as control. (B) Naïve mice (N group) were introduced in the cage of mice previously colonized by CaORAL3 (H group) and colonization load was followed in time. Results of two independent assays are shown in the graph.

Figure 7

Competition between CaORAL3 and other C. albicans strains in the murine commensalism model. (A) 10⁷ SC5314-RFP cells were inoculated in mice previously colonized by CaORAL3 cells and without antibiotic treatment for 39 days. Colonization load was followed in time by spreading stool suspension on SD-chloramphenicol medium to distinguish white/red colonies. (B,D) 10⁷ CaORAL3 cells tagged with RFP were inoculated in mice previously colonized by (B) CaORAL3 or (D) CaHG43 strains and without antibiotic treatment for 39 days. Colonization load was followed similarly. (C) At the end of the experiments, mice from groups in (B) were euthanized and the amount of each strain was quantified in the different portions of the gastrointestinal tract.