INVESTIGATION OF CANDIDA ALBICANS AND ENTEROCOCCUS FAECALIS BIOFILMS UNDER LOW IRON CONDITIONS

Enterococcus faecalis is a gram-positive coccus found in the gastrointestinal tract of both humans and animals as commensal. Through the acquisition of mobile genetic elements, this commensal bacterium can transform into a multi-drug resistant opportunistic pathogen. As a pathogen, E. faecalis can cause endocarditis, septicemia, urinary tract, and endodontic infections. Similarly, Candida albicans is the most common fungal opportunistic pathogen found in the mouth, gastrointestinal tract, genitourinary tract, and on the skin. This yeast can form hyphae, leading to invasive infections in immunocompromised patients. Iron is important for the formation of hyphae and iron levels in the host can affect Candida infection outcomes. C. albicans and E. faecalis are frequently co-isolated from various infection sites in humans, and both have been shown to form interkingdom biofilms in vitro. Specifically, we want to focus on the characterization of this dual-species interaction under low iron conditions. We used a C. albicans strain expressing mCherry from a constitutive promoter mixed with one of three strains of E. faecalis (a commensal strain OG1RF, OG1RF that contains the pheromone responsive plasmid pCF10, and an oral clinical isolate). Pheromone responsive plasmids are associated with virulence and are present in as high as 80% of clinical isolates. These strains were grown overnight in an optic bottom 24-well plate under a low iron concentration (400µM of bathophenanthrolinedisulfide acid). The biofilms were stained with Syto9 and Calcofluor White and imaged using laser scanning confocal microscopy. As expected, C. albicans produced low levels of hyphae in low iron conditions. However, C. albicans showed an increase in hyphal formation when grown with OG1RF, OG1RF with pCF10, or the clinical isolate despite being in low iron condition. All strains of E. faecalis had increased chaining and could be observed in close association with Candida hyphae. E. faecalis colony forming units (CFU) were the same whether it was grown with or without C. albicans. However, there was a significant decrease in C. albicans CFU when grown with E. faecalis under high or low iron conditions. The bacteriocin EntV that is known to inhibit C. albicans may be produced under high or low iron conditions. In addition, E. faecalis, being an efficient iron scavenger, may outcompete C. albicans for iron, reducing the fungal load regardless of available iron. We hypothesize that E. faecalis may induce hyphae formation in C. albicans under low iron conditions through multiple mechanisms. First, E. faecalis produces hydrogen peroxide. which can be observed within the bacterial chains wrapping around the hyphae and this peroxide can further induce hyphal formation. Second, during steady-state growth, E. faecalis may release N-acetylglucosamine (NAG) that is known to trigger increased hyphal growth via transcriptional induction of BRG1 and/or phosphorylation of the Mitogen Activated Protein Kinase (MAPK) Cek1. Cek1-P levels were observed to be enhanced in the presence of E. faecalis. Future experiments are focused on determining the transcriptional levels of entV and BRG1 and measuring the levels of NAG.