P105 In vitro interaction of Malassezia and commensal Staphylococcus species

Abstract Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objective Malassezia is the most abundant fungal skin commensal organism, representing 50%–80% of total fungi present on the skin. It has been associated with many skin disorders such as pityriasis versicolor (PV) and seborrheic dermatitis/dandruff (SD/D). The role of Malassezia in disease manifestation is not discerned. It is important to understand its interaction with bacterial flora such as Staphylococcus epidermidis and S. capitis in vitro. We have studied the interaction of Malassezia and Staphylococcus species isolated from skin flora. Methods Malassezia restricta, M. globosa (n = 5) isolated from patients with SD and M. furfur (n = 5) isolated from PV were sub-cultured on Modified Dixon's agar (MDA). Staphylococcus epidermidis and S. capitis were isolated from patients with SD and sub-cultured on brain heart infusion (BHI) agar. Malassezia species requires media supplemented with lipids (MDA) for its growth. Bacteria and Malassezia were quantified on MDA and BHI agar by Miles and Mishra method to perform interaction between them. For direct interaction, suspensions (100 μl) of M. restricta, M. globose, and M. furfur were prepared in normal saline and added to wells on the plates of lawn cultures containing S. epidermidis and S. capitis (107 CFU/ml). Plates were incubated for 12 h at 35°C and observed for zone of inhibition. To investigate the release of antibacterial compounds into the extracellular environment, M. furfur was inoculated in modified Dixon's broth (MDB) and incubated at 35°C for 5 days. Supernatant was collected at 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h of incubation and evaluated for antibacterial activity by agar-well diffusion assay. Effect of cell-free supernatant of Malassezia on growth of bacteria was also monitored by growth kinetics of S. epidermidis for 24 h in the absence and presence of M. furfur supernatant using Epoch-2 microplate spectrophotometer. Results MDA supported the growth of bacteria at different cell densities (107-103 CFU/ml count) and incubation time of S. epidermidis and S. capitis was similar on MDA and BHI. Zone of inhibition (ZOI) was witnessed with M. restricta (20.6 ± 3 mm, 21 ± 3 mm), M. globosa (21 ±1 mm, 22.6 ±2 mm) and M. furfur isolates (16.5 ± 1 mm, 18 ± 2 mm) against S. capitis and S. epidermidis respectively by direct interaction. Inhibition of bacteria by M. furfur was noted from 48-120 h as ZOI (21.7 ±5.1 mm) was observed on bacterial lawn cultured plate. When growth kinetics of S. epidermidis was monitored in presence of M. furfur supernatant, maximum value reached upto 0.26 ± 0.019 only from 0.01 ± 0.001 at OD600 in 9 h including lag phase of 4 h (Fig. 1). However, OD600 value reached upto 0.97 ± 0.005 in 8 h including lag phase of 1.5 h in absence of supernatant. Doubling time calculated from logistic growth equation was 76.6 ± 4.4 and 65.2 ± 2.9 minutes in the presence and absence of supernatant respectively. Conclusion Inhibition of bacteria by Malassezia species noted in our study has not been reported earlier. The possible production of antibacterial compounds by Malassezia might be responsible for dysbiosis leading to disease.