Anti-staphylococcal activity of soil-derived Penicillium citrinum extract

The increasing prevalence of methicillin resistant S. aureus (MRSA) led to higher morbidity, mortality, and treatment costs. The escalating antibiotic resistance levels and the stagnation in antibiotic discovery highlight the need for novel therapeutic alternatives. Soil fungi are a source of antimicrobial agents, given their capacity to produce diverse bioactive metabolites. Soil fungi from Alexandria, Egypt was investigated for extracts with anti-staphylococcal potential.

Soil samples were collected and fungal isolates were obtained on potato dextrose agar (PDA). Isolates were screened for antibacterial activity using agar well diffusion technique against standard bacterial strains. Minimum inhibitory concentration (MIC) of most active fungal extract was determined against S. aureus, including MRSA. Checkerboard assay was performed to assess the combination with commonly used antibiotics and MTT assay was used to evaluate the extract's cytotoxicity. Major active metabolites were characterized by gas chromatography-mass spectrometry (GC-MS) and a growth curve analysis was subsequently conducted to evaluate the antibacterial/growth inhibition activity of the most abundant metabolite.

Twenty-three fungal isolates were obtained, Penicillium citrinum extract showed the strongest anti-staphylococcal activity. Plackett-Burman analysis was used to optimize fermentation conditions to enhance anti-staphylococcal activity. The MIC of the butanolic extract ranged between 500 and 1000 µg/mL against commensal, clinical and standard S. aureus. P. citrinum extract combination with gentamicin was synergistic against all clinical MRSA isolates and its combination with ciprofloxacin was synergistic against 37.5% of the isolates. Cytotoxicity on the human skin fibroblast cell line confirmed the safety of the extract at the concentrations used in the synergistic combinations. GC-MS analysis revealed the most abundant bioactive metabolites to be dibutyl phthalate (10.58%), tridecane (7.58%) and hexadecane (7.42%). Growth curve analysis of a representative clinical MRSA isolate in the presence of dibutyl phthalate showed its growth inhibition properties but with no enhancement of the gentamicin efficacy highlighting the importance of the extract as a whole.

This study highlights Penicillium citrinum as a promising source for safe and effective antimicrobial agents, particularly as an adjunct to antibiotics against resistant staphylococcal infections.