Ultrastructural changes in methicillin-resistant Staphylococcus aureus (MRSA) induced by metabolites of thermophilous fungi Acrophialophora levis

Abstract

Staphylococcus aureus and Methicillin-resistant S. aureus (MRSA) remains one of the major concerns of healthcare associated and community-onset infections worldwide. The number of cases of treatment failure for infections associated with resistant bacteria is on the rise, due to the decreasing efficacy of current antibiotics. Notably, Acrophialophora levis, a thermophilous fungus species, showed antibacterial activity, namely against S. aureus and clinical MRSA strains. The ethyl acetate extract of culture filtrate was found to display significant activity against S. aureus and MRSA with a minimum inhibitory concentration (MIC) of 1 μg/mL and 4 μg/mL, respectively. Scanning electron micrographs demonstrated drastic changes in the cellular architecture of metabolite treated cells of S. aureus and an MRSA clinical isolate. Cell wall disruption, membrane lysis and probable leakage of cytoplasmic are hallmarks of the antibacterial effect of fungal metabolites against MRSA. The ethyl acetate extract also showed strong antioxidant activity using two different complementary free radicals scavenging methods, DPPH and ABTS with efficiency of 55% and 47% at 1 mg/mL, respectively. The total phenolic and flavonoid content was found to be 50 mg/GAE and 20 mg/CAE, respectively. More than ten metabolites from different classes were identified: phenolic acids, phenylpropanoids, sesquiterpenes, tannins, lignans and flavonoids. In conclusion, the significant antibacterial activity renders this fungal strain as a bioresource for natural compounds an interesting alternative against resistant bacteria.

Fig 1. Representative standard curves for (A) total phenolic content; (B) flavonoid content.

Fig 2. (A) Colonies of culture IBSD-19 grown on PDA for seven days, (B) image of conidia and conidiophores from a seven-day MEA culture, (C) phylogenetic tree of partial ITS-rDNA sequences of IBSD19 fungal strain.

Reference sequences were downloaded from NCBI with the accession numbers indicated in parentheses.

Fig 3. Antibacterial potential of ethyl acetate extract against S. aureus and MRSA (STR = streptomycin; CHL = chloramphenicol).

Fig 4. Effects of ethyl acetate extract on the ultrastructure of S. aureus and MRSA as observed by SEM (A, control; B, S. aureus; C, MRSA).

Fig 5

(A) DPPH radical scavenging activity of the extract. (B) ABTS+ radical scavenging activity of the extract. Values are mean ± SD; n = 3.

Fig 6. TLC photography of fungal extract.

These fractions were developed in a preselected solvent system containing chloroform /methanol (8:2, by volume) and visualized using several methods, namely, ultraviolet lamps emitting at 365 nm after heating for 3 min at 105 °C after spraying with vanillin sulphuric acid (A), ultraviolet lamps emitting at 365 nm after spraying with anisaldehyde-sulphuric acid solution (B), and 0.04 mg·mL−1 DPPH in ethanol (C).

Fig 7. GC-MS based chemical profiling of ethyl acetate extract from IBSD-19 fungi strain.

Fig 8. Chemical structures of constituents detected in IBSD-19 extract.