Fractionation and Antibacterial Activity of Penicillium commune Organic Extract Isolated from Antarctica Marine Sediments

The development of resistant pathogens and use of environmental unfriendly commercial products in agriculture are a major concern worldwide. Natural products are an important source of new active molecules and microorganisms such as Penicillium are known to produce an array of antimicrobial compounds. Marine sediments in Antarctica provide extreme conditions and novel bioactive molecules may be produced by Penicillium sp. found in the region, which can be an alternative to the current chemicals used against Xanthomonas citri subsp. citri (X. citri) the citrus canker pathogen. This study aimed to purify a bioactive extract produced in Czapek medium by Penicillium commune. CRM1540, isolated from marine sediments in Antarctica. Metabolites production was conducted in Czapek medium at 15 ºC and 150 rpm for 20 days. The crude extract was obtained by liquid-liquid extraction with ethyl acetate followed by solvent removal. After antimicrobial evaluation, the extract was fractionated by high pressure liquid chromatography (HPLC). A volume of 250 µL of sample in methanol (HPLC grade) at 30 mg/mL and filtered in PTFE (hydrophilic) 0.22 µm membrane was injected in an NST C18 100A column (250 mm x 4.6 mm x 5 µm). Methanol 95% was used as mobile phase for a 120 min run with flow of 1.0 mL/min and ten (P01 – P10) fractions were collected according to peak detection at 254 nm. The fractions were dried and dissolved in 1% dimethyl sulfoxide (DMSO) for activity in X. citri. For the antibacterial assay, 50 µL of inoculum at log phase (OD600: 0.8) diluted 1:10 in NYG medium (yeast extract 0.3% w/v, peptone bacteriological 0.5% w/v, glycerol 2% v/v) was added to microtubes containing 500 µL of the fractions at 500 µg/mL, 250 µg/mL,125 µg/mL and 62.5 µg/mL in NYG. The microtubes were incubated at 29 ºC and 200 rpm overnight followed by serial dilutions and plating in NYG agar. The plates were incubated at 29 ºC and colony forming units were counted after 24 and 48h. Growth curve profiles of the bacteria were also obtained by running a growth curve experiment with the crude extract and the bioactive fractions in a 24 well plate for 48h. The plate was incubated at 29 ºC in a Synergy H1 microplate reader and absorbance (600 nm) was read every 30 min. Kanamycin 20 µg/mL, 1% DMSO were used as positive and vehicle control, respectively, and NYG with inoculum was the negative control for the tests. Both assays showed the antibacterial activity of fractions P01 and P10. The crude extract was bioactive at 750 µg/mL for 48h while P01 presented bactericidal activity at 500 µg/mL for 48h and bacteriostatic characteristics at 250 µg/mL, delaying the growth of X. citri. In lower concentrations, P01 did not affect bacterial growth. Fraction P10 showed antibacterial action at 62.5 µg/mL, inhibiting the phytopathogen growth for 48h. The bioactive fractions are under mass spectrometry and 1H and 13C NMR analysis to verify its purity and molecule structure.
References:
LIU et al.. Marine Derived Penicillium Species as Producers of Cytotoxic Metabolites. Marine Drugs. v. 15, p. 329-372. 2017.
SVAHN et al.. Penicillium nalgiovense Laxa isolated from Antarctica is a new source of the antifungal metabolite amphotericin B. Fungal Biology and Biotechnology. v. 2, p. 1-8. 2015.