AN483, a new anti-MRSA compound from Streptomyces sp.

Gram-positive eubacteria are representative of pathogenic microorganisms. Especially, Staphylococcus aureus is the most clinically important of these pathogens because of its exceptional virulence, stress tolerance and capacity to accumulate antimicrobial resistances.^{1, 2} Antibiotic-resistant strains of pathogenic bacteria are increasingly prevalent in hospitals and the community. Of antibiotic-resistant pathogens, the one that has gained the most attention is methicillin-resistant S. aureus (MRSA) because of its high frequency of infection as well as its resistance to many other antibiotics.^{3, 4} Overall, in the United States and the United Kingdom, 40–60% of nosocomial S. aureus strains are methicillin resistant. The increased use of vancomycin, the last anti-MRSA antibiotic in the last century, resulted in the prevalence of vancomycin-resistant Enterococcus. Although some anti-MRSA drugs such as linezolid and daptomycin have been introduced in recent years,⁵ new classes of anti-MRSA agents are still needed.

The actinomycetal strain AN100483 was isolated from a soil sample that was collected at Colocasia esculenta field, Baekam Mountain, Gyeongsangbuk-do, Korea, and identified as Streptomyces sp. based on 16S DNA sequence. Fermentation was carried out in a liquid culture medium containing soluble starch 1%, glucose 2%, soybean meal 2.5%, beef extract 0.1%, yeast extract 0.4%, NaCl 0.2%, K₂HPO₄ 0.025% and CaCO₃ 0.2% (adjusted to pH 7.2 before sterilization). A portion of the strain AN100483 from a mature plate culture was inoculated into a 500-ml Erlenmeyer flask containing 80 ml of the above sterile seed liquid medium and cultured on a rotary shaker (150 r.p.m.) at 28 °C for 3 days. For the production of an active compound, 5 ml of the seed culture were transferred into 500 ml Erlenmeyer flasks containing 100 ml of the above medium, and cultivated on a rotary shaker (150 r.p.m.) for 10 days at 28 °C. After incubation, the fermented liquid cultures (3 l) were extracted with 80% acetone. The acetone extracts were concentrated in vacuo to an aqueous solution, which was then extracted with an equal volume of EtOAc three times. The EtOAc extract (1.2 g) was subjected to silica gel (56 × 200 mm, Merck Art No. 7734.9025) column chromatography, followed by stepwise elution with CHCl₃–MeOH (100:1, 50:1, 25:1). Each fraction was tested for anti-MRSA activity. The active fractions eluted with CHCl₃–MeOH (50:1) were pooled and concentrated in vacuo. The resultant residue (285 mg) was purified by preparative HPLC equipped with Waters 510 HPLC pump and Waters 996 Photodiode Array Detector. The reverse phase HPLC column (20 × 150 mm, YMC C18) was eluted with CH₃CN–H₂O (65:35) containing 0.035% trifluoroacetic acid at a flow rate of 6 ml per min to afford 3.3 mg of 1 with retention time of 28 min.

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