Antimicrobial bioassays

Target microorganisms for antimicrobial bioassays were bacteria associated with nosocomial infections, called ESKAPE pathogens (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter aerogenes). Drug-sensitive bacteria, i.e., E. faecalis DMST 2860, S. aureus DMST 8840, K. pneumoniae DMST 7592, A. baumannii DMST 10437, P. aeruginosa DMST 4739 and E. aerogenes DMST 8841, were purchased from the National Institute of Health of Thailand, whereas drug-resistant bacteria, i.e., methicillin-resistant Staphylococcus aureus (MRSA) AMH 10, extended-spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae AMH 20 and multidrug-resistant (MDR) Acinetobacter baumannii AMH 30, were kindly provided by Ananda Mahidol Hospital (AMH), Lopburi Province, Thailand.

Strain SMC 277^T was precultured in a 125 ml flask containing 10 ml of SCM medium [55] for 3 days at 28°C with 250 rpm shaking. Each 1% (v/v) of the seed culture was then transferred to each 250 ml flask containing 50 ml of SCM medium, and incubated at 28°C, 250 rpm for 7, 14 and 21 days separately. Each supernatant was added with 5 ml of Diaion^® HP-20 resin (Sigma). After leaving the suspension for 2 h under shaking, the resin was washed with 25 ml of water, filtered, and eluted with 25 ml of 80% methanol. The methanolic extract was evaporated to dryness, and dissolved with 100 μl of 67% methanol. Each 5 μl of the extract was transferred onto a paper disk (diameter of 6 mm, Whatman) to examine bioactivity. For bacterial targets, single colonies grown on tryptic soy agar (Becton Dickinson) at 37°C overnight were picked, adjusted to 10⁸ CFU/ml with tryptic soy broth (Becton Dickinson) and swabbed on the entire surface of Muller Hinton agar (MHA) (Becton Dickinson). The paper disk with extract was placed on the surface of MHA plates containing the target microorganisms. A disk with 5 μl of 67% methanol was used as a negative control. The plate was incubated at 37°C for 24 h and then measured for the diameter of zone of inhibition. Each test was performed in triplicate.