Microflex lt instrument

All samples were cultured on different media; blood, chocolate and MacConkey agars (Oxoid Ltd, Basingstoke, Hamsphire, England). Non-lactose fermenting (NLF) colonies were identified by conventional biochemical testing and antimicrobial susceptibility testing using the disc diffusion method (Modified Kirby Bauer technique) on Mueller-Hinton agar according to Clinical and Laboratory Standards Institute11 guidelines. Further identification was done by MALDI-TOF MS on a Microflex LT instrument (Bruker Daltonics GmbH, Leipzig, Germany) with FlexControl (version 3.0) software (Bruker Daltonics) for the automatic acquisition of mass spectra in the linear positive mode within a range of 2 to 20 kDa, according to the instructions of the manufacturer. For isolate identification, the row spectra were compared with those in the Biotyper 56 database and a log (score) of ≥2 was considered for a secure species identification. Spectra were acquired with a microflex LT mass spectrometer (Bruker Daltonics), recorded in the positive linear mode at a laser frequency of 20 Hz, ion source 1 voltage 59 20 kV, ion source 2 voltage 8.5 kV, and a mass range from 2000 to 20 000 KDa, as described elsewhere.12 (link) For MALDI-TOF MS, bacteria were cultured for 24h at 37°C on MacConkey agar and extracts were prepared as described previously.12 (link),13 (link)

The clinical MRSA isolate used in this study was provided by the Clinical Laboratory Department of the First Affiliated Hospital, Nanjing Medical University, Nanjing, China. The strain was isolated from blood and was resistant to many antibiotics except for linezolid, teicoplanin, and vancomycin (Supplementary Tables S16 online). Moreover, the isolate was identified using a MicroFlex LT instrument (Bruker Daltonics) according to the manufacturer's recommendations. Spectra were analyzed by Flexcontrol 3.0 software and Biotyper 2.0 database (Bruker Daltonics)31 (link). Blood agar medium was used to culture the colonies. Nutrient broth (NB) medium was used for routine culturing of the strain, while trypticase soy broth (TSB) medium was used to study the effects of natural compounds on MRSA biofilm in 96-well flat-bottom polystyrene plates (Costar 3599; Corning; USA). The resveratrol and ursolic acid used in this study were isolated from natural products in our group and their purities were confirmed to be >98% using high performance liquid chromatography methods. They were dissolved in ethanol at 30 mg/mL and 5 mg/mL, respectively. The vancomycin used in this study was purchased from Sigma and dissolved in water at 5 mg/mL. All compounds were filtered with a 0.22-μm filter in sterile conditions and then stored at 4°C.

Bacteria from pure cultures #2 and #4 were identified by Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF). The protein mass spectra were acquired with the use of an automated mass spectrometer Bruker MALDI-TOF BioTyper (Bruker Daltonics, Germany) within the range of m/z 3 000 to 15 000. The Bruker MALDI-TOF BioTyper target plate was inoculated with bacterial test standard (Bruker #8255343) and calibration was performed according to the recommendations of the manufacturer. Using the direct colony transfer method, the tested bacteria were applied to the target spot, allowed to dry and covered with 1 µl of 70% formic acid, followed by 1 µl of matrix (Bruker, HCCA #8255344). The bacterial identification was obtained using a Microflex LT instrument (Bruker Daltonics, Germany) with Flex Control (version 3.0) software (Bruker Daltonics, Germany). The mass spectra were matched with the spectral database carried out by automated software, the MALDI BioTyper automation (version 2.0) software (Bruker Daltonics, Germany). The identification was conducted by Clinical Microbiology, London Health Science Centre, London, Ontario.