CoNS were identified by standard methods (colony morphology, catalase positive, DNase negative) [17 ], and further identified to species level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the Biotyper 2.0 database (Bruker Daltronics, Bremen, Germany) [18 (link)]. A score of ≥2 was accepted for identification. All isolates were tested for antimicrobial susceptibility to cefoxitin, clindamycin, co-trimoxazole, gentamicin, and fusidic acid according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (v 5.0, www.eucast.org ). Constitutive and inducible resistance to clindamycin was determined with the D-shaped disc diffusion method (Oxoid AB, Sweden). After initial identification, isolates were stored at −80 ° C pending further analysis. Multidrug-resistance (MDR) were defined as resistance to cefoxitin and ≥3 other classes of antimicrobial agents. When estimating the MRSE prevalence and the prevalence of resistance to other antimicrobials among patients per sampling day, the S. epidermidis isolate exhibiting resistance to highest number of antimicrobials was used.
Biotyper 2.0 database
Manufactured by Bruker
Sourced in Germany, United States
The Biotyper 2.0 database is a comprehensive microbial identification system that utilizes matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology. The database contains a large collection of reference spectra for the identification of a wide range of microorganisms, including bacteria, yeasts, and fungi.
Automatically generated - may contain errors
3 protocols using biotyper 2.0 database
1
Antimicrobial Resistance of CoNS Isolates
2
MRSA Biofilm Inhibition by Natural Compounds
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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.
3
Bacterial Strain Identification Protocol
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Bacteremia-associated S. aureus strains were routinely identified on the basis of their colony morphology, Gram staining, catalase test and slide test for clumping factor (Staphytect Plus; Oxoid, UK). All identifications were confirmed by MALDI-TOF MS, using a Microflex LT instrument, Flexcontrol 3.0 software and the Biotyper 2.0 database (Bruker Daltonics, USA). Following antimicrobial susceptibility testing (see below), all isolates were stored at room temperature in agar tubes (BioRad, USA).
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