Mupirocin

Clinical isolates of S. aureus were obtained through the Network of Antimicrobial Resistance in Staphylococcus aureus (NARSA) program (Table 1). clindamycin hydrochloride monohydrate (TCI America, Portland, OR, USA, >98.0% purity) and mupirocin (pure USP) (AppliChem, St. Louis, MO, USA) powders were purchased commercially and dissolved in dimethyl sulfoxide (DMSO) (for clindamycin) or ethanol (for mupirocin) to prepare a stock solution (10 μg/mL). Lipoderm was purchased from the Professional Compounding Centers of America (Houston, TX, USA). Cation-adjusted Mueller-Hinton broth (CAMHB) (Sigma-Aldrich, St. Louis, MO, USA), Tryptic soy broth (TSB) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), mannitol salt agar (Hardy Diagnostics, Santa Maria, CA, USA), phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA), Dulbeco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA), fetal bovine serum (FBS) (USA Scientific, Inc., Orlando, FL, USA), petroleum jelly (Equate [Walmart, Inc.], Bentonville, AR, USA), and 96-well plates (CellTreat Scientific Products, Shirley, MA, USA) were all purchased from commercial vendors.

Staphylococcus strains used in this study are presented in Table 1. Mueller-Hinton broth (MHB) was purchased from Sigma-Aldrich. Trypticase soy broth (TSB), Trypticase soy agar (TSA), and Mannitol salt agar (MSA) were purchased from Becton, Dickinson (Cockeysville, MD). Ebselen was purchased from (Adipogen corp, San Diego), vancomycin hydrochloride (Gold Biotechnology), linezolid (Selleck Chemicals), mupirocin (applichem, NE), and chloramphenicol (Sigma-Aldrich)

Bifidobacteria were routinely propagated on de Man Rogosa Sharpe media (MRS; BD Difco) with 0.05% (w/v) of L-cysteine (Sigma Aldrich, St. Louis, MO, USA). Cultures were incubated at 37 °C in an anaerobic chamber maintained with a gas mix of 7% H₂, 10% CO₂, and N₂ to balance (Coy Laboratory Products, Grass Lake, MI, USA). Genomic DNA was extracted from liquid cultures using the MasterPure Gram Positive DNA Purification Kit according to the manufacturer’s protocol (Epicentre, Madison, WI, USA). In order to isolate bifidobacteria, fresh feces were mixed into 5 mL of peptone water. Serial dilutions of 10x and 100x were performed on bifidobacterial-specific media agar plates which consisted of MRS, 0.05% (w/v) L-cysteine, and 0.05% (w/v) mupirocin (AppliChem Panreac, Chicago, IL, USA). Isolated colonies were identified via the bifidobacteria-specific colorimetric fructose-6-phosphoketolase assay and PCR of the 16s and ITS rRNA gene regions (PCR primers: F-GGTGTGAAAGTCCATCGCT, R-GTCTGCCAAGGCATCCACCA; Sanger sequencing primers: F-GGTGTGAAAGTCCATCGCT, R-CATGCCCCTACGTCCAG) according to Turroni et al. and Milani et al. [21 (link),22 (link),23 (link)].

Samples were thawed, diluted 1:10 in buffered peptone water (Merck, Darmstadt, Germany), and plated on blood agar [BA; Columbia agar + 5% sheep blood (BioMérieux, Marcy l’Etoile, France)], tryptic soy agar (TSA; Biolife Italiana, Milano, Italy), Wilkins-Chalgrene anaerobe agar (WCA-M; Oxoid, Basingstoke, United Kingdom) supplemented with mupirocin (50 mg/L) (AppliChem GmbH, Germany) and Rogosa agar (ROG; pH 5.5; Merck, Darmstadt, Germany). Plates were incubated at 37°C for 72 h in aerobic (TSA) conditions or anaerobic conditions (BA, WCA-M and ROG). Colonies with various morphology were systematically picked up from TSA (15 colonies), BA (15 colonies), WCA, and ROG (up to 10 colonies) and their genus was determined by MALDI-TOF MS. Due to poor growth of bacteria on Rogosa agar, samples were re-analyzed by direct plating of 1 mL of milk sample.