Doxycycline (catalog No. HY-N0565), minocycline hydrochloride (catalog No. HY-17412), tigecycline hydrochloride (catalog

No. HY-B0117A), eravacycline (catalog No. HY-16980), and omadacycline (catalog No. HY-14865) were purchased from MedChemExpress (MCE, Shanghai, China).

Antimicrobial MICs were determined using the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) document M45-A2 [18]. Overnight bacterial cultures were diluted 1:100 in 2 mL of 1 × Mueller-Hinton broth (MHB). Following 3 hours of incubation at 37°C, 10.82 ×g, the colony suspension (corresponding to a 0.5 McFarland standard, 1.0–1.5 × 108 colony forming units [cfu]/mL) was diluted 1:100 in 1 mL 2 × MHB. Aliquots (100 μL) of the colony suspension (1.0–1.5 × 106 cfu/mL) were inoculated into polystyrene microtiter plates (Costar3599; Corning, NY, USA) containing 100-μL aliquots of tigecycline, eravacycline, or omadacycline (0.0078, 0.0156, 0.031, 0.06, 0.125, 0.25, 0.5, 1, 2, 4, or 8 mg/L). Following 24 hours incubation at 35°C, the MICs were calculated as the drug concentration in the well without obvious bacterial precipitation. At present, there are no unified criteria for determining the antimicrobial susceptibility of M. catarrhalis. Thus, we used the European Committee on Antimicrobial Susceptibility Testing (EUCAST)-recommended MIC breakpoints for M. catarrhalis. Isolates with an MIC ≤ 1 or > 2 mg/L were considered susceptible or resistant, respectively, to doxycycline or minocycline.

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