| Summary: | <i>Elizabethkingia anophelis</i> is a multidrug-resistant pathogen. This study evaluated the antimicrobial activity of minocycline, tigecycline, ciprofloxacin, and levofloxacin using in vitro time-kill assays and in vivo zebrafish animal models. The <i>E. anophelis</i> strain <i>ED853-49</i> was arbitrarily selected from a bacterial collection which was concomitantly susceptible to minocycline, tigecycline, ciprofloxacin, and levofloxacin. The antibacterial activities of single agents at 0.5–4 × minimum inhibitory concentration (MIC) and dual-agent combinations at 2 × MIC using time-kill assays were investigated. The therapeutic effects of antibiotics in<i> E. anophelis</i>-infected zebrafish were examined. Both minocycline and tigecycline demonstrated bacteriostatic effects but no bactericidal effect. Minocycline at concentrations ≥2 × MIC and tigecycline at concentrations ≥3 × MIC exhibited a long-standing inhibitory effect for 48 h. Bactericidal effects were observed at <i>ciprofloxacin and levofloxacin concentrations of ≥</i>3 × <i>MIC</i> within 24 h of initial inoculation. Rapid <i>regrowth of E. anophelis </i>occurred <i>after </i>the initial killing phase when ciprofloxacin was used<i>, regardless of the concentration. </i>Levofloxacin treatment at the concentration of ≥2 × MIC consistently resulted in the long-lasting and sustainable inhibition of bacterial growth for 48 h. The addition of minocycline or tigecycline weakened the killing effect of fluoroquinolones during the first 10 h. The minocycline-ciprofloxacin or minocycline–levofloxacin combinations achieved the lowest colony-forming unit counts at 48 h. Zebrafish treated with minocycline or a combination of minocycline and levofloxacin had the highest survival rate (70%). The results of these in vitro and in vivo studies suggest that the combination of minocycline and levofloxacin is the most effective therapy approach for <i>E. anophelis</i> infection.
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