Word counts: Body of the text (2,668 words) Abstract Purpose. Sitafloxacin (Sfx) is a new fluoroquinolone (FQ) that has shown a strong bactericidal effect against Mycobacterium tuberculosis (Mtb) in vitro. However, data on Sfx efficacy against Mtb with gyrA/B mutations and its epidemiological cut-off (ECOFF) value remain limited. Therefore, we evaluated and compared the in vitro activity of Sfx against gyrA/B-mutant Mtb to that of moxifloxacin (Mfx), levofloxacin (Lfx), and ciprofloxacin (Cfx),

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... and determined the ECOFF for Sfx. Methodology. A total of 109 clinical Mtb isolates, including 73 multidrug-resistant (MDR) isolates, were subjected to minimum inhibitory concentration (MIC) analysis in oleic-albumin-dextrose-catalase (OADC)-supplemented Middlebrook 7H9 medium. Our results showed that Sfx had lower cumulative MIC than Mfx, Lfx, and Cfx. Furthermore, we preformed direct DNA sequencing of the quinolone resistance-determining regions (QRDRs). Results. We identified the following mutations: D94G, D94A, A90V, D94H, D94N, and G88A in gyrA; and A543V, A543T, E540D, R485C, D500A, I552S, and D577A in gyrB. Based on our results, an ECOFF of 0.125µg/mL was proposed for Sfx. With this ECOFF, 15% of Lfx-resistant isolates with MIC ≥ 2 µg/mL were susceptible to Sfx. Conclusion. Sfx had the lowest cumulative MIC and a relatively low ECOFF value against Mtb, indicating that Sfx was more effective against not only gyrA-mutant isolates but also MDR isolates in Japan.