The isolates were identified using biochemical tests with triple sugar iron slants (Japan BD, Tokyo, Japan), motility–indole–lysine medium (Japan BD), and cellobiose–lactose–indole–β-d-glucuronidase medium (Kyokuto Pharmaceutical, Tokyo, Japan).

Colistin susceptibility and minimum inhibitory concentrations (MICs) of the isolates were determined using the standard broth microdilution method [16]. MICs ≥ 4 μg/mL were interpreted as resistance to colistin.

The colistin resistance gene, mcr, was assessed using multiplex PCR. In brief, DNA was extracted from the isolates by boiling the bacterial suspension in tris(hydroxymethyl)aminomethane (10 mM)–ethylenediaminetetraacetic acid (1 mM) buffer pH 8.0 (Nippon Gene, Tokyo, Japan). The presence of mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 was detected using PCR, as described previously [11].

The genetic relatedness of isolates was assessed using pulsed-field gel electrophoresis (PFGE). XbaI-digested genomic DNA of isolates embedded in agarose was analyzed using the CHEF-DR III System (Bio-Rad, Hercules, CA, USA) according to a reported method [17].

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