Agarose gel

Genomic DNA was extracted from 200 µL of the blood samples using FavorPrep™ Blood Genomic DNA Extraction Mini Kit (Favorgen, Pingtung, Taiwan) according to the manufacturer's instruction and stored at -20 ºC until further analysis.
The conventional PCR (cPCR) assays were performed by universal Mycoplasma spp. primers targeting the partial sequence of the 16 S rRNA gene [51] . Subsequently, identification of CMhm, Mhf, and CMt in PCRpositive samples was performed using a panel of three species-specific primers in cPCR [22] . PCR reactions were performed in a 25 µL volume reaction mixture containing 12.5 µL of Taq DNA Polymerase 2X Mastermix (Ampliqon, Odense, Denmark), 2 µl of the template DNA, 1 µL of 10 pmol of each forward and reverse primer (synthesized by metabion international AG, Planegg, Germany), and 8.5 µL distilled deionized water. In each run, positive DNA controls that were kindly provided by Professor Dr. Roberta Iatta (University of Bari, Italy), and distilled deionized water were used as positive and negative controls. PCR amplification were run in a SimpliAmp™ thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA) with conditions as described in Table 2. The PCR amplification products were documented using UV Imager (Transilluminator, Vilber Lourmat, France) after electrophoresis in a 1% agarose gel (SinaClon, Tehran, Iran) at 100 V for 60 min.

A multiplex PCR analysis was performed to determine the presence of OXA-type carbapenemase, bla OXA-23- like , bla OXA-24-like and bla OXA-58-like . Amplification reaction was carried out in a final volume of 25μL with 1X PCR buffer, 1 U Taq polymerase, 2mM MgCl 2 , 200μM of Deoxynucleotide triphosphate (dNTP) (Sinaclon, Tehran, Iran), 0.2μM of each primer (TAG, Copenhagen A/S Denmark), and 1μL of template DNA. PCR conditions were programmed in a Mastercycler (Eppendorf, Hamburg, Germany) as follows: initial denaturation at 94°C for 5 minutes, followed by 30 cycles at 94°C for 30 seconds, 53°C for 40 seconds, and 72°C for 50 seconds, and a final extension at 72°C for 6 minutes. PCR products were separated with electrophoresis on 1.5% agarose gel (Sinaclon), and after staining with ethidium bromide, visualized under UV gel documentation system. A. baumannii reference strains, including NCTC 13304, NCTC 13302, and NCTC 13305 were used as positive control for bla OXA-23-like , bla OXA-24-like and bla OXA-58-like , respectively. A negative control was included in each PCR reaction, containing all components except the DNA template, which was replaced by distilled water. 24 For each gene, one amplicon was sequenced (Bioneer, Daejeon, South Korea).

To determine clonal relatedness, REP-PCR was performed on all isolates with specific primers previously described, 27 that are listed in Table 1. Amplification conditions were performed using previously established protocols with some modifications. 28 Each reaction mixture for PCR contained 1X PCR buffer, 3.5mM of MgCl 2 , 300μM of Deoxynucleotide triphosphate (dNTP), 3% Dimethyl sulfoxide (DMSO) (Sinaclon), 0.5μM of each primer (TAG Copenhagen A/S, Denmark), and 1 U of Taq polymerase and 1μL of genomic DNA in a final volume of 25μL. PCR conditions included: 94°C for 10 minutes; 30 cycles of 94°C for 1 minute, annealing temperatures 45°C for 1 minute, 72 o C for 2 minutes, and 72°C for 16 minutes. Amplification products were separated by electrophoresis on 1.2% agarose gel (Sinaclon); after staining with ethidium bromide, they were observed in a UV gel documentation system, then they were photographed and compared together by visual inspection. 28 All fingerprints were observed by one observer, and fingerprints were interpreted according to previous studies. 27, 29

The PCR products were separated by electrophoresis (80 V, 40 min) using a 1% agarose gel (SinaClon, Iran) in 1× Tris/Borate/EDTA buffer. An amount of 5 μl from each of the product was run on the agarose gel. Then, the gel was stained with ethidium bromide (0.5 μg/ml) (SinaClon) and visualized by UV illuminator device (ProteinSimple, USA) and all images were saved on hard disk. A DNA marker of 100 bp (Sinaclon) was used for comparative analysis.