Xbai restriction endonuclease

An overnight-grown bacterial culture was suspended in EET buffer (100 mM EDTA, 10 mM EGTA, 10 mM Tris—HCl (pH 8)) and adjusted to an optical density of 0.9 at 600 nm. The suspension was mixed with equal volumes in a 2% solution of low-melting-temperature agarose in EET buffer. After solidification, the agarose plugs were incubated for 4 h at 37°C in EET buffer containing 1 mg lysozyme and 50 ug lysostaphin per ml. The plugs were transferred to EET buffer containing 1% sodium dodecy1 sulfate and 20 mg proteinase K per ml of buffer and incubated overnight at 50°C. Plugs were washed thoroughly with TE buffer (10mM Tris—HCl, 1 mM EDTA (pH 8)) and digested overnight with XbaI restriction endonuclease (TAKARA, Shiga, Japan). DNA separation was performed in 0.5× TBE buffer in a pulsed-field electrophoresis system (CHEF MAPPER; Bio-Rad Laboratories, California, USA) with the following conditions: temperature 14°C; voltage 6 V/cm; switch angle, 120°and switch ramp of 4–40 s for 21h. Salmonella enterica serotype Braenderup H9812 was used as a marker for PFGE. The restriction patterns were analyzed and interpreted according to Tenover et al [15 (link)].

An overnight grown bacterial culture in LB medium at 37°C was centrifuged and suspended in cell suspension buffer [100 mM EDTA, 100 mM Tris–HCl (pH 8.0)] and adjusted to an optical density (OD) of 4.0 at a wavelength of 600 nm. The suspension was mixed with equal volumes 2% solution of low melting agarose in Tris-EDTA [TE: 1 mM EDTA, 10 mM Tris–HCl (pH 8.0)]. After cooling, the agarose sections were incubated for 4 h at 54°C in cell lysis buffer [50 mM Tris–HCl, 50 mM EDTA (pH 8.0), 0.01 g/ml N-lauroyl-sarcosine, sodium salt, and 0.1 mg/ml proteinase K]. Thereafter, the sections were washed thoroughly with TE buffer and digested overnight with XbaI restriction endonuclease (Takara Bio, Inc., Otsu, Japan). Genomic DNA was separated in 0.5 × Tris/borate/EDTA (TBE) buffer in a Pulse-Field Gel Electrophoresis (PFGE) system (CHEF Mapper; Bio-Rad Laboratories, Inc., Hercules, CA, United States) at 14°C, using a voltage of 6 V/cm, a switch angle of 120°, and a switch ramp of 6–36 s for 21 h.

An overnight bacterial culture was suspended in cell suspension buffer [100 mM EDTA, 100 mM Tris-HCl (pH 8.0)] and adjusted to an optical density of 4.0 at a wave length of 600 nm. The suspension was mixed with equal volumes 2% solution of low-melting agarose in Tris-EDTA [TE: 1 mM EDTA, 10 mM Tris-HCl (pH 8.0)]. After cooling, the agarose sections were incubated for 4 h at 54°C in cell lysis buffer [50 mM Tris-HCl, 50 mM EDTA (pH 8.0), 0.01 g/ml N-lauroyl-sarcosine, sodium salt, 0.1 mg/ml proteinase K]. Thereafter, the sections were washed thoroughly with TE buffer and digested overnight with XbaI restriction endonuclease (Takara Bio, Inc., Otsu, Japan). Genomic DNA was separated in 0.5× Tris/borate/EDTA (TBE) buffer in a PFGE system (CHEF Mapper; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 14°C, using a voltage of 6 V/cm, a switch angle of 120°, and a switch ramp of 6–36 s for 21 h.

An overnight bacterial culture was suspended in cell suspension buffer [100 mM EDTA, 100 mM Tris-HCl (pH 8.0)] and adjusted to an optical density of 4.0 at the wave length of 600 nm. The suspension was mixed with equal volume of 2% solution of low-melting agarose in Tris-EDTA [TE: 1 mM EDTA, 10 mM Tris-HCl (pH 8.0)]. After cooling, the agarose sections were incubated for 4 h at 54°C in cell lysis buffer [50 mM Tris-HCl, 50 mM EDTA (pH 8.0), 0.01 g/mL N-lauroyl-sarcosine, sodium salt, 0.1 mg/mL proteinase K]. They were then washed thoroughly with TE buffer and digested overnight with XbaI restriction endonuclease (Takara Bio, Inc., Otsu, Japan). Genomic DNA separation was performed in 0.5 X Tris/borate/EDTA (TBE) buffer in a PFGE system (CHEF Mapper; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 14°C, using a voltage of 6 V/cm, a switch angle of 120° and a switch ramp of 6–36 s for 19 h. The Salmonella enterica serotype Braenderup H9812 was used as a marker for PFGE.

Molecular typing of 54 NDM-producing E. coli isolates was performed by pulsed-field gel electrophoresis (PFGE). The plugs containing genomic DNA were prepared according to the procedure described by Pereira et al. (2015 (link)). The DNA fragments digested with restriction endonuclease XbaI (TaKaRa Biotechnology, Dalian, China) were separated by PFGE on 1% SeaKem Gold agarose (Lonza, Rockland, ME, USA) using the CHEF Mapper XA PFGE system (Bio-Rad, USA) for 18 h at 14°C. The electrophoretic switch times were 6.8–35.4 s. Salmonella H9812 was used as reference marker. Dice coefficients was used to calculate the similarity of PFGE patterns. Dendrograms were constructed by the unweighted pair group method with arithmetic averages (UPGMA) using BioNumerics software version 5.10. Isolates were categorized to be of the same cluster when their dice similarity index was ≥85%. Multi-locus sequence typing of E. coli was conducted by PCR as previously described (Wirth et al., 2006 (link)). The allelic profiles and sequence types were identified by amplifying and sequencing the seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, recA) according to the reference website (https://enterobase.warwick.ac.uk/species/index/ecoli). A minimum spanning tree of 54 bla_NDM-positive isolates was also constructed by BioNumerics software version 5.10.

All isolates were analyzed by PFGE to determine their genetic relatedness. In brief, the bacterial suspension mixed with equal volumes of low-melting-temperature agarose was cleaved with 1% sodium dodecyl sulfate and proteinase K (Sigma, USA) at 54°C overnight, and then digested with restriction endonuclease Xba I (Takara, Dalian, China) at 37°C for 8 h. DNA separation was performed in 0.5× TBE buffer using a pulsed-field electrophoresis system (CHEF MAPPER; Bio-Rad Laboratories, California, USA) under the following conditions: temperature 14°C; voltage 6.0 V/cm; switch angle, 120°; and switch ramp of 2.16–54.17 s for 18 h. Comparison of pulsotypes was performed with Bionumerics software version 6.6. Isolates were allocated into genetic similarity clusters using an 80% cut-off value. MLST was performed as described previously (https://pubmlst.org/ecloacae/). New alleles and sequence types were submitted to the MLST website and approved (https://pubmlst.org/ecloacae/). EnteroBase together with GrapeTree was used to analyze population evolutionary relationship (https://enterobase.readthedocs.io/en/latest/index.html).