The β-lactamase content of all blaNDM-like-positive isolates was determined by isoelectric focusing (IEF). Bacterial extracts were obtained by sonication of bacterial cells suspended in 1% glycine buffer and clarified by centrifugation. Sonicated cell extracts were analyzed by IEF in polyacrylamide gels containing ampholytes (pH 3.5–9.5; AP Biotech, Piscataway, NJ). The separated β-lactamases were visualized by covering the gel with the chromogenic cephalosporin nitrocefin (0.2 mg/ml; Oxoid Ltd., Basingstoke, United Kingdom; Papagiannitsis et al., 2015 (link)).
On the basis of the IEF data, PCR detection of various bla genes was performed by the use of primers specific for blaTEM−1, blaOXA−1, blaSHV, blaCTX−M, and blaCMY, as reported previously (Pałucha et al., 1999 (link); Pérez-Pérez and Hanson, 2002 (link); Woodford et al., 2006 (link); Coque et al., 2008 (link)). Both strands of the PCR products were sequenced using an ABI 377 sequencer (Applied Biosystems, Foster City, CA).
On the basis of the IEF data, PCR detection of various bla genes was performed by the use of primers specific for blaTEM−1, blaOXA−1, blaSHV, blaCTX−M, and blaCMY, as reported previously (Pałucha et al., 1999 (link); Pérez-Pérez and Hanson, 2002 (link); Woodford et al., 2006 (link); Coque et al., 2008 (link)). Both strands of the PCR products were sequenced using an ABI 377 sequencer (Applied Biosystems, Foster City, CA).
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