First detection of a transferable blaCTX-M-14b gene in a Klebsiella pneumoniae clinical isolate from Tunisia and analysis of its genetic context

Klebsiella pneumoniae ML2508 was isolated from a patient at the surgery unit of the Military Hospital (Hpital Militaire de Tunis), Tunisia. It was identified as a producer of extended-spectrum beta-lactamases (ESBLs) by the double-disk synergy test. The beta-lactamases produced by the strain were characterized by isoelectric focusing, determination of the specific activities against penicillins and cephalosporins, determination of the inhibitory concentration required to inhibit 50% of enzyme activity (IC50), and the inhibition effect of EDTA on putative metallo-beta-lactamases. The crude extract of K. pneumoniae ML2508 contains five different beta-lactamases with pI 5.5, 7.3, 7.6, 8.1, and 8.6. Only the beta-lactamase with pI 8.1 was transferred by transformation and conjugation experiments. Molecular characterization of these genes was performed by PCR and sequencing. The four chromosomal beta-lactamases are TEM (pI 5.5), 2 SHV (pI 7.3 and 7.6), and CTX-M-28 (pI 8.6). The beta-lactamase with pI 8.1 was encoded by bla (CTX-M-14b) gene located on a 50-kb highly conjugative plasmid. The study of the genetic context of bla (CTX-M-14b) was realized by PCR-mapping and DNA sequencing. A novel variant of tnp (ISEcp1) designated ISEcp1C was detected upstream of the gene.