In vitro analysis of ISEcp1B-mediated mobilization of naturally occurring beta-lactamase gene blaCTX-M of Kluyvera ascorbata

Abstract

ISEcp1B has been reported to be associated with and to mobilize the emerging expanded-spectrum beta-lactamase blaCTX-M genes in Enterobacteriaceae. Thus, the ability of this insertion sequence to mobilize the blaCTX-M-2 gene was tested from its progenitor, Kluyvera ascorbata. Insertions of ISEcp1B upstream of the blaCTX-M-2 gene in K. ascorbata reference strain CIP7953 were first selected with cefotaxime (0.5 and 2 microg/ml). In those cases, ISEcp1B brought promoter sequences enhancing blaCTX-M-2 expression in K. ascorbata. Then, ISEcp1B-mediated mobilization of the blaCTX-M-2 gene from K. ascorbata to Escherichia coli J53 was attempted. The transposition frequency of ISEcp1B-blaCTX-M-2 occurred at (6.4+/-0.5)x10(-7) in E. coli. Cefotaxime, ceftazidime, and piperacillin enhanced transposition, whereas amoxicillin, cefuroxime, and nalidixic acid did not. Transposition was also enhanced when studied at 40 degrees C.

FIG. 1.

Schematic representation of ISEcp1B-mediated mobilization of the naturally occurring CTX-M-2 β-lactamase gene of Kluyvera ascorbata CIP7953. 1. Introduction of ISEcp1B in K. ascorbata by electroporation. 2. Selection of K. ascorbata strains with ISEcp1B inserted upstream of the bla_CTX-M-2 gene. ISEcp1B enhances the gene expression by bringing promoter sequences. 3. Transfer of recipient plasmid in cefotaxime-resistant K. ascorbata strains. 4. ISEcp1B mobilizes the bla_CTX-M-2 gene on the plasmid under various conditions: with or without amoxicillin, piperacillin, cefuroxime, cefotaxime, ceftazidime, or nalidixic acid at 22, 30, 37, or 40°C. 5. Plasmid-located events of transposition are isolated. White arrow with black dots, ISEcp1B; black arrow with white dots, bla_CTX-M.