In Vivo Selection of a Chromosomally Encodedβ-Lactamase Variant Conferring Ceftazidime Resistance in Klebsiellaoxytoca

Abstract

Klebsiella oxytoca clinical isolate A was recovered from the urine of a 55-year-old man with prostatic and urinary tract infections. This isolate displayed a β-lactam resistance phenotype consistent with overproduction of a chromosomally encoded class Aβ -lactamase and had decreased susceptibilities to allβ -lactams except ceftazidime, cephamycins, and carbapenems. Four weeks after treatment with an antibiotic regimen that included ceftazidime, K. oxytoca isolate B, which had a high level of resistance to ceftazidime, was isolated from the urine of the same patient. Isoelectric focusing analysis of the culture extracts of these isolates gave a pI of 5.4 for both isolates. Cloning experiments with the PCR products of the bla_OXY gene resulted in two Escherichia coli DH10B recombinant clones with resistance phenotypes mirroring those of the parental isolates. Sequencing analysis revealed that the bla_OXY-2-5 gene from K. oxytoca B had a single nucleotide substitution compared to the sequence of the bla_OXY-2 gene from K. oxytoca A, leading to a proline-to-serine substitution at position 167, according to the numbering of Ambler. Biochemical analysis of purified OXY-2-5 showed that it had the ability to hydrolyze ceftazidime. This is the first report of in vivo selection of a K. oxytoca isolate that produced a chromosomally encodedβ -lactamase conferring resistance to ceftazidime.