Collateral damage of flomoxef therapy: In vivo development of porin deficiency and acquisition of bla _DHA-1 leading to ertapenem resistance in a clinical isolate of Klebsiella pneumoniae producing CTX-M-3 and SHV-5 β-lactamases

Objectives: The study aimed to characterize the genetic basis of flomoxef and collateral ertapenem resistance in a clinical isolate of extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL-KP) after flomoxef exposure. Methods: Four ESBL-KP isolates (Lkp11-14) were recovered sequentially from four episodes of bacteraemia in an elderly patient. Laboratory investigations included genotyping by PFGE, resistance gene analysis by PCR and sequencing, and outer membrane protein analysis by SDS-PAGE. Plasmid analysis by DNA-DNA hybridization, electroporation and conjugation was also performed. Results: Lkp14 was recovered after 21 days of flomoxef therapy. It demonstrated an indistinguishable PFGE pattern when compared with those produced by Lkp11-13. However, resistance to both flomoxef and ertapenem emerged in Lkp14. Molecular characterization revealed that, in addition to the pre-existing ESBL production (CTX-M-3 and SHV-5) and OmpK35 deficiency found in Lkp11-13, Lkp14 had acquired an extra plasmid-mediated AmpC β-lactamase gene (bla_DHA-1) and failed to express OmpK36, because of insertional inactivation by an insertion sequence IS 5. Other resistance mechanisms, such as production of carbapenem-hydrolysing enzymes or expression of chromosomal efflux, were apparently not involved. Conjugational transfer of the plasmid-mediated bla_DHA-1 gene into Lkp11 resulted in a significant increase in the MICs of cephamycins and β-lactamase inhibitors, but not in those of carbapenems. Conclusions: Lkp14 was apparently derived from the previously flomoxef-susceptible isolates, Lkp11-13. After flomoxef exposure, the in vivo acquisition of the plasmid-mediated bla_DHA-1 gene has led to flomoxef resistance in Lkp14, and the concomitant depletion of OmpK36 expression has resulted in a collateral effect of ertapenem resistance and diminished susceptibilities to imipenem and meropenem.