Evolution of ceftazidime/avibactam resistance and plasmid dynamics in OXA-48-producing Klebsiella spp. during long-term patient colonization

Abstract

To prospectively monitor the evolution of the resistome of OXA-48-producing Klebsiella species in a patient with long-term colonization, with a particular focus into the plasmid dynamics and the evolution of ceftazidime/avibactam resistance. All OXA-48-producing Klebsiella spp. isolates from a single patient admitted to a hospital during seven months were prospectively collected. MICs were determined through reference broth microdilution. Multilocus sequence types, SNPs analysis, resistance mechanisms, genetic context of β-lactamases and plasmid dynamics were determined by WGS and bioinformatic analysis. The impact of β-lactamase variant obtained after ceftazidime/avibactam exposure was determined via cloning experiments. Four isolates, two before (one OXA-48-producing K. pneumoniae and one CTX-M-15-like-producing K. pneumoniae) and two after treatment with ceftazidime/avibactam (one OXA-48- and CTX-M-15-like-producing K. pneumoniae and one OXA-48- and CTX-M-15-like-producing K. aerogenes) were collected. The plasmid dynamics analysis demonstrated that the IncL and IncFIIK plasmids, in which blaOXA-48 and blaCTX-M-15-like genes were located, respectively, exhibited a high degree of conservation indicating a potential for both intra- and interspecies transmission. The K. pneumoniae isolate obtained after treatment, which differed from the previous isolate by just six SNPs, exhibited resistance to ceftazidime/avibactam through P167S substitution in CTX-M-15, which is now designated CTX-M-273. Cloning experiments demonstrated enhanced resistance to ceftazidime/avibactam. The transfer of plasmid-borne β-lactamase resistance genes between intra- and interspecies bacterial populations enables the rapid diversification of the bacterial genome. The emergence of ceftazidime/avibactam resistance through the modification of CTX-M-enzymes represents a mechanism by which OXA-48-producing Enterobacterales may evolve toward ceftazidime/avibactam resistance in vivo.