β-lactamase amplification and porin loss drive progressive β-lactam resistance in recurrent ESBL Enterobacteriaceae bacteremia

Carbapenem resistant Enterobacteriaceae (CRE) are a critical public health issue. Recent studies indicate many CRE lack carbapenemases, but contain extended spectrum β-lactamases (ESBLs). We investigated 16 longitudinal, recurrent cases of extended spectrum B-lactamase (ESBL)-positive Enterobacteriaceae bacteremia to gain insights into mechanisms underlying the emergence of non-carbapenemase producing CRE (non-CP-CRE). Using a combination of short- and long-read sequencing technologies, we identified that non-CP-CRE emerges from an ESBL background through a combination of insertion sequence mediated β-lactamase translocation, subsequent amplification of β-lactamase encoding genes such as bla_OXA-1 and bla_CTX-M, and porin inactivation. Interestingly, the β-lactamase gene amplification occurred both on plasmids and on the chromosome, including in the middle of porin-encoding genes. Additionally, overexpression of bla_OXA-1 increased resistance to the broad-spectrum β-lactam, piperacillin-tazobactam. This analysis shows mechanisms underlying non-CP-CRE emergence and demonstrates that copy number of β-lactamase genes needs to be considered to fully understand antimicrobial resistance amongst key human pathogens.