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 {beta}-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 {beta}-lactamase translocation, subsequent amplification of {beta}-lactamase encoding genes such as blaOXA-1 and blaCTX-M, and porin inactivation. Interestingly, the {beta}-lactamase gene amplification occurred both on plasmids and on the chromosome, including in the middle of porin-encoding genes. Additionally, overexpression of blaOXA-1 increased resistance to the broad-spectrum {beta}-lactam, piperacillin-tazobactam. This analysis shows mechanisms underlying non-CP-CRE emergence and demonstrates that copy number of {beta}-lactamase genes needs to be considered to fully understand antimicrobial resistance amongst key human pathogens.nnOne Sentence SummaryAmplification of {beta}-lactam genes with porin loss was associated with development of {beta}-lactam resistance in serial Enterobacteriaceae clinical isolates.