Role of β-lactamase encoding gene amplification in the development of non-carbapenemase producing, carbapenem-resistant Enterobacteriaceae

PROJECT SUMMARY Carbapenem resistant Enterobacteriaceae (CRE) are among the most urgent antimicrobial resistant (AMR) public health threats. CRE are broadly divided into carbapenemase producing and non-carbapenemase producing (i.e. CP-CRE and non-CP-CRE). Although CP-CRE has been intensely investigated, systematic studies from the United States and elsewhere show that non-CP-CRE make up some 50% of total CRE. Recently published data show that the outcomes for patients infected with non-CP-CRE are as poor as those infected with CP producing strains. Thus, strategies to mitigate non-CP-CRE development and spread are urgently needed. In the preliminary data to this application, we show that non-CP-CRE can emerge from an extended spectrum β-lactamase (ESBL) producing background by a chromosomally located transposon unit (TU) mediated amplification of β-lactamase encoding genes. Moreover, we have identified that these TUs can insert and amplify within porin encoding genes. Given that porins are key mechanisms by which carbapenems enter the bacterial cells, the TU insertion and amplification both increases β-lactamase production and decreases carbapenem entry, thereby engendering the emergence of non-CP-CRE. It is the goal of this proposal to determine the prevalence and mechanisms underlying β-lactamase gene amplification in non-CP- CRE including how often porin encoding genes are interrupted by TUs containing AMR elements. Additionally, we will use an experimental evolution system to observe how clinical ESBL Enterobacteriaceae isolates progress to non-CP-CRE and the fitness costs engendered by β-lactamase gene amplification. In specific aim 1, we will apply our recently developed combined long-read/short-read whole genome sequencing (WGS) approach to a large cohort of Escherichia coli and Klebsiella pneumoniae non-CP-CRE isolates to determine the prevalence and mechanisms of β-lactamase encoding gene amplification. Additionally, we will determine the impact of augmented expression of β-lactamase encoding on non-CP-CRE antimicrobial susceptibility using an inducible expression system. In specific aim 2, we will assess how clinical ESBL E. coli and K. pneumoniae progress to non-CP-CRE in response to various β-lactam antimicrobials using a novel microfluidics system which allows for longitudinal assessments of experimental evolution. By applying our combined WGS approach to serial isolates, we will be able to definitively assess the evolutionary trajectory by which clinical ESBL isolates develop carbapenem resistance in the absence of producing a carbapenemase. Finally, we will determine the potential fitness costs of non-CP-CRE development in clinical isolates by passaging strains in the absence of antimicrobial selective pressure. Completion of the research proposed herein will set the stage for more in-depth exploration of the role and mechanisms underlying gene amplification in a wide range of clinically important AMR pathogens.