Mechanisms and parameters controlling gene amplification leading to phenotypic heterogeneity in Enterobacter - PROJECT SUMMARY Antibiotic resistance is a growing global crisis exacerbated by a dearth of novel antibiotic classes and the rapid emergence of resistance to novel antibiotics of existing classes. In the United States, nearly 1 in 7 deaths attributed to bacterial pathogens are caused by Enterobacter or Klebsiella pneumoniae, members of the order Enterobacterales with among the highest rates of cephalosporin-and carbapenem-resistance. One form of antibiotic resistance is heteroresistance, a type of phenotypic heterogeneity in which an isolate harbor a minority resistant subpopulation within a majority susceptible population. The resistant subpopulation is dynamic: unlike other resistant subpopulations which include persisted and tolerant cells, the heteroresistant subpopulation grows during antibiotic exposure and becomes the majority population as the susceptible cells are killed but will return to baseline frequency when antibiotic exposure ends. Thus, this transient, low frequency resistant subpopulation is challenging to detect clinically and may be a cause of unexplained treatment failure. Experiments aimed to understand the mechanistic basis for heteroresistance to the novel cephalosporin cefiderocol revealed that many Gram-negative isolates harbor resistant subpopulations that encode many copies of an extended-spectrum beta-lactamase gene. The primary focus of preliminary experiments, Enterobacter isolate RS, encodes the blaSHV-5 beta-lactamase in a region flanked by identical sequences. This region undergoes homology-mediated gene amplification, creating the resistant subpopulation. While the subpopulation is resistant to cefiderocol and other cephalosporins because of the multiple copies of blaSHV-5, the underlying principles which result in multiple copies are unknown. To characterize these principles of amplification, experiments proposed here will in Aim 1 define the factors governing homology-mediated amplification in the Enterobacter chromosome, focused on understanding how the size and content of the amplified region, and the homologous sequences, influence gene amplification and subpopulation dynamics in vitro and during murine models of antibiotic therapy. Additional experiments identified that the activity of the SHV-5 enzyme dictates the extent of amplification. Chemical or genetic inhibition of SHV-5 activity resulted in greater gene copy number in the resistant subpopulation, establishing a connection between enzyme activity and subpopulation dynamics. Thus, experiments in Aim 2 will determine the effects of beta- lactamase activity on gene amplification and subpopulation dynamics in Enterobacter and K. pneumoniae in vitro and in vivo. Findings from this work will describe the factors governing gene amplification and provide insight into diverse forms of phenotypic heterogeneity that result from gene amplification, as well as enhance our understanding of the dynamics between resistant subpopulations and antibiotic treatment.
