Shift in pre-existing antibiotic heteroresistance explains AST change from susceptible to resistant during patient treatment

The development of resistance during antibiotic therapy can lead to treatment failure, negative outcomes, and even death. While bacterial development of resistance during therapy is observed relatively frequently, it is paradoxically thought to often be mediated by rare mutations (estimated frequency of ∼1 in 10^6 or less) and is generally viewed as an unpreventable event. Here, we describe the initial classification of an isolate of Klebsiella pneumoniae as susceptible to piperacillin/tazobactam (TZP) by standard broth microdilution (BMD) testing, yet the classification of a subsequent follow-up isolate after patient treatment as resistant, seemingly demonstrating de novo evolution of TZP resistance. However, we reveal that both isolates actually exhibit heteroresistance, a phenomenon in which an isolate harbors a minor subpopulation of resistant cells, co-existing with a majority susceptible population. We demonstrate that an increase in the frequency of the resistant subpopulation in the follow-up isolate, mediated by an increase in copy number of the SHV-1 beta-lactamase, led to this isolate being classified resistant. These data highlight how a relatively minor change in the frequency of the resistant subpopulation in a heteroresistant isolate, rather than the de novo evolution of classical resistance in which all cells exhibit resistance, can underlie the classification of resistance by diagnostic tests. This emphasizes the need for more sensitive diagnostics which can detect heteroresistance, and potentially help explain why results indicating resistance are often observed rapidly after the introduction of novel antibiotics into clinical practice.