Antibiotic resistance mediated by gene amplifications

Abstract

Apart from horizontal gene transfer and sequence-altering mutational events, antibiotic resistance can emerge due to the formation of tandem repeats of genomic regions. This phenomenon, also known as gene amplification, has been implicated in antibiotic resistance in both laboratory and clinical scenarios, where the evolution of resistance via amplifications can affect treatment efficacy. Antibiotic resistance mediated by gene amplifications is unstable and consequently can be difficult to detect, due to amplification loss in the absence of the selective pressure of the antibiotic. Further, due to variable copy numbers in a population, amplifications result in heteroresistance, where only a subpopulation is resistant to an antibiotic. While gene amplifications typically lead to resistance by increasing the expression of resistance determinants due to the higher copy number, the underlying mechanisms of resistance are diverse. In this review article, we describe the various pathways by which gene amplifications cause antibiotic resistance, from efflux and modification of the antibiotic, to target modification and bypass. We also discuss how gene amplifications can engender resistance by alternate mutational outcomes such as altered regulation and protein structure, in addition to just an increase in copy number and expression. Understanding how amplifications contribute to bacterial survival following antibiotic exposure is critical to counter their role in the rise of antimicrobial resistance.

Fig. 1. The formation of amplifications is a two-step process.

In the first step, a region of the genome (green rectangles) is duplicated, either by (left) homologous recombination between flanking repeat sequences (red segments) typically mediated by RecA (although alternate mechanisms may also play a role), or (right) via non-homologous recombination. Due to the resulting homology, RecA-mediated homologous recombination can then generate additional copies leading to tandem gene amplifications.

Fig. 2. Gene amplifications lead to heteroresistance.

Gene amplifications typically result in copy number variability of the amplified region in cells within a population when exposed to an antibiotic. Subsequent extended growth in high concentrations of the antibiotic will allow only the subpopulation with higher copy numbers to survive and grow, leading to heteroresistance, while extended antibiotic-free growth can result in instability and loss of the amplifications, reducing any potential fitness costs.

Fig. 3. Gene amplifications often provide high-frequency evolutionary trajectories.

In S. aureus, exposure to the antibiotic ciprofloxacin (CPX) that primarily targets the topoisomerase IV enzyme can select for resistance either via topoisomerase IV mutations or due to gene amplification of the efflux pump-encoding norA gene (blue rectangle). When norA is flanked by two identical transposable elements (red segments), homologous-recombination mediated gene amplifications provide the high-frequency evolutionary path (denoted by the thicker arrow). Similarly, exposure to the dual-targeting antibiotic delafloxacin (DLX) selects for resistance either via dual topoisomerase IV and gyrase mutations or due to the more frequent non-homologous recombination mediated amplification of the efflux pump-encoding sdrM gene (green rectangle). Thus, in both cases, gene amplifications provide the more common evolutionary trajectory instead of single or dual target mutations respectively.

Fig. 4. Mechanisms of antibiotic resistance via gene amplifications.

Gene duplications and amplifications can lead to antibiotic resistance by a variety of mechanisms including increased drug efflux, drug inactivation via overexpression of inactivating enzymes, target modification by increased expression of modifying enzymes or modifying target structure, and target bypass via overexpression of the target itself, drug-sequestering proteins, alternate low-susceptibility targets, or substrates of the antibiotic target. While amplifications most often function by increasing expression through higher copy numbers, amplifications can also result in altered regulation or protein structure, leading to resistance.