Antibiotic resistance is a growing problem worldwide. Although it has not been studied in as much depth, bacterial persistence offers another possible reason for antibiotic treatment failure. In a recent publication and webinar, Bram Van den Bergh, a postdoctoral researcher at the VIB-KU Leuven Center for Microbiology, describes using Inscripta technology to study how population bottlenecks can influence the evolution of persistence in bacterial cell population. Here are some highlights from the webinar.
Bacterial persistence is a specific type of tolerance that allows a subset of cells to survive antibiotics treatments. Treating cells with a bactericide results in a biphasic killing pattern where most cells are killed off, but a subset of the population (called persisters) survives even under prolonged treatment. Van den Bergh described persistence as a hedging strategy that protects bacteria against the disastrous effects of antibiotics. Evidence suggests that persistence may even be responsible for the chronic nature of some infections.
The mechanisms that bacteria use to develop persistence have not been studied as much as those of resistance. To gain insight into some factors that drive emergence of persistence, VIB researchers studied how population bottlenecks influence persistence evolution. They created artificial bottlenecks via serial dilutions, evolved these populations to establish a persistence phenotype and then sequenced them to estimate mutation frequencies. They found that populations that evolved from smaller bottlenecks were characterized by low-frequency mutations and more interpopulation diversity, pointing toward a rugged underlying fitness landscape. However, studying the evolution of persistence in this way was not ideal due to high variability of the starting populations. The researchers needed a more controlled setup.
This led them to Inscripta who could help them generate more controlled and reproducible libraries. For this next round of experiments, the team used libraries created with Inscripta’s genome engineering technology — which included a knockout library containing more than 4,000 E. coli mutants and a noncoding RNA library with 87 knockout mutants – to mimic the conditions of bottlenecking. They performed two rounds of laboratory evolution with six bottlenecks, using eight replicates per bottleneck. In contrast, to have the same degree of certainty, they would have to use up to 40 replicates per condition in the traditional experimental setup.
The results confirmed the previous conclusions and delivered new insights about the topography of the fitness landscape and genes implicated in the emergence persistence, with significantly reduced experimental effort. Van den Bergh and his team continue to study the evolution of bacterial persistence and are using Inscripta’s automated Onyx™ platform for genome engineering to create gene saturation libraries. If you want to learn more, watch the highlights from the webinar below or full webinar on demand.