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Environmental and intracellular stresses can perturb protein homeostasis and trigger the formation and accumulation of protein aggregates. It has been recently suggested that the level of protein aggregates accumulated in bacteria correlates with the frequency of persister and viable but nonculturable cells that transiently survive treatment with multiple antibiotics. However, these findings have often been obtained employing fluorescent reporter strains. This enforced heterologous protein expression facilitates the visualization of protein aggregates but could also trigger the formation and accumulation of protein aggregates. Using microfluidics-based single-cell microscopy and a library of green fluorescent protein reporter strains, we show that heterologous protein expression favors the formation of protein aggregates. We found that persister and viable but nonculturable bacteria surviving treatment with antibiotics are more likely to contain protein aggregates and downregulate the expression of heterologous proteins. Our data also suggest that such aggregates are more basic with respect to the rest of the cell. These findings provide evidence for a strong link between heterologous protein expression, protein aggregation, intracellular pH, and phenotypic survival to antibiotics, suggesting that antibiotic treatments against persister and viable but nonculturable cells could be developed by modulating protein aggregation and pH regulation.

Bacteriophages represent an avenue to overcome the current antibiotic resistance crisis, but evolution of genetic resistance to phages remains a concern. In vitro, bacteria evolve genetic resistance, preventing phage adsorption or degrading phage DNA. In natural environments, evolved resistance is lower possibly because the spatial heterogeneity within biofilms, microcolonies, or wall populations favours phenotypic survival to lytic phages. However, it is also possible that the persistence of genetically sensitive bacteria is due to less efficient phage amplification in natural environments, the existence of refuges where bacteria can hide, and a reduced spread of resistant genotypes. Here, we monitor the interactions between individual planktonic bacteria in isolation in ephemeral refuges and bacteriophage by tracking the survival of individual cells. We find that in these transient spatial refuges, phenotypic resistance due to reduced expression of the phage receptor is a key determinant of bacterial survival. This survival strategy is in contrast with the emergence of genetic resistance in the absence of ephemeral refuges in well-mixed environments. Predictions generated via a mathematical modelling framework to track bacterial response to phages reveal that the presence of spatial refuges leads to fundamentally different population dynamics that should be considered in order to predict and manipulate the evolutionary and ecological dynamics of bacteria–phage interactions in naturally structured environments., Bacteriophages represent a promising avenue to overcome the current antibiotic resistance crisis, but evolution of phage resistance remains a concern. This study shows that in the presence of spatial refuges, genetic resistance to phage is less of a problem than commonly assumed, but the persistence of genetically susceptible bacteria suggests that eradicating bacterial pathogens from structured environments may require combined phage-antibiotic therapies.