Individual bacteria in structured environments rely on phenotypic resistance to phage.

Erin L Attrill,Rory Claydon,Sean Meaden,Edze R Westra,Aidan T Brown,Sarah V Harding,Urszula Łapińska,Mario Recker,Stefano Pagliara,Victor Sourjik

doi:10.1371/journal.pbio.3001406

Abstract

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.

Highlights

Escherichia coli and its lytic phage T4 have been extensively employed to increase our molecular understanding of phage biology [9,10], and T4 has been considered for phage therapy [11,12,13]
In well-mixed environments T4 rapidly amplified, as expected, in the presence of E. coli BW25113 (S1 Fig and Data A in S1 File). This resulted in a rapid collapse of the bacterial population, followed by regrowth of the culture to a final density comparable to bacteria growing in the absence of phages, suggesting the emergence of genetically resistant mutants that could either be preexistent in the initial inoculum or have evolved during exposure to phage
When we used a green fluorescent protein (GFP) reporter strain for ompC expression in the presence of ephemeral spatial refuges, we found that the subpopulation of E. coli that was killed by T4 by 24 h (N = 108 susceptible cells) displayed an initial distribution of GFP fluorescence that was significantly higher than the one measured for the subpopulation of E. coli that survived T4 treatment (N = 42 surviving cells, Fig 5A, p-value = 0.006, Data S in S1 File)

Summary

Introduction

The emergence of antimicrobial resistance has caused a resurgence of interest in using phages in therapeutic settings [5,6], stimulating research on the arsenal of antiphage mechanisms that bacteria have evolved in order to survive [7,8]. Escherichia coli and its lytic phage T4 have been extensively employed to increase our molecular understanding of phage biology [9,10], and T4 has been considered for phage therapy [11,12,13]. T002522/1), and Natural Environment Research Council (NE/R010935/1).

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Conclusion