Nutrient Transitions Are a Source of Persisters in Escherichia coli Biofilms

Christophe Beloin,Stephanie M Amato,Mark P Brynildsen

doi:10.1371/journal.pone.0093110

Abstract

Chronic and recurrent infections have been attributed to persisters in biofilms, and despite this importance, the mechanisms of persister formation in biofilms remain unclear. The plethora of biofilm characteristics that could give rise to persisters, including slower growth, quorum signaling, oxidative stress, and nutrient heterogeneity, have complicated efforts to delineate formation pathways that generate persisters during biofilm development. Here we sought to specifically determine whether nutrient transitions, which are a common metabolic stress encountered within surface-attached communities, stimulate persister formation in biofilms and if so, to then identify the pathway. To accomplish this, we established an experimental methodology where nutrient availability to biofilm cells could be controlled exogenously, and then used that method to discover that diauxic carbon source transitions stimulated persister formation in Escherichia coli biofilms. Previously, we found that carbon source transitions stimulate persister formation in planktonic E. coli cultures, through a pathway that involved ppGpp and nucleoid-associated proteins, and therefore, tested the functionality of that pathway in biofilms. Biofilm persister formation was also found to be dependent on ppGpp and nucleoid-associated proteins, but the importance of specific proteins and enzymes between biofilm and planktonic lifestyles was significantly different. Data presented here support the increasingly appreciated role of ppGpp as a central mediator of bacterial persistence and demonstrate that nutrient transitions can be a source of persisters in biofilms.

Highlights

Bacterial persisters are rare, phenotypic variants, whose hallmark characteristic is a transient, yet extraordinary, ability to tolerate antibiotics while their surrounding kin are killed. [1] Biofilms contain persisters, and this phenotypic state has been hypothesized to underlie why biofilm infections often relapse. [2,3] Persisters form during biofilm growth, and despite the identification of several important mediators, including ppGpp,[4,5,6] Lon, [4] RecA, [6] and YafQ, [7] the aspects of biofilm development that generate persisters, along with their respective pathways, remain ill-defined
We found that diauxic transitions stimulate persister formation in E. coli biofilms through a pathway that involves the ppGpp synthase, RelA, and nucleoidassociated proteins (NAPs), FIS and HU. [22,23] This pathway is qualitatively similar to the one found in planktonic cultures, with the exceptions of the removal of one ppGpp synthase to alter stringent control was sufficient to eliminate persister formation and only a subset of the NAPs involved in persister formation in planktonic conditions were found to participate in persister formation in biofilms
[31] We have previously demonstrated that diauxic carbon source transitions stimulate persister formation in planktonic E. coli cultures

Summary

Introduction

Phenotypic variants, whose hallmark characteristic is a transient, yet extraordinary, ability to tolerate antibiotics while their surrounding kin are killed. [1] Biofilms contain persisters, and this phenotypic state has been hypothesized to underlie why biofilm infections often relapse. [2,3] Persisters form during biofilm growth, and despite the identification of several important mediators, including ppGpp,[4,5,6] Lon, [4] RecA, [6] and YafQ, [7] the aspects of biofilm development that generate persisters, along with their respective pathways, remain ill-defined. We identified a persister formation pathway in response to nutrient transitions in planktonic E. coli. [5,20] Together, these phenomena suggest that nutrient transitions may be a source of persisters in biofilms. Persister formation cascades, from source of stress to antibiotic tolerance, have mainly been studied in planktonic systems, [12,14,16,21] and the extent to which these pathways operate in biofilms remains an open question. Specific genes important for biofilm persistence have been identified [4,5,6,7], but their placement in formation cascades are just beginning to be elucidated.[4,5,6]

Methods

Results

Conclusion