Microevolution in response to transient heme-iron restriction enhances intracellular bacterial community development and persistence.

Rachael L Hardison,Kevin M Mason,Derek R Heimlich,Alistair Harrison,Meghan E O’Bryan,Robert P Sebra,Rachel M Wallace,Sheryl S Justice,Heather W Pinkett,Vincent T Lee

doi:10.1371/journal.ppat.1007355

Abstract

Bacterial pathogens must sense, respond and adapt to a myriad of dynamic microenvironmental stressors to survive. Adaptation is key for colonization and long-term ability to endure fluctuations in nutrient availability and inflammatory processes. We hypothesize that strains adapted to survive nutrient deprivation are more adept for colonization and establishment of chronic infection. In this study, we detected microevolution in response to transient nutrient limitation through mutation of icc. The mutation results in decreased 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in nontypeable Haemophilus influenzae (NTHI). In a preclinical model of NTHI-induced otitis media (OM), we observed a significant decrease in the recovery of effusion from ears infected with the icc mutant strain. Clinically, resolution of OM coincides with the clearance of middle ear fluid. In contrast to this clinical paradigm, we observed that the icc mutant strain formed significantly more intracellular bacterial communities (IBCs) than the parental strain early during experimental OM. Although the number of IBCs formed by the parental strain was low at early stages of OM, we observed a significant increase at later stages that coincided with absence of recoverable effusion, suggesting the presence of a mucosal reservoir following resolution of clinical disease. These data provide the first insight into NTHI microevolution during nutritional limitation and provide the first demonstration of IBCs in a preclinical model of chronic OM.

Highlights

The host sequesters essential micronutrients as a mechanism to limit bacterial infection [1]
The ability to adapt to new microenvironments is consistent with the predominance of Nontypeable Haemophilus influenzae (NTHI) as a causative agent of otitis media (OM) in children
We evaluated the microevolution of NTHI associated with adaptation and persistence in response to nutrient limitation

Summary

Introduction

The host sequesters essential micronutrients (i.e. metals) as a mechanism to limit bacterial infection [1]. Bacteria that require exogenous nutrients for growth are subjected to stresses that necessitate adaptation for survival. Bacteria can adapt to these stresses through transcriptional, epigenetic and genetic mechanisms [1]. Identification of genes subject to microevolution provides insight into the nutritional and environmental stresses incurred during infection as well as new therapeutic targets to quell infection. During ascent from the nasopharynx to the middle ear, NTHI experiences fluctuations in nutrient availability, heme-iron. The lack of sufficient heme-iron in the middle ear is indicated by the increase in the number of iron-uptake genes in NTHI strains isolated from children with otitis media [(OM); [4]]. We previously demonstrated that transient heme-iron restriction of NTHI potentiates changes in bacterial morphology, biofilm architecture, disease severity and provides a survival advantage in the preclinical model of OM [7]

Methods

Results

Discussion

Conclusion