Environmentally determined differences in the murine lung microbiota and their relation to alveolar architecture.

Yeojun Yun,Miriam Linnenbrink,John F Baines,Girish Srinivas,Safa Alnahas,Ulrich Steinhoff,Sven Kuenzel,Kenneth D Bruce,Ulrich E Schaible,Yuanpu Peter Di

doi:10.1371/journal.pone.0113466

Abstract

Commensal bacteria control the micro-ecology of metazoan epithelial surfaces with pivotal effect on tissue homeostasis and host defense. In contrast to the upper respiratory tract, the lower respiratory tract of healthy individuals has largely been considered free of microorganisms. To understand airway micro-ecology we studied microbiota of sterilely excised lungs from mice of different origin including outbred wild mice caught in the natural environment or kept under non-specific-pathogen-free (SPF) conditions as well as inbred mice maintained in non-SPF, SPF or germ-free (GF) facilities. High-throughput pyrosequencing of reverse transcribed 16S rRNA revealed metabolically active murine lung microbiota in all but GF mice. The overall composition across samples was similar at the phylum and family level. However, species richness was significantly different between lung microbiota from SPF and non-SPF mice. Non-cultivatable Betaproteobacteria such as Ralstonia spp. made up the major constituents and were also confirmed by 16S rRNA gene cloning analysis. Additionally, Pasteurellaceae, Enterobacteria and Firmicutes were isolated from lungs of non-SPF mice. Bacterial communities were detectable by fluorescent in situ hybridization (FISH) at alveolar epithelia in the absence of inflammation. Notably, higher bacterial abundance in non-SPF mice correlated with more and smaller size alveolae, which was corroborated by transplanting Lactobacillus spp. lung isolates into GF mice. Our data indicate a common microbial composition of murine lungs, which is diversified through different environmental conditions and affects lung architecture. Identification of the microbiota of murine lungs will pave the path to study their influence on pulmonary immunity to infection and allergens using mouse models.

Highlights

Human-associated microbiota have been characterized at many body sites including the gut [1] and epithelial surfaces including the skin [2], vagina [3], oral cavity [4] and upper respiratory tract (RT) [5]
Nucleic acids were extracted directly from different mouse populations originating from the wild or from animal suppliers and held under GF, SPF or non-SPF conditions. Lungs of these mice were compared to those of outbred Mus musculus domesticus populations derived from the wild, either housed under non-SPF conditions or sampled directly in the natural environment. cDNA from RNA extracts was used as template for 454 pyrosequencing using the ‘926r’ 16S rRNA primer for reverse transcription, since direct 16S rRNA gene amplification by Polymerase chain reaction (PCR) from lung tissue DNA failed, most likely due to the low amount of bacterial nucleic acid template in the tissue samples [7]
From these cDNA templates, a total of 90,662 16S rRNA gene region sequences averaging 3,022, were generated per sample from 30 excised lungs. These results revealed distinct bacterial populations in murine lungs, with Betaproteobacteria dominating over Firmicutes, Gammaproteobacteria, Epsilonproteobacteria and Actinobacteria (Figure 1)

Summary

Introduction

Human-associated microbiota have been characterized at many body sites including the gut [1] and epithelial surfaces including the skin [2], vagina [3], oral cavity [4] and upper respiratory tract (RT) [5]. Of these the gut, oral cavity and upper respiratory tract are heavily colonized by a diverse range of bacterial species in healthy individuals [6]. Despite the careful precautions taken in the previous study, a recurring concern is over the potential for contamination arising from the passage of lower respiratory tract samples through the heavily colonized regions of the upper airways and oral cavity

Methods

Results

Conclusion