The Murine Lung Microbiome Changes During Lung Inflammation and Intranasal Vancomycin Treatment

Abstract

Most microbiome research related to airway diseases has focused on the gut microbiome. This is despite advances in culture independent microbial identification techniques revealing that even healthy lungs possess a unique dynamic microbiome. This conceptual change raises the question; if lung diseases could be causally linked to local dysbiosis of the local lung microbiota. Here, we manipulate the murine lung and gut microbiome, in order to show that the lung microbiota can be changed experimentally. We have used four different approaches: lung inflammation by exposure to carbon nano-tube particles, oral probiotics and oral or intranasal exposure to the antibiotic vancomycin. Bacterial DNA was extracted from broncho-alveolar and nasal lavage fluids, caecum samples and compared by DGGE. Our results show that: the lung microbiota is sex dependent and not just a reflection of the gut microbiota, and that induced inflammation can change lung microbiota. This change is not transferred to offspring. Oral probiotics in adult mice do not change lung microbiome detectible by DGGE. Nasal vancomycin can change the lung microbiome preferentially, while oral exposure does not. These observations should be considered in future studies of the causal relationship between lung microbiota and lung diseases.

Keywords: Antibiotic; Denaturing gradient gel electrophoresis; broncho-alveolar lavage; carbon nanotubes; probiotic.

Fig. (1a)

Dendrogram based on DGGE profiles representing 16S rRNA gene PCR-derived amplicons of blue BAL and red caecum samples collected from nine-weeks-old naïve female BALB/cj (n=10). DGGE: the BAL samples form a tight cluster that can be distinguished clearly from the caecum samples. Reliably separated branches on the dendrogram have a high (green) cophenetic score.

Fig. (1b)

PCA plots based on DGGE profiles of 16S rRNA gene PCR-derived amplicons of blue BAL and red caecum samples collected from nine-weeks-old naïve female BALB/cj (n=10). DGGE: denaturing gradient gel electrophoresis after DGGE-PCR: polymerase chain reaction. There is a group to the left that consists of all BAL samples clustering separately from the caecum. The variation between the BAL samples is much lower than between the caecum samples.

Fig. (2a)

Dendrogram based on DGGE profiles representing 16S rRNA gene PCR-derived amplicons of BAL samples collected from C57Bl/6 dams exposed i.n. to either PBS (blue) or CNT particles (red) before mating (n=20). Most samples are very similar, but a few samples have intense bands that are absent or weaker in the remaining samples. These samples all belong to the 4 defining CNT samples from the CNT group. Reliably separated branches on the dendrogram have a high (green) cophenetic score.

Fig. (2b)

PCA plots based on DGGE profiles of 16S rRNA gene PCR-derived amplicons of BAL samples collected from C57Bl/6 dams exposed i.n. to either PBS or CNT particles. The colors represent red: CNT dams and blue PBS dams (n=20). DGGE: denaturing gradient gel electrophoresis after DGGE-PCR: polymerase chain reaction. There is a group to the lower left that consists of both control and CNT dams, but the outliers that cluster separately are all CNT.

Fig. (2c)

Dendrogram based on DGGE profiles representing 16S rRNA gene PCR-derived amplicons of BAL samples collected from 6 weeks old C57Bl/6 mice. The mice are both naïve male and female offspring, from C57Bl/6 dams exposed i.n. to either PBS or CNT particles before mating. The colors represent red: CNT-male, pink CNT-female and dark blue PBS-male and light- blue PBS –female.(n=20). Males and females clearly cluster separately. Reliably separated branches on the dendrogram have a high (green) cophenetic score.

Fig. (2d)

PCA plots based on DGGE profiles of 16S rRNA gene PCR-derived amplicons of BAL samples collected from six weeks old C57Bl/6 mice. The mice are both male and female offspring from C57Bl/6 dams exposed i.n. to either PBS or CNT particles. The colors represent red: CNT-male, pink CNT-female and dark blue PBS-male and light- blue PBS –female (n=20). DGGE: denaturing gradient gel electrophoresis after DGGE-PCR: polymerase chain reaction.

Fig. (3a)

Dendrogram based on DGGE profiles representing 16S rRNA gene PCR-derived amplicons of caecum samples collected from eleven-week-old female BALB/cj mice. The light- blue (O) groups received oral exposure to Vancomycin from drinking water ad libitum. The red (IN) received vancomycin 0.21 mg/35µl nasally by i.n. 2x5 days. The blue PBS group received PBS 35µl nasally by i.n. 2x5 days. The band that separetes the O-group has the highest average intensity is marked with a black box. Reliably separated branches on the dendrogram have a high (green) cophenetic score.

Fig. (3b)

shows: PCA plots based on DGGE profiles of 16S rRNA gene PCR-derived amplicons of caecum samples collected from eleven-week-old female BALB/cj mice. The light-blue (O) groups received oral exposure to Vancomycin from drinking water ad libitum. The red (IN) received vancomycin 0.21 mg/35µl nasally by i.n.for 2x5 days. The blue PBS group received PBS 35µl nasally by i.n. 2x5 days. DGGE: denaturing gradient gel electrophoresis after DGGE-PCR: polymerase chain reaction.

Fig. (3c)

Dendrogram based on DGGE profiles representing 16S rRNA gene PCR-derived amplicons of BAL samples collected from eleven-week-old female BALB/cj mice. The light-blue (O) groups received oral exposure to Vancomycin from drinking water ad libitum. The red (IN) received vancomycin 0.21 mg/35µl nasally by i.n. 2x5 days. The blue PBS group received PBS 35µl nasally by i.n. 2x5 days. All IN Vancomycin exposed except one sample cluster together. Reliably separated branches on the dendrogram have a high (green) cophenetic score.

Fig. (3d)

PCA plots based on DGGE profiles of 16S rRNA gene PCR-derived amplicons of BAL samples collected from eleven-week-old female BALB/cj mice. The light-blue (O) groups received oral exposure to vancomycin from drinking water ad libitum. The red (IN) received vancomycin 0.21 mg/35µl nasally by i.n. 2x5 days. The blue PBS group received PBS 35µl nasally by i.n. 2x5 days.