Pulmonary and intestinal microbiota dynamics during Gram-negative pneumonia-derived sepsis

Abstract

Background: The gut microbiome plays a protective role in the host defense against pneumonia. The composition of the lung microbiota has been shown to be predictive of clinical outcome in critically ill patients. However, the dynamics of the lung and gut microbiota composition over time during severe pneumonia remains ill defined. We used a mouse model of pneumonia-derived sepsis caused by Klebsiella pneumoniae in order to follow the pathogen burden as well as the composition of the lung, tongue and fecal microbiota from local infection towards systemic spread.

Results: Already at 6 h post-inoculation with K. pneumoniae, marked changes in the lung microbiota were seen. The alpha diversity of the lung microbiota did not change throughout the infection, whereas the beta diversity did. A shift between the prominent lung microbiota members of Streptococcus and Klebsiella was seen from 12 h onwards and was most pronounced at 18 h post-inoculation (PI) which was also reflected in the release of pro-inflammatory cytokines indicating severe pulmonary inflammation. Around 18 h PI, K. pneumoniae bacteremia was observed together with a systemic inflammatory response. The composition of the tongue microbiota was not affected during infection, even at 18-30 h PI when K. pneumoniae had become the dominant bacterium in the lung. Moreover, we observed differences in the gut microbiota during pulmonary infection. The gut microbiota contributed to the lung microbiota at 12 h PI, however, this decreased at a later stage of the infection.

Conclusions: At 18 h PI, K. pneumoniae was the dominant member in the lung microbiota. The lung microbiota profiles were significantly explained by the lung K. pneumoniae bacterial counts and Klebsiella and Streptococcus were correlating with the measured cytokine levels in the lung and/or blood. The oral microbiota in mice, however, was not influenced by the severity of murine pneumonia, whereas the gut microbiota was affected. This study is of significance for future studies investigating the role of the lung microbiota during pneumonia and sepsis.

Keywords: Dynamics over time; Gut microbiota; Klebsiella pneumoniae; Lung microbiota; Mice; Microbiome; Pneumonia; Sepsis; Tongue.

Fig. 1

Organ bacterial loads and cytokine levels during K. pneumoniae infection. a Experimental design. Mice received an intranasal inoculation with 10⁴ colony forming units (CFU) of K. pneumoniae. A group was killed every 6 h, until 36 h (n = 10–12) at which time blood, lung, tongue and feces were collected, t = 0 serves as the control group. This model becomes lethal approximately 40 h post-inoculation (a). Pulmonary (b) and blood (c) CFU at 0 to 36 h post-inoculation. Tumor necrosis factor (TNF) of lung homogenate (d) and blood plasma (e) at 0 to 36 h post-inoculation. Data are shown as median, the top bar denotes at which time-points the data are significantly different from the 0-h group, the stars show the range of significance, p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****)

Fig. 2

Lung microbiota dynamics after inoculation with 1 × 10⁴ CFU of K. pneumoniae. Samples were analyzed at time is 0, 6, 12, 18, 24 and 30 h, with n = 12 mice. a Shannon index for alpha diversity. b Principal coordinates analysis (PCoA) of Bray–Curtis dissimilarities of the lung microbiota. c Relative abundances of top 10 most abundant genera. d The most prevalent genera Streptococcus and Klebsiella have been plotted with their relative abundances and standard deviations over time

Fig. 3

Alpha and beta diversity of the lung, tongue and fecal microbiotas. Mice received an intranasal inoculation with 10⁴ colony forming units (CFU) of K. pneumoniae. Mice were killed at 0, 12, and 30 h post-inoculation (n = 12), lung tongue and feces were extracted for microbiota analysis. a Alpha diversity; Shannon of lung tongue and fecal samples at 0, 12 and 30 h post-inoculation. b Beta diversity; PCoA Bray analysis of lung tongue and feces over time. c Genus distribution for lung tongue and fecal microbiota at 0, 12, 30 h post-inoculation, where the top 10 genera in the lung are colored, other genera are grey. d Fast expectation–maximization for microbial source tracking (FEAST) analysis on the lung microbiota showing which percentage of the lung microbiota can be traced back to the tongue, feces and unknown source at 0, 12 and 30 h post-inoculation, in a dataset without K. pneumoniae