Gut microbes improve prognosis of Klebsiella pneumoniae pulmonary infection through the lung-gut axis

Abstract

Background: The gut microbiota plays a vital role in the development of sepsis and in protecting against pneumonia. Previous studies have demonstrated the existence of the gut-lung axis and the interaction between the gut and the lung, which is related to the prognosis of critically ill patients; however, most of these studies focused on chronic lung diseases and influenza virus infections. The purpose of this study was to investigate the effect of faecal microbiota transplantation (FMT) on Klebsiella pneumoniae-related pulmonary infection via the gut-lung axis and to compare the effects of FMT with those of traditional antibiotics to identify new therapeutic strategies.

Methods: We divided the mice into six groups: the blank control (PBS), pneumonia-derived sepsis (KP), pneumonia-derived sepsis + antibiotic (KP + PIP), pneumonia-derived sepsis + faecal microbiota transplantation(KP + FMT), antibiotic treatment control (KP+PIP+PBS), and pneumonia-derived sepsis+ antibiotic + faecal microbiota transplantation (KP + PIP + FMT) groups to compare the survival of mice, lung injury, inflammation response, airway barrier function and the intestinal flora, metabolites and drug resistance genes in each group.

Results: Alterations in specific intestinal flora can occur in the gut of patients with pneumonia-derived sepsis caused by Klebsiella pneumoniae. Compared with those in the faecal microbiota transplantation group, the antibiotic treatment group had lower levels of proinflammatory factors and higher levels of anti-inflammatory factors but less amelioration of lung pathology and improvement of airway epithelial barrier function. Additionally, the increase in opportunistic pathogens and drug resistance-related genes in the gut of mice was accompanied by decreased production of favourable fatty acids such as acetic acid, propionic acid, butyric acid, decanoic acid, and secondary bile acids such as chenodeoxycholic acid 3-sulfate, isodeoxycholic acid, taurodeoxycholic acid, and 3-dehydrocholic acid; the levels of these metabolites were restored by faecal microbiota transplantation. Faecal microbiota transplantation after antibiotic treatment can gradually ameliorate gut microbiota disorder caused by antibiotic treatment and reduce the number of drug resistance genes induced by antibiotics.

Conclusion: In contrast to direct antibiotic treatment, faecal microbiota transplantation improves the prognosis of mice with pneumonia-derived sepsis caused by Klebsiella pneumoniae by improving the structure of the intestinal flora and increasing the level of beneficial metabolites, fatty acids and secondary bile acids, thereby reducing systemic inflammation, repairing the barrier function of alveolar epithelial cells, and alleviating pathological damage to the lungs. The combination of antibiotics with faecal microbiota transplantation significantly alleviates intestinal microbiota disorder, reduces the selection for drug resistance genes caused by antibiotics, and mitigates lung lesions; these effects are superior to those following antibiotic monotherapy.

Keywords: FMT; Klebsiella pneumoniae; antibiotheraphy; drug resistance gene; gut microbes; pulmonary infection.

Figure 1

(A). Fecal microbiota transplantation appropriately restores changes in gut microbes diversity induced by pulmonary infections. Fresh feces from mice of PBS, KP, KP+PIP and KP+FMT group at 12, 24 and 48 hours were obtained for metagenomic sequencing and the diversity of the gut microbes in each group was compared using the Mothur calculation (n = 6). (B). The composition of gut microbes was significantly different after antibiotic treatment. Principal component analysis (PcoA) was performed using R vegan, thus visualizing the differences in gut microbes β-diversity between PBS, KP, KP+PIP and KP+FMT group (n = 6). (C). The gut microbes is dominated by opportunistic pathogens after antibiotic treatment at the phylum level. Based on the Bray-Curtis algorithm, QIIME was used to calculate the distance between the samples to obtain the Bray-Curtis distance matrix, based on which hierarchical clustering analysis was performed, and then unweighted group averaging algorithm was used to construct a tree structure for visual analysis (n = 6). (D). The intestinal flora is dominated by favorable flora after fecal microbiota transplantation, whereas opportunistic pathogens predominate after antibiotic therapy. Differences in the composition of gut microbes from PBS, KP, KP+PIP and KP+FMT group at the genus level were analyzed using LEfSe (LDA Effect Size) analysis to look for differential flora between the groups (n = 6).

Figure 2

Both fecal microbiota transplantation and post antibiotic fecal microbiota transplantation were effective in improving mortality rates. Prism software was used to create survival curves by Kaplan and Meier’s product limit method and to compare the survival curves of PBS, KP, KP+PIP and KP+FMT groups using the log-rank test and Gehan - Wilcoxon test(* p<0.05, ** p<0.01)(n = 6).

Figure 3

(A). Fecal microbiota transplantation and antibiotics are effective in ameliorating local pathological damage in the lungs. HE staining was used to stain lung tissue sections of PBS, KP, KP+PIP and KP+FMT groups to observe the local pathological damage of lungs in each group (n = 6), magnification: ×200, scale bar = 100 μm. (B). Faecal microbiota transplantation improves pulmonary local pathological injury more than antibiotic therapy. The Smith semi-quantitative scoring system was used to quantify five aspects of lung oedema, alveolar and interstitial hemorrhage, alveolar and interstitial inflammation, lung atelectasis and hyaline membrane formation in the HE staining results of the lung tissues of each group of mice (n = 6)(ns, P>0.05, * P<0.05, ** P<0.01).

Figure 4

(A). The ability of antibiotic treatment to reduce pro-inflammatory factors and increase anti-inflammatory factors in serum is greater than that of fecal microbiota transplantation. The levels of pro-inflammatory factors TNF-ɑ, IFN-γ, IL-6 and IL-1β and anti-inflammatory factor IL-10 in serum of mice at 12, 24 and 48 hours between PBS, KP, KP+PIP and KP+FMT groups were compared (n = 6)(ns, P>0.05, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001). (B). The ability of antibiotic treatment to reduce pro-inflammatory factors and increase anti-inflammatory factors in alveolar lavage fluid is greater than that of fecal microbiota transplantation. The levels of pro-inflammatory factors TNF-ɑ, IFN-γ, IL-6 and IL-1β and anti-inflammatory factor IL-10 in alveolar lavage fluid of mice at 12, 24 and 48 hours between PBS, KP, KP+PIP and KP+FMT groups were compared (n = 6)(ns, P>0.05, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001). (C). Both fecal microbiota transplantation and antibiotic therapy reduce the expression of apoptotic protein cleaved-caspase 3 in the airway mucosal barrier. The expression of the apoptotic protein cleaved-caspase 3 in alveolar epithelial cells of mice in PBS, KP, KP+PIP and KP+FMT groups were determined by immunohistochemistry at 12,24 and 48 hours (n = 6).Yellow color indicates positive cells and blue color indicates negative cells, magnification: ×200, scale bar = 100 μm. (D). Fecal microbiota transplantation is more effective than antibiotic therapy in reducing the expression of apoptotic protein cleaved-caspase 3 in alveolar epithelial cells. Positive cell counts were performed under a light microscope in three fields of vision in 12-, 24 and 48 hour immunohistochemical sections in PBS, KP, KP+PIP and KP+FMT groups. Positive cell count ratios were used to represent the expression level of the apoptotic protein Cleaved-Caspase3 in each group (n = 6) (ns, P>0.05, ** P<0.01, *** P<0.001, **** P<0.0001). (E). Both fecal microbiota transplantation and antibiotic therapy elevate connexin expression in the airway mucosal barrier. Immunofluorescence analysis of the airway mucosal barrier protein expression of ZO-1 and E-cadherin in PBS, KP, KP+PIP and KP+FMT groups at 48 hours (n = 6). Red labels represent ZO-1, green labels represent E-cadherin, magnification: ×200, scale bar = 100 μm. (F). Fecal microbiota transplantation is more effective than antibiotic treatment in elevating the levels of expression of ZO-1 in the airway mucosal barrier. Images were collected under a fluorescence microscope, and three fields of vision were selected at 12, 24, and 48 hours for PBS, KP, KP+PIP and KP+FMT groups. Positive cell areas were calculated, and positive area ratios were used to represent the levels of expression of ZO-1 in each group (n = 6) (ns, P>0.05, * P<0.05, ** P<0.01). (G). Fecal microbiota transplantation effectively restores the levels of expression of airway mucosal barrier connexin E-cadherin induced by lung infection. Images were collected under a fluorescence microscope, and three fields of vision were selected at 12, 24, and 48 hours for PBS, KP, KP+PIP and KP+FMT groups. Positive cell areas were calculated, and positive area ratios were used to represent the levels of expression of E-cadherin in each group (n = 6) (ns, P>0.05, * P<0.05, ** P<0.01).

Figure 5

(A). Fecal microbiota transplantation is effective in restoring levels of beneficial intestinal metabolites of fatty acids induced by lung infection relative to antibiotic therapy. Levels of beneficial intestinal metabolites fatty acids were determined by macrogenomics in PBS, KP, KP+PIP and KP+FMT groups (n = 6). Significant differences were found in acetic acid, propionic acid, butyric acid and propionic acid (* P<0.05, ** P<0.01). (B). Fecal microbiota transplantation is effective in restoring levels of beneficial intestinal metabolites of secondary bile acids induced by lung infection relative to antibiotic therapy. Levels of beneficial intestinal metabolites secondary bile acids were determined by macrogenomics in PBS, KP, KP+PIP and KP+FMT groups (n = 6). Significant differences were found in chenodeoxycholic acid 3-sulfate, isodeoxycholic acid, taurodeoxycholic acid and 3-dehydrocholic acid. (C). Fecal microbiota transplantation can increase the level of beneficial metabolites in the gut by improving the structure of the gut microbes, thereby increasing the level of beneficial metabolites in the gut. Heatmap was used to represent the correlation between specific differential gut microbes and metabolites between PBS, KP, KP+PIP and KP+FMT groups (n = 6). The horizontal axis represents differential metabolites, the vertical axis represents differential flora, green is a positive correlation, red is a negative correlation, and the darker the color, the greater the correlation (* P<0.05, ** P<0.01, *** P<0.001).

Figure 6

(A). Antibiotic treatment significantly increases levels of drug-resistant genes in the gut of mice. Violinplot was used to represent the abundance of drug-resistance genes in the total gut microbiota of each group in PBS, KP, KP + PIP and KP + FMT groups (n = 6), and the comparison of the abundance of drug-resistance genes in the gut microbiota of the individual samples in each group. (B). Combined fecal microbiota transplantation after antibiotics is effective in improving gut microbiological structure. Differences in the composition of gut microbes from KP+PIP+PBS and KP+PIP+FMT groups were analyzed using LEfSe (LDA Effect Size) analysis to look for differential flora between the groups (n = 6). (C). Combined fecal microbiota transplantation after antibiotics is effective in reducing the abundance of drug-resistance genes generated during antibiotic therapy. Violinplot was used to represent the abundance of drug-resistance genes in the total gut microbiota of KP+PIP+PBS and KP+PIP+FMT groups (n = 6), and the comparison of the abundance of drug-resistance genes in the gut microbiota of the individual samples in each group. (D). After treatment with antibiotics combined with fecal microbiota transplantation, the differential gut microbes of mice was negatively correlated with drug-resistance genes. Correlation analysis of differential gut microbes and drug-resistance genes between groups KP+PIP+PBS and KP+PIP+FMT groups using bubble connectivity plots, with positive correlation in green and negative correlation in red (n = 6). (E). Combined fecal microbiota transplantation after antibiotics had a greater ability to reduce pro-inflammatory factors as well as elevate anti-inflammatory factors in serum than antibiotic monotherapy. The levels of pro-inflammatory factors TNF-ɑ, IFN-γ, IL-6 and IL-1β and anti-inflammatory factor IL-10 in serum of mice at 12, 24 and 48 hours between KP+PIP+PBS and KP+PIP+FMT groups were compared (n = 6)(ns, P>0.05, * P<0.05, ** P<0.01, *** P<0.001). (F). Combined fecal microbiota transplantation after antibiotics had a greater ability to reduce pro-inflammatory factors as well as elevate anti-inflammatory factors in alveolar lavage fluid than antibiotic monotherapy. The levels of pro-inflammatory factors TNF-ɑ, IFN-γ, IL-6 and IL-1β and anti-inflammatory factor IL-10 in alveolar lavage fluid of mice at 12, 24 and 48 hours between KP+PIP+PBS and KP+PIP+FMT groups were compared (n = 6)(ns, P>0.05, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001). (G). Combined fecal microbiota transplantation after antibiotics reduces pulmonary local pathological damage similarly to antibiotic monotherapy. HE staining was used to stain lung tissue sections of KP+PIP+PBS and KP+PIP+FMT groups to observe the local pathological damage of lungs in each group (n = 6), magnification: ×200, scale bar = 100 μm. The Smith semi-quantitative scoring system was used to quantify five aspects of lung oedema, alveolar and interstitial hemorrhage, alveolar and interstitial inflammation, lung atelectasis and hyaline membrane formation in the HE staining results of the lung tissues of each group of mice (n = 6)(ns, P>0.05, * P<0.05, ** P<0.01). (H). Combined fecal microbiota transplantation after antibiotics reduces apoptotic proteins cleaved-caspase 3 in airway epithelial cells similarly to antibiotic monotherapy. The expression of the apoptotic protein cleaved-caspase 3 in alveolar epithelial cells of mice in KP+PIP+PBS and KP+PIP+FMT groups were determined by immunohistochemistry (n = 6), magnification: ×200, scale bar = 100 μm. Positive cell count ratios were used to represent the expression level of the apoptotic protein Cleaved-Caspase3 in each group (n = 6) (ns, P>0.05). (I). Combined fecal microbiota transplantation after antibiotics elevate connexin expression in the airway mucosal barrier similarly to antibiotic monotherapy. Immunofluorescence analysis of the airway mucosal barrier protein expression of ZO-1 and E-cadherin in KP+PIP+PBS and KP+PIP+FMT groups at 48 hours, magnification: ×200, scale bar = 100 μm. Positive cell areas were calculated, and positive area ratios were used to represent the levels of expression of ZO-1 (A) and E-cadherin (B) in each group (n = 6) (ns, P>0.05, * P<0.05).