Patient characteristics are shown in Table 1. During the study period, all patients required mechanical ventilation and received antibiotics intravenously. The antibiotic types are summarized in Table 1. Except for two patients, none of the patients were administered antibiotics within 3 months before study enrollment. None of the patients developed Clostridioides (Clostridium) difficile infection. Enteral nutrition was administered within 48 h from admission in 52 patients (73.2%), and 15 patients (21.1%) did not receive enteral feeding during the study period. The median duration of ICU stay was 17 (IQR: 9–37) days. In this study, 24 patients (34%) died from causes of death that included multiple organ failure in 10 patients, cardiac failure in 8 patients, and brain death in 6 patients.
Gut Microbiota at Admission and Progression of Dysbiosis During the Acute-Phase ICU stay
All sampled data are summarized in Additional file 2: Table S1. The taxonomic composition of the gut microbiota at phylum level of the first sample and the last sample in each patient and the healthy controls is shown in Fig. 1. Bacteroidetes and Firmicutes were the predominant phyla in most patients, and their proportions varied from patient to patient at the first sample. The composition of the gut microbiota dramatically changed during the ICU stay. The occupancy rates of the top 10 taxonomic compositions at phylum, class, order, family, and genus level of all patient samples are shown in Additional file 1: Fig. S2. The five major phyla in the patients were Bacteroidetes (44.1%), Firmicutes (34.1%), Proteobacteria (12.7%), Actinobacteria (4.2%), and Fusobacteria (3.1%). The proportion of each phylum at the first sample between patients with trauma, cardiac arrest, and sepsis is shown in Additional file 1: Table S2. There were no significant differences between diseases types except for Proteobacteria (p = 0.032).
The composition rate and absolute proportional changes of each phylum as well as changes in the number of OTUs in the healthy controls and the patient group on days 1–3, 4–7, and 8–14 from admission are shown in Fig. 2. The composition rate of each phylum was not significantly different between each sampling day. However, the absolute proportional differences of Bacteroidetes and Firmicutes significantly increased over time (p < 0.001; Fig. 2a, b), and the number of OTUs significantly decreased over time (p < 0.001; Fig. 2c). To compare the microbial community between the healthy controls and the patient group on each sampling day, we also performed UniFrac distance analysis of our 16S rRNA gene sequencing data. The distance between the healthy controls and the patient group on each sampling day significantly increased until day 7 (p < 0.05; Fig. 2d), which meant that the microbial flora structures gradually changed until day 7. The taxonomic composition of the gut microbiota at genus level during the ICU stay and that of the healthy controls is shown in Additional file 1: Fig. S3. There were significant changes in the proportions of Blautia, Clostridium, Coprococcus, Enterococcus, Faecalibacterium, Lacnospiraceae, Roseburia, Ruminococcus, and Streptococcus between the healthy controls and each sampling day.
These results suggested that each ICU patient had their own unique gut microbiota at admission, and the dysbiosis was gradually proceeding with the loss of diversity of gut microbiota during the acute phase of the ICU stay.
Convergence of Gut Microbiota at the Phylum Level by Administered Antibiotics
To assess the association between the use of broad-spectrum antibiotics and the gut microbiota of the ICU patients, the absolute proportional change from the first sample and the relative proportional change from the preceding sample were estimated between the carbapenem and non-carbapenem groups by use of a mixed-effect model.
The absolute proportional changes of Bacteroidetes and Firmicutes increased and stabilized approximately on the 7th hospital day in both groups (Fig. 3). Statistically significant differences were found between the carbapenem and non-carbapenem groups in Bacteroidetes from the 11th to 15th hospital day and in Firmicutes from the 7th to 14th hospital day (p < 0.05). The relative proportional changes of Bacteroidetes and Firmicutes converged almost to zero approximately on the 7th hospital day in both groups (Fig. 4).
Among the other phyla, Actinobacteria showed a significant difference in the absolute proportional change between the carbapenem and non-carbapenem groups (p = 0.030; Fig. 3). Significant differences in the absolute proportional changes in Proteobacteria from the 1st to 4th hospital day, in Actinobacteria from the 13th to 17th hospital day, and in Fusobacteria from the 14th to 17th hospital day (p < 0.05) were also found by testing for contrasts in interaction (Fig. 3). The relative proportional change of Proteobacteria converged almost to zero approximately on the 7th hospital day in both groups (Fig. 4).
To identify differences between other antibiotics, the absolute proportional changes of each phylum between the carbapenem group, penicillin group, and cephalosporin group were also estimated (Additional file 1: Fig. S4). Each phylum reached inflection points approximately on the 7th hospital day in each antibiotics group, but the differences did not reach statistical significance between the groups.
These results suggested that the major changes of the gut microbiota, especially of Bacteroidetes and Firmicutes, may be completed within the first week after admission to ICU, and broad-spectrum antibiotics may be associated with the change of the composition of gut microbiota from the early phase after ICU admission. Actinobacteria may be more sensitive to carbapenems than other phyla.
Association Between Disease Severity and the Transition of Gut Microbiota in the Acute-Phase ICU Patients
To estimate the association between disease severity and the gut microbiota of the ICU patients, the absolute proportional changes from the first sample were estimated by a linear mixed-effect model with interaction of initial SOFA score and changes in SOFA score. There was a statistically significant difference in the absolute proportional change in Actinobacteria with interaction of the initial SOFA score and changes in SOFA score during the patients’ ICU stay (p < 0.001; Fig. 5). This result suggested that disease severity may be associated with the change in the proportion of Actinobacteria during the acute phase of ICU stay.
Association Between Gut Dysbiosis and Mortality During the Acute-Phase ICU Stay
We explored whether gut dysbiosis during the acute-phase ICU stay could be associated with mortality by using CART and multivariate logistic regression analyses. The cutoff values of the maximum and minimum B/F ratios within seven days of admission were determined to be 8 and 1/8, respectively. The diagnostic characteristics of this tree have a sensitivity of 54.2% and specificity of 74.5% (Fig. 6a). ROC curve analysis using these B/F ratios for the outcome of ICU mortality was performed, and the AUC was 0.853 (Fig. 6b). Differences in the rates of ICU mortality were calculated by Kaplan–Meier analysis using these B/F ratios, and the hazard ratio was 2.41 (95% confidence interval [CI]: 1.04–5.51, p = 0.039) (Fig. 6c). Multivariate logistic regression analysis showed an association between an imbalance in B/F ratio and higher mortality when the B/F ratio was > 8 or < 1/8 (odds ratio: 5.54, 95% CI: 1.39–22.18, p = 0.015; Table 2). We also performed multivariate logistic regression analysis adjusted for APACHE II score on admission, sex, and B/F ratio (> 8 or < 1/8) of the fecal samples on the first three days from admission or at seven to nine days from admission to estimate ICU mortality (Additional file 1: Table S3). The B/F ratio on the first three days from admission was not associated with ICU mortality, whereas the B/F ratio one week after admission was associated with ICU mortality.
These results suggested that the B/F ratio within seven days from admission might be useful as an indicator of poor prognosis and that an extreme change in the composition of the gut microbiota may be associated with mortality in the ICU. Although it may be difficult to predict prognosis based on the value of the B/F ratio on admission, changes in the B/F ratio during the ICU stay may be more useful for the prediction of prognosis.