Patient characteristics

A single fecal sample was collected from 34 critically ill patients (mean age 64 years; 48% male) between 1 and 21 days after ICU admittance [median 4.5 (IQR 2.8–7) days; ESM Tables S1 and S2]. Of these 34 patients, 25 were admitted because of sepsis and one had a non-infectious diagnosis (ESM Table S1). Fifteen healthy subjects served as controls.

Microbial composition of fecal samples

Compared to healthy subjects, the stool microbiota showed a highly abnormal composition in most patients, based on both the highest bacterial taxonomic ranks (phylum level) and lowest detectable ranks (genus level).

Phylum level

To obtain a global view of the fecal microbiota in critically ill patients, we first investigated microbiota composition at the phylum level (Fig. 1). Firmicutes and Bacteroidetes are the predominant phyla in a stable intestinal microbiota, which was confirmed in our healthy subjects with these two phyla combined constituting 89–98% of all bacteria. However, in 12 of the 34 patients, Firmicutes and Bacteroidetes constituted <89% of all bacteria and even constituted as low as <1% in one sample (p < 0.0001, healthy controls vs. ICU patients). This was the case in both septic and non-septic patients. A relative high abundance of Proteobacteria was observed in approximately one-third of patients (p = 0.02, healthy controls vs. ICU patients). This phylum of Gram-negative bacteria includes many pathogenic and lipopolysaccharide-containing bacteria, such as Escherichia. There was a trend towards an increased ratio of Gram-negative to Gram-positive bacteria in ICU patients (p = 0.0658, healthy vs. ICU patients).

Fig. 1 High interindividual diversity in fecal microbiota composition at the phylum level in both septic and non-septic critically ill patients. A single fecal sample was collected from septic and non-septic intensive care unit (ICU) patients and healthy subjects. Total bacterial 16S rDNA was isolated and sequenced to investigate the bacterial composition of these samples. Each bar represents the microbiota composition of one individual patient (patient number indicated at bottom of each bar) at the phylum level, which is the highest bacterial taxonomic rank. Data are presented as the percentage of total 16S rDNA reads in each sample; colors indicate different phyla. Patients are grouped according to their main diagnosis. Table shows which classes of antibiotics each patient received during their stay in the intensive care unit prior to fecal sampling. Topical P/T/A Topical application of polymyxin E, tobramycin, and amphotericin B. None of the healthy subjects had received antibiotics for at least 12 months prior to fecal sampling Full size image

While it is known that intestinal microbiota is highly personalized, some very striking individual patterns were observed. The fecal sample of patient no. 12 (who had an abdominal Escherichia coli sepsis) showed a high abundance of Synergistetes and those of patient nos. 4 and 18 revealed a high abundance of Verrucomicrobia (discussed in more detail further in the text.

Genus level

Our analysis of bacterial composition at the genus level (ESM Fig. S1) revealed an even greater interpatient diversity than that seen at the phylum level. Septic and non-septic patients did however share some common characteristics when compared to healthy subjects. Most notably, several key bacterial genera that each represent around 5% of the microbiota in healthy subjects had disappeared almost completely, including Faecalibacterium, Blautia, Ruminococcus, Subdoligranulum and Pseudobutyrivibrio (all p < 0.0001, healthy controls vs. ICU patients).

No single bacterial genus colonized >50% of the microbiota in the healthy subjects, whereas this did occur in 13 of the 34 patients, with one genus comprising even >75% of the microbiota in four patients (ESM Fig. S1). These dominating genera included the usual suspects, such as Enterococcus (p = 0.0002 healthy controls vs. ICU patients). Members of Staphylococcus and Escherichia/Shigella were also significantly more abundant in the ICU patients, especially in the abdominal sepsis group (p = 0.042 and p = 0.049, respectively, healthy controls vs. ICU patients; p = 0.0048 and p = 0.001, respectively, healthy controls vs. ICU patients with abdominal sepsis). A canonical correspondence analysis to deduce the major driving forces in the variability within all microbial data indicated that fecal microbiota from abdominal sepsis patients differed the most from that of the other patient groups (Fig. 2).

Fig. 2 High interindividual diversity in septic critically ill patients with sepsis. a Pearson correlations were calculated to investigate the level of similarity in microbiota composition between patients with a critical illness of different origin (sepsis originating from lung, abdomen, urinary tract, or other location and non-septic critical illness). Patient groups are indicated by colors. Healthy individuals are depicted as additional control group. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.001 vs. healthy controls; p = 0.0005 for all septic patients vs. healthy controls. b A canonical correspondence analysis to deduce the major driving forces (Axis 1, Axis 2) in the variability within the microbial data of all ICU patients, thereby indicating whether samples are alike (in close proximity to each other) or not (increased distance). The variance is indicated as percentages. Symbols/colors represent patient groups as indicated (see a) Full size image

Surprisingly, in some patients bacteria belonging to Lactobacillus spp. (p = 0.051, healthy controls vs. ICU patients with pulmonary sepsis; p = 0.06, healthy controls vs. ICU patients with abdominal sepsis) and Akkermansia spp. (of the phylum Verrucomicrobia; no statistically significant correlation with sepsis) were abundantly present. Notably, A. muciniphila is known for immune stimulation and barrier function improvement [21, 22]. The phylum Synergistetes consists of anaerobic Gram-negative bacteria that have been associated with periodontal disease; those in sample 12 belong to the genus Pyramidobacter. The significance of Pyramidobacter in fecal microbiota is as yet unknown, but the oral isolate P. piscolens is known to produce hydrogen sulphide, which is implied in compromising gut barrier function [22, 23]. Overall, the similarity between samples from septic patients (Pearson correlation) was significantly lower than that of controls (p = 0.0005 healthy controls vs. septic patients; Fig. 2).

In patients undergoing allogeneic HSCT, intestinal domination by vancomycin-resistant enterococci precedes bloodstream infection with these bacteria [24]. We did not observe any correspondence between intestinal abundance and clinical infections (data not shown). Likewise, no cases of C. difficile were recorded.

Antibiotics

All patients received SDD consisting of an oral paste and solution containing non-absorbable polymyxin E, tobramycin, and amphotericin B as well as a third-generation cefalosporin administered intravenously, unless the latter was deemed unnecessary by the treating physician as the patient was already being treated with another broad-spectrum antibiotic (Fig. 1). All septic patients were treated with additional antibiotics, varying from one to ten different classes of antimicrobial agents (Fig. 1; detailed description in ESM Table S2). Septic patients were treated with a significantly higher number of antibiotic classes than were non-septic critically ill patients (p < 0.01), but no differences in microbiota composition were observed between these two groups, both at the phylum and genus levels.

Association between microbiota composition and clinical outcome parameters

To explore whether microbiota-related markers could be related to outcome parameters, we used three parameters that are commonly used to describe the microbiota, namely, bacterial diversity, the Firmicutes/Bacteroidetes ratio (F/B-ratio), and the Gram-positive/Gram-negative bacteria ratio [5, 11, 25, 26].

We expected ultra-low bacterial diversity in our patient cohort based on the exposure of these patients to antibiotics and data reported from earlier studies [5, 13]. The critically ill patients in our study had significantly lower bacterial diversity than the control subjects (p < 0.0001; Fig. 3). There was no correlation between the number of antibiotic classes that a patient had received and bacterial diversity (Fig. 3), nor between the number of days between admission and fecal sampling and bacterial diversity (data not shown). When the cohort was divided into two groups with low bacterial diversity (Shannon index < 4.0) and normal diversity (Shannon index > 4.0), there was no intergroup difference in the occurrence of complications such as AKI and ARDS or length of ICU and hospital stay (Fig. 3; ESM Table S3). In accordance with these results, both short- and long-term mortality were not associated with microbiota diversity. Both patient groups were comparable in terms of baseline characteristics, severity of disease, and classes of antibiotics received prior to fecal sampling (Table 1).

Fig. 3 Decreased intestinal microbiota diversity in critically ill patients is not associated with survival in an exploratory setting. a Microbiota diversity, presented as the Shannon index, was calculated from 15 healthy subjects, as well as from all 34 critically ill patients. Data are presented as dot plot with the mean (solid horizontal line). ****p < 0.0001. b Shannon diversity from all 34 patient samples plotted against the number of different antibiotic classes (categorized as in Fig. 1) that a patient had received prior to fecal sampling. c Based on the range in diversity in healthy subjects, the patient cohort was split into two groups: Shannon index <4 (normal diversity) and Shannon index >4 (high diversity), for which a 90-day Kaplan–Meier survival plot is shown. Numbers below the curve Patients at risk per group Full size image

Table 1 Baseline characteristics of patients with normal or high intestinal microbiota diversity and use of antibiotics prior to fecal sampling Full size table

The F/B-ratio was very recently reported to be associated with survival in critically ill patients [12]. The F/B-ratio is known to vary widely in healthy subjects [27], as was the case in our control subjects whose F/B-ratio ranged between 0.5 and 8.4 (median 2.4, IQR 1.1–5.0; Fig. 4). In 11 of our 34 patients the F/B-ratio was <0.5, indicating a relative increase in Bacteroidetes; in six patients, Firmicutes were relatively increased (F/B > 8.4). In five patients, no Bacteroidetes were detected at all, and the F/B-ratio could not be calculated. There was no difference in F/B-ratio between survivors and non-survivors when plotted at different time points (Fig. 4).

Fig. 4 Firmicutes/Bacteroidetes ratio (F/B-ratio) and Gram-positive/Gram-negative bacteria ratio are not associated with short- and long-term survival in an exploratory setting. a The F/B-ratio was calculated from healthy subjects (dotted horizontal lines range of F/B-ratio in healthy subjects) and from all 34 critically ill patients. and plotted against survival at 4 time points. b Survival data were collected at four time points: during ICU stay (ICU) and at 30 and 90 days and 1 year (d30, d90, 1y, respectively). c The ratio of total Gram-positive/Gram-negative bacteria was calculated from healthy subjects (dotted horizontal lines range of ratio of Gram-positive/Gram-negative bacteria in health subjects. d Survival data collected at same time points as for b. b, d Data are presented as box-and-whisker plots, with median (horizontal line in box), interquartile range (ends of box), and range (whiskers). Black boxes Healthy subjects, white boxes surviving ICU patients, gray boxes non-surviving ICU patients Full size image

Similar to our results for the F/B-ratio, the Gram-positive/Gram-negative bacteria ratio varied widely in healthy subjects, ranging between 0.5 and 8.0. In 14 of the 34 patients this ratio was <0.5, indicating a relative increase in Gram-negative bacteria (Fig. 4). In two patients, no Gram-negative bacteria were detected. No differences were found between survivors and non-survivors with respect to the Gram-positive/Gram-negative bacteria ratio (Fig. 4).