Profiling gut microbiota and bile acid metabolism in critically ill children

Abstract

Broad-spectrum antimicrobial use during the treatment of critical illness influences gastrointestinal fermentation endpoints, host immune response and metabolic activity including the conversion of primary to secondary bile acids. We previously observed reduced fermentation capacity in the faecal microbiota of critically ill children upon hospital admission. Here, we further explore the timecourse of the relationship between the microbiome and bile acid profile in faecal samples collected from critically ill children. The microbiome was assayed by sequencing of the 16S rRNA gene, and faecal water bile acids were measured by liquid chromatography mass spectrometry. In comparison to admission faecal samples, members of the Lachnospiraceae recovered during the late-acute phase (days 8-10) of hospitalisation. Patients with infections had a lower proportion of Lachnospiraceae in their gut microbiota than controls and patients with primary admitting diagnoses. Keystone species linked to ecological recovery were observed to decline with the length of PICU admission. These species were further suppressed in patients with systemic infection, respiratory failure, and undergoing surgery. Bile acid composition recovers quickly after intervention for critical illness which may be aided by the compositional shift in Lachnospiraceae. Our findings suggest gut microbiota recovery can be readily assessed via measurement of faecal bile acids.

Figure 1

Primary and secondary bile acids (a) primary bile acids (Cholic acid (CA), Chenodeoxycholic acid (CDCA), Glycochenodeoxycholic acid (GCDCA), and Taurocholic acid (TCA)) in faecal water measured by LC–MS as a ratio of total measured bile acid. (b) secondary bile acids (Deoxycholic Acid (DCA), Lithocholic acid (LCA), Isolithocholic acid (ILCA), Ursodeoxycholic acid (UDCA), 3α-Hydroxy,12-Ketolithocholic acid (3a-H,12-KLCA), Taurohyocholic acid (THCA), and Glycoursodeoxycholic acid (GUDCA)) in faecal water measured by LC–MS as a ratio of total measured bile acid. A significant reduction to the relative concentration of total secondary bile acids versus the total measured bile acid pool was observed for patients sampled at day 1–3 and day 4–7 compared to the control. The median proportion of these secondary bile acids at days 8–10 was comparable to that of the aged-matched controls, but we observed a significant increase of patient bile acids at days 8–10 above the median for patients at day 1–3. (c) the ratio of CA to CDCA, two species of primary bile acids in the human gut. The control samples had a median CA:CDCA ratio of 0.9; the median CA:CDCA ratios on days 1–3, 4–7, an 8–10 in the faecal samples of the critically patients were 4.4, 4.4, and 5.9, respectively. (d) Ratio of primary bile acids to secondary bile acids. (e) ratio of CA to DCA in faecal water. The ratio of CA to DCA was elevated in across all patient timepoints compared to the control. Patient samples collected on days 8–10 had lower ratios of CA:DCA than patients at day 1–3. (f) ratio of CDCA to LCA and ILCA. CDCA:LCA ratios were increased compared to controls in patient samples from day 1–3 and day 4–7, returning to control levels at days 8–10. Conover-Iman significance ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05, p > 0.05 = not shown.

Figure 2

Bacterial diversity, and abundance of beneficial bacteria. (a) Shannon diversity of sampling groups, bars indicate 1st 2nd and 3rd quartiles. Due to the broad range of alpha-diversity at day 1–3, day 4–7 and days 8–10, only the median alpha diversity of samples collected at days 8–10 was significantly lower than that of the control group after correcting for multiple comparisons. (b) NMDS clustering of beta diversity of bacterial populations compared between sampling groups. PERMANOVA p-value = 0.001. Ellipses indicate 60% similarity. Conover-Iman significance ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05, p > 0.05 = not shown.

Figure 3

Relative proportion of bacteria identified by 16S rRNA gene sequencing. Bacterial 16S rRNA gene counts are represented as proportional abundance at Family level for each sample. The majority of analysed 16S rRNA gene counts are within 12k to 16k reads. The ten most abundant families in the control microbiomes were the Bacteroidaceae, Ruminococcaceae, Prevotellaceae, Lachnospiraceae, Tannerellaceae, Veillonellacea, Peptostreptococcaceae, Coriobacteriaceae, Rikenellaceae and Acidaminococcaceae. These ten families represent a median of 82.8% (IQR 12.7%) of the control faecal microbiota and a median of 45.3% (IQR 57%) of the patient faecal microbiota. The ten most abundant bacterial families present in the patient faecal microbiome, which were not highly represented in the control samples were the Enterococcaceae, Enterobacteriacea, Bifidobacteriaceae, Streptococcaceae, Staphylococcaceae, Corynebacteriaceae, Clostridiaceae, Lactobacillaceae, Christensenellaceae and the Sutterellaceae. These families represented a median of 7.9% (IQR 8.8%) of the control microbiome, and a median of 31.1% (IQR 64.8%) of the patient samples.

Figure 4

Relative abundance of recovery associated bacteria (RAB) and Lachnospiraceae. (a) Proportion of RAB present in microbiomes. Patient samples from day 1–3, day 4–7 and days 8–10 were significantly reduced from the median level of the control group. RAB at days 8–10 were also significantly reduced from the median RAB level at day 4–7. (b) Proportion of Lachnospiraceae present in microbiomes. No significant difference was observed between groups after controlling for multiple comparisons. Conover-Iman significance ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05, p > 0.05 = not shown.