The human gut microbiome is a key modulator of human health. It is known to play important roles in the outcome of treatments and diseases, including acute lymphoblastic leukemia (ALL). Although survival rates of ALL exceed 90%, infectious complications still remain a challenge to disease prognosis and quality of life. Recent studies suggest a link between infection events, hematological diseases such as ALL, and gut microbiome. To investigate how the gut microbiome varies between ALL patients with infectious and no infectious complications we imported publicly available sequencing data originally collected by Nearing et al. to the CosmosID-HUB. This included gut metagenomic samples from 19 pediatric ALL patients with infectious complications and 25 without infectious complications (Nearing et al. 2019). We performed a comparative analysis using the CosmosID-HUB Microbiome.
Plotting a heatmap (Figure 1) for samples based on the relative abundances of bacterial species in the gut microbiomes of ALL patients with and without infectious complications illustrates clear clustering of samples by their infectious complication status. The heatmap highlights that an unknown Bacteroides species and Bacteroides fragilis are almost ubiquitously absent in the gut microbiome of ALL patients with infectious complications. Bacteroides are normally highly abundant in the healthy gut microbiome. However, Agrobacterium, Achrobacter and Rhizobium species, which are abundant in the gut microbiome of ALL patients with infectious complications, are almost always absent in patients without infectious complications. This implies a shift away from a healthy gut composition in ALL patients with infectious complications.
Figure 1: A heatmap illustrating clustering of gut microbiome samples from ALL patients with and without infectious complications based on the relative abundance of the most abundant 25 bacterial species; in blue (with infectious complications) and green (without infectious complications)
Performing a Wilcoxon Rank Sum Test on the CHAO1 indices of diversity of the cohorts with and without infectious complications gave a significant difference (p-value = 0.033, Figure 2), suggesting that the gut microbiome of ALL patients without infectious complications have higher species richness than the gut microbiome of ALL patients with infectious complications. However, no significant difference in alpha diversity was identified using Shannon’s index, highlighting a lack of difference in species evenness.
Figure 2: A box plot illustrating the mean CHAO1 alpha diversity index of gut microbiomes of ALL patients with and without infectious complications
We next investigated whether the gut microbiomes of ALL patients with and without infectious complications represented dissimilar community compositions. To do that, we compared the Bray-Curtis dissimilarity matrices of the cohorts using a principal coordinate analysis, which illustrated that the two cohorts were dissimilar. We confirmed the significance of community composition dissimilarity between the two cohorts using a PERMANOVA test (p-value = 0.002, Figure 3). The samples show three major groups of clusters; one with mostly non-infectious samples, one with mostly infectious samples, and a mixed group that includes more non-infectious samples. This suggests that overall community composition is significantly changed by infectious complications.
Figure 3: A 3D visualization of principal coordinates analysis results performed on the Bray-Curtis dissimilarity matrix of the gut microbiomes of ALL patients with and without infectious complications (p-value = 0.002).
To elucidate differentially abundant taxa that may drive the significant beta diversity results, we performed a linear discriminant analysis of effect size (LefSe) over the microbiome data of the two cohorts. We visualized only the taxa which were highly likely to be reproduced by filtering out the taxa with linear discriminant analysis (LDA) effect size below 4 (Figure 4). There were 8 differentially abundant taxa with an LDA effect size > 4. In the infectious complications cohort, the differentially abundant taxa were two Agrobacterium species, an opportunistic pathogen of immunocompromised patients and patients with venous catheters (Alnor et al. 1994). Achromobacteri marplatensis and an unclassified Rhizobium species were also significantly enriched. Achromobacter marplatensis is known to carry AMR genes and has been isolated from 10% of patients with cystic fibrosis in France, Serbia, Denmark, Argentina, and the UK, (Chan et al. 2020). Certain Rhizobium species may be causative agents of bloodstream infections in immunocompromised and venous catheter patients (Chen and Hansen 2008). Four other species were significantly enriched in the non-infectious complications cohort, including Bacteroides fragilis, an unclassified Bacteroides species, Alistipes putredinis, and an unclassified Parabacteroides. Bacteroides species are dominant in the healthy gut and metabolize polysaccharides and oligosaccharides to provide beneficial nutrients to the host (Zafar and Saier Jr. 2021). Bacteroides fragilis specifically is associated with protection from antibiotic-induced diarrhea (Zhang et al. 2018), induction of B and T cells that protect against viral encephalitis (Ramakrishna et al. 2019), and protection of gut integrity and against Graft Versus Host Disease (Sofi 2021), to name a few health benefits. Parabacteroides species are closely related to Bacteroides, are also dominant in the gut, and have similar health benefits, including an association with lower blood pressure (Jennings et al. 2021). The Alistipes genus is also part of the healthy gut microbiome but its role in disease is less well studied. Alistipes putredinis has been associated with cruciferous vegetable intake (Li et al. 2009), possibly due to its ability to metabolize fiber and glucosinolates, which may, in turn, contribute to the production of nutrients beneficial to the host. Overall, taxa significantly enriched in non-infectious ALL are more frequently associated with health benefits, whereas those enriched in infectious ALL are associated with disease. These findings indicate significant differences in the ALL gut microbiome in infectious and non-infectious complications and imply that infectious complications may shift the community towards a disease-associated state and higher risk of poor outcomes.
Figure 4: A bar plot visualization of LDA effect sizes of taxa with an effect size of above 4 in infectious and non-infectious ALL complications
Key findings: The gut microbiome in pediatric ALL patients with and without infectious complications is significantly different by alpha and beta diversity indices. Taxa enriched in non-infectious complications are generally associated with a healthy gut microbiome while those enriched in infectious complications are associated with disease. This may indicate that infectious complications significantly shift the ALL gut microbiome and increase the patient risk of poor outcomes.
