Potential for Monitoring Gut Microbiota for Diagnosing Infections and Graft-versus-Host Disease in Cancer and Stem Cell Transplant Patients

Abstract

Background: Gut microbiota, the collective community of microorganisms inhabiting the intestine, have been shown to provide many beneficial functions for the host. Recent advances in next-generation sequencing and advanced molecular biology approaches have allowed researchers to identify gut microbiota signatures associated with disease processes and, in some cases, establish causality and elucidate underlying mechanisms.

Content: This report reviews 3 commonly used methods for studying the gut microbiota and microbiome (the collective genomes of the gut microorganisms): 16S rRNA gene sequencing, bacterial group or species-specific quantitative polymerase chain reaction (qPCR), and metagenomic shotgun sequencing (MSS). The technical approaches and resources needed for each approach are outlined, and advantages and disadvantages for each approach are summarized. The findings regarding the role of the gut microbiota in the health of patients with cancer and stem cell transplant (SCT) patients (specifically in modulating the development of gut-derived bacterial infections and a posttransplant immune-mediated complication known as graft-vs-host-disease) are reviewed. Finally, there is discussion of the potential viability of these approaches in the actual clinical treatment of cancer and SCT patients.

Summary: Advances in next-generation sequencing have revolutionized our understanding of the importance of the gut microbiome to human health. Both 16S rRNA gene sequencing and MSS are currently too labor-intensive or computationally burdensome to incorporate into real-time clinical monitoring of gut microbiomes. Yet, the lessons learned from these technologies could be adapted to currently used methods (e.g., qPCR) that could then be rigorously tested in the clinical care of these patients.

Fig. 1.. Overview of 16S rRNA gene sequencing.

Genomic DNA is extracted from a stool sample. The 16S rRNA gene variable region (e.g., V4) is amplified from each sample using a composite forward primer and a reverse primer containing a unique 6- to 12-base barcode that is used to tag PCR products from respective samples. Amplicon libraries are pooled together and sequenced simultaneously. 16S rRNA gene sequencing data undergoes quality control and filtering and is demultiplexed. Sequences are clustered into phylotypes termed operational taxonomic units (OTUs). Representative sequences of each OTU are then interrogated against a reference database of validated 16S rRNA gene sequences. Relative abundances of bacterial taxa can then be calculated and presented in graphical representation (e.g., pie-chart).

Fig. 2.. Overview of MSS.

Genomic DNA is extracted from a stool sample, quantified, and assessed for quality. High-quality gDNA then undergoes massive parallel sequencing, allowing the sequencing of thousands of organisms simultaneously. MSS sequencing data undergo quality control and removal of human sequences. For taxonomic identification, unique clade-specific marker genes or lowest common ancestor positioning approaches are commonly used. Functional insight can be ascertained by utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology database, with each sequence directly mapped to a KEGG orthology representative sequence. Furthermore, antibiotic resistance gene abundance of the entire metagenome can also be determined by mapping to antibiotic resistance gene databases.

Fig. 3.. Overview of bacterial group or species-specific qPCR.

Standardization of Enterobacteriaceae qPCR assay (A). Enterobacteriaceae group-specific primers are used to amplify a segment of the 16S rRNA gene using a representative strain’s (e.g., E. coli) gDNA. This PCR fragment is subcloned into a vector and transformed into E. coli. Plasmids are recovered from transformed E. coli and sequenced for verification. Varying concentrations of linearized plasmid are then used to perform Enterobacteriaceae-specific qPCR, and standard curves are created. Enterobacteriaceae qPCR on an unknown sample (B). An unknown sample (e.g., patient stool specimen) can then be assessed for Enterobacteriaceae concentrations by performing Enterobacteriaceae qPCR on gDNA extracted from feces. Bacterial numbers are then determined from the previously determined standard curves. qPCR measures the number of 16S rRNA gene copies per sample, not the actual bacterial numbers or colony-forming units. Gene copies per sample are then normalized to both stool gDNA concentration and weight.