Combination of molecular imaging with microbiome analysis: infection landscapes in clinical samples

Question

Next-generation sequencing (NGS) is a powerful tool, which is broadly used for microbiome analysis. Its use for diagnostic applications, however, is hampered by technical challenges, such as lack of sensitivity and risk of contamination. Therefore, data might be difficult to interpret in the clinical setting. Whereas NGS allows the overall assessment of species involved, Fluorescence in situ hybridization (FISH) shows the abundance and localization of specific microbial populations. This way, key microbial species, which are directly associated with the host epithelial surfaces or invasive within the host tissue, can be identified. However, for proper FISH probe selection, prior knowledge on potential microbial candidates is required.

Methods

We developed MG-FISH (Microbiome-guided fluorescence in situ hybridization), which synergistically combines FISH with NGS out of consecutive tissue sections. Consequently, microorganisms (bacteria and fungi) are both identified and localized. We used MG-FISH to identify the bacterial species involved in a case of an aspirated, endoscopically removed peanut from a 78-year-old patient.

Results

We amplified and sequenced microbial DNA out of methacrylate-embedded tissue sections and identified Staphylococcus, Streptococcus, Veillonella dispar and Actinomyces as most abundant microorganisms. Using specific FISH probes for the respective target species, we visualized, quantified, and localized the microorganisms.

Conclusions

MG-FISH gives microbiome data a spatial dimension in situ: It allows quantification and distribution of the microorganisms involved, localization at and in the tissue, and identification of key pathogens. This will further the use of NGS for diagnostic applications.