Molecular Accounting and Profiling of Human Respiratory Microbial Communities: Toward Precision Medicine by Targeting the Respiratory Microbiome for Disease Diagnosis and Treatment

Abstract

The wide diversity of microbiota at the genera and species levels across sites and individuals is related to various causes and the observed differences between individuals. Efforts are underway to further understand and characterize the human-associated microbiota and its microbiome. Using 16S rDNA as a genetic marker for bacterial identification improved the detection and profiling of qualitative and quantitative changes within a bacterial population. In this light, this review provides a comprehensive overview of the basic concepts and clinical applications of the respiratory microbiome, alongside an in-depth explanation of the molecular targets and the potential relationship between the respiratory microbiome and respiratory disease pathogenesis. The paucity of robust evidence supporting the correlation between the respiratory microbiome and disease pathogenesis is currently the main challenge for not considering the microbiome as a novel druggable target for therapeutic intervention. Therefore, further studies are needed, especially prospective studies, to identify other drivers of microbiome diversity and to better understand the changes in the lung microbiome along with the potential association with disease and medications. Thus, finding a therapeutic target and unfolding its clinical significance would be crucial.

Keywords: diagnosis; lung; microbiome; molecular; personalized medicine; precision medicine; pulmonary medicine; respiratory diseases; therapeutic targets; treatment.

Figure 1

Schematic representation of the Sanger sequencing process. The Sanger sequencing method uses a high-fidelity DNA-dependent polymerase to create a single strand of DNA that is complementary to the DNA template [66,69]. The synthesis reaction commences at the 3′ end with a single primer that is complementary to the template. Deoxynucleotide (or nucleotide) monomers, which are the building blocks of DNA, are sequentially added in a template-dependent manner. These form phosphodiester bonds between the 3′ hydroxyl of the primer and the 5′ triphosphate of the next nucleotide to be added to the sequence. The reaction mixture also includes A, C, G, and T di-deoxynucleotides, which mimic DNA monomers sufficiently to be incorporated into the sequence. Yet, unlike deoxynucleotides, the absence of the critical 3′ hydroxyl means there is no binding facility for incoming nucleotides, preventing further elongation. Furthermore, a fluorescent tag is incorporated into the di-deoxynucleotides enabling the DNA sequence to be automatically detected [66,67]. Each reaction is based on multiple copies of DNA fragments of different lengths, but always terminating in a di-deoxynucleotide at the comparable nucleotide position of the template molecule. The automatic process loads reaction mixtures onto sequencing machines with capillaries and uses electrophoresis to separate the DNA molecules based on their molecular weight, which varies according to the point at which the fragments terminate. As the di-deoxynucleotides pass through the gel, the fluorescent emission is read to determine the DNA sequence. As each of the four nucleotides fluoresces with a different colour, a four-colour chromatogram can be used to interpret the sequence. Capillary-based automated electrophoresis forms the basis of modern-day Sanger sequencing, and the devices are able to simultaneously analyse 8–96 sequencing reactions [67].