Organisms that are present within a certain material can be identified by analysis of the genetic material present in a sample. When the complete genetic information is assessed, identification of the genes encoded on the DNA makes it possible to determine which tasks are potentially being performed by the microorganisms for the host
The composition of the bacterial community within a sample can be defined by analyzing variable regions of the 16S rRNA gene. Additional genetic information is not needed for these evaluations.
As opposed to simple targeted community analyses, shotgun metagenomics identifies all different kinds of microorganisms that are part of the microbiota, such as viruses fungi and archaea, not just bacteria. Genes present within the microorganisms are classified, and the gene categories are used to draw conclusions on the tasks performed by the microbiota.
Samples are subjected to quality control and DNA is subsequently isolated, which will be prepared for sequencing according to application requirements. Sequencing is performed using modern high-throughput technologies. Data analysis can be limited to microbiota composition or extended to include functionality.
New possibilities in diagnostics
A 14 year old patient was hospitalized for the third time due to fever, headache, diffuse weakness, muscle pains, nausea, and vomiting. Standard tests could not identify the cause of disease. As MRI showed involvement of the brain, a biopsy was performed, which also failed to identify the cause of disease. Due to the MRI results and negative outcome of infectious disease tests, treatment for a potential candidate disease was attempted; however, the patient’s condition continued to worsen and he was finally placed in a medically-induced coma. Metagenomic analysis of cerebrospinal fluid eventually identified leptospira as the cause of infection. Treatment was altered according to these results and the patient fully recovered from meningoencephalitis. (Wilson et al. 2014 N Engl J Med)
Patients suffering from cystic fibrosis are regularly screened for infections of the respiratory tract. In addition to the standard culture-based method, samples were simultaneously analyzed by metagenomics. Cultures were negative for Pseudomonas aeruginosa, commonly causing infections in these patients. P. aeruginosa was, however, detected by metagenomics analysis, allowing early treatment and thus preventing chronic infection.
Metagenomic analysis brings new possibilities to diagnostics, enabling selection of suitable therapy to cure patients or prevent worsening of disease.
Identification of treatment possibilities
Some type 2 diabetes patients are missing certain bacteria within their gut microbiome that support the barrier function of the gut. This leads to increased translocation of substances from the gut into the body, resulting in increased immune reaction. A metagenomics analysis of a stool sample of a type 2 diabetes patient can give valuable hints on such potentially missing bacteria. If this is the case, the supporting bacterial species can be assisted by adjusting the patient’s diet or administration of probiotics, leading to increased insulin sensitivity and reduction of blood sugar levels.
A patient suffering from chronic inflammatory bowel disease stopped responding to treatment. A Clostridium difficile infection (cause of severe, life threatening diarrhea) was excluded, but analysis of the intestinal microbiome revealed divergence from a healthy microbiome (fewer anti-inflammatory bacteria). Adjusting the patient’s diet (e.g. reduction of fat, lactose carbohydrates and fiber) changed the composition of the intestinal microbiome and relieved symptoms. Medication that was taken prior to and during the diet could also be reduced.
Monitoring of therapeutic outcome
A patient with an infected jawbone was treated with antibiotics for several months. The therapy led to recurring diarrhea due to Clostridium difficile infection, which in turn was treated with antibiotics according to the standard procedure. After the 6th relapse, fecal transplantation was performed. After this the patient was free of symptoms. The composition of the transplanted microbiome was monitored over several years by metagenomic analysis. No further C. difficile infection occurred. (Broecker et al. 2013 Digestion)