Advances in Microbiome Diagnostics
The organisms that are a part of our normal biology are essential for our survival and well-being. The genetic material of these organisms collectively makes up the human microbiome. However, there are many scientists that use the term microbiome interchangeably with microbiota (the actual normal microorganism in a particular environment, such as the human body). For the purposes of this article, microbiome will be reserved for indicating the total genome of human microbiota.
Various illnesses have been found to be linked to disturbances in the microbiota. Dysbiosis, an imbalance in the normal flora in or on the body, is known to be characterized by a reduction in the levels of normal and beneficial flora and an overgrowth of pathogenic microorganisms. This microbiota status has been associated with a number of health conditions such as obesity, inflammatory bowel disease (IBD), and even cancer (1,2,3).
Diagnostic tools that detect changes and adverse effects on the microbiome provide answers regarding the etiology and basis of many medical and health conditions. The NIH’s multidisciplinary Human Microbiome Project was established with the goal to create comprehensive information on the human microbiome in healthy and disease states. This information can ultimately be used to determine the role of various microbiome differences in human disease.
Microbiome-Associated Disease and Diagnostics
Scientists search for diagnostic methods that are noninvasive, cost-effective, and accurate. The identification of biomarkers in the saliva would provide a means to easily and quickly obtain actionable information to support disease diagnosis. Advances in this regard include the use of high-throughput sequencing methods to identify pathogenic organisms in the saliva of patients with periodontal disease and absent in non-affected individuals (4). Also, the U.S. FDA has approved an ELISA-based HIV test that uses saliva samples, and it is available over-the-counter (5).
Research findings regarding the microbiome of the gastrointestinal (GI) tract in healthy and select disease states have the potential to provide noninvasive and accurate methods to diagnosis and choose treatment plans for systemic diseases associated with an altered microbiome. The disease states most studied are cardiovascular disease, IBD, and diabetes. These and other disease states have been linked to alterations in the microbiota.
The link between microbiota and cardiovascular disease is based on findings that the microorganisms metabolize dietary phosphatidylcholine into trimethylamine-N-oxide (TMAO), a metabolite associated with the development of atherosclerosis. When healthy individuals were provided phosphatidylcholine, TMAO levels were increased. This increase was inhibited with antibiotic treatment (6).
Studies (using 16S sequence analysis) of the microbiome of patients with IBD showed that they had a lower diversity and increased abundances of specific bacteria including, for example, Enterobacteriaceae, Pasteurellaceae, Clostridiales, and Bacteroidales. They also found that antibiotic treatment exacerbated the microbial dysbiosis common with IBD (7). Diabetes is also linked to dysbiosis. In a study involving the transplantation of nonobese-male fecal microbiota in males with metabolic syndrome, improvement in insulin sensitivity was observed (8).
Microbial translocation-Associated Disease
Translocation of the GI tract microorganisms to the systemic circulation is normally prevented by the physiological environment and components that allow normal gut function. However, translocation through and intact barrier and its link to sepsis has been observed. Microbial translocation has also been found to be associated with increased infections in critically ill patients (9).
Brenchley et al. showed that microbial translocation to the circulatory system from the GI tract is responsible for immune system activation that causes progressive HIV infection (10). The mechanism of the microbial translocation–related disease process is yet to be fully understood (9). Next-generation sequencing has emerged as a vital tool for uncovering information to decipher the microbiome-disease links and mechanisms.
Next-Generation Sequencing and Microbiome Diagnostics
The availability of high-throughput massive parallel sequencing methods such as NGS has opened the door to new and massive amounts of information regarding the human microbiome and its link to health and disease. It has proven very valuable in providing information to study microbiome-health relationships. It also has already been used to help make diagnoses and to detect drug resistance.
Candida albicans, a member of the healthy human microbiome, is a pathogenic in those who are immunocompromised. Drug resistance to common treatments for C. albicans infection has been observed. Using NGS, Ford et al. identified new mutations such as single-nucleotide polymorphisms (SNPs) and loss-of-heterozygosity events and are believed to contribute to the resistance (10).
Mutla et al. used NGS to detect GI mucosal microbiome dysbiosis in HIV patients. They also found that these alterations correlated with immune activation in HIV. The alterations noted were a decrease in microbiome diversity and loss of commensals. Further, there was an increase in the pathogenic microorganisms (11).
To determine the role of microbiota changes in infants with necrotizing enterocolitis, Leach et al. analyzed 16SrDNA using NGS. They found that although the fecal microbiota did not change significantly in comparison to controls, more of potentially pathogenic bacteria were found in those infants (12). However, a potential biomarker for the disease was identified (S100A12) and found to increase after diagnosis.
The massive data generated by NGS, the low cost of application, and the speed to obtain data has changed how genomic etiology of disease is used to diagnose and develop treatments for many diseases. The emerging information is beginning to provide insight into mechanisms of disease and how to apply this to personalized medicine efforts. This is particularly important for diseases currently without known mechanisms of their development. Therapies that then target the microbiome can possibly reverse related disease states.
- Turnbaugh, Peter J; Ruth E Ley; Michael A Mahowald; Vincent Magrini; Elaine R Mardis; Jeffrey I Gordon (2006-12-21). “An obesity-associated gut microbiome with increased capacity for energy harvest”. Nature. 444 (7122): 1027–1031.
- Seksik, P. (2010). “Gut microbiota and IBD”. Gastroentérologie Clinique et Biologique. 34 (Suppl 1): S44–51.
- Castellarin M, Warren RL, Freeman JD, Dreolini L, Krzywinski M, Strauss J, Barnes R, Watson P, Allen-Vercoe E, Moore RA, Holt RA (2012). Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome Research. 22 (2): 299–306.
- Sakamoto M, Umeda M, Ishikawa I, Benno Y. Comparison of the oral bacterial flora in saliva from a healthy subject and two periodontitis patients by sequence analysis of 16S rDNA libraries. Microbiol Immunol. 2000;44(8):643-52.
- US Food and Drug Administration. July 2012. First rapid home-use HIV kit approved for self-testing. FDA Consumer Health Information. http://www.fda.gov/downloads/ForConsumers/ConsumerUpdates/UCM311690.pdf. Accessed 6 May 2013.
- Tang WH, Wang Z, Levison BS, et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. The New England journal of medicine. Apr 25; 2013 368(17):1575–84.
- Gevers D, Kugathasan S, Denson LA, et al. The treatment-naive microbiome in new-onset Crohn’s disease. Cell host & microbe. Mar 12; 2014 15(3):382–92.
- Hartstra AV, Bouter KE, BÃ¤ckhed F, Nieuwdorp M. Insights into the role of the microbiome in obesity and type 2 diabetes. Diabetes Care. 2015 Jan;38(1):159-65.
- Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S, Kazzaz Z, Bornstein E, Lambotte O, Altmann D, Blazar BR, Rodriguez B, Teixeira-Johnson L, Landay A, Martin JN, Hecht FM, Picker LJ, Lederman MM, Deeks SG, Douek DC. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med. 2006 Dec;12(12):1365-71.
- Ford CB, Funt JM, Abbey D, Issi L, Guiducci C, Martinez DA, Delorey T, Li BY, White TC, Cuomo C, Rao RP, Berman J, Thompson DA, Regev A. The evolution of drug resistance in clinical isolates of Candida albicans. Elife. 2015 Feb 3;4:e00662.
- Mutlu EA, Keshavarzian A, Losurdo J, et al. A Compositional Look at the Human Gastrointestinal Microbiome and Immune Activation Parameters in HIV Infected Subjects. Relman DA, ed. PLoS Pathogens. 2014;10(2):e1003829.
- Leach ST, Lui K, Naing Z, Dowd SE, Mitchell HM, Day AS. Multiple Opportunistic Pathogens, but Not Pre-existing Inflammation, May Be Associated with Necrotizing Enterocolitis. Dig Dis Sci. 2015 Dec;60(12):3728-34.
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