In recent years, there has been a dramatic increase of obesity among all ages. It is considered to be one of the life threatening conditions for individuals (World Health Organization, 2016). Indeed, it is the first step for body deterioration by increasing the chance of the onset of other serious health conditions such as diabetes and heart diseases (World Health Organization, 2016). Recently, a number of studies have been looking into a preventative measures to tackle this phenomena. One strong link has been found between the gut bacteria and losing weight. It has been proven that obese people gut community are more efficient in extracting energy from food than lean people (Turnbaugh et al. 2006).
However, the gestural intestinal tract has a numerous bacterial species, which means the exact species associated either with obese people or lean people needs to be determined, in order to understand the differences between the two groups and finding a suitable treatment. It has been stated that conventional culturing technique can grow only few species from environmental samples, which gives unclear view of what the sample contain and what other unknown species are hidden. Thus, ribosomal RNA isolation from feces sample is highly recommended for identifying the associated microorganisms with both groups of people (Hutchison, 2007).
Ribosomal 16s RNA sequencing technology
Ribosomal 16s RNA is an RNA of the subunit 30 of the ribosome molecule in prokaryotic cells. It is a highly important molecule because it has the specific complimentary code of Shine-Dalgarno sequence. This sequence has been found by John Shine and Lynn Dalgarno in 1975 (Shine and Dalgarno, 1975). It is located on the messenger RNA strand prior the starting point of translation by 8 bases. Furthermore, this code is important for ribosome to recognize and send a signal to start the translation process and forming proteins. However, each organism has its own unique Shine-Dalgarno sequence and highly conservative ribosomal RNA sequence, which facilitate the annealing of universal primers to initiate the sequencing process (Chakravorty et al, 2007). Moreover, it contain hyper variable regions which makes it a helpful tool in studying the microbiome of humans to distinguish the bacterial species from each other (Case et al, 2007). Notably, the length of the full ribosomal RNA genome is about 1,550 base pair long, yet it has enough bases to identify the organism in a rapid and cost effective way. Despite all these advantages of ribosomal 16s RNA sequencing, the cost considerations makes it hard to be performed as a routine diagnostic test in clinical laboratories (Clarridge, 2004).
Admittedly, ribosomal 16s RNA sequencing technique has both negative and positive aspects. The most important limitation in this technique is that some databases have inaccuracy in terms of the availability of genetic information, in regards the lower heterogenetic ribosomal 16s RNA sequence in bacteria, which lead to faulty results and difficulties in matching and comparing data (Clarridge, 2004).
Ribosomal 16s RNA sequencing of microbial gut
One proposed solution to determine the difference of bacterial species present in the gut of obese and lean people is by using ribosomal 16s RNA sequencing technology. This can be done by collecting feces sample from obese and lean individuals. Then, these samples will undergo an isolation process, this involve adding certain chemicals with specific amounts and following several steps ended by centrifugation. Thus, all the unwanted fragments in the sample will be excluded and the desired ones collected from the supernatant and ready for sequencing process. The sequencing start with amplifying the ribosomal 16s RNA by using universal primers in polymerase chain reaction followed by sample segregation into gel electrophoresis (Smith et al, 2011). After doing the sequencing process and comparing the sequence with the existing bacterial genomes on the database, the final result will be a full screen of existing microorganisms for both groups. In addition to, the most predominant bacteria will be known from the sequencing results as it will be represented by the higher number of copies of the same gene per sample.
From the findings the following conclusion can be deduced. Even though the most predominant bacterial species genes in the sample of lean and obese people can be known by using this sequencing technology, there is still a missing information is needed to give a brief understanding of the metabolic process associated with these bacteria and their exact role in regulating the degradation of food. From the provided data and the sequencing findings it can be assumed that there is a certain metabolic mechanism within the predominant species in the gut of lean people associated with less energy extraction which needs a further investigation to know the exact characteristics of those predominant bacterial species. However, if the exact characteristic has been identified, it can be used for developing a phage therapy.
It is also known as gene therapy. The main concept of this type of therapy is to carry genes and introduce it into cells in order to replace mutated or abnormal gene with a copy of normal one. This will make the cell produce normal proteins rather that faulty ones (Genetics Home Reference, 2016). Usually genes that has been inserted directly to a given cell does not work. In this case a carrier is needed to insert those genes and a capable carrier would be a virus (Genetics Home Reference, 2016).
It is well known that viruses require a host cell to be active, in other words they need a living cell to attach on its surface and release their own infectious genes. They can infect a wide range of living organisms including bacteria and human. However, in order to infect a host cell there must be a specific chemical receptor site on that cell wall a virus can recognize by their outer protein layer (Tortora et al, 2010).
Bacteriophages are the type of viruses that infect bacteria and it can be found and isolated from any area rich in bacteria. Recently, it has been used to treat from bacterial infections instead of antibiotics. Moreover, these bacteriophages has two cycle for multiplication lytic and lysogenic. The lytic one makes the host cell dead immediately at the end, while the lysogenic cycle is an integration and combination of virus genomes with the host bacterial genome, it occurs inside the bacterial cell and no action happen until external stimulation motivate the viral genome to do their infectious effect. For instance, the bacterial cell divisions continue normally with passing the viral genome to the new cells as a recombinant DNA. One of the lysogenic cycle useful characteristic is the phage conversion, which has the ability to make the bacterial cell exhibit new properties. For example, Corynebacterium diphtheriae can cause diphtheria disease that produce toxin and it is not able to produce toxin unless it has a lysogenic phage. As the lysogenic phage has the specific genes that encode for producing this toxin (Tortora et al, 2010). Furthermore, phages have the ability to increase in number while they are in host cell area, which gives additional feature for the patient of giving auto dosing (Loc-Carrillo, 2011).
Notably, the outer protein layer of a virus and its inner genomes both can be modified and engineered in a laboratory. For this reason, a proposed therapy for obese people would be an oral phage therapy. This can be achieved by cloning the exact bacterial characteristic of extracting less energy from food that are associated with the gut of lean people and design it in a plasmid, then inserted into a modified bacteriophage, which will target a selected predominant species in the gut of obese people. This will make the existing predominant bacteria in the gut of obese people obtain the additional characteristic of extracting less energy from food. Subsequently, the targeted bacterial cell will continue their cell divisions, which will colonize the gut with the same modified genes and characteristic. However, in the meantime a highly suggested treatments for obese people would be fecal microbiota transplantation and/or probiotics.
Fecal microbiota transplantation
Feces transplantation is a cost effective and harmless method of treatments that has been mainly used to maintain the balance of bacterial gut in human since 1950s. It has been used to treat obesity and other certain serious diseases that are strongly associated with microbiota disruption such as Clostridium difficile infection, Ulcerative colitis and autoimmune conditions. The treatment preparation protocol involve collecting a feces sample from a healthy donor in a certain amount. The donor must pass a full medical diagnostic procedure prior the donation, to make sure it is free from diseases. Most importantly the sample should be treated with anti-clostridium drug prior transplantation in addition to mixed with saline solution. The dose can be given either through enema, colonoscope or nasogastric and all of these routes had shown the same effectiveness in patient’s gastrointestinal tract. The amount of the dose and the duration varies upon the disease or condition of the patient. Although there is no health risk concern reported from using this method as a treatment, it is still classified as an experimental treatment (The Fecal Transplant Foundation, 2016).
However, this treatment method can be considered as an effective way for colonizing the gastrointestinal tract of obese people by collecting fecal samples from lean people. This will transfer the desired bacteria species from lean to obese people in a less risk and low cost manner.
They are simply a combination of several non-infectious bacterial species added to an edible product such as yogurt, or in the form of food supplements, which can be prepared in powder compound, liquid or pills. It is mainly used to help re-colonize the gut with microbiota species and restore it, which contribute in maintaining the balance of these disrupted bacteria species and prevent from serious health conditions (California Dairy Research Foundation, 2011).
However, knowing the exact bacterial species that are predominant in the gut of lean people can also be used to colonize this type and introduce it to a probiotic supplement product and make it specific for obese people treatment.
Ethical issues for human microbiome cloning
In the light of starting a human microbiome sequencing and cloning there are a number of points should be considered as follows:
- The group that are participating in the project should be fully informed with all the risks, benefits and procedure. However, the limited information that it has been found and already known about the human microbiome is very limited and this is not enough to cover and express all the risk factors. Another important measure is providing information for those participant about future researches that they would use their results in. It is a challenging point in any research to predict what can be done in future and the informed consent should be taken at the beginning. Furthermore, informed consent has been a center of controversy, yet some has stated that it is acceptable once the approval was based on uncertainty, risks and future research besides well governance (McGuire, 2008).
- The hesitancy of whether to inform participants with their results and to what extent. This has a risk on the person behaviors. For example, the results for a person shown that their gut microbiome are associated with risk of obesity the person by knowing this information would change their lifestyle without further consultation (McGuire, 2008).
- Privacy is the third point for any genetic research. These data should be de-identifying prior publishing on a database for public. However, newly advancements in technology has made it easier to determine the individuals without knowing their identity through the genetic information and this has increased controversy about privacy issue which ended up by blocking the access to unauthorized people (McGuire, 2008).
- Taking the minimum sample with less invasion as possible. For example, taking a feces sample is better than taking a biopsy from the gut. This will limit the risk exposure and progress (McGuire, 2008).
- Considering the sample size and diversity in relation to what exactly is being seeking for. For example, taking a sample from children and elderly people should be excluded in a study about microbiome, because there are concerns about the changes in microbiome in these two stages of life (McGuire, 2008).
By: Rasha Bajbaa
California Dairy Research Foundation. (2011). Probiotics Basics. Available at: http://cdrf.org/home/checkoff-investments/usprobiotics/probiotics-basics/ [Accessed 23/11/2016].
Clarridge, J.E. (2004). Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clinical microbiology reviews, 17(4), pp.840-862.
Case, R.J., Boucher, Y., Dahllöf, I., Holmström, C., Doolittle, W.F. and Kjelleberg, S. (2007). Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies. Applied and environmental microbiology, 73(1), pp.278-288.
Chakravorty, S., Helb, D., Burday, M., Connell, N. and Alland, D. (2007). A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. Journal of microbiological methods, 69(2), pp.330-339.
Franca, L.T., Carrilho, E. and Kist, T.B. (2002). A review of DNA sequencing techniques. Quarterly reviews of biophysics, 35(02), pp.169-200.
Hutchison, C.A. (2007). DNA sequencing: bench to bedside and beyond. Nucleic acids research, 35(18), pp.6227-6237.
Loc-Carrillo, C., and Abedon, S.T. (2011). Pros and cons of phage therapy. Bacteriophage, 1(2), pp.111-114.
McGuire, A.L., Colgrove, J., Whitney, S.N., Diaz, C.M., Bustillos, D. and Versalovic, J. (2008). Ethical, legal, and social considerations in conducting the Human Microbiome Project. Genome research, 18(12), pp.1861-1864.
Smith, B., Li, N., Andersen, A.S., Slotved, H.C. and Krogfelt, K.A. (2011). Optimising bacterial DNA extraction from faecal samples: comparison of three methods. The open microbiology journal, 5(1).
Shine, J. and Dalgarno, L. (1975). Determinant of cistron specificity in bacterial ribosomes. Nature, 254(5495), p.34.
The Fecal Transplant Foundation. (2016). What is FMT?. Available at: http://thefecaltransplantfoundation.org/what-is-fecal-transplant/ [Accessed 24/11/2016].
Turnbaugh, P.J., Ley, R.E., Mahowald, M.A., Magrini, V., Mardis, E.R. and Gordon, J.I. (2006). An obesity-associated gut microbiome with increased capacity for energy harvest. nature, 444(7122), pp.1027-131.
Pictures : https://www.researchgate.net/figure/Variable-regions-of-the-16S-ribosomal-RNA-Secondary-structure-of-the-16S-rRNA-of_fig4_264798721