CH391L/S13/Probiotics

Introduction

A probiotic (from the Latin, pro-, "in favor, for" and the Greek , biōtikós, "pertaining to life")refers to live microorganism that provides a benefit to the host, either directly or indirectly, by via interactions with the hosts cells or the host's microbiota. Such microorganism will interact by producing bioactives, biological compounds and macromolecules, that will produce such benefit.Although the concept of a probiotic has evolved since the the last century and the the first years of the current century that it acquired the current definition and we can see the health benefits humans can gain from he understanding of such interactions. Some research currently ongoing includes the Human Microbiome Project that hopes to elucidate broad characteristicts of the microbiota in large populations[], and the use on single organisms to prevent disease. For example, the use of fecal transplantation for antibiotic-associated diarrea [1].

Definition of a Probiotic Trough Time

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Probiotics

The Time Before Probiotics

The medical importance of the human microbiome, or the diverse microbial communities that has co-evolved with us, is to understand that a mammal has an 'extended genome' and finding ways to study this extended genome. For more than a century, we have been taking a look at this complex system of microbe-microbe and host-microbe interactions that allows the stable in which co-exist. Starting with simple evidence that the lack of such microbiota that is passed on to us by our mother and gained through out the years through the study of gnobiotic animal models vs. the ones reared conventionally gave some resistance to infections to the host. In addition in the past century when antimicrobials and antibiotics became common in treatment of diseases but in turn generated a state that we call dysbiosis or a state of microbial imbalance in the gut microbiota. In summary, as our lifestyles changed so has our microbiome.

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The Advent of Probiotics

Although not the concept is not new, it was not until recently that we are taking a second look at probiotics. Although originally most of the reserach has focused on the gut microbiome, we are expanding the scope to the whole of the human host microbiome. With our current understanding of the microbiome has expanded to encompass: 1) the relation of the gut microbiome and drug metabolism , 2) energy metabolism , 3) immune system conditioning/reponse, 4)post-surgical recovery.[2] Based on this understanding we can start understanding how can we alter the microbiome to move from the state of dysbiosis to homeostasis. An ideal probiotic would try to achieve reestablishing the benefits in the context of a diet that includes a probiotic either as a supplement or a treatment to a disease. Nowadays this approach can take the form of a rudimentary fecal transplantation from diseased individuals to healthy ones(83), combined treatments of Lactobacillus paracasei and Lactobacillus rhamnosus and a set of prebiotics allowed a second set of Bifidobacteria to increase and led to Clostridium perfrigens to decrease that lead to a different energy metabolism profile in mice. Demonstratating the benefits of two distinct approches to probiotic treatment, bateriotherapy and bioecological approach. Both ideas argue in general that adding pre-morbid gut microbiota (83) or a conbination of prebiotics,prebiotics and synbiotics may be beneficial to our health.

Current Probiotic Preclinical Studies

• Studies on Probiotic Photoprotection
  • As current evidence indicates the composition of the intestinal microbiota is important beyond for the immune response at the local and systemic levels and provide beneficial effects in the gut can be expanded to the skin, where probioticts might also exert a benefit through the immune system modulation. In a recent study suggests that intake of Lactobacillus johnsonii NCC 533 was shown in a randomized double blind placebo-controlled clinical trial it could modulate the cutaneous immune stability after UV exposure rebalancing indirectly the skin's immune system response. [3]

• • Another approach has been to genetically modify Streptococcus mutants by deleting lactate dehydrogenase gene and making it defficient in lactic acid production. In turn this same strain became an effector used to produce mutacin which provided an advantage to other strains of S. mutans. This strain was tested in gnobiotic rats was not reported to affect other indigenous flora except for other indigenous S. mutants strains that are associated with dental caries. [4]

Frontiers in Probiotics : Genetic Modification

Since probiotics are now being considered as alternative to antibiotics as antiobiotic-resistant strains of bateria become more common and bacteria that present in diary products are not native to the human microbiota find a hostile environment where these are eliminated rapidly despite any benefit provided. Despite their beneficial therapeutic benefits that range from simple maintenance of the gut flora to causing tumor to regress this have not been widely implemented clinically. Their acceptance do have risks as we are still gaining knowledge about specific interactions in vivo. Still it we could initially employ natural and artificial Gram positive strains, non-spore forming lactic acid bacteria to develop necessary approaches that could result in probiotics to treat diseases by genetically engineering or synthetically engineering new safe strains that could survive.

• Food Grade Genetic Modification Systems

Included in these are markers that permit their selection such as pVS40 plasmids(wide-host-range) or pWV01 plasmids (narrow-host-range) that contain a selectable marker that confers a new phenotype, i.e. utilizing a new sugar, or conferring metal resistance. A second way is to directly modify the probiotic at the chromosome level as it allows for stable genetic modifications leaving no foreign DNA by employing thermosensitive plasmids. Finally also a genetic expression system has been developed for L. lactis. An example of such an engineered strain is described [5] in which an L.lactis strain in which by inactivating the aldB α-acetolactate decarboxylase gene increases α-acetolactate that is converted to diacetyl, which is the chemical responsible for butter's flavor. Other examples include of a LAB strain modification in which an heterologous gene from another one was introduced to increase the production of α-ketogluterate from glutamate in that is present in high levels in cheese. An so on so forth, yet examples like these are just baby steps in the advent of synthetic biology where much more could be done.[5]

Oversight of Probiotics

United States Regulation of Probiotics

Probiotics are currently regulated by the Food and Drug Administration (FDA)in one of the following ways; As a dietary supplement, in which case only a premarket notice to the FDA is necessary or as a drug in which case a premarketing safety, efficacy and approval by the FDA are required. Currently, most of the probiotics on the market fall under the umbrella of a dietary supplement, but situations where the number of infections and the severity of such cases are causing clinicians to evaluate their use as drug, as it's happening for Clostridium difficile infections. In such cases, Florastor (Saccharomyces boulardii) a probiotic currently marketed as a drug is beneficial as it demonstrated its efficacy in reducing the recurrence of C. difficile when used in combination with standard treatment methods. Although cases in which Florastor has lead to fungemia,yeast present in the blood, have been reported, mostly in patients that were not receiving the treatment via introduction of live yeast from contaminated hands of a technician to a catheter site.

Global Standardization of Guidelines of Probiotics

The first effort to establish global guidelines were made in 2001 when a Joint Food and Agriculture Organization of the United Nation and the World Health Organization meeting was organized. The following guidelines were proposed:

• Testing and Clinical Guidilines
  • 1. Identify the genus and species of the probiotic strain, phenotipically and genotipically.
    2. Perform in vitro testing of the probiotic mechanism of action.
    3. Do clinical trials pre- and postmarketing to provide substantiation to the claim it makes.

• Safety Assesment Guidilines
  • 1. Identify the patterns of antimicrobial resistance of the probiotic.
    2. Characterize its metabolism.
    3. Identify possible side effects in humans which could include:
    • Production of a toxin that causes adverse health effects
    • Test its hemolytic potential
    • Test for lack of infectivity.

iGEM 2009: Stanford's Approach to Probiotics

The 2009 Stanford iGEM Team project centered on probiotics and Inflammatory Bowel Disease (IBD). IBD, as explained, is caused by an imbalance of two types of T-cells, Treg cells that immunosuppres the Th17 cells that cause the inflammation seen in patients. They suggest that an novel theraputic mechanism can be achieved by in vivo regulation of these cells. Their approach focuses in constructing two different Escherichia coli(E.coli) strains, each that would contain a distinct input/output cassette , each that is referred as a device. The first device would detect as input Nitric Oxide(NO), a byproduct of inflammation and Th17 proliferation, produces retinoic acid, that blocks further CD4+ T-cells differentiation into Th17 cells. The second device detects 5-Methyl tryptophan (5MT) as an input and produces Interleukin-6 to regulate Treg proliferation to regulate their immunosuppression response. Ideally depending on the balance between these two markers, if too much NO is sensed by Device 1 it would prevent inflammation. The opposite would also be true if the second device sences to much 5MT that would immunosuppress Th17 cell by blocking their differentiation.

Probiotics and the Media links

The Media perspective on Probiotics

• http://www.npr.org/2011/09/02/140146780/probiotic-bacteria-chill-out-anxious-mice
• http://www.telegraph.co.uk/health/healthnews/8261808/Designer-probiotic-yogurts-could-help-people-lose-weight.html

References

6. Romero PA and Arnold FH. Exploring protein fitness landscapes by directed evolution. Nat Rev Mol Cell Bio, 2009.

7. Crameri A, Whitehorn EA, Tate E, Stemmer WP. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol, 1996.

8. Giver L, Gershenson A, Freskgard PO, Arnold FH. Directed evolution of a thermostable esterase. Proc Natl Acad Sci USA, 1998.

9. Cadwell RC and Joyce GF. Randomization of genes by PCR mutagenesis. Genome Res, 1992.

10. Abou-Nader M and Benedik MJ. Rapid generation of random mutant libraries. Bioeng Bugs, 2010.

11. Stemmer WP. Rapid evolution of a protein by in vitro DNA shuffling. Nature, 1994.

12. Lin H and Cornish VW. Screening and selection methods for large-scale analysis of protein function.