Bacterial cells in the human body out number the human cells by factor of ten. The microbial flora in the human body perform many functions like fermenting unused energy substrates, improve immune system, preventing growth of pathogenic bacteria, regulating the development of the gut, producing vitamins for the host (such as biotin and vitamin K). However, in certain conditions, some species are thought to be capable of causing disease by producing infection or increasing cancer risk for the host. Constipation is one of the major disease in the worldwide. More than 2.5-million Pakistanis have frequent constipation, accountinf for 1-million physion visits a year. Constipation occurs when bowel movements become difficult or less frequent. The normal length of time between bowel movements ranges widely from person to person. It is assumed that change in the gut flora may leads to constipation. In this Project we are looking to dissect the Gut flora of constipation patients and to compare it with the normal and healthy microbial flora, to detect the possible microorganism associated with the constipation by the aid of Molecular technique like Polymerase Chain Reaction based Denaturing Gradient Gel Electrophoresis (DGGE). Secondly, by making constipation animal model through diet and then screen some natural compounds or probiotic strains of lactobacillus and bifidobacterium species which helps to regain normal healthy microbila flora. This Dissection of Constipation microbial niche helps us to develop new strategy to treat constipation condition and develops new drugs to maintain healthy gut flora to reverse constipation condition.
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Our body is home to a wide variety microorganisms estimated about three hundred and one thousand different kinds of bacteria inhabiting the gut. Gut flora predominantly contain Bacteria, some fungi and viruses. Among them approximately thirty or forty different species (e.g. Bifidobacterium and Lactobacillus Species etc.) make up the bulk of the inhabitants. Microorganisms in the gut flora outnumbered the total number of cells in the body by a large number. By far, most of these bacteria are found in the colon. Each bowel movement contains a large amount of these critters, with one to two-thirds the dry weight of stool being solely bacteria (1). Together these creatures play a crucial role in the health and welfare of your body. Gut Microorganisms live symbiotic relationship with the host. Gut flora helps the host by boosting immune system, digestion of carbohydrate, fats and protein, absorbtion of vitamins and balance water in the gut and keeps pathogenic microorganisms in check. Gut problems likegas, bloating, constipation, Crohn's, irritable bowel syndrome (IBS), and ulcerative colitis can all be improved by maintaining healthy gut ecosystem (6). Constipation is one of the most common gastrointestinal complaints in the United State as well as in Pakistan. More than 2.5-million Pakistanies have frequent constipation. Constipation occurs when bowel movements become difficult or less frequent. Healthy intestinal flora is also vital for prevention of constipation. Constipation is one of the most prominent signs, especially when the stools are dry or hard. This means there is too little bacteria to loosen up the formed feces and keep them moist, because, unlike other stool components, bacterial cells retain moisture. So, the complete profiling of gut bacterium in constipated person is very important to evaluate for better prevention of disease. Prevailance of anitibiotic resistance among the Microbial world, asking us to find alternatives lik eprobiotics and natural compounds to cure diseases. So in this project by using animal model we screen some probiotcs and natural compounds which promotes healthy gut flora which in turn keep the gut moistened to revert the condition of constipation. So, this research might helps the constipation related problem among human population. Constipation is a global phenomenon, physicians are frequently faced with patients whose main concern is constipation. Constipation occurs when bowel movements become difficult or less frequent. Healthy intestinal flora is also vital for prevention of constipation. Constipation is one of the most prominent signs, especially when the stools are dry or hard. This means there is too little bacteria to loosen up the formed feces and keep them moist, because, unlike other stool components, bacterial cells retain moisture. Human gut contains a vast array of microorganisms. Intestinal microbiota is one of the complex ecological niche performing critical functions (3-5). Advances in the field of metagenomics allowed us to dissect the microflora of the ecological niche like gut. Patients with chronic constipation may feature changes in the microï¬‚ora of the large bowel, which are characterized by a relative decrease in obligate bacteria and a parallel increase in potentially pathogenic microorganisms and fungi . These alterations in the intestinal microï¬‚ora could alter the metabolic milieu of the colon with resultant changes in the concentration of physiologically active substances that may inï¬‚uence the motor and secretory functions of the bowel [8, 9]. Still the Microflora associated to constipation is not well characterized. There is growing interest in the use of probiotics in organic and functional gastrointestinal disorders. Probiotics are live microbial food ingredients which are reported to be effective in the treatment of IBD, travellers diarrhea and constipation [6-8]. Probiotics, such as Bifidobacteria and lactobacilli, both produce lactic, acetic and other acids resulting in a lowering of pH in the colon. A lower pH enhances peristalsis of the colon and subsequently decreases colonic transit time which is beneficial in the treatment of constipation [9, 10]. Natural compounds extracted from plants (flower, fruits and vegetables) can also be used as an alternative to boost good and healthy microbial flora.
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Stool samples will be collected from constipated patients prescribed by the doctors from 2-3 different hospital. A questionnare will be filled by the patients (attached). Blood samples were also collected and serum was seperated from the same patients. Serum samples will be stored for its use.Subjects which are on antibiotic therapy or probiotic therapy will be excluded from the study.
DNA extraction From the stool samples will be carried out by using DNA Extraction Kit according to manufacturere protocol.
Polymerase Chain Reaction -Denaturing Gradient Gel Electrophoresis (DGGE):
Primers targeting the variable V3 region of 16S rRNA gene were applied, The GC clamp was attached to the 5 end of the forward primer to allow detection of the corresponding PCR products with DGGE . PCR will be done using PCR kit The size of the obtained amplicons was checked through agarose gel electrophoresis. DGGE analysis was performed by a Universal Mutation Detection System with an 8 % polyacrylamide gel containing a 35-65 % gradient of urea and formamide (a 100 % denaturing solution contained 40 % [v/v] formamide and 7.0 mM urea) as reported . The electrophoresis will be done at 200 V for 10 min, followed by a ï¬xed voltage at 85 V at 60oC for 8 h. After Electrophoresis the gels were stained with 0.5 lg/ml ethidium bromide solution for 60 min, washed by deionised water, and viewed by Gel Documentation System.
Analysis of DGGE Profile:
The PCR-DGGE proï¬les will be analyzed by using Phoretix 1D (Single Gel Dendrogram) software . Analysis will be done on number of bands, their gray intensity and the similarity of DGGE proï¬les. The Shannon-Weaver index of diversity (H 0 ) and Evenness will be calculated[4, 5]. Statistical analysis will be performed by using SPSS.
To identify some separated and strong bands, a sterile scalpel will be used to cut out the bands from polyacrylamide gel under UV illumination. Cut bands will be washed and keep in sterile water overnight at 4oC for diffusion. The diffused DNA will be served as DNA template to re-amplifying by PCR using the same primers without GC clamp. After puriï¬cation, PCR products will be cloned into the PMD18-T Easy vector, transformed into competent Escherichia coli Nova blue cells, and will screened for positive plasmid insertions. A second PCR will be conducted to conform the successful construction of the recon. The obtained PCR products will be puriï¬ed and sent for sequencing followed by GeneBank by Blast search (NCBI). (6)
Real Time PCR Assay:
Enterobacteriaceae, Enterococcus, Clostridium, Bacteroids, Lactobacillus and Biï¬dobacterium genus, the important member groups with their unique characteristics in the intestinal ï¬‚ora as their abundance determine the healthy or unhealthy gut flora, will be quantiï¬ed by real-time PCR using group-speciï¬c primers. A tenfold dilution series of plasmid DNA containing target species DNA will be made and used in each real-time PCR assay to generate standard curves for quantitation of target DNA in test samples. (7)
Serum samples from the blood were tested for the B and T lymphocyte, T lymphocyte subpopulation, Bacterial antibodies titre to determine the immune status of constipated patients.
Antibiotic and Diet based Constipaption was induced in Rat to evaluate the role of natural compounds for the cure of constipation.
Microflora of rat before and after induction of constipation was evaluated using DGGE as mentioned above as well as level of bacteria mentioned above will also be evaluated using real time PCR.
Natural Compounds: Different Natural compounds idolated form Plants from different regions of Pakistan was screen for the lexative or anticonstipation properties in the re establishment of Healthy Microbial Flora.
Probiotics: different strains of probiotcs like Lactobacillus acidophillus, Lactobacillus salivarius, Lactobacillus casei etc will be purchased from bacterial culture collection unit. and tested for their probiotic ability in terms of coaggregation, tolerate acidic condition, bile salt concentration etc.
After brobiotics test the best among all will be tested to treat the constipation and will be given daily thrice orally for a week then the level of constipation was evaluated in terms of stool passage and healthy microbial flora re establishment.
Immune Status: After the treatment of Natural compounds and Probiotics the immune profile were measured for the same paramenter as discussed above.
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