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The term normal flora illustrates microorganisms that are normally found in different body site in average, healthy persons. The constituent and figures of the flora diverge in unlike areas and sometimes at different ages and physiologic states. They contain microorganisms whose morphologic, physiologic, and genetic properties forbid them to colonize and proliferate under the circumstances to facilitate, subsist in particular sites, to coexist with other colonizing organisms, and to hinder rival intruder. Therefore, every reachable area of the body presents a meticulous ecologic niche, colonization require a particular set of property of the migrating microbe.
The amount of organisms in the flora is likely to surpass the amount of cells in the body by a factor of 10[Bengmark 1998]. The members of the normal flora basically are of two types, may stay for highly variable periods. Residents are strains that have an establish role at one of the various body sites, which they reside in forever. Transients acquire as of the environment and ascertain themselves temporarily but are apt to be barred by rivalry from inhabitants or by the host's immune defense mechanisms. The word carrier state is used when potentially pathogenic organisms are involved. For e.g., Streptococcus pneumoniae, a cause of pneumonia, and Neisseria meningitidis, a cause of meningitis, may be isolated from the throat of 5 to 40% of healthy people.
The gastrointestinal tract (GIT) also consists of various normal flora which place an important role. The GIT consists of several anatomically and functionally distinct regions - the oral cavity, the pharynx (which is also part of the respiratory tract), esophagus, stomach, small intestine (duodenum, jejunum, and ileum) and the large intestine (cecum, colon, and rectum). Essentially, it is continuous tube extending from the mouth to the anus [Michael W 2008]. The ecological determinants within each region of the GIT are very different, and each region. The relationship between Homo sapiens and the native microbiota of the GIT is an exceptional case of a symbiosis. In the upper regions of the GIT (the stomach, duodenum, and jejunum), the host provides an environment that is not favorable to wide colonization by microbes, and it is in these regions that the host extracts many of the nutrients from the constituent of an individual's diet. In the terminal ileum and the colon, the environment is appropriate for the founding of large and varied microbiota. Within the region, the host takes advantage of the enormous metabolic potential of its native microbiotato degrades dietary constituents that it is itself unable to digest.
Many of the ensuing products are then riveted by the colon, along with vitamins synthesized by some microorganisms. The microbes, in turn, are abounding with surroundings that are appropriate for their development, and a steady supply of nutrients. The indigenous microbiota plays an important role in the development of the intestinal mucosa and immune system of its host. The environment of the GIT varies considerably along its length, resulting in distinct microbial communities in each of the main regions. In addition, there is also a horizontal stratification at any point within the GIT. The organisms that are most frequently cultivated from the GIT belong to the genera Bacteroides, Eubacterium, Clostridium, Bifidobacterium, Streptococcus, Lactobacillus, Peptostreptococcus, Peptococcus, Ruminococcus, Fusobacterium, Veillonella, Enterococcus, Propionibacterium, Actinomyces, Desulfovibrio, Helicobacter, Porphyromonas, Prevotella, Escherichia, Enterobacter, Citrobacter, Serratia, Candida, Gemella, and Proteus.[ Michael W 2008]
Several species of bacteria exist and grow in the individual gastrointestinal tract. An individual contains 300-500 diverse species of bacteria [Simon GL., et al 1984; Borriello SP., et al 1986]. The number of microbial cells within the gut lumen is about 10 times larger than the number of eukaryotic cells in the human body [Bengmark 1998]. The gastrointestinal tract of baby starts Colonization instantly after birth and occurs within a few days. The preliminary colonisation is very relevant to the final composition of the permanent flora in adults [Ducluzeau 1993]. The genera bacteroides, bifidobacterium, eubacterium, clostridium, peptococcus, peptostreptococcus, and ruminococcus are mainly found in human beings [Salminen 1998]. While aerobes (facultative anaerobes) such as escherichia, enterobacter, enterococcus, klebsiella, lactobacillus, proteus, etc are among the genera found. Each person has several hundreds of species belonging to these genera, with a particular mixture of main species that is different from to found in other persons [Salminen 1998].
The individual's flora can vary under some conditions, for example acute diarrhoeal illnesses, antibiotic treatment, or to lesser extent induced by dietary interventions, but individuals' flora composition pattern usually remain constant[ Moore et al 1995]. A major metabolic role of colonic microflora is the fermentation of non-digestible dietary residue and endogenous mucus produced by the epithelia [Roberfroid et al., 1995]. Gene diversity in the microbial community provides different enzymes, biochemical pathways that are dissimilar from the host's own constitutive resources. Fermentation of carbohydrates is a main basis of energy in the colon. A colonic microorganism helps in vitamin synthesis and in absorption of calcium, magnesium, and iron [Roberfroid et al., 1995; Miyazawa et al., 1996; Younes et al 2001]. The intestinal mucosa interfaces between the immune system and the outer environment. Microbial colonisation of the gastrointestinal tract affect the composition of gut related lymphoid tissue.
In adults, immunity might frequently reshape by constant interactions between the host and its bacteria that take place in the gut. A commensal microbe tries to avoid the immune response [Krinos., et al 2001]. In microbes the immune response relies on adaptive and innate component. Nearly all bacteria in human faeces are coated by specific IgA units [Van der Waaij., et al 2001]. Innate responses are mediate not only by white blood cells that can phagocytes and kill pathogens, also by intestinal epithelial cells, which manage host response by synthesizing a broad series of inflammatory mediators and transmitting signals to underlying cells in the mucosa [Kagnoff MF 1997]. Resident bacteria act a crucial line of resistance to colonisation by exogenous microorganism so, as it prevents of the invasion of tissues by pathogens. Sterilized animals are vulnerable to infection [Baba E., et al 1991; Taguchi H., et al 2002]. Colonisation resistances apply to opportunistic bacteria present in the gut but have limited growth. The stability between species of resident bacteria provides constancy in the microbe's population in the similar individual under usual conditions. However, use of antibiotics can disturb the natural balance and allow overgrowth of species with latent pathogencity [Van der Waaij., 1989].
The host provides a nutrient that the bacterium requires, and the bacterium aggressively indicates how much it needs to the host. This symbiotic relationship prevents surplus production of the nutrient; Bacteria can inhibit the growth of their rival by producing antimicrobial substances called bacteriocins [Brook I 1999; Lievin V 2000]. The ability to synthesize bacteriocins is generally distributed among microbial communities of the gastrointestinal tract; the role of bacteriocins is mainly restricted to localized niches. The passage of feasible bacteria from the gastrointestinal tract via the epithelial mucosa is known bacterial translocation [Van Leeuwen 1994]. Translocation of endotoxins from viable or dead bacteria in little amount perhaps constitute a physiologically significant; enhance to the reticulo endothelial system, particularly to the Kupffer cells in the liver. Though, dysfunction of the gut mucosal barrier can effect in translocation of numerous viable microorganisms, typically belonging to gram-negative aerobic genera (escherichia, proteus, klebsiella). After passage to the epithelial barrier, bacteria can go via the lymph to extraintestinal sites, such as the spleen, mesenteric lymph nodes, and liver.
The three main mechanisms in promotion of bacterial translocation in animals are overgrowth of bacteria in the small intestine; deficiencies in host immune defenses and increased permeability of the intestinal mucosal barrier [Van Leeuwen 1994]. Bacterial translocation can arise in human beings in various disease processes [Lichtman SM 2001]. Normal gastrointestinal bacteria have been cultured from the mesenteric lymph nodes of patients undergoing laparotomy, disorders such as multisystem organ failure, acute severe pancreatitis, advanced liver cirrhosis, intestinal obstruction, and inflammatory bowel diseases [Lichtman SM 2001]. Bacterial translocation is linked with a increase in advance of post operative sepsis in patients undergoing operation. The molecular mechanisms of colorectal cancer are well recognized, however factors such as diet may too have role in advance of sporadic colon cancer. Dietary fat and high consumption of red meat (processed meat) are related with high risk of colon cancer [Bingham 1999 ] Comparative a high consumption of fish, calcium, fruits, vegetables, whole grain cereals, has been related with reduced risk [Rafter 1998] .
The outcome of diet on the carcinogenic development might be mediate by change in metabolic activity and composition of the colonic microflora. Intestinal bacteria might play a part in the development of colon cancer through production of carcinogens. In normal individual, meal rich in fat and meat deprived in vegetables raise the faecal excretion of N-nitroso compounds [Hughes 2001]. In additional, group of carcinogens of dietary are the heterocyclic aromatic amines are produced in meat while it is cooked. A few intestinal microbes strongly damage to DNA in colon cells induced by heterocyclic amines, whereas other intestinal bacteria can uptake and detoxify such compounds [ Wollowski 2001]. Bacteria of the bacteroides and clostridium genus raises the occurrence and growth rate of colonic tumours induce in animals, while other genera such as lactobacillus and bifidobacteria avoids tumorigenesis [Mahony et al 2001]. A descriptive account on human study compared the composition of the faecal flora of an individual with diverse risks of colon cancer. The presence of Bacteroides vulgatus and Bacteroides stercoris showed the high risk of colon cancer Lactobacillus acidiphilus, Lactobacillus S06 and Eubacterium aerofaciens showed the Low risk. Although the evidence is not definite, colonic flora appears to be a main factor that modulates risk of colonic cancer in human beings.
Bacteria can also be used to improve human health. It provides specific health benefits as consumed as a food component or supplement would be called a probiotic. Probiotics do not necessarily reside in the intestine. Several probiotics are helpful in prevention and cure of acute diarrheal conditions. Consumption of probiotics to patients on antibiotics significantly reduced antibiotic-associated diarrhea in children and adults [Saavedra 1994] .Probiotics can be used to prevent such antibiotic-associated diarrhea [Oberhelman 1999]. Lactobacillus rhamnosus strain GG has been helpful as in prevention of disease of diarrhea in undernourished children, particularly not breastfed [Guandalini 2000]. Children with acute gastroenteritis who received a probiotic supplement (either L rhamnosus, L reuteri, or L casei) too had notably decreased extent of diarrhea [Guandalini 2000].
Probiotics are mainly effectual in acute diarrhea by rotavirus infection. Bacteria as a starter culture in yoghurt improve digestion of lactose and eradicate symptom of intolerance in an individuals who do not absorb lactose well. This useful outcome is due to presence of microbial galactosidase (lactase) in the fermented milk product. Live bacteria are vital for the effect, since heated or pasteurized yoghurts do not avoid lactose malabsorption and symptoms of intolerance [ Labayen 2001]. People who are intolerant to lactose tend to reduce dairy products lacking calcium in their body. These make yoghurt bacteria very consistent to accomplish sufficient calcium intakes through dairy products in adults. Orally intake probiotics can enhance specific IgA responses to rotavirus in infected children or to Salmonella typhi in individuals undergoing vaccination with an attenuated strain [Link-Amster 1994].
In healthy individuals, diverse probiotics developed in a fermented milk product briefly colonized their gut and improved phagocyte action [Schiffrin.,et al 1995].Probiotics and prebiotics have been shown to prevent colon cancer in several animals, but their role in reduction of risk of colon cancer in human beings is not established [Burns AJ., et al 2000 ].However, probiotics have been shown to reduce the faecal activity of enzymes known to produce genotoxic compounds that act as tumour initiators in human beings.[ Spanhaak et al.,1998 ]
The extensive presence of resistance elements in microbes is mainly the result of human action is controversial. [Kirandeep Bhullar., et al 2012] Any chemical substance either of microbial, synthetic or semi-synthetic origin, used in the clinical treatment of infections, is called antibiotic., the resistant strains was reported Within four years following the introduction of penicillin during the Second World War. [Saswati Sengupta; Madhab K Chattopadhyay 2012] Resistance genes exist not only in infection -causing organisms, it is present in commensal organism such as E. coli or enterococcus. In a study of faecal flora from an ambulatory community, it was found that 40% of individual relying on antibiotics conceded two or more resistances in 10% of their E. coli, 25% had three or more resistances, 10% had four or more [Levy etal 1988]. Individual excrete resistant E. coli at the 50% level, even while not consuming antibiotics [Levy et al 1988].
Maximum cases of resistant faecal flora have been reported from Holland [Bonten et al 1992], and elsewhere [Calva etal 1992, Leistevuo et al 1996]. Food is one of the sources of antibiotic resistant; Gram negative bacteria are related with uncooked foods [Levy 1984]. A study from France showed the role of food bacteria to the intestinal flora by studying the same individual when consuming the normal or sterilized food [Corpet 1988]. Tetracycline resistance in the faecal flora was high while an individual were eating normal food for 21 days, but dropped dramatically when the diet was shifted to sterilized food for 17 days. Resistant variant, antibiotics can influence the natural balance by altering the types of organisms.
A study of a varied Bifidobacteria, one of the predominant genus in the gut flora, are Gram-positive bacteria commonly present in human and animal intestines [Depeint et al., 2008; Guarner and Malagelada, 2003; Matsuki et al., 2004]. Although Bifidobacteria are rarely linked with infections, the concern for transferable antimicrobial resistant factors has been raised upon their use in probiotic products [Borriello et al., 2003; Levy and Marshall, 2004; Teuber et al., 1999].Bifidobacterium bifidumstrain Yakult YIT 4007 is a commercial strain which is utilized to produce a fermented milk beverage. This strain was isolated sequentially by stepwise screenings of mutants resistant to erythromycin, neomycin, and streptomycin from its progenitor strain B. Thus, genetic bases of these antibiotic resistances are very important to elucidate the nature of the antibiotic resistance of B. bifidum YIT 4007 because the product containing this strain is ingested by consumers on a daily basis, the genetic basis of these antibiotic resistances of B. bifidumYIT4007, and found that these antibiotic resistances were acquired by mutations of chromosomal genes. The antibiotic-resistance in bacteria is a emerging global problem which needs to be addressed at the earliest. The various aspects and the mechanism in the study in antibiotic-resistance of bacteria remain unexplored till now.
Rationale and Scope of the Study
An individual is said to healthy when he is physical, mentally and socially fit and free from various infections or disease , an individual often fall sick and seek medical attention, our bodies are tends to infections and disease. To overcome diseased condition, globally diverse practices are followed the modern medical science and the traditional methods, the aim is to cure illness and sustain good health. Medical systems are a vital part of all cultures, which influence the health status of the people. The medical system includes the sum of health facts, idea, skill and practices of the every group. Essentially, there are two systems of health care in the developing world: one is traditional and the modern medical science. The medical traditions in the customary system are varied in their chronological background, hypothetical logic and practices, their modern social realities and their dynamics [Veena Bhasin 2007].
The many of Sikkim's population are into traditional meditation. It has a rich medicinal plant, 424 different plants habitats in Sikkim due to this high altitude variation and climate [Panda 2009]. Sikkim is prevalent of olden medical system which is still popular nurtured by the Buddhist groups for the traditional Tibetan pharmacopoeia [Panda 2009].The three ethnic groups (Lepcha, Bhutia and Nepali communities) traditionally practice folk medicine in different ways which are related with ayurveda and Tibetan system of medicine [Panda 2009].Association between modern and traditional medicine can come in to being in four different ways-monopolistic, tolerant, parallel and integrated [Veena Bhasin 2007]. Indian Medical Council Act has formally established the traditional system- Ayurvedic, Unani and Siddha-as official components of national health care in India. In Ladakh, a traditional medical system Amchi has been incorporated into health planning. It is based on Tibetan medical system, and is holistic, cost effective and locally available [Bhasin, 1997].
The World Health Organization (WHO) estimates that 4 billion people, 80 percent of the world population, presently use herbal medicine for some aspect of primary health care. Herbal medicine is a major component in all indigenous peoples' traditional. The traditional healer, as defined by the W.H.O. (1976), is a person who is recognized by the community in which he lives as competent to provide health care by using vegetable, animal and mineral substances and certain other methods based on the social, cultural and religious background, as well as on the knowledge, attributes and beliefs that are prevalent in the community, regarding physical, mental and social well-being and the causation of disease and disability. Traditional remains an important source of medical care in the developing countries even though it is not officially recognized by the government health care programs [Jaspan, 1969; Kleinman, 1980].
In state, modern medical system, which tends to be synonymous with a monopolistic medical "establishment" and a doctor-dependent, hospital-based, curative health care model, does not generally recognize, cooperate with, or adjust to the traditional medical systems. The two exist side by side; yet remain functionally unrelated in any organizational sense. The combined use of both types of expertise provides an optimal broad-spectrum response to health problems. "Medical pluralism offers a variety of treatment options that health seekers may choose to utilize exclusively, successively, or simultaneously" -(Stoner, 1986).
People may try a variety of practitioners and treatments, from the same or different systems, until a cure results. Patients may accept some aspects of the scientific health care system as presented to them by a government physician, and they may supplement this with information gathered in consultation with traditional healers. The systems differ in availability, quality of care, levels of technology, and social adaptability; yet, ideally, both are intended to serve the same population in need. It persists in urban as well as rural settings despite the availability of allopathic health services. In traditional medical systems worldwide, afflictions that beset body and mind can be explained in both naturalistic and super naturalistic terms. When a wound does not heal, when a sickness does not respond to treatment, and when the normally expected and predictable does not happen, other explanations beyond the organic are sought [Scheper-Hughes, 1978].
In Sikkim till date, some many individuals are into traditional medicine, and avoid the use of antibiotics.
People from the state, often complain about gastric problems, which have not been address largely.
Research Problem/Objectives and Hypothesis
An individual practicing traditional medication also harbor antibiotic resistance gene, which supports a growing understanding that antibiotic resistance is natural, ancient, phenomenon. There will be difference in the normal gut flora in the person on the basis of preference of treatment or meditation.i.e. Traditional medication and the modern medical science
To check the dominant microflora of healthy persons of different age groups.
To find out the dominant microflora of people who are less frequently exposed to antibiotics then those who are frequently exposed.
To find out the types of antibiotics resistant genes present in individuals who are less frequently exposed to antibiotics then those who are frequently exposed.
To study the relative no of copies of antibiotics genes present in gut are less frequently exposed to antibiotics then those who are frequently exposed.
Research Methods and Materials
Field Survey: the field study will be conducted to identification the people with the less frequent antibiotics use and the frequent antibiotics user ,will be considered for the study
Isolation of gut normal flora by using selective / non -selective media
2 g of sample dissolved in 18ml peptone water (0.1%) and the sample will be homogenized followed by Serial dilution and plating into suitable Selective/Non-Selective media and Incubation at 37 0C; aerobic/anaerobic will be done , Enumeration of colony forming units and isolation of microorganism will be followed .
MEDIA USED TO STUDY THE BACTERIAL POPULATIONS
Selective and non -selective media
Selective media permits certain types of microbes to grow, and inhibit the growth of other organisms. Non-selective media are used to grow the wide range of microorganism. A variety of selective and non-selective media are used in clinical, diagnostic centers and laboratories, and in food and dairy laboratories.
Bacteroides ; Bacteroides Bile Esculin agar - (Engelkirk et al., 1992)
Lactobacillus; cultured on LAMVAB medium. - (Hartemink et al., 1997)
Enterococci and Enterobacteriaceae
(Enterococcus Agar) -(Audicana et al., 1995)
(TSA and VRBGA/ Mc Conckey agar.)
Bifidobacterium; Bifidobacterium Agar
Plate count Agar -Total Viable Count - (Alander et al., 1997)
Characterization of normal microflora Biochemical test
A drop of distilled water will be taken on a clear glass slide, colony emulsified by a sterilized inoculating loop to make a thin smear, made by spreading specimen uniformly. This smear will be heat fixed. Smear was covered with crystal violet stain for 30 -60 second it is also known as differential staining, as it difference between Gram positive and gram negative. - [MWFHS Laboratory Procedures Manual 2004].
Semisolid agar will be used for this purpose. The organism will be inoculated by stabbing a directly. After overnight inoculation motility was exposed by dispersion turbidity as of the stab line throughout the medium - [MWFHS Laboratory Procedures Manual 2004].
Triple sugar iron agar (TSI)
This medium will be used for initial identification of gram negative bacilli, mainly member of the Enterobacteriaecae. Three main characteristics of a bacterium detect by this medium include ability to produce gas, the production of hydrogen sulfide gas and the ability to ferment carbohydrate. This tube will be inoculated at 350C for overnight. [MWFHS Laboratory Procedures Manual 2004].
Indole test: Indole production tests some bacteria, which have the ability to degrade tryptophan to indole. The test organism will be cultured in peptone broth which contains tryptophan. This broth was inoculated at 350C for overnight and Kavac.s reagent was added. Development of red colour surface in the broth within 10 minutes indicates positive test [MWFHS Laboratory Procedures Manual 2004].
Methyl red/ and Voges-Proskauer tests
These two tests use the same medium (MR-VP Broth).MR-VP broth contain peptone, glucose, and a partial phosphate buffer. The Enterobacteriaceae ferment glucose produces acid when grown in MR-VP broth. Some organisms produced fermentation products mostly acids as others produce mixed acids or alcohols. To detect those organisms that make typically acid fermentation products, a pH indicator Methyl Red (which turns red at pH 4.5, yellow at pH 6.0, and orange in between) is added after 2-5 days of growth.
To detect the alcohol by-products, add the VP reagents A (Î±-naphthol) and B (KOH or NaOH) and then oxygen after 2-5 days of growth. The VP reagents react specifically with acetoin (acetylmethylcarbinol), an intermediate in the 2, 3-butanediol pathway to give a red color. To perform the tests inoculate one tube of MR-VP broth and incubate at 37Â°C for at 2-5 days. After the incubation, half of the broth will be transferred to a second test tube. To one tube, add 5 drops of Methyl Red and to the other, add 5 drops of VP reagent A followed by 5 drops of VP reagent B.
Coagulase in the presence of enzyme coagulase, the addition of commercial rabbit plasma produces a "clumping" reaction. This test is used to differentiate pathogenic staphylococcus aureus (positive) from staphylococcus epidermidis (negative). -[Bailey and Scott 2002]
Hydrogen peroxide is a by-product of respiration and is lethal if it accumulates in the cell. The enzyme catalase degrades the hydrogen peroxide in the cell before it can do any cell damage. It splits the H2O2 to free oxygen (bubbles) and water. Generally, the test reaction is very fast and obvious bubbles will be seen. Catalase is made by your own cells, as well as a variety of other cells, including many bacteria. This test is particularly important for the gram + bacteria. For example, the genus Staphylococcus is a + catalase reaction, whereas the genus Streptococcus is a reaction. [Fall 2011]
Fermentation is an anaerobic process i.e. it does not require oxygen but can occur in its presence. Oxidation is an aerobic process that only occurs in the presence of oxygen. This reaction is restricted to those organisms that process the cytochrome oxidase enzyme i.e. oxidase positive [Hugh R: E. Leifson 1953]
Growth at different pH, NaCl conc, temperatures and Requirement of the growth factors will be consider in the study.
Bergy's Manual of Systematic Bacteriology-will be referred for identification
Biolog system for confirmation
Phenotypic Identification of normal microflora:
Use of Biolog Identification system based on the utilization of 95 substrates in 96 welled plate. The metabolism of the substrates in the wells of the microplates results in reduction of tetrazonium dye producing a color change, and a specific "metabolic fingerprint" was obtained for each strain and have to be compared with the data of the Biolog Microlog database software (Biolog Inc.).
ANTIBIOTIC RESISTANCE CHARACTERISTICS
Antibiotic resistance/susceptibility will be screened by Biolog
Detection of types of antibiotics resistance genes by nested PCR and semi-quantitative PCR -( A. Mothershed et al., 2005)
Nested PCR is a conventional PCR method that amplifies a target region of DNA with an outer primer pair in an initial reaction, followed by a second amplification using an internal primer pair. It is helpful for pathogen detection in medical specimens it has superior sensitivity over a single amplification, but can be problematic due to carryover contamination from the first reaction to the second [ Apfalter P., 2002]. It has also been used for the detection of the 16S and 23S rRNA genes of bacteria's and provides multiple overlapping amplicons for accurate sequencing of these genes.
Relative copy number of antibiotic resistant genes will be checked by real time PCR. Real-time PCR use fluorescently labeled probe with two flanking primers in the reaction. Real-time PCR has increased sensitivity and rapidity, broader dynamic range, elimination of post amplification handling steps, and higher throughput conducive to automation .Though real-time PCR is in widespread use because of its enhanced sensitivity, the overriding limitation of this technology is the high cost of special reagents and instrumentation. - (A. Mothershed et al., 2005)
POSSIBLE OUT COME OF THE STUDY
The antibiotics resistant genes of microorganisms from individuals never exposed to antibiotics and from individuals exposed to antibiotics will be known.
Difference in microflora among individuals.
The dominant microflora of people who are not less frequently exposed to antibiotics will be known.
The erosion rate of beneficial microorganisms due to indiscriminate use of antibiotics will be known.
We would know, if there is increase in antibiotic resistant microorganisms in people consuming antibiotics.