Isolation And Identification Of Enterococcus Faecium Biology Essay

Enterococci are widely distributed in the environment ; within the human organic structure, they are normal commensals of the unwritten pit, GI piece of land and vagina. Enterococcus faecium is a Gram-positive bacteria group D, alpha hemolitic or nonhemolitic in the genus Enterococcus ( Chingwaru W. , 2003 ) . It can be a commensal ( non-harmful coexisting being ) , in the human bowel, but it may besides be a pathogen — doing diseases like neonatal meningitis.

Ordinary lactobacillus average including: MRS medium, Luria-Bertani ( LB ) medium, high-sterilized stored at 4 a„? icebox spare. In the unfertile console, the yoghurt incorporating lactic acid bacterium was diluted with unfertile saline to a consecutive dilution ( 10-3,10-4,10-5 ) of 50I?l suspension and coated them on the home base incorporating MRS pick medium and marked. The home base was placed at 37a„? warm box without O for 36 ~ 48 hours. Single settlement was picked and repeatedly crossed to obtain the pure strains.

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Bacterial designation methods include the common ( tradition ) classified designation, numerical value designation and mechanization identificationaˆ‚The common ( tradition ) classified designation needed to observe many points. It is so boring. But numerical value designation is harmonizing to the designation mark. It uses the trial card. The consequence is expressed by the manner of figure and compared with the informations of database to acquire an designation consequence. Automation designation can make whole process mechanization in the measure of vaccination, cultivation, reading and study. It can provide a powerful instrument in rapid clinical micro-organism diagnosing. No affair what sort of designation methods, it all needs to place bacteriums harmonizing to the morphology, physiological features and biochemical features.

Enterococcus faecium is a sort of ellipse cell. Its diameter is 0.5 ~ 1.0 I?m. It can demo a short-chain agreement often in braces or in liquid medium. It has slight motion. In the blood home base it can organize a milklike white home base or white, fringy unity, cardinal bulge, big and smooth settlement. Its diameter is 1-2mm, and has rare colourss, largely around the settlements without haemolysis. After the Gram discoloration, bacterium shows purple. It is Gram-positive coccobacteria. The chemical science character of Enterococcus faecium is contact-negative, and it can turn in 6.5 % stock, bile Esculin agar home base and 0.1i?…methylene bue. It besides can turn in 10a„? or 45a„? , pH 9.6 stock medium, and can digest 65a„? 30min status. It can ferment lactose, sorbitol and Osmitrol and can utilize arginine, but ca n’t utilize ferment arabinose.

2.3.2 Biochemical designation

Designation by lactic acid bacteriums and biochemical designation trials, severally taking the exercise-induced, adjacency enzyme, anaerobiotic growing, l0a„? and 45a„? , 60a„? 30 min, 6.5i?…NaCl salt-tolerant growing, pH 9.6 alkalic environment growing, methyl ruddy, benzopyrrole, sugar intoxicant acid agitation ( glucose, galactose, maltose, lactose, raffinose, manicol and so on ) . Comparing the experiment consequence with “ Berger ‘s Manual of Determinative Bacteriology ” ( the 9th edition ) to find comprehensively.

2.3.3 The growing curve of Enterococcus faecium

The growing of bacteriums includes increasing volume, measure and the betterment of metabolic map. Different bacteriums have their ain different growing form. Under the unchanged civilization conditions, calculate the figure of bacteriums oriented to clip, clip as abscissa, the figure of bacteriums as perpendicular axis we can acquire a curveaˆ‚The growing curve includes the stagnancy period, logarithmic stage, stationary stage and diminution stage. The length of each period is different harmonizing to the medium composing and civilization conditions due to bacteria ‘s ain features. In a comparative survey of different bacteriums, growing curve can assist to take bacteria with the same growing stat and this can do a dependable experiment consequence. Detect the bacteria-growth curve chiefly use two methods, such as turbidness method and feasible being dilution count. Turbidity method is to put the different phase and different concentration bacterial civilization to the spectrophotometer, and number the bacteriums by turbidness method. We can utilize the turbidness to stand for the output of bacteria and step entire dacterial count ( including the dead bacteria ) . It is simple, installation comparing with the turbidness method. But it merely can bespeak the bacterial comparative growing information. Feasible being dilution method of numeration is to thin the bacterial liquid to different dilution of 10-1aˆ?10-2 and so on. Every dilution can be cultured in the home base and settlement is counted with colony events-per-unit-time metre. The entire measure of feasible being by utilizing this method can stand for the true growing information of bacteria.

3 Application of Enterococcus faecium

3.1 Supplement of dood with enterococcus faecium ( SF68 ) stimulates immune maps

The enteric microflora play a important function in host defence as demonstrated by their ability to modulate both innate and acquired unsusceptibility at the local every bit good as systemic degrees. Due to these immunological belongingss, specific strains of lactic acid bacteriums ( LAB ) ,3 defined as probiotics, have raised considerable involvement in recent old ages. When ingested as a provender addendum in sufficient Numberss, probiotics are unrecorded micro-organisms that beneficially affect the GI balance, traveling far beyond the conventional nutritionary consequence. The mechanisms underlying the immune modulating belongingss of probiotics are non to the full understood. However, they may be due indirectly to the ability of probiotics to equilibrate the enteric microflora and/or be a effect of a direct accessory consequence on the production of immune factors, such as cytokines. In fact, several strains of LAB were shown to heighten nonspecific unsusceptibility in vitro every bit good as in vivo, including the release of tumour mortification factor-I± and interleukin 6 ( 4 ) , increased phagocytosis in mice and worlds ( Perdigon, G. et Al, 1988 ) ( Schiffrin, E. J. et Al, 1995 ) and stirred natural slayer cell activity ( Matsuzaki, T. , 1998, aller, D. et Al, 2000 ) . Their ability to specifically modulate the host ‘s immune responses to pathogens was besides demonstrated ( Meydani, S. N. & A ; Ha, W. K. , 2000 ) . An addition in rotavirus-specific antibodies was detected in kids with acute rotavirus diarrhoea who received Lactobacillus rhamnosus ( Kaila, M. et Al, 1992 ) . Furthermore, it was shown that disposal of L. johnsonii to human voluntaries boosted the specific Ig IgA response to the Salmonella typhi vaccinum Ty21a ( Link-Amster, H. et Al, 1994 ) .

3.2 Application in intervention

Enterococcus fececium can ferment a assortment of sugar, and merchandise acid. It has high chemical forbearance, and is widely distributed in nature, but besides human and carnal intestine microbial. It has extremely detected in animate being fecal matters ( Archimbaud, C. et al. , 2003 ) . Ultilizing non-toxic, facultative anaerobic Enterococcus, in concurrence with other carnal probiotics ( Animal Microecologicai Agent, AMEA ) can forestall and handle many digestive piece of land diseases of animate beings by replaceing antibiotics.

Intestinal vegetations can be regarded as a normal human organic structure variety meats, these normal vegetations straight involved in the activities of the host metamorphosis is normal physiological activities of the host indispensable constituent ( Baldassarri, L. et al. , 2001 ) . Endogenous Enterococcus as one of the normal vegetations of the human organic structure, chiefly the Ministry of colonisation in the ileum and colon, and some strains may be straight involved in the metamorphosis of the host stuff. Enterococcus lactic acid fecal matters as enteric vegetations, with a bio-barrier map, from healthy people or animate beings that have separate physiological maps of bacteriums to re-giving taking it right people are harmless and safe, but such microorganisms in the organic structure and settled and reproduction Ecology is a really complex issue. Made feasible by the agent is likely to play a biological activity within a certain scope, or portion of engagement in host metabolic activity, ensuing in a certain physiological function and hence have the disease, seting the significance of the ecological balance ( Chow, J. W. et al. , 1993 ) .

Intestinal vegetations can be regarded as normal human organic structure variety meats. These normal vegetations straight involved in the activities of the host metamorphosis, and are normal physiological activities of the host indispensable constituent. Endogenous Enterococcus as one of the normal vegetations in human organic structure, chiefly lasting works in the ileum and colon, and some strains may be straight involved in the metamorphosis of the host stuff. Enterococcus faecium as enteric vegetation, has a bio-barrier map. Enterococcus faecium separated from healthy people or animate beings besides have physiological maps Re-giving to people is harmless and safe, but works and reproduction in the organic structure is a really complex ecology issue. Feasible being praeparatum is likely to play a biological activity within a certain scope, or portion of engagement in host metabolic activity. It merchandises a certain physiological function and therefore can be used in the disease provention, intervention and the ecological balance accommodation.

Presently, the application of enterococci is chiefly in Enterococcus faecium. The earliest illustration of the usage of Enterococcus faecium is out of Japan. It was foremost developed for the intervention of assorted enteric diseases. Yogurt merchandises on the market are incorporating Enterococcus faecium in Japan or pharmaceutical drugs, such as Lascpan, Lacton, Ruosu, Lebenin, Biosmin. “ Mummy Love ” that merchandise in South Korea contains 108 / g fecal matters enterococci and it can handle gastro-intestinal diseases in babies probiotic readyings ( Coque, T. M. et al. , 1995 ) .

3.3 Safety of Enterococcus faecium

Endogenous enteric bacteriums, particularly the colonisation of lactic acid bacteriums in the intestine healthy people, the people are safe, because a big figure of endogenous enterococci colonisation in the human enteric piece of land under the spot, which may impact the host enteric digestion and soaking up, and other map. It has been reported ( Dicuonzo, G. et al. , 2001 ) that yoghurt incorporating Enterococcus faecium can forestall diarrhoea. By double-blind trial, the incidence of antibiotic group was 27.2 % , while taking the probiotic group was merely 3.7 % . So it is non merely proved to be effectual, but did non happen any side effects. In 1989 Food and Drug Administration ( FDA ) and the American Association of Feed Public ( AAFCO ) has besides introduced the safety 42 probiotic bacteriums, including Enterococcus faecium.

3.4 Oral application of Enterococcus faecium strain EE3

Probiotic disposal is being used progressively in human and veterinary medical specialty. A assortment of micro-organisms, particularly lactic acid bacteriums ( LAB ) , such as lactobacilli, bifidobacteria, and enterococci nonpathogenic LAB are associated with a broad assortment of beginnings, such as works stuff and assorted nutrients. Based on the definition of probiotics, equal Numberss of feasible beings must make the enteric piece of land to hold a good consequence. For this to go on, probiotic beings must be able to last theodolite through the acidic environment of the tummy and persist in the presence of gall. They have to multiply at the site of finish in the bowel. In add-on, they must be able to bring forth the counter metabolites against ruling saprophytic microflora ensuing in competitory growing. Antimicrobial activity of LAB is due to the production of organic acids ( lactic and acetic acids in peculiar ) , C dioxide, ethyl alcohol, H peroxide, biacetyl and bacteriocins. These abilities are ofen found among lactobacilli and enterococci, which are most often used as carnal provender addendums or straight as probiotic readyings. Among enterococci, merely the species of Enterococcus faecium of hunman beginning was used as a commercial probiotic. E.faecium used as probiotics expeditiously protect animate beings from diseases caused by E.coli, salmonellae or clostridium ; it is besides reported ( Eaton, T. J. & A ; Gasson, M. J. , 2001 ) that the best preventative effectiveness of E.faecim from commercial probiotic readying Vitacanis against Salmonella enterica serovar Typhimurium in the GI piece of land of gnotobiotic mice.

E.faecium EE3 train was able to last theodolite through the laniary enteric piece of land, taking to enrichment of the colonic microflora and local and systematic effects. This strain besides persisted in the digestive piece of land for about 3 months after surcease of its disposal. The new eyetooth probiotic strain of Lactobacillus fermentum AD1 persisted in the fecal matters of Canis familiariss even for 5 months after surcease, nevertheless, it reached a lower concluding concentration ( 103~105 CFU/g ) than the EE3 strain ( 105~108 CFU/g ) . On the other manus, the well-known probiotic strain Lactobacillus fermentum GG survived in the healthy Canis familiariss merely 8d after surcease of its disposal. From the clinical point of position, continuity during disposal should be less of import than colonisation. However, the possible probiotic micro-organism should be able to colonise the digestive piece of land at least temporarily. Decrease of entire enterococci can be explained likely by competitory interactions of the EE3 strain with other enterococci. Although enterococci usually producted lactic acid, its higher sums ( as the other enterococci ) could be achieved by the concentration of inoculated EE3 strain ; in add-on, differences in lactic acid production were found among enterococci isolated from different beginnings. Local effects of the EE3 strain application included an addition of LAB and a important lessening of Pseudominas-like bacteriums and staphylococcus with drawn-out consequence up to the terminal of the experiment It is reported that a important addition in faecal LAB during disposal of nutrient supplemented with probiotic strain L.acidophilus DSM 13241. However, the action of the probiotic, in footings of its consequence towards the figure of lactobacilli, was lost after surcease of nutrient supplemented with the same probiotic strain ( L.acidophilus DSM 13241. ) . E.faecium EE3 strain survived the theodolite through the digestive piece of land of Canis familiariss. Our consequences suggested that bacteriums and staphylococcus. Furthermore, the degrees of entire lipoids, proteins and cholesterin in blood of healthy Canis familiariss were besides influenced to make physiological bounds.

3.5 Application of Enterococcus faecium strain M-74

The value of pull offing coronary artery disease hazard factors has been confirmed in the past few decennaries. Hypercholesterolemia, high blood pressure and smoke are still considered to be the “ classical ” hazard factors. Intervention tests have by and large shown that take downing “ hazard factors ” reduces the subsequent rate of cornonary bosom disease, shot, and other cardiovascular diseases. The Framingham Study was the major survey, which verified that cholesterin increases the hazard of developing bosom disease. Data end product was clear: the hazard of clinically evident coronary bosom disease is a uninterrupted nonlinear map of blood cholesterin degrees. Each addition in the serum cholesterin concentration by 1 % consequences in 2~3 % addition in the hazard of CHD. The find that hypercholesteremia plays a major function in the formation and development of coronary artery disease has led to a figure of pharmacological and non-pharmacological ( including dietary ) approaches resulted in its riddance. Fact that the serum cholesterin concentration is influenced by nutrient has been known for a long clip ago. Subsequently, the first grounds of impacting cholesterin degree by act uponing enteric microflora was found. One manner to make this is by utilizing probiotics. Up to now, the inquiry of supposed hypocholesterolemic consequence of probiotics has non been decidedly established. While some surveies documented it good, others bring contradictary findings or even doubt it. Another unsolved issue is an bing antecedently mentioned consequence from the long term point of position. In available beginnings, we have non found any study where probiotics were administrated for one twelvemonth. Some experimental and clinical surveies have documented the hypocholesterolemic consequence of rpobiotic civilizations. Not all of the probiotics seem to hold the same belongingss and same consequence on serum cholesterin degrees. The purpose of our survey was to look into the impact of long-run orally administered probiotic strain Enterococcus faecium M-74 enriched with Se on lipid profile ( entire cholesterin, LDH, HDL and triglycerides ) in worlds.

Originally, the term “ probiotics ” was used for a phenomenon, when each of both together cultivated beings had produced substances exciting the growing of the another 1. The significance of the term “ probiotic ” has been germinating since. Many definitions have been suggested. The most normally used, scientifically valid and hence acceptable one is that postulated by Fuller. Harmonizing to him, probiotics are populating micro-organisms that favorably influence the heath of the host by bettering composing of its enteric microflora. The FAO ( Food and Agriculture Organisation for the United Nations ) and WHO ( World Health Organisation ) refer to probiotics as life micro-organisms which when ingested in sufficient sum, beneficially act upon the wellness of the host. Before a certain bacterial strain can be classified probioticm it must run into some standards. The most of import one is its good wellness consequence. The cognition of probiotic intercession in lipid metamorphosis is non new. In the 1970s, research workers observed hypocholesterolemic consequence of yogurt eaten day-to-day in big sum in worlds. Similar observations have been seen in carnal surveies. Many tests aimed to turn outing the hypocholesterolemic consequence of milk and fermented milk merchandises come from early observation. It is found that a low incidence of clinical coronary bosom disease in some African folks with low cholesterin degrees in malice of their diet rich in concentrated fats, meat and cholesterin but abundant in milk and fermented milk merchandises along. In some surveies it is observed that a gradual lessening of serum cholesterin concentration in topics having capsules with Enterococcus faecium M-74 from baseline up to hebdomad 23 when the lessening exceeded important degree ( P & lt ; 0.001 ) . This decrease remained of import besides one month after the followup, i.e. 4 hebdomads after surcease of capsules application ( -0.81 mmo ; /l, i.e. by 13.6 % ) . The nonpharmacological attacks to the direction of coronary arteria disease hazard factors still have non been exhausted, even in the modern pharmacological and interventional intervention epoch. In our survey, the disposal of the E.faecium M-74 probiotic strain was associated with the decrease of serum cholesterin concentration by 12 % after 56 hebdomads. This alteration was chiefly due to the lessening in LDL cholesterin, as no important changes in HDL and triglycerides were no ted. With semilunar sum of facts it is sensible to speculate that probiotics will happen a topographic point as a curative option. Alteration of the interstine microflora ( viz. by probiotics ) may represents a new challenge in the intervention of assorted disease, i.e. cardiovascular upsets.


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