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[Experimental evaluation of the biological safety of gene-engineered bacteria using a model strain Bacillus subtilis interferon-producing strain].

The in vitro and in vivo evaluation of the biological and ecological safety of genetically modified bacteria (GMB) was carried out on B. subtilis recombinant strain 2335/105, capable of producing human interferon alpha-2, used as experimental model. As shown in this investigation made with the use of bacteriological analysis and polymerase chain reaction, the oral administration of GMB to calves, chickens and white mice produced no disturbances in the microbial ecology of the gastrointestinal tract of warm-blooded animals and did not lead to the appearance of spontaneous transformants. The present work is the first experimental evaluation of the biological safety of genetically modified microorganisms, used as the component of Subalin, a probiotic preparation intended for use in veterinary practice.

Animals↗

[Dysbacteriosis--infectious processes of mixed etiology].

At present dysbacteriosis or altered normal and sometimes very individual proportion of microorganisms in various macroorganism microbiotopes is considered as an infectious disease since it results from pathogenic action of microbes, is characterized by a set of features relating to pyoinflammatory processes and even can be contagious.

Adult↗

[Effect of intravenous and intragastric feeding on the microflora of the rat intestine].

During intravenous feeding, the contents of the cecum showed a dramatic fall in the amount of anaerobic lactobacilli and the growth of the content of enterobacteria and enterococci. The level of staphylococci and streptococci remained unchanged. These changes did not depend on the presence of fatty component in the nutritive mixture. The switch over of the animals from oral to intragastric feeding did not produce any changes in the microbile profile of the cecum. However, a distinct relationship was noticed between the overall amount of all the representatives of the microflora and the feeding pattern. Apart from an increase in the content of the microorganisms caused by administering a full-value nutritive mixture to the stomach, there was an appreciable rise in the cecum mass.

Amino Acids↗

Hepcidin--a peptide hormone at the interface of innate immunity and iron metabolism.

Hepcidin is a cationic amphipathic peptide made in the liver, released into plasma and excreted in urine. Hepcidin is the homeostatic regulator of intestinal iron absorption, iron recycling by macrophages, and iron mobilization from hepatic stores, but it is also markedly induced during infections and inflammation. Under the influence of hepcidin, macrophages, hepatocytes, and enterocytes retain iron that would otherwise be released into plasma. Hepcidin acts by inhibiting the efflux of iron through ferroportin, the sole known iron exporter that is expressed in the small intestine, and in hepatocytes and macrophages. As befits an iron-regulatory hormone, hepcidin synthesis is increased by iron loading, and decreased by anemia and hypoxia. Hepcidin is also rapidly induced by cytokines, including IL-6. The resulting decrease in plasma iron levels eventually limits iron availability to erythropoiesis and contributes to the anemia associated with infection and inflammation. The decrease in extracellular iron concentrations due to hepcidin probably limits iron availability to invading microorganisms, thus contributing to host defense.

Amino Acid Sequence↗

Immune responses of specific pathogen-free and gnotobiotic mice to antigens of indigenous and nonindigenous microorganisms.

Strains of indigenous Escherichia coli, Bacteroides, and Lactobacillus were isolated from the gastrointestinal tracts of specific pathogen-free (SPF) mice. Nonvaccinated SPF mice exhibited in their spleens low numbers of plaque-forming cells (PFC) and rosette-forming cells reacting with antigens of these andigenous bacteria. PFC reacting with these bacterial antigens were not detected in infant SPF mice until 7 days after birth. Compared with nonvaccinated controls, SPF mice vaccinated parenterally with indigenous E. coli or Bacteroides produced a moderate increase in the numbers of specific PFC. Thus, the SPF mouse is capable of responding immunologically after vaccination with microbes indigenous to its intestinal tract. However, more PFC reacting with homologous vaccine antigens were detected after parenteral vaccination of SPF mice with nonindigenous E. coli O127:B8, E.coli O14, or B. fragilis than after parenteral vaccination with indigenous E. coli or Bacteroides. Gnotobiotic mice orally monoassociated with these nonindigenous bacteria exhibited greater immune responses to antigens of the bacteria used to monoassociation than did gnotobiotes monoassociated with the indigenous microbes. The results are consistent with the hypothesis that mice are more responsive immunologically to antigens of nonindigenous bacteria than they are to antigens of certain microbes indigenous to their gastrointestinal tracts.

Animals↗

Effects of daily oral administration of rifaximin and neomycin on faecal aerobic flora in rats.

The aim of this study was to compare the influence of rifaximin and neomycin on faecal flora in rats. The study was performed on 18 Wistar rats (three groups of six male animals). Group 1 received rifaximin (50 mg kg(-1)/day), group 2, neomycin (50 mg kg(-1)/day) and group 3 was used as control. Drugs were administered orally, once daily for 3 days. Faecal specimens, collected from each rat on day 3, were cultured for the quantitative and qualitative determination of aerobic microorganisms. Rifaximin treatment produced a marked reduction in the number of total aerobic bacteria and Salmonellae; neomycin caused reduction in Salmonellae, but did not cause statistically significant changes in total aerobic bacterial count. The binding of neomycin with faeces could explain this limited activity, which does not correlate with the in vitro susceptibility of the organism affected. These results confirm that rifaximin is suitable for topical treatment to reduce selected bacterial load in the gut intestines.

Administration, Oral↗

Clinical uses of microorganisms and their products.

Although antibiotics and conventional vaccines are the two most familiar examples of man's exploitation of microorganisms as clinical allies, microorganisms and their products are assuming an increasingly prominent role in the diagnosis, treatment and prevention of human diseases. This report attempts to give a brief overview of the status of the use of microorganisms in clinical medicine and to identify potentially fertile areas for future progress in their clinical application, concentrating on areas other than the already extensively reviewed ones of antibiotics and classic immunization.

Antigens, Bacterial↗

Mechanisms of lipid-body formation.

Most organisms transport or store neutral lipids as lipid bodies - lipid droplets that usually are bounded by specific proteins and (phospho)lipid. Neutral-lipid bodies vary considerably in their morphology and are associated with an extremely diverse range of proteins. However, the mechanisms by which they are generated in plants, animals and microorganisms appear to share many common features: lipid bodies probably arise from microdomains of the endoplasmic reticulum (or the plasma membrane in prokaryotes) that contain lipid-biosynthesis enzymes, and their synthesis and size appear to be controlled by specific protein components.

Adipocytes↗

Fate of cyst(e)ine synthesized by microbial activity in the ruminant caecum.

Cyst(e)ine synthesized by microorganisms in the caecum of sheep was labelled following the infusion of Na2 35SO4 into the terminal ileum. [35S]Cyst(e)ine activity was detected in the faeces, but not in plasma or wool. Appreciable absorption of 35S, presumably in the form of sulphide, into the circulation occurred, and its presence in saliva was demonstrated. It was concluded that nutritionally negligible quantities of cyst(e)ine are likely to be absorbed from the ovine large intestine.

Animals↗

[Infectious diarrhea study in Castellón, Spain (EDICS): population incidence of sporadic cases in 2004 and comparison with the year 2000].

BACKGROUND AND OBJECTIVE: Infectious intestinal diseases are highly prevalent, and among them sporadic cases are the most common processes. The aim of this study was to estimate the incidence of sporadic cases diagnosed in a healthcare district of Castellón (Spain) in the year 2004, and to compare them with those of the year 2000. MATERIAL AND METHOD: Based on routine coprocultures and individual demographic data of each patient, we calculated the population rates by age for the more frequent microorganisms, especially detailed for children up to 5 years of age. We report the results of the year 2004, and compare these with the previous study of the year 2000. RESULTS: Campylobacter showed the highest rate (1.01 x 103), followed by Salmonella (0.75), each without significant differences regarding the year 2000. The rate of rotavirus cases (0.65) was smaller than in 2000; and for adenovirus 2004 was an atypical seasonal year. In children younger than 3 years, rates were 26.54; 10.23 and 11.75. As in the previous study, the age-distribution in children < 5 years was very particular. An U inverted shape was seen for Campylobacer, and the rates for rotavirus decreased after the peak observed in the youngest group (< 6 months). Among hospitalized patients rotavirus was the most common and Campylobacter the less common. CONCLUSIONS: The epidemiological traits of the rates by age until the age of five are characteristic for each microorganism, as are the proportions of hospitalization. Rates of Campylobacter and Salmonella in the year 2004 were similar to the year 2000, but a decrease in the rotavirus rates was observed.

Adolescent↗

[A method of determining the effect of breast milk on the adhesive properties of microorganisms].

The authors have modified the method for studies of the microorganism adhesive properties in the presence of breast milk; this modification is based on the ability of bacterial cells to adhere to the surface of human red cells of I (0) Rh (+) blood group. The study has revealed significant differences in the adhesion characteristics of S. aureus 209, B. bifidum I, and B. adolescentis MC-42 strains in the presence of breast milk of women with normal babies and of those whose babies suffered from intestinal dysfunctions.

Bacterial Adhesion↗

Metal speciation dynamics and bioavailability: bulk depletion effects.

Under conditions of bulk depletion, the speciation and bioavailability of trace metals must be considered at two different time scales: (i) the time scale of the biouptake flux, as determined by diffusion of the bioactive free metal, dissociation of the bioinactive complex species, and the internalization rate; and (ii) the time scale of depletion of the bulk medium. The implications of these two time scales for the speciation dynamics are discussed in terms of experimental conditions. The geometry of the system is taken into accountvia a spherical cellular model. It considers a spherical organism depleting a spherical volume in a nonstirred medium and assumes linear adsorption of the metal atthe biointerface and first-order internalization kinetics. In cases where the rate of biouptake is fully controlled by the internalization step, concentration gradients in the medium are insignificant. Then the biouptake becomes independent of the geometry of the system, and the model has a much simpler solution. Examples of trace metal uptake by microorganisms are analyzed: (i) cobalt uptake by Prochlorococcus in the presence of NTA, under conditions where bulk depletion is the controlling process due to the large number of organisms and high internalization rates, (ii) silver uptake by Chlamydomonas reinhardtii with significant effects of bulk depletion, due to the high internalization rate; (iii) lead uptake by Chlorella vulgaris with pratically negligible bulk depletion due to the low internalization rate of the metal; and (iv) lead uptake by intestinal Caco-2 cells, illustrating the simplification of the bulk depletion model for a system with different geometry where internalization is the rate-controlling step.

Animals↗

Probiotics in human disease.

Western civilization is facing a progressive increase in immune-mediated, gut-related health problems, such as allergies and autoimmune and inflammatory diseases, and genetic factors are an unlikely explanation for these rapid increases in disease incidence. Two environmental factors that relate to the modern lifestyle in Western societies are hygiene and nutrition. There has been a decline in the incidence of microbial stimulation by infectious diseases as a result of improved hygiene, vaccination, and antimicrobial medication. In the past, methods of food preservation involved either the natural fermentation or drying of foods; thus, the human diet once contained several thousand times more bacteria than it does today. The development of probiotic, functional foods aims to "kill two birds with one stone," which is accomplished by providing a microbial stimulus to the host immune system by means of beneficial live microorganism cultures that are characteristic of the healthy, human gut microflora, ie, probiotics. Probiotic bacteria were shown to reinforce the different lines of gut defense, which are immune exclusion, immune elimination, and immune regulation. They were also shown to stimulate nonspecific host resistance to microbial pathogens, thereby aiding in pathogen eradication. Consequently, the best documented clinical application of probiotics is in the treatment of acute diarrhea. In humans, documented effects were reported for the alleviation of intestinal inflammation, normalization of gut mucosal dysfunction, and down-regulation of hypersensitivity reactions. These data show that probiotics promote endogenous host defense mechanisms. Thus, modification of gut microflora by probiotic therapy may offer a therapeutic potential in clinical conditions associated with gut-barrier dysfunction and inflammatory response.

Diet↗

Recombinant Saccharomyces cerevisiae expressing P450 in artificial digestive systems: a model for biodetoxication in the human digestive environment.

The use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs. A system that mimics the human gastrointestinal tract was combined with a computer simulation to evaluate the survival rate and cinnamate 4-hydroxylase activity of a recombinant model of Saccharomyces cerevisiae expressing the plant P450 73A1. The yeasts showed a high level of resistance to gastric and small intestinal secretions (survival rate after 4 h of digestion, 95.6% +/- 10.1% [n = 4]) but were more sensitive to the colonic conditions (survival rate after 4 h of incubation, 35.9% +/- 2.7% [n = 3]). For the first time, the ability of recombinant S. cerevisiae to carry out a bioconversion reaction has been demonstrated throughout the gastrointestinal tract. In the gastric-small intestinal system, 41.0% +/- 5.8% (n = 3) of the ingested trans-cinnamic acid was converted into p-coumaric acid after 4 h of digestion, as well as 8.9% +/- 1.6% (n = 3) in the stomach, 13.8% +/- 3.3% (n = 3) in the duodenum, 11.8% +/- 3.4% (n = 3) in the jejunum, and 6.5% +/- 1.0% (n = 3) in the ileum. In the large intestinal system, cinnamate 4-hydroxylase activity was detected but was too weak to be quantified. These results suggest that S. cerevisiae may afford a useful host for the development of biodrugs and may provide an innovative system for the prevention or treatment of diseases that escape classical drug action. In particular, yeasts may provide a suitable vector for biodetoxication in the digestive environment.

Computer Simulation↗

On the use of antibiotics in Crohn's disease.

It is difficult to understand how a disease process characterized by ulcerations, fissures, abscesses, fistulas, lymphangitis, and granulomas has not attracted greater use of antibiotics, particularly as the sites are constantly contaminated by intestinal bacteria. I have had a favorable experience with broad-spectrum antibiotics in the treatment of a variety of forms of ileocolitis in animals and now advocate that Crohn's disease be treated at length with these drugs. Microbiologic culture of serosa, mesenteric lymph nodes, and fistulas has demonstrated that bacterial species are present in a significant proportion of cases, and serology has shown that patients have elevated antibody levels to many of these same microorganisms. Now immunocytochemistry provides documentation of Escherichia coli and streptococcal antigen within the lesions of a majority of patients. That these bacteria may be secondary invaders should not decrease our need to address them. Several chronic granulomatous diseases that were once thought to be intractable now yield to long-term antibiotic treatment, including Whipple's disease, malakoplakia, and granulomatous colitis of Boxer dogs. Many of the perianal lesions of Crohn's disease respond to short-term metronidazole, and the medium-term (3-6 months) use of broad-spectrum antibiotics, most recently ciprofloxacin, has shown promising results. In view of the increasing evidence of bacterial participation in this disease, it is now important that physicians test some of our newer broad-spectrum antibiotics, in a controlled format, and over an extended time.

Animals↗

Genotoxic activity in vivo of the naturally occurring glucoside, cycasin, in the Drosophila wing spot test.

Cycasin, methylazoxymethanol-beta-glucoside, is a naturally occurring carcinogenic compound. The genotoxicity of cycasin was assayed in the Drosophila wing spot test. Cycasin induced small single and large single spots on feeding at 10 mumol/g medium. The presence of these spots indicates that cycasin is genotoxic in Drosophila melanogaster. Microorganisms which showed beta-glucosidase activity for cleaving cycasin to toxic aglycon were isolated from gut flora of the Drosophila larvae. Consequently, the Drosophila wing spot test would be useful for mutagenicity screening of other naturally occurring glucosides.

Animals↗

Increased proliferation and apoptosis of colonic epithelial cells in dextran sulfate sodium-induced colitis in rats.

We have evaluated morphologic alterations and epithelial cell apoptosis and proliferation of colonic mucosa in the acute and chronic phases of DSS-induced colitis. Colitis was induced in Sprague-Dawley rats by 7 days of 4% DSS oral administration followed by 7 days of tap water for one, two, and three cycles. Control rats receved tap water only. Morphological changes in colonic mucosa were evaluated and scored by light and scanning electron microscopy. Apoptosis was studied by TUNEL assay and cell proliferation by Ki-67 immunoreaction. The expression of both proapoptotic (Fas, FasL, Bax, p53) and antiapoptotic (Bc12) cellular proteins was determined by immunohistochemistry. Morphologic assessment showed the most severe colonic epithelial lesions and inflammation in the distal colon with a trend to increasing severity from the first to the third DSS cycle. In DSS rats, the epithelial apoptotic index increased 20-fold after the first cycle and 120-fold after the second and third cycles compared with the controls; in the same way, the expression index of proapoptotic proteins (Fas, FasL, Bax, p53) dramatically increased. The proliferative index increased about 40 to 60-fold compared to controls, with no difference among the three DSS cycles. In conclusion, DSS-induced colitis in rats, which has many structural and ultrastructural features similar to those seen in human ulcerative colitis, is a suitable model for studying increased epithelial apoptosis and proliferation. Further studies employing this model will permitt two hypotheses to be tested. (1) Increased apoptosis may lead to a breakdown of the epithelial barrier function and facilitate the mucosal invasion of intraluminal microorganisms and/or antigens. (2) Abnormal and persistent epithelial hyperproliferation could be causally related to the development of colorectal cancers in the setting of chronic colonic inflammation.

Animals↗

Microorganisms and parasites in chronic infective diarrhea.

AIM: To reveal the pattern of microorganisms in chronic infective diarrhea cases. METHODS: We examined all patients suffering from chronic infective diarrhea over a six year period The patients were examined physically and at the same time laboratory tests,colon enema X-ray and colonoscopy, ileoscopy, upper GI endoscopy and small bowel X-ray were performed. RESULTS: We found 138 (66. 7%) chronic infective diarrhea from 207 chronic diarrhea patients. Parasitic causes were Candida albicans (48.55%), Blastocystis hominis (6.52%), Entamoeba histolytica (3.62%), and Giardia lamblia (3.62%) etc. Bacterial causes were Pathogenic E. coli(34.78%), Aerobacter aerogenes (3.62%), Mycobacterium tuberculosis (3.62%), Geotrichum (1.45%), Shigella sonnei(0. 72%), Salmonella paratyphi (2.89%)etc. CONCLUSION: The most frequent microorganisms and parasites found in chronic infective diarrhea were pathogenic E.coli and Candida albicans.

Adult↗

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