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Design and evaluation of a Lactobacillus group-specific ribosomal RNA-targeted hybridization probe and its application to the study of intestinal microecology in pigs.

The potential importance of anaerobic bacteria belonging to the Lactobacillus group has been well documented. Appropriate methods to rapidly evaluate species diversity and fluctuations in their population levels within the Lactobacillus group are being developed. Molecular tools such as hybridization probes based on rRNA sequences are well suited to these studies. The work reported here was undertaken to test the specificity of an hybridization probe to specifically recognize microorganisms of the Lactobacillus group and assess its usefulness as a quantitative tool to study fluctuations of the Lactobacillus population relative to the total bacterial population in gastrointestinal contents of pigs. We have designed a 25-mer oligonucleotide that targets a region common to and specific for the Lactobacillus group 16S rRNA sequences within the available database. The optimal wash temperature of the probe was experimentally determined to be 54 degrees C. The results obtained using the Lactobacillus group-specific probe (LGP) shows that Lactobacillus populations vary along the different segments of the gastrointestinal tract (GIT). In weaning piglets, the relative Lactobacillus signal intensity obtained constituted 100% of the relative RNA index in the stomach contents as determined by a bacterial domain probe (BDP), and between 90 to 100% in the duodenum. The signal of the Lactobacillus population decreased and reached its minimum in the distal part of the GIT. The same trend was observed in adult pigs, but in the stomach they constituted no more than 30% as determined by the BDP, and were present at lower levels in the other parts of the GIT. These studies document the quantitative importance of the lactobacilli in the stomach and small intestine of pigs. Further studies to investigate the role of lactobacilli in promoting the ecological balance of gut bacteria for probiotic therapy are being undertaken.

Animals↗

Hepcidin--a regulator of intestinal iron absorption and iron recycling by macrophages.

Hepcidin is a recently discovered peptide made in the liver, distributed in plasma and excreted in urine. This peptide hormone is the homeostatic regulator of intestinal iron absorption, iron recycling by macrophages, and iron mobilization from hepatic stores. Hepcidin acts by inhibiting the efflux of iron through ferroportin, the sole known iron exporter of enterocytes, macrophages and hepatocytes. As befits an iron-regulatory hormone, hepcidin synthesis is increased by iron loading and decreased by anemia and hypoxia. Hepcidin is markedly induced during infections and inflammation, causing iron to be sequestered in macrophages, hepatocytes and enterocytes. The resulting decrease in plasma iron levels eventually contributes to the anemia associated with infection and inflammation. These alterations in iron metabolism probably have a role in host defense by limiting the availability of iron to invading microorganisms. At the opposite extreme, early studies indicate that hepcidin deficiency--due to the dysregulation of its synthesis or mutations in the hepcidin gene itself--is the immediate cause of most forms of hemochromatosis.

Anemia↗

[Antibiotic treatment of perforated appendicitis in childhood--a prospective study].

A prospective randomised study (I) was carried out on 130 children undergoing laparotomy for perforated appendicitis. The present study evaluates the therapeutic effectiveness of different antibiotic regimens in modifying the rate of post-operative complications (intraabdominal abscess, ileus, wound infiltration or abscess). During the first year of this study two treatment groups were used. Group A consisted of 29 children treated with Sulfometrol/Trimethoprim; the rate of postoperative complications was 44.8%. Group B consisted of 36 children treated with mezlocillin, the rate of complications being 13.8%. However, the same treatment with mezlocillin during the second year of the prospective study showed an increase of this rte to 39% (28 children--group C). The final group of 37 children was treated during the second year with mezlocillin and metronidazole (group D). The postoperative rate of complications was 10.8%. A retrospective analysis of further 80 children (study II) with perforated appendicitis treated with mezlocillin and metronidazole showed a consistent low rate of postoperative complications at 10.2%. In 6.8% of children studied, an operative intervention was necessary (four cases of ileus, four wound abscesses). The mean postoperative hospitalisation period decreased from 22.7 days in group A to 15.2 days in group D and finally to 14 days in study II. The present study shows that an effective and persistent attenuation of the rate of postoperative complications after perforated appendicitis in children depends on an early onset of therapy and on the appropriate choice of antibiotic drugs that are effective against aerobic and anaerobic microorganisms.

Anti-Bacterial Agents↗

Disseminated toxoplasmosis as a cause of diarrhea.

Disseminated toxoplasmosis in patients with acquired immunodeficiency syndrome is often a central nervous system disease but may be seen as myocarditis or pneumonitis. Toxoplasmic colitis is a rarity. We report a case of toxoplasma colitis in a patient with diarrhea and hypokalemia. The microorganisms were identified in the colonic mucosa on H&E sections and confirmed by immunoperoxidase study.

Adult↗

[DNA analyses in diagnostic pathology].

Since the publication of the polymerase chain reaction (PCR) technique in 1985, there has been an explosion of reports on its use in medicine and science. In this overview the use and limitations of PCR in diagnostic pathology are discussed. As biopsies get smaller, molecular technology will play an ever greater role in improving diagnostic accuracy. PCR is useful in tumour characterisation by assessment of clonality and detection of specific chromosomal rearrangements. This way better targeting of therapeutic intervention is possible. The value of PCR in the diagnosis of cancer, HLA typing and the detection of microorganisms is illustrated by some case reports.

Adult↗

Microbial risks in mild hot smoking of fish.

Hot smoked fish should fulfill the requirements set up for ready-to-eat foods. The total bacterial count on the raw material is 10(3) to 10(5) CFU per cm2 of skin and 10(2) to 10(9) CFU per gram of intestines. Contamination with pathogenic bacteria is very low, mainly with Clostridium botulinum, Listeria monocytogenes, Staphylococcus aureus, and Vibrio parahaemolyticus. In the premises of low hygiene, the contamination may increase due to unsanitary procedures, rotation of assigned duties of workers, and airborne microorganisms during packing of the product. Hot smoking in mild conditions at temperature in the fish not exceeding 65 degrees C and low concentration of salt does not inactivate all pathogens or inhibit bacteria during storage. Thus the required safety can be obtained only by using very fresh fish handled in hygienic conditions, controlling the processing and the plant hygiene in critical control points, and chilling of the product to about 2 degrees C. Most critical are the hygienic conditions in handling of the product after smoking. The use of preservatives for extending the shelf life of smoked fish is being investigated. High-quality shelf life of mild hot smoked mackerel at about 2 degrees C is at least 3 weeks.

Animals↗

Severe abdominal infections in neutropenic patients.

Abdominal infections are an important cause of morbidity and mortality in neutropenic patients. We present a retrospective series of 16 patients, mostly with acute leukemia, who developed severe abdominal infections during chemotherapy-induced neutropenia between 1991 and 1997. The frequency among patients with acute leukemia was 2.35% (13 of 553). Thirteen patients presented with enterocolitis and 3 patients presented with cholecystitis. Eight patients died. Bacteremia was present in 6 patients, 4 patients suffered from proven or strongly suspected fungal infections, and 1 patient suffered from cytomegalovirus infection. Early surgical management was required in a patient with intestinal obstruction, whereas other patients could be managed conservatively. Two patients with acute cholecystitis were treated with antibiotics until the end of neutropenia and then were resected. Severe abdominal injections in neutropenic patients, which are often fatal, were caused by nonbacterial microorganisms in one-fourth of the cases and could be managed conservatively in most instances.

Abdomen↗

Interaction of microorganisms, epithelium, and lymphoid cells of the mucosa-associated lymphoid tissue.

Differentiation of specific epithelial cell lineages during development, as well as epithelial plasticity in response to heterologous cell-to-cell cross talk during adult life, accounts for the large variety of functions which are performed by the mucosal surfaces found in the human body. Among its functions, the digestive mucosa is able to sample antigens and microorganisms through M cells of Peyer's patches' follicle-associated epithelium, in order to trigger the development of either tolerance or immune responses. At least in the gut, M-cell formation is immunoregulated. Close contact between immune cells and intestinal epithelium modifies the permeability of the epithelial barrier by inducing the conversion of enterocytes into M cells, offering at the same time an opportunistic way of invasion for pathogens. These lympho-epithelial interactions triggering M-cell formation have now been modeled in culture.

Adult↗

Induction of Listeria monocytogenes infection by the consumption of ponderosa pine needles.

An infectious microorganism, identified as Listeria monocytogenes, has been isolated from the bloodstream of pregnant mice fed a diet containing Pinus ponderosa needles. When the isolate was injected into pregnant mice, reproductive dysfunction and other changes, including speckled livers, spleen atrophy, and hemorrhagic intestines, appeared to mimic the signs of the disease in pregnant mice fed pine needles. Moreover, these pathological changes are similar to those observed in cattle and other mammals experiencing abortions or toxemia, or both, attributed to the ingestion of P. ponderosa needles, suggesting that L. monocytogenes may be a part of the etiology of "pine needle abortion."

Abortion, Veterinary↗

Probiotics as biotherapeutic agents: present knowledge and future prospects.

Since the early observations of Elie Metchnikoff, a wealth of experiments have described the use of selected microorganisms, mainly belonging to the lactic acid bacteria family, for the prevention or treatment of a variety of pathological situations. Nevertheless, the mechanisms underlying the proposed actions remain vastly unknown, partly as a consequence of the complexity of the gastro-intestinal ecosystem with which these biotherapeutic agents are expected to interact, but also because of the increasing variety of strains considered to have potential probiotic characteristics. During the past decades, however, the beneficial effect of specific strains in preventing or treating intestinal disorders has been substantiated by well-controlled clinical trials. Increasing evidence, including human studies, is also supporting the immunomodulatory role attributed to given lactic acid bacterial strains. The desire by consumers to use natural methods for health maintenance rather than long-term chemotherapeutic agents (i.e. antibiotics), linked to their expectation that food becomes a source of prolonged well-being, supports the speculation that the probiotic market will expand rapidly. Much of this growth will also depend on the reliability of claims that these products will bare. Therefore, the legislator will have to provide clear rules and regulations which will depend on measurable biomarkers and criteria based on scientific evidence. These commercial and legislative needs will hopefully provide scientists with the resources necessary to conduct the multidisciplinary research required to establish facts and mechanisms of action for carefully selected probiotic strains. These research results will probably be as essential for the positioning of probiotic preparations as either a food, a food supplement or as pharmaceutical preparation.

Animals↗

Bacteriology of anal fistulae.

OBJECTIVE: To evaluate by bacteriological analysis the possible relationship between the chronicity of perianal fistulae and the presence of probable permanent infection of the fistulae. MATERIAL AND METHODS: The study included 27 patients, 21 men and 6 women who had undergone surgery for chronic perianal fistulae type II (according to the Parks classification). A total of 27 samples of their fistulous tracts were sent to the microbiology department for the identification of germs. All samples were taken from the theatre and delivered to the laboratory in the same manner. They were then processed under de same conditions in terms of dilutions, inoculation and culture mediums used for the identification of germs in the tracks of the fistulae. RESULTS: A total of 45 were isolated from 21 different species of microorganisms. Most samples (78.26%) had polymicrobic growth. The predominant species were: Escherichia coli (45.45%), Bacteroides fragilis (16.66%), Staphylococcus aureus (12.12%) and Streptococcus viridans (12.12%). No Mycobacteria were found in any specimen. CONCLUSIONS: Anal fistulae can be colonized by poli- or mononormal intestinal or skin microbacterial flora or a combination of both. This report suggests that neither the type or the number of germs its related to the chronicity of cryptoglandular anal fistulae.

Anal Canal↗

Review article: Intestinal epithelia and barrier functions.

The mucosal epithelia of the digestive tract acts as a selective barrier, permeable to ions, small molecules and macromolecules. These epithelial cells aid the digestion of food and absorption of nutrients. They contribute to the protection against pathogens and undergo continuous cell renewal which facilitates the elimination of damaged cells. Both innate and adaptive defence mechanisms protect the gastrointestinal-mucosal surfaces against pathogens. Interaction of microorganisms with epithelial cells triggers a host response by activating specific transcription factors which control the expression of chemokines and cytokines. This host response is characterized by the recruitment of macrophages and neutrophils at the site of infection. Disruption of epithelial signalling pathways that recruit migratory immune cells results in a chronic inflammatory response. The adaptive defence mechanism relies on the collaboration of epithelial cells (resident sampling system) with antigen-presenting and lymphoid cells (migratory sampling system); in order to obtain samples of foreign antigen, these samples must be transported across the barriers without affecting the integrity of the barrier. These sampling systems are regulated by both environmental and host factors. Fates of the antigen may differ depending on the way in which they cross the epithelial barrier, i.e. via interaction with motile dendritic cells or epithelial M cells in the follicle-associated epithelium.

Adaptation, Physiological↗

Typhlitis. An 18-year experience and postmortem review.

A review of pediatric autopsy results at the Texas Children's Hospital, Baylor College of Medicine from 1970 through 1987 was conducted. Thirty-three cases of typhlitis were identified in patients with acute leukemia and two cases each in patients with lymphoblastic lymphoma and aplastic anemia. Patients ranged in age from 10 months to 17 years. Fifty-seven percent were male and 43% were female. All were myelosuppressed. A postmortem incidence rate of 24% was determined for patients with acute leukemia. Common symptoms included abdominal pain and distention in 78% of patients and acute lower gastrointestinal bleeding in 35%. Abdominal radiographs varied in spectrum from a nonspecific bowel gas pattern to frank right colonic pneumatosis intestinalis. Thirty-three patients received chemotherapy within 30 days before onset of abdominal symptoms. All patients were febrile (greater than 38.5 degrees C), and 33 received broad-spectrum antibiotics. Three patients received amphotericin B. Premortem, 84% of organisms cultured from blood were bacterial whereas 16% were fungal. Fungal pathogens accounted for 53% of new microorganisms seen at autopsy. Postmortem examination showed typhlitis in the following anatomic distributions: (1) confined to the cecum; (2) involving the cecum and ileum; (3) involving the cecum, ileum, and ascending colon; or (4) involving the cecum, with sporadic ulcers throughout the intestine. This review includes clinical and postmortem features of typhlitis and current strategies for diagnosis and management.

Adolescent↗

Peroxidase activity in the epithelium of the digestive tract of the bullfrog, Rana catesbeiana.

Peroxidase activity was examined cytochemically in the mucosal epithelium along the length of the digestive tract from the esophagus through the large intestine during the development of the bullfrog, Rana catesbeiana. In the tadpole of this species, cells with peroxidase activity were found abundantly in the esophagus, stomach, and large intestine; and the types of such cells differed according to the region: ciliated cells and mucous cells in the esophagus; ciliated cells in the stomach; and brush cells, absorptive cells, and goblet cells in the large intestine, respectively. After metamorphosis, however, peroxidase activity was observed exclusively in absorptive cells and goblet cells in the large intestine. Peroxidase activity was commonly demonstrated in apical vesicles or granules, to some degree in rough endoplasmic reticulum, and in some elements of the Golgi apparatus. Furthermore, reaction product was also found in mucus covering the luminal surface of such epithelial cells. These findings indicate that peroxidase-positive cells, which may have the ability to synthesize peroxidase as a secretory product, were distributed mainly in three regions of the digestive tract in tadpoles (esophagus, stomach, and large intestine), but were centered in one specific region, the large intestine, after metamorphosis. Concomitantly, the variety of types of peroxidase-positive cells decreased during metamorphosis. Our results indicate that some of the peroxidase in the digestive tract may have a secretory origin and may play a role in the defense against microorganisms.

Animals↗

Romanomermis culicivorax: penetration of larval mosquitoes.

In the presence of second larval instars of three mosquito species the preparasites of Romanomermis culicivorax swam near the water surface in an orthokinetic manner. When the preparasites were ca. 1 mm from the host, they stopped and swam klinotactically toward the host. During this phase, the preparasites secreted a small amount of a putative adhesive material from the anterior region and host contact was completed. The adhesive appeared to aid in attachment of the preparasites to the host and initiation of the search-boring phase. The preparasites glided over the host until a suitable penetration site was found. The penetration phase was initiated by probing with the odontostyle. This was followed by partial paralysis, decreased intestinal peristaltic movement, and temporary cardiac arrest in all host mosquitoes which was probably related to injection of esophageal secretions. SEM observations showed that the abdominal walls were the most frequent site for penetration. As the preparasites entered through the penetration hole, microorganisms adhering to the cuticle of the preparasites were retained by the adhesive which accumulated around the penetration site. Thus, microbial contamination of the host was avoided by a mechanical cleansing mechanism. Penetration was usually completed in less than 10 min.

Animals↗

Carbohydrates: a limit on bacterial diversity within the colon.

The human large intestine is recognised as a physiologically important organ responsible for the conservation of water and salts. Through its resident bacteria, it is also capable of complex, enzyme catalysed, hydrolytic-digestive functions that have a high biological impact on the host. These microorganisms metabolise dietary components, principally complex carbohydrates that are not hydrolysed or absorbed in the upper gastrointestinal tract, and in this way, sequester energy for the host, through fermentation. This process involves a series of anaerobic, energy-yielding, catabolic reactions which complete digestive processes in the gut, resulting in end products that in turn influence the distribution of microbial species present as well as having some systemic effects. Some of the bacteria are thought to possess important health-promoting activities, especially with respect to their influence on mucosal and systemic immune responses to disease. These bioactivities can be modulated by substrates that support and influence microbial development, growth and survival. For these reasons, it is necessary to review dietary factors that may delimit bacterial diversity, to be able to predict responses and sensitivities to various environmental pressures and manipulations that occur in this area of human microbiology.

Bacteria↗

[Intestinal bacterial microflora study in chronic ulcerohemorrhagic colitis].

Bacteriological tests on feces of patients with ulcerative-hemorrhagic colitis, in exacerbation, were carried out. The same patients were dynamically followed up as regards clinical picture and paraclinical indices, rectoromanoscopy include. Quantitative and qualitative analysis of the fecal microflora was made according to the following indices: total number of mesophili aerobic microorganisms, coli-bacteria, proteus, enterococci, staphylococci, Salmonella-bacteria. The pure cultures isolated were tested according to 30 biochemical indices, their precise classification position being determined on that base. The production of the following enzymes was studied in the cultures isolated: protease, oxyreductase -- catalase and peroxidase, aminotransferase -- COT and GPT; phosphatase -- acid and alkaline, disaccharidase and urease. The data obtained from the bacteriological studies were compared with those of a control group of healthy subjects. Elevated enzyme constellation was found among the patients with exacerbated ulcerative-hemorrhagic colitis.

Adult↗

Green fluorescent protein for detection of the probiotic microorganism Escherichia coli strain Nissle 1917 (EcN) in vivo.

Probiotic microorganisms are defined as viable nutritional agents conferring benefit to the health of the human host. Especially, Escherichia coli strain Nissle 1917 (EcN) was shown to be equally effective as mesalazine in the maintenance of remission in ulcerative colitis (UC). Presumably, the therapeutic effect of EcN is linked to the presence of the strain in the region of interest; however, it remains difficult to follow the orally administered strain on its passage through the complex microbial environment of the intestine in vivo, inhabited dominantly by various E. coli strains, using traditional culturing methods. In this study we transformed EcN and a wild-type E. coli from a laboratory rat (EcR) with a plasmid carrying a gfp gene (pUC-gfp) to obtain EcN- and EcR-GFP to allow in vivo detection without alteration of strain-specific characteristics. Analysis of different strain-specific characteristics included the measurement of stimulation of IL-8 secretion and adhesion in vitro using the epithelial cell line HT-29. The kinetics of intestinal distribution in mice and colonization properties in rats following oral administration was studied in vivo. Detectability of the strain in histologic specimens was analysed using fluorescence microscopy and immunohistochemistry. The identity of fluorescent E. coli strains isolated from stool samples, Peyer's patches (PP) and mesenteric lymph nodes (MLN) was determined by REP-PCR. We were able to demonstrate that EcN and EcN-GFP do not differ in stimulation of IL-8 secretion or adhesion to HT-29 cells. In vivo, EcN-GFP colonies were readily detectable by fluorescence microscopy in luminal samples and also by immunohistochemistry in histological sections allowing analysis of the kinetics of the intestinal passage following oral administration. Translocation of fluorescent and non-fluorescent bacteria into PP and MLN was noted at 6 h post oral administration. EcN-GFP was detectable initially for 14 days in faecal samples of rats, while EcR-GFP was detectable throughout the whole experiment (45 days). Challenge with ampicillin at day 45 demonstrated continuing presence of EcN-GFP in small numbers by reappearing fluorescent colonies. The plasmid was not stable in vivo since non-fluorescent EcN colonies were detected also in faecal samples by REP-PCR. In summary, transformation of EcN to obtain EcN-GFP in our study had no detectable influence on the probiotic microorganism regarding adhesion on and induction of IL-8 secretion of HT-29 cells and allows the detection in mixed microbial environments in vivo but the stability of EcN-GFP in vivo is limited.

Ampicillin↗