Microorganisms Responsible for Controlling the Populations of Escherichia coli and Enterococcus and the Consistency of Cecal Contents in the Chicken

A study was made to clarify what kinds of intestinal organisms might be responsible for controlling the populations of Escherichia coli and Streptococcus faecalis var. liquefaciens in the cecum and the consistency of the cecal contents of chickens. Germ-free chickens were inoculated orally with various mixtures of bacterial cultures alone or in combination, different dilutions of the cecal contents of chickens, different dilutions of the cecal contents treated by heating or with chloroform, the supernatant of diluted cecal contents, and dilutions of human feces. Factors controlling the E. coli population, enterococcal population, and consistency of the cecal contents were shown to be independent of one another. The ecosystem controlling the E. coli or enterococcal population was more complex than that controlling the consistency of the cecal contents. The former was composed of anaerobic and facultatively anaerobic bacteria isolated and heat- or chloroform-resistant organisms, and the latter of heat- or chloroform-resistant organisms alone, which were inferred not to be prevailing in the cecal contents of chickens. Discussion is made on ecological systems controlling the intestinal flora.     

Oral yeast flora and its ITS sequence diversity among a large cohort of medical students in Hainan,China

The most prevalent fungal infection of humans is candidiasis which is caused by species of Candida that are typical members of the commensal microbial flora of the oral mucosa and other body surfaces. Since species of Candida differ in virulence properties and susceptibilities to anti-fungal drugs, understanding the human commensal yeast flora will have a significant impact on designing treatment and prevention strategies against yeast infections. However, although there is a global interest in Candida species, the global distributions of Candida species remain largely unknown, especially among healthy hosts. Here we report the oral yeast flora from the surveys of over 1,000 medical students in China. Our results showed that this population had a yeast carriage rate (4.5%) much lower than other population samples reported previously from Mainland China (40–70%). In addition, C. albicans was isolated at a much higher frequency than those from other Chinese samples, with a frequency (80.9%) more similar to those in developed regions such as North America. The oral yeast carriage rates and yeast species compositions were similar between male and female students and between the hosts borne and raised on Hainan Island and those borne and raised on Mainland China. Furthermore, the sequence variation at the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene cluster was analyzed for strains of the dominant species, C. albicans. Our analysis identified 14 ITS types among the 41 Hainan isolates of C. albicans. However, only four of the 14 ITS types were identical to those in reference strains from Europe and North America. Taken together, our analyses suggest that the oral yeast flora among host populations in China is highly heterogeneous and that there is a high ITS sequence diversity in the Hainan population of C. albicans.     

Physiological state of Escherichia coli BJ4 growing in the large intestines of streptomycin-treated mice.

Growth rates of Escherichia coli BJ4 colonizing the large intestine of streptomycin-treated mice were estimated by quantitative hybridization with rRNA target probes and by epifluorescence microscopy. The ribosomal contents in bacteria isolated from the cecal mucus, cecal contents, and feces were measured and correlated with the ribosomal contents of bacteria growing in vitro at defined rates. The data suggest that E. coli BJ4 grows at an overall high rate in the intestine. However, when taking into account the total intestinal volume and numbers of bacteria present in cecal mucus, cecal contents, and feces, we suggest that E. coli BJ4 in the intestine consists of two populations, one in the mucus which has an apparent generation time of 40 to 80 min and one in the luminal contents which is static.     

Bacterial community in the intestine of the sea urchin Strongylocentrotus intermedius during digestion of Macrocystis pyrifera

Denaturing gradient gel electrophoresis (DGGE) was used to study the bacterial community changes in the intestine of the sea urchin Strongylocentrotus intermedius during the digestion of Macrocystis pyrifera. The distinct bands in DGGE gels were sequenced, and the results indicated that the bacterial community in the large and small intestine varied at different periods of digestion. Samples from the large intestine included six specific bands belonging to the genus Psychromonas, whereas samples from the small intestine included eight specific bands representing Psychromonas, Shewanella, Saccharophagus degradans, and Nitrosomonas eutropha. The bacterial flora differed at different periods of digestion. The increase in the microbial community species in the large intestine was not obvious compared with that in the small intestinal microbial community. Several microbes involved in degradation of M. pyrifera were found in the intestine of sea urchin.     

Fecal fungal flora of pediatric healthy volunteers and immunosuppressed patients

Most hematogenous candidiasis originates from endogeneous host flora. Fungal flora of gastrointestinal system are important source of infection especially in immunosupressed patients. The purpose of this study was to investigate the fecal fungal flora of pediatric patients with hematologic malignancy or disorders and to compare the results with healthy volunteers. For this purpose, fungal etiological agents were investigated retrospectively in stool samples of 80 patients followed in Bone marrow transplantation and Hematology–Oncology units. The diagnosis of patients were as follows: 26 acute myelogeneous leukemia, 19 acute lymphocytic leukemia, 5 lymphoma, 3 chronic myelogeneous leukemia, 2 solid tumor, 4 neuroblastoma and 21 hematologic disorders. In patients, totally 102 fungal growth was detected and 42 (41.2%) C. albicans and 51 (50%) non-albicans Candida species and 9 (8.8%) yeast other than Candida and mould was isolated. The results were compared prospectively with growth in stool samples of 61 healthy children. C. albicans was detected in 16 (43.2%) and non-albicans Candida species in 15 (40.5%) and yeasts other than Candida and mould in 6 (16.2%) of 37 fungal growth in controls. Non-albicans Candida species growth was found significantly higher and C. glabrata was more prevelant in patients than in controls (p < 0.001).     

Production and characterization of monoclonal antibodies to a Veillonella species from a continuous-flow culture system of chicken cecal bacteria

Administering native intestinal flora to newly hatched chicks protects against cecal Salmonella colonization, and is known as competitive exclusion. Continuous-flow culture systems have been used to maintain defined competitive exclusion cultures. We have recently demonstrated that such a stable continuous-flow culture, CF3, contains 29 bacterial strains representing ten genera. Broiler chicks treated with CF3 are protected against Salmonella colonization of the ceca. Such protection is correlated with elevated concentrations of proprionic acid in the cecal contents of treated chicks. In this study we report on the preparation and characterization of monoclonal antibodies to one of the proprionic acid producing anaerobes contained in CF3, namely Veillonella CF3. Five different monoclonal antibodies were characterized with respect to: (1) isotype; (2)Veillonella specificity as judged by cross-reactivity profiles with other bacteria; (3) sensitivity as measured by the limit of detection of the number of colony forming units of Veillonella; and (4) antigen recognition of Veillonella by Western Blot analysis. These antibodies have been used to enumerate Veillonella in both the CF3 cultures and in the ceca of young chicks.     

Susceptibility of <Emphasis Type="Italic">Candida albicans</Emphasis> from Cystic Fibrosis Patients

Candida albicans is a common microbe, colonizer and potential pathogen found in respiratory cultures of cystic fibrosis (CF) patients. Because of possible development of resistance in patient isolates resulting from residence in the abnormal milieu of CF patient airways, or from exposure to antifungals, and considering the possibility of patient-to-patient spread of microbes and reports of elevated resistance to other fungal pathogens, it was important to assay the susceptibility of isolates of Candida and compare that profile to isolates from the community. In our center, and unlike another fungal pathogen, no increase in resistance of Candida isolates of the CF cohort was found.     

Influence of a cecal volume-reducing intestinal microflora on the excretion and entero-hepatic circulation of steroids and bile acids

From mouse fecal material we have isolated four strictly anaerobic bacteria which, when associated with germfree mice or rats, reduced the cecal volume by 80 and 60%, respectively. This cecal volume-reducing flora did not metabolize estrone-3-sulfate, taurolithocholate-3-sulfate or taurolithocholate but gnotobiotic rats associated with this particular flora (CRF-rats) excreted these compounds faster in feces plus urine than did germfree rats. The time needed for 50% excretion (t1/2) of orally administered estrone-3-sulfate was 32 h in germfree rats versus 13 h in CRF rats; for intraperitoneally injected taurolithocholate-3-sulfate the t1/2 was 63 h in germfree versus 17 h in CRF rats and for taurolithocholate the t1/2 was 199 h in germfree and 96 h in CRF rats. Association of germfree rats with the cecal volume-reducing flora did not change the cecal absorption rate of estrone-3-sulfate, but shortened the 50% small intestinal transit time of [14C]PEG from 10 to 3 h; a value also found in conventional rats. These results stress the important influence of the intestinal microflora on the absorption and excretion of steroids via its effect on the physiology of the whole intestinal tract and point to the deficiencies inherent to the use of germfree animals in excretion studies.     

Establishment of specific pathogen-free guinea-pig colonies using limited-flora guinea-pigs associated with conventional guinea-pig flora, and monitoring of their cecal flora.

Six groups of limited flora (LF) Hartley guinea-pigs were produced by inoculation of hysterectomy-derived GF guinea-pigs with various combinations of cecal bacteria of conventional (CV) guinea-pigs to determine the effective bacterial cocktails for the establishment of a specific pathogen free (SPF) colony. Bifidobacterium magnum (Bif) isolated from CV guinea-pigs was used for pretreatment. The mortality of LF guinea-pigs inoculated with only Bif was 75%, and that of those inoculated with Bif plus chloroform-treated cecal suspension (CHF) or Bif plus CHF plus 32 isolates from CV guinea-pigs was 40 to 66.7%. These three groups were in an unhealthy condition with mucoid enteritis-like diarrhea. However, the mortality of LF guinea-pigs inoculated with the anaerobic growth on EG plates injected with 10(-5) dilution of cecal contents (CF) or inoculated with Bif plus CF was 6.3 and 15%, respectively. These latter two groups of LF guinea-pigs were transferred to separate barrier rooms and some of the LF guinea-pigs were maintained in isolators as a source of intestinal flora for SPF guinea-pigs. The composition of cecal flora of LF guinea-pigs was stable for a long time, and bacteroidaceae and peptococcaceae were maintained as predominant components. The basic composition of the cecal flora of SPF guinea-pigs originated from LF guinea-pigs, which consists mainly of the anaerobic bacteria, was not changed over a long period, and the flora composition became similar to that in CV guinea-pigs. Guinea-pig-specific pathogens from the SPF colonies were not detected during experiments.     

Acid proteinase,phospholipase, and biofilm production of <Emphasis Type="Italic">Candida</Emphasis> species isolated from blood cultures

Three virulence factors comprising proteinase, phospholipase, and biofilm among 68 Candida albicans and 31 non-albicans Candida strains (11 C. tropicalis, 8 C. parapsilosis, 6 C. glabrata, 4 C. guillermondii, 2 C. krusei) isolated from blood cultures were analyzed. In total, 61 (89.7%) C. albicans strains were detected as proteinase positive whereas eight (25.8%) non-albicans Candida strains were proteinase positive (P < 0.05). Phospholipase production was detected in 41 (60.3%) C. albicans strains. All non-albicans Candida strains were phospholipase negative. Biofilm production was determined by both visual and spectrophotometric methods. Eight (11.8%) of C. albicans strains and 13 (41.93%) of 31 non-albicans Candida strains were biofilm positive with two of the methods (P < 0.05). According to our results, we may suggest that detection of hydrolytic enzyme and biofilm production abilities of the Candida isolates in clinical mycology laboratories may warn the clinican for a possible hematogenous infection.     

Bacteria,phages and pigs: the effects of in-feed antibiotics on the microbiome at different gut locations

Disturbance of the beneficial gut microbial community is a potential collateral effect of antibiotics, which have many uses in animal agriculture (disease treatment or prevention and feed efficiency improvement). Understanding antibiotic effects on bacterial communities at different intestinal locations is essential to realize the full benefits and consequences of in-feed antibiotics. In this study, we defined the lumenal and mucosal bacterial communities from the small intestine (ileum) and large intestine (cecum and colon) plus feces, and characterized the effects of in-feed antibiotics (chlortetracycline, sulfamethazine and penicillin (ASP250)) on these communities. 16S rRNA gene sequence and metagenomic analyses of bacterial membership and functions revealed dramatic differences between small and large intestinal locations, including enrichment of Firmicutes and phage-encoding genes in the ileum. The large intestinal microbiota encoded numerous genes to degrade plant cell wall components, and these genes were lacking in the ileum. The mucosa-associated ileal microbiota harbored greater bacterial diversity than the lumen but similar membership to the mucosa of the large intestine, suggesting that most gut microbes can associate with the mucosa and might serve as an inoculum for the lumen. The collateral effects on the microbiota of antibiotic-fed animals caused divergence from that of control animals, with notable changes being increases in Escherichia coli populations in the ileum, Lachnobacterium spp. in all gut locations, and resistance genes to antibiotics not administered. Characterizing the differential metabolic capacities and response to perturbation at distinct intestinal locations will inform strategies to improve gut health and food safety.     

Bacterial flora of newly caught Antarctic fish Notothenia neglecta

Summary The heterotrophic bacterial flora of Antarctic fish Notothenia neglecta was studied in Potter Cove (King George Island, South Shetland Islands). Quantitative and qualitative analysis of aerobic bacteria from sea water, skin, stomach and intestine were carried out. In addition, anaerobic flora of stomach and intestine was studied and compared. Pseudomonas was dominant in sea water samples but, neither skin nor digestive tract samples showed to be rich in this genus. Stomach flora showed variable results between samples. The gut flora was composed almost exclusively of Vibrio. Despite extreme environmental conditions this Antarctic fish show an intestinal indigenous microflora very similar to warmer waters fish. Several factors, that we discussed, could be determining the Vibrio dominance in the gut of marine teleosts.     

Glycosphingolipid patterns of the gastrointestinal tract and feces of germ-free and conventional rats

Acid and non-acid glycosphingolipids of stomach, small and large intestine, and stimulated feces of germ-free and conventional rats of the same stain have been isolated and characterized. The glycosphingolipid patterns of the intestinal organs were chemically and immunologically very similar between the two groups of rats and relatively unaffected by the presence of an intestinal microbial flora. The major exception was the presence of hematoside with N-glycoloylneuraminic acid (NeuGc) (NeuGc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) in the stomach of conventional rats not found in the stomach of germ-free animals. Glycosphingolipids of stimulated feces of germ-free animals were derived from epithelial cells mainly of the small intestine and showed no signs of degradation. Glycosphingolipids of feces of conventional rats completely retained the pattern of blood group A-, B-, and H-active glycolipids as found in sterile feces but contained less of hematoside and more of lactosylceramide. This effect was probably due to degradation by bacteria, as demonstrated in vitro with the production of lactosylceramide after treatment of the isolated acid glycolipids of sterile feces with neuraminidase from Clostridium perfringens. The amount of total non-acid glycosphingolipids per dry weight was similar for stomach, was 50% higher for small intestine, and 300% higher for large intestine of germ-free animals compared to conventional animals. Due to the presence of large amounts of mucins the dry sterile feces contained 12% less non-acid glycolipids than conventional feces. However, calculated per rat per day the germ-free animal excreted more of non-acid glycosphingolipids (1.8 and 1.2 mg, respectively).     

Biomaterial-Associated Infection with Candida albicans in Mice

Candida yeasts are frequently isolated from patients with continuous ambulatory peritoneal dialysis peritonitis or other biomaterial-associated infections. The mouse model of candidal peritonitis was used to study the interaction of Candida cells with end-point attached heparinized polyethylene (H-PE) and with polymorphonuclear leukocytes (PMNs) or macrophages (Mφ). Two Candida strains differing in cell surface hydrophobicity and in expression of fibronectin (Fn) binding were used for the study. Cells of both Candida strains adhered at higher numbers to H-PE surfaces preadsorbed with Fn or with human dialysis fluid (HDF) than to non-modified H-PE, supporting a role of Fn in mediating adhesion. C. albicans 4016 cells expressing low hydrophobicity and low binding of soluble Fn demonstrated stronger adhesion to PMNs than the more hydrophobic C. albicans 3248 yeasts, which express high binding of soluble Fn. However, C. albicans 4016 cells were more resistant to phagocytic killing and were hardly eradicated in intraperitoneally infected mice. The animals depleted in PMNs by treatment with CY were neither able to eradicate C. albicans 3248 (rapidly eliminated by normal mice) nor C. albicans 4016 yeasts (with a tendency to persist in the tissues of normal mice).     

Antifungal activity of hypocrellin compounds and their synergistic effects with antimicrobial agents against Candida albicans

Candida albicans is a common human fungal pathogen. The previous study revealed that quinone compounds showed antimicrobial activity against C. albicans by inhibiting cell growth. However, it was unclear whether quinones have other antifungal effects against C. albicans in addition to fungicidal effects. In this study, we assessed the inhibitory activity of a total of 25 quinone compounds against C. albicans morphological transition, which is essential for the pathogenicity of C. albicans. Several quinones exhibited strong inhibition of mycelium formation by C. albicans SC5314. Three leading compounds, namely hypocrellins A, B and C, also exhibited marked attenuation of C. albicans SC5314 virulence in both human cell lines and mouse infection models. These three compounds significantly suppressed the proliferation of C. albicans SC5314 cells in a mouse mucosal infection model. Intriguingly, hypocrellins not only attenuated the cytotoxicity of a nystatin-resistant C. albicans strain but also showed excellent synergistic effects with antifungal agents against both wild-type C. albicans SC5314 and the drug-resistant mutant strains. In addition, hypocrellins A, B and C interfered with the biological functions and virulence of various clinical Candida species, suggesting the promising potential of these compounds for development as new therapeutic agents against infections caused by Candida pathogens.     

Some aspects of the gastrointestinal microflora of germfree mice associated with cultured microfloras

Attempts are described to 'normalize' germfree mice by association with 3, 21 and 71 different intestinal bacterial cultures isolated from mice with an SPF flora. Germfree mice associated naturally with an SPF flora served as controls. Vital bacterial counts were determined by aerobic and anaerobic culture. Stomach and small intestine contained fewer bacteria per gram than caecum and large intestine. Aerobic vital counts from caecum and large intestine were higher in the experimental groups than in control mice. The aerobic and anaerobic flora in stomach and small intestine comprised mainly Gram-positive non-fusiform shaped rods. In the caecum and colon Gram-positive cocci predominated in the aerobic culture while in the anaerobic culture fusiform-shaped rods were prominent. Scanning electron microscopy of oesophagus, ileum, caecum and faeces demonstrated colonization of the oesophageal epithelium only after association with 71 bacterial strains; the filamentous bacteria present in the ileum of SPF mice were not found in the experimental groups and caecum and faeces contained mainly fusiform-shaped bacteria. Non-bacterial matter decreased in the caecum and faeces with increase in the complexity of the flora.     

Studies on defense mechanisms against Candida albicans infection in congenitally athymicnude (nu/nu) mice

The defense mechanisms against Candida albicans infection were studied by using a mouse thigh lesion model in congenitally athymic nude (nu/nu) mice and their normal littermates (nu/+). Nu/nu mice were more resistant to C. albicans infection than nu/+ mice judging from the course of the thigh lesion, the results of CFUs (colony-forming units) of C. albicans in the lesion, and histopathological observations. Histopathological and serological studies revealed that granulocytic cellular infiltration was predominant, and there were few indications of development of cell-mediated immunity to protect Candida infection in Candida-infected nu/nu and nu/+ mice. These results confirmed that lower susceptibility of nu/nu mice to C. albicans infection as compared with nu/ + mice was due to accelerated non-specific defense mechanisms in nu/nu mice, and that cell-mediated or humoral immunity played a minor role in the defense against Candida infection in this experimental model.Furthermore, treatment with high titer of rabbit anti-C. albicans serum was effective to control the number of Candida cells in thigh lesions of BALB/c mice.Above experimental results seem to clearly indicate the great variability of defense manifestation according to the experimental model exployed.     

The Dynamic Distribution of Porcine Microbiota across Different Ages and Gastrointestinal Tract Segments

Metagenome of gut microbes has been implicated in metabolism, immunity, and health maintenance of its host. However, in most of previous studies, the microbiota was sampled from feces instead of gastrointestinal (GI) tract. In this study, we compared the microbial populations from feces at four different developmental stages and contents of four intestinal segments at maturity to examine the dynamic shift of microbiota in pigs and investigated whether adult porcine fecal samples could be used to represent samples of the GI tract. Analysis results revealed that the ratio of Firmicutes to Bacteroidetes from the feces of the older pigs (2-, 3-, 6- month) were 10 times higher compared to those from piglets (1-month). As the pigs matured, so did it seem that the composition of microbiome became more stable in feces. In adult pigs, there were significant differences in microbial profiles between the contents of the small intestine and large intestine. The dominant genera in the small intestine belonged to aerobe or facultative anaerobe categories, whereas the main genera in the large intestine were all anaerobes. Compared to the GI tract, the composition of microbiome was quite different in feces. The microbial profile in large intestine was more similar to feces than those in the small intestine, with the similarity of 0.75 and 0.38 on average, respectively. Microbial functions, predicted by metagenome profiles, showed the enrichment associated with metabolism pathway and metabolic disease in large intestine and feces while higher abundance of infectious disease, immune function disease, and cancer in small intestine. Fecal microbes also showed enriched function in metabolic pathways compared to microbes from pooled gut contents. Our study extended the understanding of dynamic shift of gut microbes during pig growth and also characterized the profiles of bacterial communities across GI tracts of mature pigs.     

Presence of Bacillus coagulans spores and vegetative cells in rat intestine and feces and their physiological effects

ABSTRACT

This study was aimed to investigate the presence of Bacillus coagulans vegetative cells in the intestine and fecal samples in rats fed B. coagulans spores as well as to estimate the ratios of spores and vegetative cells in these samples. A two-step process has been developed to enumerate B. coagulans in different mixed bacterial samples, specifically (1) observation of yellow ring formation on modified GYEA medium upon incubation at 55°C, (2) microscopic examination of spore formation after 7 d of incubation. Our results have demonstrated the presence of vegetative cells in the intestinal and fecal samples in rats fed B. coagulans spores. The ratios of B. coagulans spores and vegetative cells in cecal fluid, colonic content, and feces were approximately 2:8, 2:8, and 4:6, respectively. The existence of B. coagulans vegetative cells improved the intestinal milieu through an elevated short-chain fatty acid concentrations, higher fecal moisture, and lower fecal pH.     

Characterization of Mucosal Candida albicans Biofilms

C. albicans triggers recurrent infections of the alimentary tract mucosa that result from biofilm growth. Although the ability of C. albicans to form a biofilm on abiotic surfaces has been well documented in recent years, no information exists on biofilms that form directly on mucosal surfaces. The objectives of this study were to characterize the structure and composition of Candida biofilms forming on the oral mucosa. We found that oral Candida biofilms consist of yeast, hyphae, and commensal bacteria, with keratin dispersed in the intercellular spaces. Neutrophils migrate through the oral mucosa and form nests within the biofilm mass. The cell wall polysaccharide β-glucan is exposed during mucosal biofilm growth and is more uniformly present on the surface of biofilm organisms invading the oral mucosa. We conclude that C. albicans forms complex mucosal biofilms consisting of both commensal bacterial flora and host components. These discoveries are important since they can prompt a shift of focus for current research in investigating the role of Candida-bacterial interactions in the pathogenesis of mucosal infections as well as the role of β-glucan mediated signaling in the host response.