Rapid and bacteria-dependent in vitro hydrolysis of iodothyronine-conjugates by intestinal contents of humans and rats

Faecal suspensions from healthy humans, conventional (CV), germ-free (GF) and intestine-decontaminated (ID) rats were tested for the in vitro hydrolysis of 125I-labelled iodothyronine sulphates and 3,3',5-triiodothyronine glucuronide (T3G). Whereas 20-fold diluted human and CV rat faecal suspensions hydrolyzed up to 90% of the sulphates, no hydrolysis was observed in 5 times diluted faecal suspensions of GF and ID rats. These results add further weight to the assumption that intestinal iodothyronine sulphatase activity is of bacterial origin. Twenty times diluted human and CV rat faecal suspensions hydrolyzed approximately 80% of the T3G. In the 5 times diluted faecal suspensions of GF and ID rats up to 15% hydrolysis of T3G was still observed. It was concluded that the major part of the gastrointestinal iodothyronine glucuronidase activity is produced by bacteria. The remaining activity presumably originates from gastrointestinal mucosal cells.     

Iodothyronine sulfate-hydrolyzing anaerobic bacteria isolated from human fecal flora

Abstract 23 Bacterial strains isolated from human fecal flora were screened for hydrolysis of iodothyronine sulfates. Three obligate anaerobic bacterial strains possessed sulfatase activity. Two strains were identified as Peptostreptococcus productus , the other strain probably belonged to the genus Eubacterium or Lachnospira . In anaerobic incubations with growing bacteria up to 78% of the iodothyronine sulfates were deconjugated in 24 h. Hydrolysis was dependent on the bacterial strains and on the iodothyronine sulfates tested. This study extents previous observations of similar iodothyronine sulfatase activities associated with bacteria from rat intestinal microflora [5]. By analogy, hydrolysis of iodothyronine sulfates by anaerobic bacteria from human intestinal microflora probably represents an exo-enzymatic process.     

Hydrolysis of iodothyronine sulfates by sulfatase activity of anaerobic bacteria from the rat intestinal flora

Abstract 2 Obligately anaerobic bacteria isolated from rat cecal flora have previously been shown to possess sulfatase activity towards 3,3'-diiodothyronine sulfate [5]. These strains have now been tested for their ability to hydrolyze the sulfate conjugates of other iodothyronines, including the thyroid hormones thyroxine and 3,3',5-triiodothyronine. In anaerobic incubations at 37°C with approximately 10⁷ bacteria per ml, variable amounts of the conjugated substrates, ranging from 15–90%, were hydrolysed in 24 h. These results showed a potent iodothyronine sulfate hydrolysing capacity of rat intestinal microflora. The strains were characterized by carbohydrate fermentation tests. One strain belonged to the genus Lactobacillus , the other strain probably to Eubacterium or Lachnospira .     

The use of rats associated with a human faecal flora as a model for studying the effects of diet on the human gut microflora

The activities of four bacterial biotransformation enzymes (β-glucosidase, β-glucuronidase, nitrate reductase and nitroreductase) were measured in the caecal contents of conventional flora rats or germ-free rats contaminated with a mixed, human faecal flora and compared with activities present in a fresh human stool preparation. Both the conventional flora rats and the rats inoculated with a human flora exhibited an enzyme profile generally similar to that of human faeces, although the conventional rat flora exhibited negligible nitrate reductase activity. The enzyme profile remained essentially unaltered in both human flora preparations following supplementation of the diet with pectin, whereas the conventional rat flora responded to this plant cell wall carbohydrate with a significant increase in nitrate reductase activity. The results demonstrate that enzymic activities of the human faecal microflora can be simulated in rats associated with a mixed population of human intestinal bacteria.     

The use of rats associated with a human faecal flora as a model for studying the effects of diet on the human gut microflora

The activities of four bacterial biotransformation enzymes (beta-glucosidase, beta-glucuronidase, nitrate reductase and nitroreductase) were measured in the caecal contents of conventional flora rats or germ-free rats contaminated with a mixed, human faecal flora and compared with activities present in a fresh human stool preparation. Both the conventional flora rats and the rats inoculated with a human flora exhibited an enzyme profile generally similar to that of human faeces, although the conventional rat flora exhibited negligible nitrate reductase activity. The enzyme profile remained essentially unaltered in both human flora preparations following supplementation of the diet with pectin, whereas the conventional rat flora responded to this plant cell wall carbohydrate with a significant increase in nitrate reductase activity. The results demonstrate that enzymic activities of the human faecal microflora can be simulated in rats associated with a mixed population of human intestinal bacteria.     

Molecular monitoring of the intestinal flora by denaturing high performance liquid chromatography

Gut flora analysis is hampered by the complexity of the intestinal microbiota and by inherent limitations of culture-based approaches. Therefore, culture-independent molecular methods based upon 16S rRNA gene analysis were applied successfully for the analysis of complex microbial communities. However, generally accepted and validated profiling methods such as denaturing and temperature gradient gel electrophoresis (DGGE/TGGE) are still laborious and time consuming. Thus, we adapted the separation of amplified bacterial 16S rRNA gene fragments by denaturing high performance liquid chromatography (DHPLC) using the WAVE Microbial Analysis System as a rapid and convenient means to display complex intestinal bacterial communities and to monitor changes in the gut flora. The separation of 16S rRNA gene fragments amplified from reference strains representing main gut bacterial populations and from human stool samples revealed that DHPLC analysis effectively detects bacterial groups predominant in the human gut flora. The investigation of faecal samples from hospitalized patients before, during and after antibiotic therapy showed that PCR-based DHPLC can be used to monitor gut flora changes. Results from DHPLC analysis were comparable with DGGE profiles generated from the same samples, demonstrating that the adapted DHPLC protocol is well suited for the analysis of complex microbial communities.     

Study of murine faecal microflora by cellular fatty acid analysis; effect of age and mouse strain

Analysis of bacteria-derived cellular fatty acids was applied to study differences in faecal floras of inbred mice. The bacterial composition of the faecal flora clearly changes with age, whereas the sex does not affect it. Most interestingly, different mouse strains were found to have different faecal floras. This was particularly observed at the age of 17–19 weeks for stool samples of four different mouse strains; the mice were handled identically in identical environments, and the two congenic strains used were different from each other only by the major histocompatibility complex (MHC). These results suggest that composition of the faecal flora is genetically regulated.     

The influence of incubation pH on the activity of rat and human gut flora enzymes

The activities of three bacterial biotransformation enzymes (β-glucuronidase, β-glucosidase, nitrate reductase) were determined in suspensions of rat caecal contents or human faeces over the pH range 6–8. All three enzymes were influenced by pH, as exemplified by β-glucosidase activity which diminished as pH increased. In other instances the rat and human flora showed distinct profiles, with nitrate reductase activity undetectable in human faeces below pH 6–6, whereas the rat caecal flora displayed optimal reduction of nitrate around neutrality. The most pronounced host-species difference was found with β-glucuronidase, which showed maximal activity at pH 6–0 in human faecal bacteria, while the rat caecal flora expressed greatest activity at pH 8–0. All three enzyme activities were associated with that fraction of rat caecal or human faecal material sedimented by centrifugation at 5000 g for 15 min, with little or no metabolism occurring in the 11000 g supernatant fluid. The results demonstrate that pH has a pronounced effect on the enzymic activity of bacterial preparations from rat and human sources.     

一种基于PCR分析多样性的小鼠肠道微生物宏基因组提取方法

目的建立一种基于PCR分析分子多样性的小鼠肠道菌群宏基因组提取方法。方法比较、综合国内外小鼠肠道菌群宏基因组的提取方法后建立一种新方法,小鼠肠道内容物经丙酮洗涤,差速离心,溶菌酶、SDS裂解,CTAB处理,酚／氯仿抽提后可得到高质量的DNA,通过紫外分光光度计、琼脂糖凝胶电泳、细菌通用引物PCR和扩增核糖体限制性酶切片段分析（ARDRA）等检测该方法的实用性。结果该方法获得的小鼠肠道菌群宏基因组DNA大小在23kb左右,A260／A280在1．8—2．0,经细菌通用引物PCR后能得到适用于ARDRA的目的产物。结论该方法经济适用性较强,具备一定的应用价值。     

The influence of incubation pH on the activity of rat and human gut flora enzymes

The activities of three bacterial biotransformation enzymes (beta-glucuronidase, beta-glucosidase, nitrate reductase) were determined in suspensions of rat caecal contents or human faeces over the pH range 6-8. All three enzymes were influenced by pH, as exemplified by beta-glucosidase activity which diminished as pH increased. In other instances the rat and human flora showed distinct profiles, with nitrate reductase activity undetectable in human faeces below pH 6.6, whereas the rat caecal flora displayed optimal reduction of nitrate around neutrality. The most pronounced host-species difference was found with beta-glucuronidase, which showed maximal activity at pH 6.0 in human faecal bacteria, while the rat caecal flora expressed greatest activity at pH 8.0. All three enzyme activities were associated with that fraction of rat caecal or human faecal material sedimented by centrifugation at 5000 g for 15 min, with little or no metabolism occurring in the 11,000 g supernatant fluid. The results demonstrate that pH has a pronounced effect on the enzymic activity of bacterial preparations from rat and human sources.     

Improvement of human faecal flora-associated mouse model for evaluation of the functional foods

AIMS: Animal models are required for evaluation of the functional foods such as pro/prebiotics exerting effects through the metabolism of the intestinal microflora. The object of this study was to establish new human flora-associated mice reflecting the environment of the human intestinal tract. METHODS AND RESULTS: We inoculated a human faecal suspension into segmented filamentous bacteria (SFB) monoassociated mice as a model system. In both human flora (HF) and SFB-associated mouse (HF-SFB mouse), intestinal characteristics such as the composition of intraepithelial lymphocytes, the expression of major histocompatibility complex (MHC) class II molecules and the number of immunoglobulin A-producing cells in the mucosa was closer to those of conventionally reared mice than was case with human flora-associated mice (HF mice) lacking SFB. Several predominant bacterial groups except lactobacilli in human flora were found in faeces of HF-SFB mice. Lactobacilli established small populations in the gut of HF-SFB mice when administered before inoculation with the human flora. Faecal enzymatic activities and organic acid concentration of HF-SFB mice proportionally reflected those of the donor subject. CONCLUSION: We established a new human flora-associated mouse (HF-SFB mouse), in which intestinal characteristics are normally developed and their major microbial composition reflect the human. SIGNIFICANCE AND IMPACT OF THE STUDY: HF-SFB mice are a valuable model for studying pro/prebiotic effects on the human intestine.     

Effect of sulphasalazine on the human intestinal flora

Sulphasalazine is commonly used to treat patients with ulcerative colitis but the mode of action is still unexplained. To investigate a possible antibacterial effect of sulphasalazine, the faecal flora composition was studied in patients with ulcerative colitis and in mice harbouring a human intestinal flora. Patients on sulphasalazine were found to have an intestinal flora that was completely resistant to sulphapyri-dine. Oral administration of sulphasalazine did not influence the composition of the human flora in mice.     

In vivo activity of nifurzide and nifuroxazide in intestinal bacteria in man and gnotobiotic mice

Although Gram-negative enteropathogenic bacteria are the target strains of nifurzide and nifuroxazide treatments, neither drug affected faecal counts of in vitro -susceptible Enterobacteriaceae in healthy volunteers. This absence of activity was shown to be due to the poor solubility of the drugs tested. Therefore, effect of high doses of nifurzide was investigated in gnotobiotic mice. Activity against in vitro susceptible enteropathogens was then observed. Normal bacterial cells were replaced in the faeces by elongated, nonseptate and unflagellated mutants. Moreover, the resistance to colonization by enterotoxigenic Escherichia coli and Shigella flexeri of an anaerobic flora of human origin was sharply decreased.     

In vivo activity of nifurzide and nifuroxazide in intestinal bacteria in man and gnotobiotic mice

Although Gram-negative enteropathogenic bacteria are the target strains of nifurzide and nifuroxazide treatments, neither drug affected faecal counts of in vitro-susceptible Enterobacteriaceae in healthy volunteers. This absence of activity was shown to be due to the poor solubility of the drugs tested. Therefore, effect of high doses of nifurzide was investigated in gnotobiotic mice. Activity against in vitro susceptible enteropathogens was then observed. Normal bacterial cells were replaced in the faeces by elongated, nonseptate and unflagellated mutants. Moreover, the resistance to colonization by enterotoxigenic Escherichia coli and Shigella flexeri of an anaerobic flora of human origin was sharply decreased.     

Hydrolysis of iodothyronine conjugates by intestinal bacteria

Glucuronide and sulfate conjugation are important pathways in the peripheral metabolism of thyroid hormone. These reactions occur predominantly in the liver, and especially the glucuronides are excreted in the bile. Although an enterohepatic circulation after intestinal hydrolysis of iodothyronine conjugates is suggested by several authors, substantial proof has not been presented so far. In the present paper experimental work from our group is reviewed. The studies showed that fecal suspensions of human or rat origin hydrolysed iodothyronine conjugates, whereas oral administration of antibiotics to rats strongly reduced this capacity. Obligately anaerobic intestinal bacteria were found to be responsible for the hydrolysis and several species belonging to the major residents of the intestinal flora of man and rat could be isolated and identified. Recent studies with conventional and decontaminated rats produced strong support for the existence of an enterohepatic circulation of thyroid hormone. Our findings are discussed in connection with other relevant studies on this subject.     

Hydrolysis of iodothyronine conjugates by intestinal bacteria

Abstract Glucuronide and sulfate conjugation are important pathways in the peripheral metabolism of thyroid hormone. These reactions occur predominantly in the liver, and especially the glucuronides are excreted in the bile. Although an enterohepatic circulation after intestinal hydrolysis of iodothyronine conjugates is suggested by several authors, substantial proof has not been presented so far.
In the present paper experimental work from our group is reviewed. The studies showed that fecal suspensions of human or rat origin hydrolysed iodothyronine conjugates, whereas oral administration of antibiotics to rats strongly reduced this capacity. Obligately anaerobic intestinal bacteria were found to be responsible for the hydrolysis and several species belonging to the major residents of the intestinal flora of man and rat could be isolated and identified.
Recent studies with conventional and decontaminated rats produced strong support for the existence of an enterohepatic circulation of thyroid hormone. Our findings are discussed in connection with other relevant studies on this subject.     

Biosurfactant production by Pseudomonas aeruginosaGS3 from molasses

Patterns of long-chain faecal fatty acids were studied by gas–liquid chromatography in 55 newborn infants in a neonatal intensive care unit. Decreased fractions of fatty acid C16 : 1⁹ and increased fractions of C16 : 0 and C17 : 0 were associated with the occurrence of abdominal distension. Decreased fractions of C16 : 1⁹ and C18 : 2^9,1 were associated with diarrhoea. Flatulence was found in infants who had relatively smaller amounts of fatty acids C17 : 0D and C15 : 0 in their faecal samples. The differences in the patterns of faecal fatty acids are due to differences in bacterial flora. The results support the hypothesis that the initial intestinal colonization plays a role in the later gastrointestinal signs of newborn infants.     

艾滋病患者肠道微生态与抗逆转录病毒治疗及耐药相关性研究新进展

艾滋病(AIDS)是一种由人类免疫缺陷病毒(HIV)引起的免疫缺陷性疾病,因其高传染性和不可治愈而备受关注。本文综述了HIV患者肠道菌群的变化与其发病机制、抗逆转录病毒耐药及肠道免疫激活的相关性。抗逆转录病毒治疗(ART)是降低肠道黏膜免疫和导致持续炎症的独立风险因素。肠道菌群参与或干扰抗病毒药物的代谢,造成肠道黏膜的通透性增加,微生物移位,激活不同的T细胞亚群的免疫活性,产生炎性因子导致CD4+细胞数量持续低下。某些特定的益生菌及粪菌移植(FMT)可调节HIV患者的肠道微生态的平衡,延缓AIDS的发病进程。     

Comparative assessment of human and farm animal faecal microbiota using real-time quantitative PCR

Pollution of the environment by human and animal faecal pollution affects the safety of shellfish, drinking water and recreational beaches. To pinpoint the origin of contaminations, it is essential to define the differences between human microbiota and that of farm animals. A strategy based on real-time quantitative PCR (qPCR) assays was therefore developed and applied to compare the composition of intestinal microbiota of these two groups. Primers were designed to quantify the 16S rRNA gene from dominant and subdominant bacterial groups. TaqMan^® probes were defined for the qPCR technique used for dominant microbiota. Human faecal microbiota was compared with that of farm animals using faecal samples collected from rabbits, goats, horses, pigs, sheep and cows. Three dominant bacterial groups ( Bacteroides/Prevotella, Clostridium coccoides and Bifidobacterium ) of the human microbiota showed differential population levels in animal species. The Clostridium leptum group showed the lowest differences among human and farm animal species. Human subdominant bacterial groups were highly variable in animal species. Partial least squares regression indicated that the human microbiota could be distinguished from all farm animals studied. This culture-independent comparative assessment of the faecal microbiota between humans and farm animals will prove useful in identifying biomarkers of human and animal faecal contaminations that can be applied to microbial source tracking methods.     

Probiotics: from myth to reality. Demonstration of functionality in animal models of disease and in human clinical trials

The enteric flora comprise approximately 95% of the total number of cells in the human body and are capable of eliciting immune responses while also protecting against microbial pathogens. However, the resident bacterial flora of the gastrointestinal tract (GIT) may also be implicated in the pathogenesis of several chronic conditions such as inflammatory bowel disease (IBD). The University College Cork-based Probiotic Research Group has successfully isolated and identified lactic acid bacteria (LAB) which exhibit beneficial probiotic traits. These characteristics include the demonstration of bile tolerance; acid resistance; adherence to host epithelial tissue; and in vitro antagonism of potentially-pathogenic micro-organisms or those which have been implicated in promoting inflammation. The primary objective of this report is to describe the strategy adopted for the selection of potentially effective probiotic bacteria. The study further describes the evaluation of two m embers of the resulting panel of micro-organisms (Lactobacillus salivarius subsp. salivarius UCC118 and Bifidobacterium longum infantis 35624) under in vitro conditions and throughout in vivo murine and human feeding trials. Specifically, an initial feeding study completed in Balb/c mice focused upon (i) effective delivery of the probiotic micro-organisms to the GIT and evaluation of the ability of the introduced strains to survive transit through, and possibly colonise, the murine GIT; (ii) accepting the complexity of the hostile GIT and faecal environments, development of a method of enumerating the introduced bacterial strains using conventional microbiological techniques; and (iii) assessment of the effects of administered bacterial strains on the numbers of specific recoverable indigenous bacteria in the murine GIT and faeces. Additional research, exploiting the availability of murine models of inflammatory bowel disease, demonstrated the beneficial effects of administering probi otic combinations of Lactobacillus salivarius UCC118 and Bifidobacterium longum infantis 35624 in prevention of illness-related weight loss. A further ethically-approved feeding trial, successfully conducted in 80 healthy volunteers, demonstrated that yoghurt can be used as a vehicle for delivery of Lactobacillus salivarius strain UCC118 to the human GIT with considerable efficacy in influencing gut flora and colonisation.