Low Concentration of Fecal Valeric Acid at 1 Year of Age Is Linked with Eczema and Food Allergy at 13 Years of Age: Findings from a Swedish Birth Cohort.

BACKGROUND: Short-chain fatty acids (SCFAs) are abundant bacterial metabolites in the gut, with immunomodulatory properties. Hence, they may influence allergy development. Previous studies have linked fecal SCFA pattern during infancy with allergy. However, the association of SCFAs to allergic outcomes in adolescence is not well established. Here, we examined how the fecal SCFA pattern at 1 year of age related to allergy at 13 years of age. METHODS: Levels of 8 SCFAs in fecal samples collected at 1 year of age from 110 children were quantified using gas chromatography. The same individuals were evaluated at 13 years of age for allergic symptoms, allergy diagnosis and allergy medication by questionnaire, and for sensitization using skin prick test against egg, milk, fish, wheat and soy, cat, dog, horse, birch, and timothy grass. RESULTS: The concentration of fecal valeric acid at 1 year of age was inversely associated with eczema at 13 years of age (OR 0.6, 95% CI: 0.4-1.0, p = 0.049) and showed a trend for inverse association with food allergy at 13 years of age (OR 0.6, 95% CI: 0.4-1.0, p = 0.057). In a sub-group analysis of children with eczema at 1 year of age, a higher concentration of fecal valeric acid was linked with reduced risk of their eczema remaining at 13 years of age (OR 0.2, 95% CI: 0.0-1.5), although this latter analysis did not reach statistical significance (p = 0.12). CONCLUSIONS: Our findings lend further support to the notion of early childhood as a critical period when allergy may be programmed via the gut microbiota. Higher levels of fecal valeric acid may be characteristic of a protective gut microbiota and/or actively contribute to protection from eczema and food allergy.

The protective role of commensal gut microbes and their metabolites against bacterial pathogens.

Multidrug-resistant microorganisms have become a major public health concern around the world. The gut microbiome is a gold mine for bioactive compounds that protect the human body from pathogens. We used a multi-omics approach that integrated whole-genome sequencing (WGS) of 74 commensal gut microbiome isolates with metabolome analysis to discover their metabolic interaction with Salmonella and other antibiotic-resistant pathogens. We evaluated differences in the functional potential of these selected isolates based on WGS annotation profiles. Furthermore, the top altered metabolites in co-culture supernatants of selected commensal gut microbiome isolates were identified including a series of dipeptides and examined for their ability to prevent the growth of various antibiotic-resistant bacteria. Our results provide compelling evidence that the gut microbiome produces metabolites, including the compound class of dipeptides that can potentially be applied for anti-infection medication, especially against antibiotic-resistant pathogens. Our established pipeline for the discovery and validation of bioactive metabolites from the gut microbiome as novel candidates for multidrug-resistant infections represents a new avenue for the discovery of antimicrobial lead structures.

Faecal short-chain fatty acid pattern in childhood coeliac disease is normalised after more than one year's gluten-free diet.

OBJECTIVE: Recent work indicates that the gut microflora is altered in patients with coeliac disease (CD). Faecal short-chain fatty acids (SCFAs) are produced by the gut microflora. We have previously reported a high SCFA output in children with symptomatic and asymptomatic CD at presentation, as well as in CD children on a gluten-free diet (GFD) for less than 1 year, indicating deviant gut microfloral function. In this report, we focus on faecal SCFA production in coeliacs on GFD for more than 1 year. MATERIALS AND METHODS: Faecal samples were collected from 53 children with CD at presentation, 74 coeliac children on GFD for less than 1 year, and 25 individuals diagnosed with CD in childhood and on GFD for more than 1 year. The control group comprised 54 healthy children (HC). The faecal samples were analysed to show the SCFA pattern taken as a marker of gut microflora function. We applied a new fermentation index, reflecting the inflammatory activity of the SCFAs (amount of acetic acid minus propionic acid and n-butyric acid, together divided by the total amount of SCFAs). RESULTS: In coeliacs on GFD for more than 1 year, the individual SCFAs, total SCFA, and fermentation index did not differ significantly from the findings in controls. In contrast, the faecal SCFA level was clearly higher in coeliacs treated with GFD for less than 1 year compared to those more than 1 year. CONCLUSIONS: This is the first study on SCFA patterns in faecal samples from individuals with CD on GFD for more than 1 year. Our study indicates that the disturbed gut microflora function in children with CD at presentation and after less than 1 year of GFD, previously demonstrated by us, is normalised on GFD for more than 1 year.

Sensitive quantification of short-chain fatty acids combined with global metabolomics in microbiome cultures.

The microbiome has been identified to have a key role for the physiology of their human host. One of the major impacts is the clearance of bacterial pathogens. We have now developed a chemoselective probe methodology for the absolute quantification of short-chain fatty acids at low nM concentrations, with high reproducibility and spiked isotope labelled internal standards. Immobilization to magnetic beads allows for separation from the matrix and the tagged metabolites upon bioorthogonal cleavage can be analyzed via UHPLC-MS. The major advantage of our sensitive method is the simple combination with global metabolomics analysis as only a small sample volume is required. We have applied this chemical metabolomics strategy for targeted SCFA analysis combined with global metabolomics on gut microbiome co-cultures with Salmonella and investigated the effect of antibiotic treatment.

Effect of exclusive enteral nutrition on gut microflora function in children with Crohn's disease.

OBJECTIVE: Exclusive enteral nutrition (EEN) is a first-line treatment in children with active Crohn's disease (CD) but is seldom used in adults with active disease. The mode of action of EEN in suppressing mucosal inflammation is not fully understood, but modulation of intestinal microflora activity is one possible explanation. The aim of this study was to investigate the effect of 6-week EEN in children with active CD, with special reference to intestinal microflora function. MATERIALS AND METHODS: Fecal samples from 18 children (11 boys, 7 girls; median age 13.5 years) with active CD (13 children with small bowel/colonic and 5 with perianal disease) were analyzed for short chain fatty acid (SCFA) pattern as marker of gut microflora function. The children were studied before and after EEN treatment. Results from 12 healthy teenagers were used for comparison. RESULTS: Eleven (79%) of the children with small bowel/colonic CD responded clinically positively to EEN treatment showing decreased levels of pro-inflammatory acetic acid as well as increased concentrations of anti-inflammatory butyric acids and also of valeric acids, similar to the levels in healthy age-matched children. In children with active perianal CD, however, EEN had no positive effect on clinical status or inflammatory parameters. CONCLUSIONS: The authors present new data supporting the hypothesis that the well-documented anti-inflammatory effect of EEN in children with active small bowel/colonic CD is brought about by modulation of gut microflora activity, resulting in an anti-inflammatory SCFA pattern. By contrast, none of the children with perianal disease showed clinical or biochemical improvement after EEN treatment.

Fecal transplant against relapsing Clostridium difficile-associated diarrhea in 32 patients.

Clostridium difficile-associated disease (CDAD) with frequent watery stools, sometimes with painful bowel movements, fever and sickness, is probably the major known cause of antibiotic-associated diarrhea and colitis, most probably depending on a disruption of the normal intestinal balance in the microbiome. In this study, we have inoculated a mixture of fecal microbes--as an enema--originating from a healthy Scandinavian middle-aged donor, regularly re-cultivated under strict anaerobic conditions for more than 10 years, to 32 patients. Twenty-two patients (69%) were durably cured. In those patients receiving the transplant by colonoscopy, four out of five were cured. To the best of our knowledge, this is the first time a fecal culture of microbes has retained the possibility for years to cure a substantial number of patients with CDAD.

Determinants of the human infant intestinal microbiota after the introduction of first complementary foods in infant samples from five European centres.

Although it is well established that early infant feeding has a major influence on the establishment of the gut microbiota, very little is understood about how the introduction of first solid food influences the colonization process. This study aimed to determine the impact of weaning on the faecal microbiota composition of infants from five European countries (Sweden, Scotland, Germany, Italy and Spain) which have different lifestyle characteristics and infant feeding practices. Faecal samples were collected from 605 infants approximately 4 weeks after the introduction of first solid foods and the results were compared with the same infants before weaning (6 weeks of age) to investigate the association with determining factors such as geographical origin, mode of delivery, previous feeding method and age of weaning. Samples were analysed by fluorescence in situ hybridization and flow cytometry using a panel of 10 rRNA targeted group- and species-specific oligonucleotide probes. The genus Bifidobacterium (36.5 % average proportion of total detectable bacteria), Clostridium coccoides group (14 %) and Bacteroides (13.6 %) were predominant after weaning. Similar to pre-weaning, northern European countries were associated with a higher proportion of bifidobacteria in the infant gut microbiota while higher levels of Bacteroides and lactobacilli characterized southern European countries. As before weaning, the initial feeding method influenced the Clostridium leptum group and Clostridium difficile+Clostridium perfringens species, and bifidobacteria still dominated the faeces of initially breast-fed infants. Formula-fed babies presented significantly higher proportions of Bacteroides and the C. coccoides group. The mode of birth influenced changes in the proportions of bacteroides and atopobium. Although there were significant differences in the mean weaning age between countries, this was not related to the populations of bifidobacteria or bacteroides. Thus, although the faecal microbiota of infants after first complementary foods was different to that before weaning commenced, many of the initial influences on microbiota composition were still evident.

How normal is a "normal" flora in animal or man?

When investigating different disturbances of the normal intestinal flora causing disorders and/or diseases in man and animal, these studies include comparisons of results with control materials, i.e., materials from conventional laboratory animals or healthy human beings. However, how "normal" is the control group's flora? In this paper two different examples will be discussed; one investigation with laboratory animals and one from a human study.

Screening-detected and symptomatic untreated celiac children show similar gut microflora-associated characteristics.

OBJECTIVE: The aim of this study was to investigate the metabolic function of intestinal microflora in children with screening-detected celiac disease (CD) to see if there is an aberrant gut flora in screening-detected CD similar to symptomatic CD and contrary to healthy controls. MATERIALS AND METHODS: As part of a Swedish multicenter screening for CD, 912 12-year-old children were screened with serum anti-human tissue transglutaminase-IgA. Small bowel biopsy specimens from children with positive serology revealed 17 individuals with CD. The functional status of the intestinal microflora was evaluated by gas-liquid chromatography of short chain fatty acids (SCFAs) in fecal samples. Our previously published findings in children with symptomatic CD and healthy controls were used as comparison. RESULTS: The children with screening-detected CD had a similar fecal SCFA profile to children with symptomatic CD, but differed significantly from that in healthy children. CONCLUSIONS: This is the first study on SCFA patterns in fecal samples from children with screening-detected CD. The similarity of the fecal SCFA profile in screening-detected and symptomatic CD indicates common pathogenic mechanisms. This could open the way for new therapeutic or prophylactic measures based on novel biological principles.

Intestinal microbiota of 6-week-old infants across Europe: geographic influence beyond delivery mode, breast-feeding, and antibiotics.

OBJECTIVES: : There are many differences in diet and lifestyle across Europe that may influence the development of the infant gut microbiota. This work aimed to assess the impact of geographic area, mode of delivery, feeding method, and antibiotic treatment on the fecal microbiota of infants from 5 European countries with different lifestyle characteristics: Sweden, Scotland, Germany, Italy, and Spain. PATIENTS AND METHODS: : Fecal samples from 606 infants (age 6 weeks) recruited within the European project INFABIO were analyzed by fluorescent in situ hybridization combined with flow cytometry using a panel of 10 rRNA targeted group- and species-specific oligonucleotide probes. Information on factors potentially affecting gut microbiota composition was collected with questionnaires and associations were evaluated with multivariate analyses. RESULTS: : The Bifidobacterium genus was predominant (40% average proportion of total detectable bacteria), followed by Bacteroides (11.4%) and enterobacteria (7.5%). Northern European countries were associated with higher proportions of bifidobacteria in infant feces, whereas a more diverse microbiota with more bacteroides characterized southern countries. Bifidobacteria dominated the microbiota of breast-fed infants, whereas formula-fed babies had significantly higher proportions of Bacteroides and members of the Clostridium coccoides and Lactobacillus groups. Newborns delivered by cesarean section or from mothers treated with antibiotics perinatally had lower proportions of Bacteroides and members of the Atopobium cluster. CONCLUSIONS: : Delivery mode and feeding method influenced the fecal microbiota of European infants at 6 weeks, as expected, but the effect of country of birth was more pronounced, with dominant bifidobacteria in northern countries and greater early diversification in southern European countries.

Intestinal microflora functions in laboratory mice claimed to harbor a "normal" intestinal microflora. Is the SPF concept running out of date?

For many years, laboratory animal breeders have used a mixture of eight bacterial strains, the so-called Altered Schaedler Flora (ASF) to inoculate Caesarian derived offspring when establishing colonies of Specific Pathogen Free (SPF) rodents fulfilling the criteria worked out by regulatory agencies as AALAS, FELASA, etc. However, recently it was shown in this journal that such SPF animals harbored a fecal flora far different from that of feral mice. Over the years, we have worked with functional aspects of host-microbe interactions(s) and the aim of the present study was to analyze some intestinal microbial biochemical activities in mice harboring an ASF flora. In the five parameter studied, the ASF mice showed a pattern similar to what is found in germfree mice and rats, demonstrating an absence of microorganisms capable of performing these reactions. These findings call for a re-considering of the SPF concept. Presence of important microbiological functions should be taken into consideration when rodents are used in biomedical research.

Antibiotic or bacterial therapy in post-giardiasis irritable bowel syndrome.

OBJECTIVE. Intestinal infection with Giardia lamblia may lead to therapy-resistant, long-lasting post-giardiasis irritable bowel syndrome (IBS). We report two open pilot studies aiming to treat this condition, using either antibiotics or bacterio-therapy. MATERIAL AND METHODS. Twenty-eight patients with persistent abdominal symptoms, following clearance of G. lamblia infection, were investigated. Eighteen received treatment with rifaximin plus metronidazole (8-10 days) whereas 10 received a suspension of live faecal flora, installed into the duodenum during gastro-duodenoscopy. Customary abdominal symptoms and symptoms following a lactulose breath test were quantified by questionnaires. Hydrogen and methane production after lactulose were analysed in expired air and excretion of fat and short-chain fatty acids (SCFAs) was examined in faeces. RESULTS. As compared with pre-treatment values, total customary symptom scores were barely significantly reduced (p = 0.07) after antibiotics, but were highly significantly reduced (p = 0.0009) after bacterio-therapy. However, symptom improvement following bacterio-therapy did not persist 1 year later. Hydrogen breath excretion was slightly reduced after antibiotics, but not after bacterio-therapy. Compared with healthy persons, faecal excretion of fat was significantly increased in Giardia-cured patients. SCFAs were increased in the bacterio-therapy group, and were not influenced by therapy. CONCLUSIONS. Both antibiotics and bacterio-therapy were ineffective with respect to cure of post-giardiasis IBS. High faecal excretion of fat and SCFAs suggests that intestinal malabsorption of fat and carbohydrates may play a role in the IBS-like complaints of these patients.

Carbohydrate intolerance in patients with self-reported food hypersensitivity: comparison of lactulose and glucose.

OBJECTIVE: Malabsorption of low-digestible carbohydrates is physiological, but poorly tolerated in some patients. We investigated symptom anticipation and microbial fermentation as possible mechanisms of carbohydrate intolerance in patients with self-reported food hypersensitivity. MATERIAL AND METHODS: In a randomized, double-blind, cross-over study, 27 consecutive patients with unexplained, self-reported food hypersensitivity were given 10 g lactulose and 10 g glucose (placebo). Symptoms and pulmonary excretion of hydrogen and methane were assessed. Short-chain fatty acids (SCFAs), lactate and prostaglandin E(2) (PGE(2)) were analyzed in rectal dialysis fluid, and compared to dialysates from nine healthy volunteers. RESULTS: Post-lactulose symptom scores were correlated with habitual symptom scores (r = 0.6, p = 0.001), significantly higher than post-glucose symptom scores (p = 0.01) and significantly higher in patients than controls (p = 0.0007). Levels of SCFAs, lactate and PGE(2) in rectal dialysates were not significantly different after lactulose and glucose, or between patients and controls. Hydrogen excretion was not correlated with symptom scores. CONCLUSIONS: The findings suggest that self-reported food hypersensitivity is related to microbial fermentation of malabsorbed carbohydrates and not to symptom anticipation solely. Levels of SCFAs, lactate and PGE(2) in rectal dialysates could not explain the fermentation-associated hypersensitivity.

Faecal short chain fatty acid pattern and allergy in early childhood.

AIM: To investigate whether functional changes of the gut flora over time were related to sensitization and allergic symptoms at four years of age. METHODS: The levels of short chain fatty acids (SCFAs) in faecal samples at one (n = 139) and four (n = 53) years of age were related to the development of positive skin prick tests (SPT) and allergic symptoms during the first four years of life. RESULTS: Faecal acetic (p < 0.01) and propionic (p < 0.01) acids decreased from one to four years of age, while valeric acid (p < 0.001) increased. Low levels of i-butyric (p = 0.01), i-valeric (p = 0.03) and valeric acids (p = 0.02) at one year were associated with questionnaire-reported symptoms of food allergy at four years. Positive SPTs and allergic symptoms at four years were associated with low faecal levels of i-butyric, i-valeric and valeric acids. At one year of age, infants with, as compared to without older siblings had higher median levels of valeric acid. CONCLUSION: A slow functional maturation of the gut microflora, as measured by faecal levels of SCFAs is associated with allergy both at one and four years. The findings lend further support to an association between allergy and the development of microbial diversity.

Intestinal cholesterol conversion in adults and elderly from four different European countries.

The growing healthy aging population in Europe is a heterogeneous group with varying nutritional needs, especially in association with problems like decreased sense of smell and taste, saliva production, chewing and swallowing problems besides all disease problems that put specific demands on the dietary intake and the role of the intestinal metabolism. To investigate this, one must clarify which functions are related to the host itself and which actions are related to the microflora. In short, the host's side of the ecosystem can be defined as milieu interieur (MI), the non-host side as milieu exterieur (ME) and MI plus ME together as milieu total (MT). We have investigated the role of the intestinal flora and its functions in adults and elderly from different European countries and this publication is concentrated on the microbial conversion of intestinal cholesterol to coprostanol, aiming to investigate the hepatic-intestinal flora co-cross-talk.