Eczema-protective probiotic alters infant gut microbiome functional capacity but not composition: sub-sample analysis from a RCT.

Probiotic Lactobacillus rhamnosus HN001 given in early life has been shown to reduce infant eczema risk, but its effect on gut microbiota development has not been quantitatively and functionally examined. The aim of this study was to investigate the impact of early life probiotic exposure on the composition and functional capacity of infant gut microbiota from birth to 2 years considering the effects of age, delivery mode, antibiotics, pets and eczema. We performed shotgun metagenomic sequencing analysis of 650 infant faecal samples, collected at birth, 3, 12, and 24 months, as part of a randomised, controlled, 3-arm trial assessing the effect of L. rhamnosus HN001, Bifidobacterium animalis subsp. lactis HN019 supplementation on eczema development in 474 infants. There was a 50% reduced eczema risk in the HN001 probiotic group compared to placebo. Both mothers (from 35 weeks gestation until 6 months post-partum if breastfeeding) and infants (from birth to 2 years) received either a placebo or one of two probiotics, L. rhamnosus HN001 (6×109 cfu), or B. animalis subsp. lactis HN019 (9×109 cfu). L. rhamnosus HN001 probiotic supplementation was associated with increased overall glycerol-3 phosphate transport capacity and enrichment of L. rhamnosus. There were no other significant changes in infant gut microbiota composition or diversity. Increased capacity to transport glycerol-3-phosphate was positively correlated with relative abundance of L. rhamnosus. Children who developed eczema had gut microbiota with increased capacity for glycosaminoglycan degradation and flagellum assembly but had no significant differences in microbiota composition or diversity. Early life HN001 probiotic use is associated with both increased L. rhamnosus and increased infant gut microbiota functional capacity to transport glycerol-3 phosphate. The mechanistic relationship of such functional alteration in gut microbiota with reduced eczema risk and long-term health merits further investigation.

A functional analysis of the formyl-coenzyme A (frc) gene from Lactobacillus reuteri 100-23C.

AIM: To examine the role of the Lactobacillus reuteri 100-23C frc gene product in oxalate metabolism, host colonization and the acid stress response. METHODS AND RESULTS: Genes encoding putative formyl-CoA transferase (frc) and oxalyl-CoA decarboxylase (oxc) enzymes are present in the genome sequences of Lact. reuteri strains. Two strains isolated from humans harboured an IS200 insertion sequence in the frc ORF and a group 2 intron-associated transposase downstream of the frc gene, both of which were lacking in two strains of animal origin, which contained intact frc and oxc genes. An frc(-) insertional mutant of Lact. reuteri 100-23C was compared with the parent strain with respect to oxalate degradation, colonization of an RLF-mouse host model and growth in the presence of acids. Neither parent nor mutant degraded oxalate in vitro or in vivo. However, the parent outcompeted the frc(-) mutant in the mouse intestine during co-colonization and the frc(-) mutant showed a reduced growth rate in the presence of hydrochloric acid. CONCLUSIONS: Intact oxc and frc genes do not ensure oxalate degradation under the conditions tested. The frc gene product is important during host colonization and survival of acid stress by Lact. reuteri 100-23C. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxalate metabolism by oxalate-degrading intestinal bacterial strains may be important in preventing urolithiasis and might lead to the derivation of probiotic products. To produce safe and efficacious probiotics, however, an understanding of the genetic characteristics of potential oxalate degraders must be obtained, together with knowledge of their functional ramifications.

Treatment and secondary prevention effects of the probiotics Lactobacillus paracasei or Bifidobacterium lactis on early infant eczema: randomized controlled trial with follow-up until age 3 years.

BACKGROUND: Allergic disease has been associated with altered intestinal microbiota. Therefore, probiotics have been suggested as a potential treatment for eczema. OBJECTIVE: We investigated whether dietary supplementation of infants with eczema at age 3-6 months with Lactobacillus paracasei CNCM I-2116 or Bifidobacterium lactis CNCM I-3446 had a treatment effect or altered allergic disease progression. METHODS: Primary outcome included eczema severity (SCORing Atopic Dermatitis, SCORAD) 3 months post-randomization. Secondary: SCORAD (other visits); infant dermatitis quality of life (IDQoL); gastrointestinal permeability; urinary eosinophilic protein X; allergen-sensitization; allergic symptoms (age 12, 18, 36 months). A total of 208 infants aged 3-6 months with physician-diagnosed eczema were recruited; 137/208 (SCORAD ≥ 10, consuming ≥ 200 mL standard formula/day) were randomized to daily supplements containing L. paracasei or B. lactis or placebo for a 3-month period, while receiving extensively hydrolysed whey-formula (dairy-free diet). There were two open observational groups, one group exclusively breastfed (n = 22) and the other, standard formula-fed (n = 49). TRIAL NUMBER: ISRCTN41490500. RESULTS: Eczema severity decreased significantly over time in all groups. No significant difference was observed between randomized groups after 12-week treatment-period (SCORAD-score pre-/post-intervention: B. lactis 25.9 [95% CI: 22.8-29.2] to 12.8 [9.4-16.6]; L. paracasei 25.4 [22.1-29] to 12.5 [9.2-16.4]; placebo 26.9 [23.4-30.6] to 11.8 [9.6-14.3]; P = 0.7). Results were similar when analysis was controlled for allergen-sensitization, or when only sensitized infants were analysed. No differences were found for secondary outcomes. No difference was observed in SCORAD-score between randomized and observational groups. CONCLUSION AND CLINICAL RELEVANCE: We found no benefit from supplementation with B. lactis or L. paracasei in the treatment of eczema, when given as an adjunct to basic topical treatment, and no effect on the progression of allergic disease from age 1 to 3 years.

Molecular analysis of the intestinal microflora in IBD.

Nucleic acid-based (molecular) analytical methods have utility in discriminating between bowel microbiota of altered compositions. This has been demonstrated in studies involving both experimental animals and humans with inflammation of the bowel. Although alterations in the composition of the microbiota can be demonstrated, future studies need to provide functional links between the candidate proinflammatory agents and disease processes.

Relationship of dietary antimicrobial drug administration with broiler performance, decreased population levels of Lactobacillus salivarius, and reduced bile salt deconjugation in the ileum of broiler chickens.

Straight-run broiler chickens were raised either in floor pens or wire-floored cages (trial 1) or in floor pens only (trials 2, 3, and 4). Birds raised in floor pens had lower BW and feed intakes than those raised in cages. The administration of bacitracin in the feed increased feed intake from d 12 to d 35, decreased the feed conversion ratio during the same period in trial 2, and improved the weight gain of broilers from d 0 to 10 in trial 3. The concentrations of conjugated bile salts (taurocholic and taurochenodeoxycholic acids) were higher in the ileal contents of broilers administered the antimicrobials compared with untreated birds. Supplementation of the feed with monensin increased fat digestibility in the ileum of the birds. Although total numbers of bacteria in ileal contents were the same regardless of whether antimicrobials were administered or not, the bacterial community differed qualitatively. Populations of Lactobacillus salivarius were reduced in birds fed antimicrobials relative to untreated broilers. A representative ileal isolate of L. salivarius deconjugated bile salts in pure culture in the laboratory and in the ileal contents of ex-Lactobacillus-free chickens maintained in a protective environment and colonized by the Lactobacillus isolate. These observations provide a link between bile salt deconjugation in the ileum by L. salivarius and decreased weight gain of broilers. Lactobacillus salivarius populations could be targeted in future studies aimed at modification of the ileal bacterial community to achieve growth promotion of broilers without the administration of antimicrobial drugs.

Investigating the effects of commercial probiotics on broiler chick quality and production efficiency.

A study was undertaken to test the effect of 2 commercially available probiotics on the production efficiency of broiler chickens hatched from the same breeder flock at 3 different ages (28, 43, and 57 wk). At each of the 3 breeder flock ages, 1,600 broiler chickens were hatched and randomly allocated to 1 of 4 treatments: 1) no probiotics (control), 2) probiotic 1 administered in the drinking water, 3) probiotic 1 administered as a spray, and 4) probiotic 2 administered in the feed. A coccidiostat was included in the feed, but no other antimicrobial agents were given. Broilers were then reared on straw litter in identical floor pens for a period of 6 wk. There were no significant differences in broiler BW, feed conversion, or mortality between the probiotic treatments and the control group in any of the trials. The 43-wk-old breeder flock had the highest fertility and hatchability and the lowest percentage of chicks culled at hatching. Throughout the broiler production period, the broilers from the 43- and 57-wk-old breeder flocks had higher BW and weight gains than the broilers produced at 28 wk of breeder flock age. Broiler feed conversion over the 6-wk production period decreased as the breeder flock aged. Probiotics had no effect on chick quality or production efficiency in broilers produced by the breeder flock ages examined.

PCR/DGGE and 16S rRNA gene library analysis of the colonic microbiota of HLA-B27/beta2-microglobulin transgenic rats.

AIMS: To determine the phylogenetic composition of the colonic microbiota of transgenic (TG) HLA-B27 rats using 16S ribosomal RNA (rRNA) gene sequences obtained from denaturing gradient gel electrophoresis (DGGE) gels and sequences from a 16S rRNA gene library. METHODS AND RESULTS: Colonic microbiota of TG and nontransgenic (NT) rats harboured by 10-week-old and 6-month-old animals was screened using PCR/DGGE. Six months old TG rats had marked inflammation of the colon compared with 10-week-old TG and NT rats. The DGGE profiles of rats with inflamed colon were similar from rat to rat (Dice's Similarity Coefficient proximal colon 73%, distal colon 83%) whereas profiles from animals without inflammation were dissimilar (52-64%). Identifications of bacterial origins of 16S rRNA gene sequences obtained from DGGE gels (200 bp) and from 16S rRNA clones (450 bp) of the colonic microbiota of diseased rats gave sequences most closely phylogenetically affiliated with uncultured or unknown bacteria. CONCLUSIONS: PCR/DGGE was shown to be an effective method to compare the colonic microbiota composition of TG and NT rats relative to the progression of inflammatory disease. Sequencing of 16S rRNA gene fragments from DGGE gels or 16S rRNA gene clones from a random library showed that uncultured or unknown bacteria were most commonly detected by both methods. It can be concluded that it would be better in future studies to search for the antigens produced by the gut microbiota against which the dysfunctional immune system reacts rather than seek phylogenetic associations. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR/DGGE can be used as a rapid initial screening method to compare the composition of bacterial communities of initially unknown composition that are associated with the development of intestinal disease.

Fecal microbiota in sensitized wheezy and non-sensitized non-wheezy children: a nested case-control study.

BACKGROUND: It has been suggested that intestinal microbiota of allergic and non-allergic children differs in composition, and that microbiota-immune system interactions may predispose children to develop sensitization. Previous studies have examined fecal microbiota of allergic children with atopic dermatitis, but little is known about that of atopic wheezy children. OBJECTIVE: To investigate the composition of the fecal microbiota of young sensitized wheezy and non-sensitized non-wheezy children, using molecular methods. METHODS: Within the context of a prospective birth cohort, we carried out a nested case-control study of sensitized wheezy children (cases) and non-sensitized non-wheezy controls. Cases and controls were matched for age, sex, parental atopy, allergen exposure, and pet ownership. We evaluated the composition of fecal microbiota by nucleic acid-based methods (PCR combined with denaturing gradient gel electrophoresis and quantification of bifidobacteria by fluorescent in situ hybridization). RESULTS: Thirty-three case-control pairs (mean age 4.4 years) provided stool samples. Comparison of total bacterial community profiles showed that each child had a unique fecal microbiota (mean Dice's similarity coefficient 22%, range 3.3-60.8%). There was no difference between the groups in prevalence of Lactic Acid bacteria (12/33 vs. 11/33, P=0.8) or bifidobacteria (30/33 vs. 31/33, P=1.00, cases vs. controls). The bifidobacterial species detected were similar in both groups. The percentage of bifidobacteria in total fecal microflora was no different between cases (median 1.7%, range 0-20.8%) and controls (1.9%, 0-18.2%, P=0.7). However, cases with eczema had significantly fewer bifidobacteria (median 1.6%, range 0-4.8%) than their controls (4.0%, 1.9-18.2%, P=0.05). CONCLUSION: We found no differences in fecal microbiota composition between sensitized wheezy and non-sensitized, non-wheezy children aged 3-5 years using nucleic acid-based methods. Differences appear to be isolated to those allergic children with eczema.

Analysis of the large bowel microbiota of colitic mice using PCR/DGGE.

AIM: To test combined polymerase chain reaction amplification of 16S rRNA gene sequences and denaturing gradient gel electrophoresis (PCR/DGGE) as an analytical method to investigate the composition of the large bowel microbiota of mice during the development of colitis. METHODS AND RESULTS: The colonic microbiota of formerly germfree interleukin 10 (IL-10)-deficient mice that had been exposed to the faecal microbiota of specific pathogen-free animals was screened using PCR/DGGE. The composition of the large bowel microbiota of IL-10-deficient mice changed as colitis progressed. DNA fragments originating from four bacterial populations ('Bacteroides sp.', Bifidobacterium animalis, Clostridium cocleatum, enterococci) were more apparent in PCR/DGGE profiles of colitic mice relative to non-colitic animals, whereas two populations were less apparent (Eubacterium ventriosum, Acidophilus group lactobacilli). Specific DNA:RNA dot blot analysis showed that bifidobacterial ribosomal RNA (rRNA) abundance increased as colitis developed. CONCLUSIONS: PCR/DGGE was shown to be an effective method to demonstrate changes in the composition of the large bowel microbiota of mice in relation to progression of inflammatory disease. The intensity of staining of DNA fragments in DGGE profiles reflected increased abundance of bifidobacterial rRNA in the microbiota of colitic animals. As bifidobacterial fragments in PCR/DGGE profiles generated from microbiota DNA showed increased intensity of fragment staining, an increase in bifidobacterial numbers in colitic mice was indicated. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR/DGGE analysis demonstrated an altered composition of the large bowel microbiota of colitic mice. This work will allow specific groups of bacteria to be targeted in future research concerning the pathogenesis of colitis.

Effects of feeding a probiotic preparation (SIM) containing inulin on the severity of colitis and on the composition of the intestinal microflora in HLA-B27 transgenic rats.

An overly aggressive immune response to the intestinal microflora in a genetically susceptible host background has been implicated in the pathogenesis of inflammatory bowel diseases. We measured the impact of a probiotic preparation (SIM) containing inulin on the severity of colitis and on intestinal microflora profiles of HLA-B27-beta(2)-microglobulin transgenic (TG) rats. SIM is a mixture of lactobacilli, bifidobacteria, and inulin. Two-month-old TG rats received either SIM or water. Control TG rats received metronidazole, alone or in combination with SIM, for 8 weeks. Nontransgenic rats received SIM or water. The cecal content was removed for analysis of the intestinal microflora by PCR combined with denaturing gradient gel electrophoresis. The colon was scored for histological evidence of inflammation, colonic myeloperoxidase activity and interleukin-1beta RNA levels were measured photometrically or by real-time quantitative PCR. At 4 months, the colonic inflammation of TG rats treated with SIM was histologically diminished compared to that in untreated TG rats (2.2 +/- 0.2 versus 2.9 +/- 0.1; P

Molecular methods for exploring the intestinal ecosystem.

Molecular methods have provided renewed impetus for the analysis of the composition of the intestinal microflora in health and disease. The polymerase chain reaction coupled with denaturing gradient gel electrophoresis provides a method whereby the bacterial communities in large numbers of samples can be compared efficiently and effectively. Altered bacterial populations associated with disease states can then be targeted for further investigation. In the long-term, an 'abnormal microflora' might be rectified by the use of probiotics or prebiotics.

Analysis of the intestinal microflora using molecular methods.

A large and complex bacterial community inhabits the distal intestinal tract of humans. This collection, known as the intestinal microflora, is dominated numerically by obligately anaerobic bacterial species. Many of these species have never been cultivated under laboratory conditions. Nucleic acid-based techniques now permit, however, the analysis of even the non-cultivable members of the bacterial community. Polymerase chain reaction (PCR) coupled with denaturing gradient gel electrophoresis (DGGE) provides a useful technique for comparisons of the composition of faecal or intestinal microfloras. PCR/DGGE has been shown to be useful in demonstrating changes that occur in the composition of the faecal microflora of infants administered antibacterial drugs. This research is important because treatment with oral antibiotics during the first 2 y of life has been identified as a predictor of subsequent atopic disease. The treatment of young children with broad spectrum oral antibiotics might produce perturbations in the composition of the intestinal microflora such that bacteria important in promoting Th1 mechanisms are depleted at a crucial age. This could result in Th2 dominance over Th1 immune responses to environmental antigens and an increased incidence of atopic disorders. PCR/DGGE provides a useful screening method to determine the impact of antibiotic treatment on the composition of the intestinal microflora of children and to identify the bacterial groups that are most affected.

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.

Molecular assessment of intestinal microflora.

The application of molecular methodologies to intestinal microflora analysis should enable the development of a detailed knowledge of the microbial ecology of the human colon. This knowledge is essential to derive scientifically valid probiotics. Molecular typing (genetic fingerprinting) methods, eg, ribotyping and pulsed field gel electrophoresis of DNA digests, provide a means of distinguishing bacterial strains inhabiting the intestinal tract. Analysis of lactobacillus, bifidobacterial, and enterobacterial populations with the use of these methods has shown that human and porcine subjects harbor a characteristic collection of bacterial strains. Additionally, perturbations and transitions that occur in these populations and are caused by antibiotic administration or by autogenic or allogenic factors can be detected by molecular analysis of the intestinal microflora. In future studies, molecular typing methods could be used to analyze the composition of bacterial populations before, during, and after the administration of the probiotic product. This experimental approach would provide information on the effect of the probiotic on indigenous strains inhabiting the intestinal tract of humans and other animals.

Cholic acid is accumulated spontaneously, driven by membrane deltapH, in many lactobacilli.

Many lactobacilli from various origins were found to apparently lack cholic acid extrusion activity. Cholic acid was accumulated spontaneously, driven by the transmembrane proton gradient. Accumulation is a newly identified kind of interaction between intestinal microbes and unconjugated bile acids and is different from extrusion and modification, which have been described previously.

The intestinal microflora: potentially fertile ground for microbial physiologists.

The intestinal microflora provides opportunities for microbial physiological research. The metabolic interactions of bacterial inhabitants of the intestinal community, bacterial bioenergetics, preferential utilization of substrates as energy sources by specific bacterial species, and intercellular signalling are among the topics of challenging research awaiting the attention of microbial physiologists.

Analysis of the fecal microflora of human subjects consuming a probiotic product containing Lactobacillus rhamnosus DR20.

The composition of the fecal microflora of 10 healthy subjects was monitored before (6-month control period), during (6-month test period), and after (3-month posttest period) the administration of a milk product containing Lactobacillus rhamnosus DR20 (daily dose, 1.6 x 10(9) lactobacilli). Monthly fecal samples were examined by a variety of methods, including bacteriological culture analysis, fluorescent in situ hybridization with group-specific DNA probes, denaturing gradient gel electrophoresis of the V2-V3 region of 16S rRNA genes amplified by PCR, gas-liquid chromatography, and bacterial enzyme activity analysis. The composition of the Lactobacillus population of each subject was analyzed by pulsed-field gel electrophoresis of bacterial DNA digests in order to differentiate between DR20 and other strains present in the samples. Representative isolates of lactobacilli were identified to the species level by sequencing the V2-V3 region of their 16S rRNA genes and comparing the sequences obtained (BLAST search) to sequences in the GenBank database. DR20 was detected in the feces of all of the subjects during the test period, but at different frequencies. The presence of DR20 among the numerically predominant strains was related to the presence or absence of a stable indigenous population of lactobacilli during the control period. Strain DR20 did not persist at levels of >10(2) cells per g in the feces of most of the subjects after consumption of the product ceased; the only exception was one subject in which this strain was detected for 2 months during the posttest period. We concluded that consumption of the DR20-containing milk product transiently altered the Lactobacillus and enterococcal contents of the feces of the majority of consumers without markedly affecting biochemical or other bacteriological factors.

Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers.

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database.

Influence of different functional elements of plasmid pGT232 on maintenance of recombinant plasmids in Lactobacillus reuteri populations in vitro and in vivo.

Plasmid pGT232 (5.1 kb), an indigenous plasmid of Lactobacillus reuteri 100-23, was determined, on the basis of nucleotide and deduced protein sequence data, to belong to the pC194-pUB110 family of plasmids that replicate via the rolling-circle mechanism. The minimal replicon of pGT232 was located on a 1.7-kb sequence consisting of a double-strand origin of replication and a gene encoding the replication initiation protein, repA. An erythromycin-selectable recombinant plasmid containing this minimal replicon was stably maintained (>97% erythromycin-resistant cells) without antibiotic selection in an L. reuteri population under laboratory growth conditions but was poorly maintained (<33% resistant cells) in the L. reuteri population inhabiting the murine gastrointestinal tract. Stable maintenance (>90% resistant cells) of pGT232-derived plasmids in the lactobacillus population in vivo required an additional 1.0-kb sequence which contained a putative single-strand replication origin (SSO). The SSO of pGT232 is believed to be novel and functions in an orientation-specific manner.