Long-term colonisation with donor bacteriophages following successful faecal microbial transplantation.

BACKGROUND: Faecal microbiota transplantation (FMT) is used in the treatment of recurrent Clostridium difficile infection. Its success is typically attributed to the restoration of a diverse microbiota. Viruses (including bacteriophages) are the most numerically dominant and potentially the most diverse members of the microbiota, but their fate following FMT has not been well studied. RESULTS: We studied viral transfer following FMT from 3 donors to 14 patients. Recipient viromes resembled those of their donors for up to 12 months. Tracking individual bacteriophage colonisation revealed that engraftment of individual bacteriophages was dependent on specific donor-recipient pairings. Specifically, multiple recipients from a single donor displayed highly individualised virus colonisation patterns. CONCLUSIONS: The impact of viruses on long-term microbial dynamics is a factor that should be reviewed when considering FMT as a therapeutic option.

The long-term effects of faecal microbiota transplantation for gastrointestinal symptoms and general health in patients with recurrent Clostridium difficile infection.

BACKGROUND: Faecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection. In short-term the treatment has been shown to be safe, however, there are no large, long-term follow-up studies looking into the potential adverse effects. AIM: To analyse the long-term effect of FMT treatment in patients with recurrent C. difficile infection and to compare the outcome to antibiotic treated patients. METHODS: Altogether 84 patients of which 45 received a FMT treatment and 39 served as controls receiving antibiotics for the infection were followed on average for 3.8 years. Their recovery and medical status was evaluated using a retrospective questionnaire, determining their quality of life, gastrointestinal symptoms and new diseases potentially related to the FMT. RESULTS: There was no difference in the incidence of severe diseases (inflammatory bowel disease, cancer, autoimmune disease, allergy, neurological diseases) between the patient groups. In addition, weight gain did not differ between treatment groups. The FMT treated patients reported that their bowel habits improved significantly faster, they had less irregular bowel function and less symptoms of upper GI-tract when compared to the patients treated with antibiotics. Significantly more patients in FMT-group reported that their mental health improved after the treatment. The willingness to receive FMT treatment for potential new C. difficile infection was significantly higher in both treatment groups compared to other treatment options. CONCLUSION: Our study highlights that FMT is a durable, safe and acceptable treatment option for patients with recurrent C. difficile infection also in long term, and it shows potential benefits over antimicrobial treatment.

Consensus report: faecal microbiota transfer - clinical applications and procedures.

BACKGROUND: Faecal microbiota transplantation or transfer (FMT) aims at replacing or reinforcing the gut microbiota of a patient with the microbiota from a healthy donor. Not many controlled or randomised studies have been published evaluating the use of FMT for other diseases than Clostridium difficile infection, making it difficult for clinicians to decide on a suitable indication. AIM: To provide an expert consensus on current clinical indications, applications and methodological aspects of FMT. METHODS: Well-acknowledged experts from various countries in Europe have contributed to this article. After literature review, consensus has been achieved by repetitive circulation of the statements and the full manuscript among all authors with intermittent adaptation to comments (using a modified Delphi process). Levels of evidence and agreement were rated according to the GRADE system. Consensus was defined a priori as agreement by at least 75% of the authors. RESULTS: Key recommendations include the use of FMT in recurrent C. difficile infection characterised by at least two previous standard treatments without persistent cure, as well as its consideration in severe and severe-complicated C. difficile infection as an alternative to total colectomy in case of early failure of antimicrobial therapy. FMT in inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS) and metabolic syndrome should only be performed in research settings. CONCLUSIONS: Faecal microbiota transplantation or transfer is a promising treatment for a variety of diseases in which the intestinal microbiota is disturbed. For indications other than C. difficile infection, more evidence is needed before more concrete recommendations can be made.

Simple faecal preparation and efficacy of frozen inoculum in faecal microbiota transplantation for recurrent Clostridium difficile infection--an observational cohort study.

BACKGROUND: Faecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection (rCDI). The finding of suitable donor, donor screening and preparation of faecal transplants are challenging in clinical work. AIM: To develop a practical protocol for preparing frozen transplants and to compare the efficacy of previously frozen and fresh faeces in treating rCDI. METHODS: Two healthy volunteers acted as universal donors for the frozen faecal preparations, which were prepared by suspending faeces into physiological saline, adding glycerol to a final concentration of 10% and storing at -80 °C. We compared the outcomes of patients with rCDI who had undergone FMT at colonoscopy and received infusion of previously prepared, freeze-stored faeces (n = 23) or fresh faeces from individual (n = 15) or universal donors (n = 11) (total n = 49). Clinical failure was defined as persistent or recurrent symptoms with a positive C. difficile toxin stool test, and a need for new therapy. RESULTS: At 12 weeks post-FMT, symptoms were resolved in 22 of 23 patients receiving previously frozen faeces, and in all 11 or 14 of 15 patients receiving fresh faeces from the universal or individual donors respectively (totally 25 of 26; P = ns, success rate 96%). Mild transient fever appeared for two patients receiving frozen faeces, but no other significant side effects were observed. 42 patients were followed up for a year post-FMT and the success rate was 88% in both fresh and frozen faeces groups. CONCLUSIONS: Preparation of frozen transplants simplifies the practical aspects of faecal microbiota transplantation without loss of efficacy or safety.

Severity of atopic disease inversely correlates with intestinal microbiota diversity and butyrate-producing bacteria.

The reports on atopic diseases and microbiota in early childhood remain contradictory, and both decreased and increased microbiota diversity have been associated with atopic eczema. In this study, the intestinal microbiota signatures associated with the severity of eczema in 6-month-old infants were characterized. Further, the changes in intestinal microbiota composition related to the improvement of this disease 3 months later were assessed. The severity of eczema correlated inversely with microbiota diversity (r = -0.54, P = 0.002) and with the abundance of butyrate-producing bacteria (r = -0.52, P = 0.005). During the 3-month follow-up, microbiota diversity increased (P < 0.001) and scoring atopic dermatitis values decreased (P < 0.001) in all infants. This decrease coincided with the increase in bacteria related to butyrate-producing Coprococcus eutactus (r = -0.59, P = 0.02). In conclusion, the high diversity of microbiota and high abundance of butyrate-producing bacteria were associated with milder eczema, thus suggesting they have a role in alleviating symptoms of atopic eczema.

Interaction of Campylobacter spp. and human probiotics in chicken intestinal mucus.

Campylobacter is the most common cause of bacterial food-borne diarrhoeal disease throughout the world. The principal risk of human contamination is handling and consumption of contaminated poultry meat. To colonize poultry, Campylobacter adheres to and persists in the mucus layer that covers the intestinal epithelium. Inhibiting adhesion to the mucus could prevent colonization of the intestine. The aim of this study was to investigate in vitro the protective effect of defined commercial human probiotic strains on the adhesion of Campylobacter spp. to chicken intestinal mucus, in a search for alternatives to antibiotics to control this food-borne pathogen. The probiotic strains Lactobacillus rhamnosus GG and Propionibacterium freudenreichii ssp. shermanii JS and a starter culture strain Lactococcus lactis ssp. lactis adhered well to chicken intestinal mucus and were able to reduce the binding of Campylobacter spp. when the mucus was colonized with the probiotic strain before contacting the pathogen. Human-intended probiotics could be useful as prophylactics in poultry feeding for controlling Campylobacter spp. colonization.

Bifidobacterium and Lactobacillus DNA in the human placenta.

AIMS: Bifidobacteria and lactobacilli are part of the human normal intestinal microbiota and may possibly be transferred to the placenta. It was hypothesized that intestinal bacteria or their components are present in the placenta and that the foetus may be exposed to them. We investigated the presence of bifidobacteria and lactobacilli and their DNA in the human placenta. METHODS AND RESULTS: We studied 34 human placentae (25 vaginal and nine caesarean deliveries) for the presence Bifidobacterium spp. and Lactobacillus rhamnosus. Cultivation was used for the detection of viable cells and genus and species-specific PCR for the detection of DNA. No bifidobacteria or lactobacilli were found by cultivation. Bifidobacterial DNA was detected in 33 and L. rhamnosus DNA in 31 placenta samples. CONCLUSIONS: DNA from intestinal bacteria was found in most placenta samples. The results suggest that horizontal transfer of bacterial DNA from mother to foetus may occur via placenta. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial DNA contains unmethylated CpG oligodeoxynucleotide motifs which induce immune effects. Specific CpG motifs activate Toll-like receptor 9 and subsequently trigger Th-1-type immune responses. Although the newborn infant is considered immunologically immature, exposure by bacterial DNA may programme the infant's immune development during foetal life earlier than previously considered.

Polymerase chain reaction and denaturing gradient gel electrophoresis monitoring of fecal bifidobacterium populations in a prebiotic and probiotic feeding trial.

A culture-independent approach based on genus-specific PCR and denaturing gradient gel electrophoresis (DGGE) was used to monitor qualitative changes in fecal bifidobacterial communities in a human feeding trial. DNA was extracted directly from feces and bifidobacterial 16S rDNA sequences were amplified using genus-specific PCR. The PCR fragments were subsequently separated in a sequence-specific manner by DGGE in order to obtain a profile of bifidobacterial fragments. The DGGE profiles revealed that in general, administration for two weeks of galactooligosaccharide and/or Bifidobacterium lactis Bb-12 (8 g and 3 x 10(10) cfu per day, respectively) did not affect the qualitative composition of the indigenous Bifidobacterium population, while B. lactis Bb-12 transiently colonised the gut.

Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis.

We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074(T) exhibited microheterogeneity differing in eight positions over almost the total length of the gene.

Probiotic bacteria and intestinal health: new methods of investigation.

This paper highlights some new methods in the probiotic research based on the use of colonic biopsies and molecular biological techniques for strain identification.

Identification of pediococci by ribotyping.

Pediococci are among the most prevalent microbial contaminants in breweries and they can cause ropiness and the accumulation of high levels of diacetyl in beer. The accurate identification of pediococci is important, because different species do not possess equal spoilage potential. In this study, 18 Pediococcus strains, mainly of brewery origin, were first identified using phenotypical characterization (API 50 CHL and SDS-PAGE profiling), and then ribotyped using a RiboPrinterR System. Six Pediococcus type strains and three other Pediococcus strains were used as references. Ribotyping showed higher discriminative capacity than phenotypical identification methods. Strains could be identified to species level and in many cases, differentiated even at strain level using this genetic fingerprinting method. The identifications performed by ribotyping were confirmed by 16S rDNA sequencing of selected strains. Automated ribotyping was found to be a rapid and reliable method for identifying pediococci, but requires the construction of a comprehensive fingerprint library.

Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei strains.

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively.

Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption.

Lactobacillus rhamnosus GG is one of the most thoroughly studied probiotic strains. Its advantages in the treatment of gastrointestinal disorders are well documented. The aim of the present study was to demonstrate with colonic biopsies the attachment of strain GG to human intestinal mucosae and the persistence of the attachment after discontinuation of GG administration. A whey drink fermented with strain GG was fed to human volunteers for 12 days. Fecal samples were collected before, during, and after consumption. L. rhamnosus GG-like colonies were detected in both fecal and colonic biopsy samples. Strain GG was identified by its characteristic colony morphology, a lactose fermentation test, and PCR. This study showed that strain GG was able to attach in vivo to colonic mucosae and, although the attachment was temporary, to remain for more than a week after discontinuation of GG administration. The results demonstrate that the study of fecal samples alone is not sufficient in evaluating colonization by a probiotic strain.

Detection of beer spoilage bacteria Megasphaera and Pectinatus by polymerase chain reaction and colorimetric microplate hybridization.

Anaerobic bacteria of the genera Megasphaera and Pectinatus cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. A polymerase chain reaction (PCR) method and a colorimetric microplate hybridization assay were developed for the rapid and specific detection of Megasphaera cerevisiae and Pectinatus spp. A biotinylated primer pair was designed for the amplification of a 403 base pair (bp) fragment of the M. cerevisiae 16S rRNA gene and a primer pair from literature was used for the amplification of an 816 bp fragment of Pectinatus 16S rRNA gene. Amplified PCR products were analyzed by the colorimetric microplate hybridization method in which a biotinylated PCR product was captured by streptavidin and hybridized with a digoxigenin-labelled oligonucleotide probe. In the final step an enzyme-linked antibody and a colorimetric reaction were utilized. A simple and rapid sample treatment was set up for the PCR detection of contaminants in beer. Detection of M. cerevisiae (> or = 5 x 10(3) colony forming units [cfu]/100 ml) and Pectinatus frisingensis (> or = 5 x 10(5) cfu/100 ml) in beer was successful, but the sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.