Cultivation and Genomics Prove Long-Term Colonization of Donor's Bifidobacteria in RecurrentClostridioides difficilePatients Treated With Fecal Microbiota Transplantation

Fecal microbiota transplantation (FMT) is an effective treatment for recurrentClostridioides difficileinfection (rCDI) and it's also considered for treating other indications. Metagenomic studies have indicated that commensal donor bacteria may colonize FMT recipients, but cultivation has not been employed to verify strain-level colonization. We combined molecular profiling ofBifidobacteriumpopulations with cultivation, molecular typing, and whole genome sequencing (WGS) to isolate and identify strains that were transferred from donors to recipients. SeveralBifidobacteriumstrains from two donors were recovered from 13 recipients during the 1-year follow-up period after FMT. The strain identities were confirmed by WGS and comparative genomics. Our results show that specific donor-derived bifidobacteria can colonize rCDI patients for at least 1 year, and thus FMT may have long-term consequences for the recipient's microbiota and health. Conceptually, we demonstrate that FMT trials combined with microbial profiling can be used as a platform for discovering and isolating commensal strains with proven colonization capacity for potential therapeutic use.Peer reviewe

The Potential of Gut Commensals in Reinforcing Intestinal Barrier Function and Alleviating Inflammation

The intestinal microbiota, composed of pro- and anti-inflammatory microbes, has an essential role in maintaining gut homeostasis and functionality. An overly hygienic lifestyle, consumption of processed and fiber-poor foods, or antibiotics are major factors modulating the microbiota and possibly leading to longstanding dysbiosis. Dysbiotic microbiota is characterized to have altered composition, reduced diversity and stability, as well as increased levels of lipopolysaccharide-containing, proinflammatory bacteria. Specific commensal species as novel probiotics, so-called next-generation probiotics, could restore the intestinal health by means of attenuating inflammation and strengthening the epithelial barrier. In this review we summarize the latest findings considering the beneficial effects of the promising commensals across all major intestinal phyla. These include the already well-known bifidobacteria, which use extracellular structures or secreted substances to promote intestinal health. Faecalibacterium prausnitzii, Roseburia intestinalis, and Eubacterium hallii metabolize dietary fibers as major short-chain fatty acid producers providing energy sources for enterocytes and achieving anti-inflammatory effects in the gut. Akkermansia muciniphila exerts beneficial action in metabolic diseases and fortifies the barrier function. The health-promoting effects of Bacteroides species are relatively recently discovered with the findings of excreted immunomodulatory molecules. These promising, unconventional probiotics could be a part of biotherapeutic strategies in the future.Peer reviewe

Cultivation and Genomics Prove Long-Term Colonization of Donor's Bifidobacteria in Recurrent Clostridioides difficile Patients Treated With Fecal Microbiota Transplantation

Fecal microbiota transplantation (FMT) is an effective treatment for recurrentClostridioides difficileinfection (rCDI) and it's also considered for treating other indications. Metagenomic studies have indicated that commensal donor bacteria may colonize FMT recipients, but cultivation has not been employed to verify strain-level colonization. We combined molecular profiling ofBifidobacteriumpopulations with cultivation, molecular typing, and whole genome sequencing (WGS) to isolate and identify strains that were transferred from donors to recipients. SeveralBifidobacteriumstrains from two donors were recovered from 13 recipients during the 1-year follow-up period after FMT. The strain identities were confirmed by WGS and comparative genomics. Our results show that specific donor-derived bifidobacteria can colonize rCDI patients for at least 1 year, and thus FMT may have long-term consequences for the recipient's microbiota and health. Conceptually, we demonstrate that FMT trials combined with microbial profiling can be used as a platform for discovering and isolating commensal strains with proven colonization capacity for potential therapeutic use

Minor Effect of Antibiotic Pre-treatment on the Engraftment of Donor Microbiota in Fecal Transplantation in Mice

Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection (rCDI) and is also considered a potential treatment for a wide range of intestinal and systemic diseases. FMT corrects the microbial dysbiosis associated with rCDI, and the engraftment of donor microbiota is likely to play a key role in treatment efficacy. For disease indications other than rCDI, FMT treatment efficacy has been moderate. This may be partly due to stronger resilience of resident host microbiota in patients who do not suffer from rCDI. In rCDI, patients typically have undergone several antibiotic treatments prior to FMT, depleting the microbiota. In this study, we addressed the effect of broad-spectrum antibiotics (Ab) as a pre-treatment to FMT on the engraftment of donor microbiota in recipients. We conducted a pre-clinical study of FMT between two healthy mouse strains, Balb/c as donors and C57BL/6 as recipients, to perform FMT within the same species and to mimic interindividual FMT between human donors and patients. Microbiota composition was assessed with high-throughput 16S rDNA amplicon sequencing. The microbiota of Balb/c and C57BL/6 mice differed significantly, which allowed for the assessment of microbiota transplantation from the donor strain to the recipient. Our results showed that Ab-treatment depleted microbiota in C57BL/6 recipient mice prior to FMT. The diversity of microbiota did not recover spontaneously to baseline levels during 8 weeks after Ab-treatment, but was restored already at 2 weeks in mice receiving FMT. Interestingly, pre-treatment with antibiotics prior to FMT did not increase the overall similarity of the recipient's microbiota to that of the donor's, as compared with mice receiving FMT without Ab-treatment. Pre-treatment with Ab improved the establishment of only a few donor-derived taxa, such as Bifidobacterium, in the recipients, thus having a minor effect on the engraftment of donor microbiota in FMT. In conclusion, pre-treatment with broad-spectrum antibiotics did not improve the overall engraftment of donor microbiota, but did improve the engraftment of specific taxa. These results may inform future therapeutic studies of FMT.Peer reviewe

Long-term effects on luminal and mucosal microbiota and commonly acquired taxa in faecal microbiota transplantation for recurrent Clostridium difficile infection

Background: Faecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection (rCDI). It restores the disrupted intestinal microbiota and subsequently suppresses C. difficile. The long-term stability of the intestinal microbiota and the recovery of mucosal microbiota, both of which have not been previously studied, are assessed herein. Further, the specific bacteria behind the treatment efficacy are also investigated. Methods: We performed a high-throughput microbiota profiling using a phylogenetic microarray analysis of 131 faecal and mucosal samples from 14 rCDI patients pre- and post-FMT during a 1-year follow-up and 23 samples from the three universal donors over the same period. Results: The FMT treatment was successful in all patients. FMT reverted the patients' bacterial community to become dominated by Clostridium clusters IV and XIVa, the major anaerobic bacterial groups of the healthy gut. In the mucosa, the amount of facultative anaerobes decreased, whereas Bacteroidetes increased. Post-FMT, the patients' microbiota profiles were more similar to their own donors than what is generally observed for unrelated subjects and this striking similarity was retained throughout the 1-year follow-up. Furthermore, the universal donor approach allowed us to identify bacteria commonly established in all CDI patients and revealed a commonly acquired core microbiota consisting of 24 bacterial taxa. Conclusions: FMT induces profound microbiota changes, therefore explaining the high clinical efficacy for rCDI. The identification of commonly acquired bacteria could lead to effective bacteriotherapeutic formulations. FMT can affect microbiota in the long-term and offers a means to modify it relatively permanently for the treatment of microbiota-associated diseases.Peer reviewe

Binding of fecal bacteria to Caco-2 cell line as a pre-selection step in isolating commensals with potential to inhibit Clostridium difficile adhesion

Clostridium difficile -infektio kehittyy tyypillisesti antibioottihoidon yhteydessä, kun suolistomikrobisto ei kykene ylläpitämään kolonisaatioresistenssiä mikrobiston koostumuksen häiriinnyttyä. Terve mikrobisto voidaan palauttaa ja C. difficile -bakteeri häätää ulosteensiirrolla, mikä on yksi harvoja toimivia hoitokeinoja vaikeissa uusiutuvissa infektioissa, joihin antibioottihoito ei tehoa. Ulosteensiirto on tutkitusti tehokas hoitokeino, mutta on vielä epäselvää mitkä ulosteen bakteerilajit ja mitkä niiden ominaisuudet ovat hoitotehon edellytys. Tämä tutkimus perustui hypoteesiin, jonka mukaan jotkut suoliston epiteelisoluihin kiinnittyvät normaalimikrobiston bakteerit voivat kilpailemalla estää C. difficile -bakteerin kiinnittymistä ja kolonisaatiota suolistoon. Noiden bakteereiden eristäminen ulosteesta mahdollistaisi tarkemmat mekanistiset tutkimukset ja C. difficile -infektion bakteeriterapiahoitoon sopivan tuotteen kehittämisen niistä. Ulostebakteerien kiinnittymistä Caco-2-solulinjaan käytettiin esi-selektiovaiheena epiteelisoluihin kiinnittyvien bakteerien eristämisessä luovutetusta ulosteesta. Mikrobiston koostumus ulosteessa ja siitä enterosyytteihin sitoutuneissa sekä viljellyissä bakteeripopulaatioissa määritettiin osittaisten 16S rDNA -juosteiden sekvensoimisella käyttäen MiSeq -menetelmää. Esi-selektio osoittautui toimivaksi lähestymistavaksi, sillä eristettyjen bakteeripopulaatioiden koostumus poikkesi alkuperäisestä sekä epiteeliin sitoutumisen että viljelyn jälkeen. Lisäksi eristetyistä populaatioista puhdistetut bakteerikannat kiinnittyivät hyvin enterosyytteihin. Myös ulostebakteerien kykyä kilpailla C. difficile -bakteerin kanssa kiinnittymisestä suoliston epiteelisoluihin in vitro tutkittiin. Caco-2-soluihin kiinnittyneistä populaatioista eristettyjä bakteereita kilpailutettiin C. difficile -bakteerin kanssa kilpailu- ja ekskluusiomenetelmiä käyttäen sekä monilajisina populaatioina että puhdistettuina kantoina. Osa bakteerikannoista ja -populaatioista vähensivät C. difficile -bakteerin sitoutumista epiteelisoluihin. Käytetyt menetelmät vaativat kuitenkin vielä kehittelyä ja tulosten varmistaminen enemmän toistoja. Saadut tulokset ovat kuitenkin lupaavia ja hyödyllinen perusta tulevalle työlle C. difficile -infektion bakteeriterapiahoidossa käytettävän tuotteen kehittämisessä.Clostridium difficile infection (CDI) is a microbiota-related disease. Typically, antibiotic-induced perturbation of gut microbiota precedes the infection, while a healthy gut microbiota provides protection, i.e. colonization resistance, against it. Furthermore, in the case of recurrent CDI that is not resolved by antibiotics, restoring the gut microbiota with a fecal microbiota transplantation (FMT) is among the few treatment options that really work. Although FMT is effective in the treatment of CDI, the factors behind treatment success remain unclear. Both, the key species and the functions that are necessary to restore the healthy microbiota and eradicate C. difficile, are a matter of speculation. This study was based on the hypothesis that the adherence of some commensal bacteria to the gut epithelial cells could play a role in eradicating C. difficile by competing for epithelial binding with it. Furthermore, the isolation of those bacteria from the donor feces would enable more detailed mechanistic studies and development of a bacterial product for the treatment of CDI in the future. As a pre-selection step, bacterial adhesion to Caco-2 cells was utilized to isolate and cultivate epithelium-adherent bacteria from the donor feces. Microbiota composition of fecal sample, and the adhered and cultured sub-populations thereof, was determined by partial 16S rDNA amplicon sequencing using MiSeq method. The pre-selection approach was successful, since the obtained populations were different, both after the adhesion and cultivation, as compared to the original fecal sample. In addition, most obtained pure isolates adhered well to enterocytes. The ability of fecal bacteria to compete with C. difficile for binding to gut epithelial cells in vitro was also studied. Isolated bacteria from Caco-2-adhered populations were applied in competition and exclusion assays with C. difficile as purified or multi-species cultures, and reduction in C. difficile binding was observed due to the certain bacteria or bacterial populations. These assays still need developing and the results must be confirmed with more repetitions. However, the results are promising and a useful ground for future work in developing bacteriotherapeutic formulations for the treatment of CDI