Molecular identification and characterisation of bifidobacteria and lactobacilli in the human gastrointestinal tract

Bifidobacteria and lactobacilli are considered to be members of the beneficial microbiota in the human gastrointestinal (GI) tract. The present study describes the development and validation of new molecular methods for the detection and analysis of bifidobacteria and lactobacilli and the application of new techniques to study Bifidobacterium and Lactobacillus populations in the human intestine. A method based on genus-specific PCR of 16S rDNA and denaturing gradient gel electrophoresis (DGGE) was developed and validated for profiling Bifidobacterium populations in human faeces. The PCR-DGGE method is a qualitative tool for assessing species/strain composition of complex communities by a single PCR reaction and subsequent resolution of the amplification products by DGGE in a sequence-dependent manner. The approach greatly facilitates the monitoring of faecal samples from large numbers of subjects to reveal bifidobacterial diversity and shifts occurring in it. The identification of DGGE fragments can be done by subsequent cloning and sequencing of the PCR products. Genotypic methods were developed and evaluated for the identification and characterisation of Lactobacillus casei -group lactobacilli ( L. casei, L. paracasei, L. rhamnosus , and L. zeae ). L. rhamnosus species-specific PCR was developed and validated. The discriminatory power of the three fingerprinting techniques, pulsed field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) and ribotyping, was compared. All three techniques were highly effective in differentiating strains below the species level and they can be placed in the following order with respect to their discriminatory power: PFGE > ribotyping > RAPD. Newly developed molecular methods were used to trace ingested probiotic strains L. rhamnosus GG (LGG) and B. lactis Bb12 in the GI-tract. The identity of LGG colonies was verified using a species-specific PCR and Bb12 was detected using the PCR-DGGE method. Both probiotic strains colonised the gut transiently and they were no longer detected in the faeces one week after the end of the administration in most subjects. The synbiotic approach with galactooligosaccharide (GOS) did not prolong the persistence of Bb12. Furthermore, LGG was found to attach in vivo to colonic mucosae and, although the attchment was temporary, to remain for more than a week after discontinuation of LGG administration. PCR-DGGE method was used to monitor qualitative changes in adult faecal Bifidobacterium populations in response to B. lactis Bb12 and/or GOS administration. In most subjects two weeks administration of Bb12 and/or GOS did not affect the qualitative composition of indigenous bifidobacterial populations, while Bb12 transiently colonised the gut. Qualitative molecular analysis was used to study the bacterial, bifidobacterial and lactobacilli populations in faeces of breast-fed and formula-fed infants before and after weaning. Genus and group-specific PCRs combined with DGGE and subsequent sequencing of the amplified 16S rDNA fragments revealed no difference in the prevalence or species distribution of Bifidobacterium and Lactobacillus between the two groups of infants. In general, DGGE patterns of 16S rDNA showed equal complexity of bacterial communities in breast-fed and formula-fed infants. Equally intensive changes occurred in the faecal microbiota in infants of both groups due to weaning.</p

Isolation of Anti-Inflammatory and Epithelium Reinforcing Bacteroides and Parabacteroides Spp. from A Healthy Fecal Donor

Altered intestinal microbiota is associated with systemic and intestinal diseases, such as inflammatory bowel disease (IBD). Dysbiotic microbiota with enhanced proinflammatory capacity is characterized by depletion of anaerobic commensals, increased proportion of facultatively anaerobic bacteria, as well as reduced diversity and stability. In this study, we developed a high-throughput in vitro screening assay to isolate intestinal commensal bacteria with anti-inflammatory capacity from a healthy fecal microbiota transplantation donor. Freshly isolated gut bacteria were screened for their capacity to attenuate Escherichia coli lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) release from HT-29 cells. The screen yielded a number of Bacteroides and Parabacteroides isolates, which were identified as P. distasonis, B. caccae, B. intestinalis, B. uniformis, B. fragilis, B. vulgatus and B. ovatus using whole genome sequencing. We observed that a cell-cell contact with the epithelium was not necessary to alleviate in vitro inflammation as spent culture media from the isolates were also effective and the anti-inflammatory action did not correlate with the enterocyte adherence capacity of the isolates. The anti-inflammatory isolates also exerted enterocyte monolayer reinforcing action and lacked essential genes to synthetize hexa-acylated, proinflammatory lipid A, part of LPS. Yet, the anti-inflammatory effector molecules remain to be identified. The Bacteroides strains isolated and characterized in this study have potential to be used as so-called next-generation probiotics

Isolation of Anti-Inflammatory and Epithelium Reinforcing Bacteroides and Parabacteroides Spp. from A Healthy Fecal Donor

Altered intestinal microbiota is associated with systemic and intestinal diseases, such as inflammatory bowel disease (IBD). Dysbiotic microbiota with enhanced proinflammatory capacity is characterized by depletion of anaerobic commensals, increased proportion of facultatively anaerobic bacteria, as well as reduced diversity and stability. In this study, we developed a high-throughput in vitro screening assay to isolate intestinal commensal bacteria with anti-inflammatory capacity from a healthy fecal microbiota transplantation donor. Freshly isolated gut bacteria were screened for their capacity to attenuate Escherichia coli lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) release from HT-29 cells. The screen yielded a number of Bacteroides and Parabacteroides isolates, which were identified as P. distasonis, B. caccae, B. intestinalis, B. uniformis, B. fragilis, B. vulgatus and B. ovatus using whole genome sequencing. We observed that a cell-cell contact with the epithelium was not necessary to alleviate in vitro inflammation as spent culture media from the isolates were also effective and the anti-inflammatory action did not correlate with the enterocyte adherence capacity of the isolates. The anti-inflammatory isolates also exerted enterocyte monolayer reinforcing action and lacked essential genes to synthetize hexa-acylated, proinflammatory lipid A, part of LPS. Yet, the anti-inflammatory effector molecules remain to be identified. The Bacteroides strains isolated and characterized in this study have potential to be used as so-called next-generation probiotics

BopA does not have a major role in the adhesion of bifidobacterium bifidum to intestinal epithelial cells, extracellular matrix proteins, and mucus

he ability of bifidobacteria to adhere to the intestine of the human host is considered to be important for efficient colonization and achieving probiotic effects. Bifidobacterium bifidum strains DSM20456 and MIMBb75 adhere well to the human intestinal cell lines Caco-2 and HT-29. The surface lipoprotein BopA was previously described to be involved in mediating adherence of B. bifidum to epithelial cells, but thioacylated, purified BopA inhibited the adhesion of B. bifidum to epithelial cells in competitive adhesion assays only at very high concentrations, indicating an unspecific effect. In this study, the role of BopA in the adhesion of B. bifidum was readdressed. The gene encoding BopA was cloned and expressed without its lipobox and hydrophobic signal peptide in Escherichia coli, and an antiserum against the recombinant BopA was produced. The antiserum was used to demonstrate the abundant localization of BopA on the cell surface of B. bifidum. However, blocking of B. bifidum BopA with specific antiserum did not reduce adhesion of bacteria to epithelial cell lines, arguing that BopA is not an adhesin. Also, adhesion of B. bifidum to human colonic mucin and fibronectin was found to be BopA independent. The recombinant BopA bound only moderately to human epithelial cells and colonic mucus, and it failed to bind to fibronectin. Thus, our results contrast the earlier findings on the major role of BopA in adhesion, indicating that the strong adhesion of B. bifidum to epithelial cell lines is BopA independent

Maternal gut and breast milk microbiota affect infant gut antibiotic resistome and mobile genetic elements

The infant gut microbiota has a high abundance of antibiotic resistance genes (ARGs) compared to adults, even in the absence of antibiotic exposure. Here we study potential sources of infant gut ARGs by performing metagenomic sequencing of breast milk, as well as infant and maternal gut microbiomes. We find that fecal ARG and mobile genetic element (MGE) profiles of infants are more similar to those of their own mothers than to those of unrelated mothers. MGEs in mothers' breast milk are also shared with their own infants. Termination of breastfeeding and intrapartum antibiotic prophylaxis of mothers, which have the potential to affect microbial community composition, are associated with higher abundances of specific ARGs, the composition of which is largely shaped by bacterial phylogeny in the infant gut. Our results suggest that infants inherit the legacy of past antibiotic consumption of their mothers via transmission of genes, but microbiota composition still strongly impacts the overall resistance load

A novel Bifidobacterium infantis-mediated TK/GCV suicide gene therapy system exhibits antitumor activity in a rat model of bladder cancer

Bladder cancer is the ninth most common malignancy in the world. Successful clinical management remains a challenge. In order To search for novel targeted and efficacious treatment, we sought to investigate anti-tumor activity of BI-TK suicide gene therapy system in a rat model of bladder tumors. We first constructed and tested an anaerobic Bifidobacterium infantis-mediated thymidine kinase (BI-TK) suicide gene therapy system. To test the in vivo efficacy of this system, we established a rat model of bladder tumors, which was induced by N-methyl-nitrosourea perfusion. Bifidobacterium infantis containing the HSV-TK (i.e., BI-TK) were constructed by transformation of recombinant plasmid pGEX - TK. The engineered BI-TK was injected into tumor-bearing rats via tail vein, followed by intraperitoneal injection of ganciclovir (GCV). Using the rat model of bladder tumors, we found that bladder tumor burdens were significantly lower in the rats treated with BI-TK/GCV group than that treated with normal saline control group (p <0.05). While various degrees of apoptosis of the tumor cells were detected in all groups using in situ TUNEL assay, apoptosis was mostly notable in the BI-TK/GCV treatment group. Immunohistochemical staining further demonstrated that the BI-TK/GCV treatment group had the highest level of caspase3 protein expression than that of the empty plasmid group and normal saline group (p < 0.05). Thus, our results demonstrate that the Bifidobacterium infantis-mediated TK/GCV suicide gene therapy system can effectively inhibit rat bladder tumor growth, possibly through increasing caspase 3 expression and inducing apoptosis

SARS-CoV-2 vaccines and donor recruitment for FMT

Non peer reviewe

International consensus conference on stool banking for faecal microbiota transplantation in clinical practice

Although faecal microbiota transplantation (FMT) has a well-established role in the treatment of recurrent Clostridioides difficile infection (CDI), its widespread dissemination is limited by several obstacles, including lack of dedicated centres, difficulties with donor recruitment and complexities related to regulation and safety monitoring. Given the considerable burden of CDI on global healthcare systems, FMT should be widely available to most centres. Stool banks may guarantee reliable, timely and equitable access to FMT for patients and a traceable workflow that ensures safety and quality of procedures. In this consensus project, FMT experts from Europe, North America and Australia gathered and released statements on the following issues related to the stool banking: general principles, objectives and organisation of the stool bank; selection and screening of donors; collection, preparation and storage of faeces; services and clients; registries, monitoring of outcomes and ethical issues; and the evolving role of FMT in clinical practice, Consensus on each statement was achieved through a Delphi process and then in a plenary face-to-face meeting. For each key issue, the best available evidence was assessed, with the aim of providing guidance for the development of stool banks in order to promote accessibility to FMT in clinical practice.Peer reviewe

Reorganisation of faecal microbiota transplant services during the COVID-19 pandemic

The COVID-19 pandemic has led to an exponential increase in SARS-CoV- 2 infections and associated deaths, and represents a significant challenge to healthcare professionals and facilities. Individual countries have taken several prevention and containment actions to control the spread of infection, including measures to guarantee safety of both healthcare professionals and patients who are at increased risk of infection from COVID-19. Faecal microbiota transplantation (FMT) has a well-established role in the treatment of Clostridioides difficile infection. In the time of the pandemic, FMT centres and stool banks are required to adopt a workflow that continues to ensure reliable patient access to FMT while maintaining safety and quality of procedures. In this position paper, based on the best available evidence, worldwide FMT experts provide guidance on issues relating to the impact of COVID-19 on FMT, including patient selection, donor recruitment and selection, stool manufacturing, FMT procedures, patient follow-up and research activities

Reorganisation of faecal microbiota transplant services during the COVID-19 pandemic

The COVID-19 pandemic has led to an exponential increase in SARS-CoV-2 infections and associated deaths, and represents a significant challenge to healthcare professionals and facilities. Individual countries have taken several prevention and containment actions to control the spread of infection, including measures to guarantee safety of both healthcare professionals and patients who are at increased risk of infection from COVID-19. Faecal microbiota transplantation (FMT) has a well-established role in the treatment of Clostridioides difficile infection. In the time of the pandemic, FMT centres and stool banks are required to adopt a workflow that continues to ensure reliable patient access to FMT while maintaining safety and quality of procedures. In this position paper, based on the best available evidence, worldwide FMT experts provide guidance on issues relating to the impact of COVID-19 on FMT, including patient selection, donor recruitment and selection, stool manufacturing, FMT procedures, patient follow-up and research activities.Peer reviewe