Influence of the bile acid/microbiota axis in ileal surgery: a systematic review.

AIM: The gastrointestinal bile acid (BA)/microbiota axis has emerged as a potential mediator of health and disease, particularly in relation to pathologies such as inflammatory bowel disease (IBD) and colorectal cancer. Whilst it presents an exciting new avenue for therapies, it has not yet been characterized in surgical resection of the ileum, where BA reabsorption occurs. The identification of BA/microbiota signatures may provide future therapies with perioperative personalized medicine. In this work we conduct a systematic review with the aim of investigating the microbiome and BA changes that are associated with resection of the ileum. METHOD: The databases included were MEDLINE, EMBASE, Web of Science and Cochrane libraries. The outcomes of interest were faecal microbiome and BA signatures after ileal resection. RESULTS: Of the initial 3106 articles, three studies met the inclusion/exclusion criteria for data extraction. A total of 257 patients (46% surgery, 54% nonsurgery controls) were included in the three studies. Two studies included patients with short bowel syndrome and the other included patients with IBD. Large-scale microbiota changes were reported. In general, alpha diversity had decreased amongst patients with ileal surgery. Phylum-level changes included decreased Bacteroidetes and increased Proteobacteria and Fusobacteria in patients with an intestinal resection. Surgery was associated with increased total faecal BAs, cholic acid and chenodeoxycholic acid. There were decreases in deoxycholic acid and glycine and taurine conjugated bile salts. Integrated BA and microbiota data identified correlations with several bacterial families and BA. CONCLUSION: The BA/microbiota axis is still a novel area with minimal observational data in surgery. Further mechanistic research is necessary to further explore this and identify its role in improving perioperative outcomes.

The Impact of Dietary Interventions on the Microbiota in Inflammatory Bowel Disease: A Systematic Review.

BACKGROUND AND AIMS: Diet plays an integral role in the modulation of the intestinal environment, with the potential to be modified for management of individuals with inflammatory bowel disease (IBD). It has been hypothesised that poor "Western-style" dietary patterns select for a microbiota that drives IBD inflammation and that through dietary intervention, a healthy microbiota may be restored. This study aimed to systematically review the literature and assess current available evidence regarding the influence of diet on the intestinal microbiota composition in IBD patients and how this may affect disease activity. METHODS: MEDLINE, EMBASE, Scopus, Web of Science and Cochrane Library were searched from January 2013 to June 2023, to identify studies investigating diet and microbiota in IBD. RESULTS: Thirteen primary studies met the inclusion criteria and were selected for narrative synthesis. Reported associations between diet and microbiota in IBD were conflicting due to the considerable degree of heterogeneity between studies. Nine intervention studies trialled specific diets and did not demonstrate significant shifts in the diversity and abundance of intestinal microbial communities or improvement in disease outcomes, whilst the remaining four cross-sectional studies did not find a specific microbial signature associated with habitual dietary patterns in IBD patients. CONCLUSIONS: Diet modulates the gut microbiota, and this may have implications for IBD; however, the body of evidence does not currently support clear dietary patterns or food constituents that are associated with a specific microbiota profile or disease marker in IBD patients. Further research is required with a focus on robust and consistent methodology to achieve improved identification of associations.

Beyond Eosinophilic Esophagitis: Eosinophils in Gastrointestinal Disease-New Insights, "New" Diseases.

Functional dyspepsia (FD) is a highly prevalent disorder. Upper endoscopy is normal, and according to the Rome IV criteria, there is no established pathology. Data accumulated over the last 15 years has challenged the notion FD is free of relevant pathology, and in particular, increased duodenal eosinophils have been observed. Intestinal eosinophils play important roles in microbial defence, immune regulation, tissue regeneration and remodelling, and maintaining tissue homeostasis and metabolism; degranulation of eosinophils releases toxic granule products (e.g., major basic protein, eosinophil-derived neurotoxin) which can damage nerves. Normal cut-offs for eosinophil infiltration into the duodenum histologically are less than five eosinophils per high power field (<25 per five high power fields). In clinical practice there is evidence that pathologically increased intestinal eosinophils may often be overlooked. In a meta-analysis duodenal eosinophils were significantly increased in FD although there was substantial heterogeneity; degranulation of duodenal eosinophils was also significantly higher in FD without significant heterogeneity. In addition, increased duodenal permeability, systemic immune activation, and an altered mucosa-associated duodenal microbiome have been identified that may help explain why symptoms arise, often occur after food with exposure to food antigens, and typically fluctuate. Several potentially reversible risk factors for FD have now been identified. We evaluate the current evidence linking duodenal microinflammation and immune activation with FD and disorders of gut-brain interactions that overlap with FD. We propose a two-hit disease model for eosinophilic functional dyspepsia (EoFD) with management implications.

A synbiotic mixture of selected oligosaccharides and bifidobacteria assists murine gut microbiota restoration following antibiotic challenge.

BACKGROUND: Typically, animal models studying gastrointestinal microbiotas compromised in early life have employed either germ-free animals or mice treated with a cocktail of antibiotics. Such studies intend to mimic scenarios of infants born by caesarean section and/or subjected to antibiotic treatment. However, the antibiotics used in these studies are rarely prescribed to infants. Therefore, an early life model was developed in which the murine gastrointestinal microbiota was severely disrupted by clindamycin treatment. RESULTS: In this mouse model, we investigated the extent supplementation with a synbiotic mixture of prebiotics, being scGOS/lcFOS with the human milk oligosaccharide 2'-Fucosyllactose (2'-FL), in combination with or without single strain or mix of "infant type" bifidobacteria, can rescue an antibiotic-compromised microbiota. Shotgun metagenomic sequencing showed that the microbiota was severely disrupted by the clindamycin challenge. No recovery was observed 3 weeks post-challenge in the scGOS/lcFOS/2'FL group, while the group that received the synbiotic treatment of scGOS/lcFOS/2'-FL with Bifidobacterium breve NRBB01 showed partial recovery. Strikingly in the scGOS/lcFOS/2'-FL group receiving the mixture of bifidobacteria resulted in a recovery of the microbiota disruption. Histological analyses showed that the clindamycin-treated animals at the end of the experiment still suffered from mild oedema and villi/colonic crypt irregularities which was ameliorated by the synbiotic intervention. CONCLUSION: Our study demonstrates that supplementation of synbiotic mixture of scGOS/lcFOS/2'-FL in combination with a specific mix of infant-type bifidobacterial strains is able to partially revive an antibiotic-perturbed gastrointestinal microbiota. Video Abstract.

Clinical and Pathologic Factors Associated With Colonic Spirochete (Brachyspira pilosicoli and Brachyspira aalborgi) Infection: A Comprehensive Systematic Review and Pooled Analysis.

OBJECTIVES: This study aims to determine what pathologic and clinical factors differentiate Brachyspira species that may be useful to clinicians and pathologists. METHODS: We identified 21 studies of Brachyspira infection with individual patient information (n = 113) and conducted a pooled analysis comparing each species. RESULTS: There were differences in the pathologic and clinical profiles of each Brachyspira species. Patients infected with Brachyspira pilosicoli infection were more likely to have diarrhea, fever, HIV, and immunocompromised conditions. Those patients infected with Brachyspira aalborgi were more likely to have lamina propria inflammation. CONCLUSIONS: Our novel data provide potential insights into the pathogenic mechanism(s) and the specific risk factor profile of Brachyspira species. This may be clinically useful when assessing and managing patients.

A pilot study: intraoperative 16S rRNA sequencing versus culture in predicting colorectal incisional surgical site infection.

BACKGROUND: Surgical Site Infection (SSI) of the abdominal incision is a dreaded complication following colorectal surgery. Identifying the intraoperative surgical site microbes may provide clarity in the pathogenesis of SSIs. Genomic sequencing has revolutionized the ability to identify microbes from clinical samples. Utilization of 16S rRNA amplicon sequencing to characterize the intraoperative surgical site may provide the critical information required to predict and prevent infection in colorectal surgery. METHODS: This is a pilot, prospective observational study of 50 patients undergoing elective colorectal resection. At completion of surgery, prior to skin closure, swabs were taken from the subcutaneous tissue of the abdominal incision to investigate the microbial profile. Dual swabs were taken to compare standard culture technique and 16S rRNA sequencing to establish if a microbial profile was associated with postoperative SSI. RESULTS: 8/50 patients developed an SSI, which was more likely in those undergoing open surgery (5/15 33.3% versus 3/35, 8.6%; P = 0.029). 16S rRNA amplicon sequencing was more sensitive in microbial detection compared to traditional culture. Both culture and 16S rRNA demonstrated contamination of the surgical site, predominantly with anaerobes. Culture was not statistically predictive of infection. 16S rRNA amplicon sequencing was not statistically predictive of infection, however, it demonstrated patients with an SSI had an increased biodiversity (not significant) and a greater relative abundance (not significant) of pathogens such as Bacteroidacaea and Enterobacteriaceae within the intraoperative site. CONCLUSIONS: 16S rRNA amplicon sequencing has demonstrated a potential difference in the intraoperative microbial profile of those that develop an infection. These findings require validation through powered experiments to determine the overall clinical significance.

Isolation and characterisation of novel Methanocorpusculum species indicates the genus is ancestrally host-associated.

BACKGROUND: With an increasing interest in the manipulation of methane produced from livestock cultivation, the microbiome of Australian marsupials provides a unique ecological and evolutionary comparison with 'low-methane' emitters. Previously, marsupial species were shown to be enriched for novel lineages of Methanocorpusculum, as well as Methanobrevibacter, Methanosphaera, and Methanomassiliicoccales. Despite sporadic reports of Methanocorpusculum from stool samples of various animal species, there remains little information on the impacts of these methanogens on their hosts. RESULTS: Here, we characterise novel host-associated species of Methanocorpusculum, to explore unique host-specific genetic factors and their associated metabolic potential. We performed comparative analyses on 176 Methanocorpusculum genomes comprising 130 metagenome-assembled genomes (MAGs) recovered from 20 public animal metagenome datasets and 35 other publicly available Methanocorpusculum MAGs and isolate genomes of host-associated and environmental origin. Nine MAGs were also produced from faecal metagenomes of the common wombat (Vombatus ursinus) and mahogany glider (Petaurus gracilis), along with the cultivation of one axenic isolate from each respective animal; M. vombati (sp. nov.) and M. petauri (sp. nov.). CONCLUSIONS: Through our analyses, we substantially expand the available genetic information for this genus by describing the phenotypic and genetic characteristics of 23 host-associated species of Methanocorpusculum. These lineages display differential enrichment of genes associated with methanogenesis, amino acid biosynthesis, transport system proteins, phosphonate metabolism, and carbohydrate-active enzymes. These results provide insights into the differential genetic and functional adaptations of these novel host-associated species of Methanocorpusculum and suggest that this genus is ancestrally host-associated.

A Systematic Review and Meta-Analysis of Intra-Operative Surgical Site Sampling: Culture versus Culture-Independent Techniques in Predicting Downstream Surgical Site Infection.

Background: Surgical site infection remains a significant cause of morbidity and mortality. Traditionally, the causation has been inferred from the organism(s) detected in the post-operative setting. However, the intra-operative surgical site and the bacteria it harbors have been scarcely studied. Compared with culture-dependent methods, the development of genomic technology provides a new sensitive tool that could aid in characterizing the bacteria within the surgical site. The purpose of this literature review is to establish if there is a predictive role of sampling the intra-operative surgical site. Methods: A systematic literature review was conducted identifying relevant literature reporting on studies that sampled the intra-operative surgical site of any specialty, using either traditional culture or a culture-independent genomic sequencing-based technique and correlation with infection was attempted. The review identified studies between 1959 and 2021 in MEDLINE, EMBASE and Cochrane. Results: The initial search identified 7,835 articles; 36 remained after screening. Thirty-one articles focused on culture-dependent techniques, five on culture-independent. Subgroup meta-analysis demonstrates that a positive intra-operative culture carries a risk of downstream infection with an odds ratio of 8.6, however limited by a high false-positive and inability to correlate the intra-operative culture with the post-operative infection. In contrast, culture-independent studies through genomic sequencing are not predictive but suggest that the surgical incision is a complex microbial community with a shift toward dysbiosis in certain patients. Conclusion: The intra-operative surgical site clearly harbors bacteria. Both techniques give rise to separate explanations underpinning the role of bacteria in surgical site infection. It is possible there is a more complex dynamic community within the incision that makes a patient susceptible to infection. Characterizing this microbial community in large scale studies, including patients with infections may enhance our ability to predict and prevent incisional surgical site infections in patients undergoing surgical procedures.

Does the microbiome play a role in the pathogenesis of colonic diverticular disease? A systematic review.

BACKGROUND AND AIMS: The role of the microbiota in diverticulosis and diverticular disease is underexplored. This systematic review aimed to assess all literature pertaining to the microbiota and metabolome associations in asymptomatic diverticulosis, symptomatic uncomplicated diverticular disease (SUDD), and diverticulitis pathophysiology. METHODS: Seven databases were searched for relevant studies published up to September 28, 2022. Data were screened in Covidence and extracted to Excel. Critical appraisal was undertaken using the Newcastle Ottawa Scale for case/control studies. RESULTS: Of the 413 papers screened by title and abstract, 48 full-text papers were reviewed in detail with 12 studies meeting the inclusion criteria. Overall, alpha and beta diversity were unchanged in diverticulosis; however, significant changes in alpha diversity were evident in diverticulitis. A similar Bacteroidetes to Firmicutes ratio compared with controls was reported across studies. The genus-level comparisons showed no relationship with diverticular disease. Butyrate-producing microbial species were decreased in abundance, suggesting a possible contribution to the pathogenesis of diverticular disease. Comamonas species was significantly increased in asymptomatic diverticulosis patients who later developed diverticulitis. Metabolome analysis reported significant differences in diverticulosis and SUDD, with upregulated uracil being the most consistent outcome in both. No significant differences were reported in the mycobiome. CONCLUSION: Overall, there is no convincing evidence of microbial dysbiosis in colonic diverticula to suggest that the microbiota contributes to the pathogenesis of asymptomatic diverticulosis, SUDD, or diverticular disease. Future research investigating microbiota involvement in colonic diverticula should consider an investigation of mucosa-associated microbial changes within the colonic diverticulum itself.

Eosinophils, Hypoxia-Inducible Factors, and Barrier Dysfunction in Functional Dyspepsia.

Functional dyspepsia (FD) is a highly prevalent disorder of gut-brain interaction (DGBI), previously known as a functional gastrointestinal disorder. Characterized by early satiety, postprandial fullness, and/or epigastric pain or burning, diagnosis depends on positive symptomatology and exclusion of obvious structural diseases. A subtle inflammatory phenotype has been identified in FD patients, involving an increase in duodenal mucosal eosinophils, and imbalances in the duodenal gut microbiota. A dysregulated epithelial barrier has also been well described in FD and is thought to be a contributing factor to the low-grade duodenal inflammation observed, however the mechanisms underpinning this are poorly understood. One possible explanation is that alterations in the microbiota and increased immune cells can result in the activation of cellular stress response pathways to perpetuate epithelial barrier dysregulation. One such cellular response pathway involves the stabilization of hypoxia-inducible factors (HIF). HIF, a transcriptional protein involved in the cellular recognition and adaptation to hypoxia, has been identified as a critical component of various pathologies, from cancer to inflammatory bowel disease (IBD). While the contribution of HIF to subtle inflammation, such as that seen in FD, is unknown, HIF has been shown to have roles in regulating the inflammatory response, particularly the recruitment of eosinophils, as well as maintaining epithelial barrier structure and function. As such, we aim to review our present understanding of the involvement of eosinophils, barrier dysfunction, and the changes to the gut microbiota including the potential pathways and mechanisms of HIF in FD. A combination of PubMed searches using the Mesh terms functional dyspepsia, functional gastrointestinal disorders, disorders of gut-brain interaction, duodenal eosinophilia, barrier dysfunction, gut microbiota, gut dysbiosis, low-grade duodenal inflammation, hypoxia-inducible factors (or HIF), and/or intestinal inflammation were undertaken in the writing of this narrative review to ensure relevant literature was included. Given the findings from various sources of literature, we propose a novel hypothesis involving a potential role for HIF in the pathophysiological mechanisms underlying FD.

The microbiota in eosinophilic esophagitis: A systematic review.

Eosinophilic esophagitis (EoE) is an atopic disease of the esophagus that has shown a significant increase in incidence and prevalence in the last 20 years. The etiology of EoE is unclear, and few studies explore the esophageal microbiota in EoE. The local microbiome has been implicated in the pathogenesis of several allergic and inflammatory diseases, such as asthma and eczema. In this study, we performed a systematic review to evaluate differences in the microbiota profile of patients with EoE compared with controls. MEDLINE, Embase, Cochrane Library, Scopus, and CINAHL (Cumulative Index to Nursing and Allied Health Literature) databases were searched to identify studies investigating the microbiota composition in EoE. Three reviewers screened the articles for eligibility and quality. Seven articles underwent full-text review, and a narrative synthesis was undertaken. The microbiota of the mouth and esophagus are correlated. Patients with active EoE present increased esophageal microbial load and increased abundance in particular species, such as Haemophilus and Aggregatibacter. On the other hand, EoE patients present a decrease in Firmicutes. High microbial load and abundance of Haemophilus are observed in EoE patients, but little evidence exists to demonstrate their influence on inflammation and disease. Understanding microbial signatures in EoE might contribute to the development of novel therapeutic strategies.

Secreted NF-κB suppressive microbial metabolites modulate gut inflammation.

Emerging evidence suggests that microbiome-host crosstalk regulates intestinal immune activity and predisposition to inflammatory bowel disease (IBD). NF-κB is a master regulator of immune function and a validated target for the treatment of IBD. Here, we identify five Clostridium strains that suppress immune-mediated NF-κB activation in epithelial cell lines, PBMCs, and gut epithelial organoids from healthy human subjects and patients with IBD. Cell-free culture supernatant from Clostridium bolteae AHG0001 strain, but not the reference C. bolteae BAA-613 strain, suppresses inflammatory responses and endoplasmic reticulum stress in gut epithelial organoids derived from Winnie mice. The in vivo responses to Clostridium bolteae AHG0001 and BAA-613 mirror the in vitro activity. Thus, using our in vitro screening of bacteria capable of suppressing NF-κB in the context of IBD and using an ex vivo organoid-based approach, we identify a strain capable of alleviating colitis in a relevant pre-clinical animal model of IBD.

Role of the duodenal microbiota in functional dyspepsia.

BACKGROUND: Functional dyspepsia (FD) is a common and debilitating gastrointestinal disorder attributed to altered gut-brain interactions. While the etiology of FD remains unknown, emerging research suggests the mechanisms are likely multifactorial and heterogenous among patient subgroups. Small bowel motor disturbances, visceral hypersensitivity, chronic microinflammation, and increased intestinal tract permeability have all been linked to the pathogenesis of FD. Recently, alterations to the gut microbiome have also been implicated to play an important role in the disease. Changes to the duodenal microbiota may either trigger or be a consequence of immune and neuronal disturbances observed in the disease, but the mechanisms of influence of small intestinal flora on gastrointestinal function and symptomatology are unknown. PURPOSE: This review summarizes and synthesizes the literature on the link between the microbiota, low-grade inflammatory changes in the duodenum and FD. This review is not intended to provide a complete overview of FD or the small intestinal microbiota, but instead outline some of the key conceptual advances in understanding the interactions between altered gastrointestinal bacterial communities; dietary factors; host immune activation; and stimulation of the gut-brain axes in patients with FD versus controls. Current and emerging treatment approaches such as dietary interventions and antibiotic or probiotic use that have demonstrated symptom benefits for patients are reviewed, and their role in modulating the host-microbiota is discussed. Finally, suggested opportunities for diagnostic and therapeutic improvements for patients with this condition are presented.

Human intestinal spirochetosis, irritable bowel syndrome, and colonic polyps: A systematic review and meta-analysis.

Human colonic spirochetosis (CS) is usually due toBrachyspira pilosicolior Brachyspira aalborgiinfection. While traditionally considered to be commensal bacteria, there are scattered case reports and case series of gastrointestinal (GI) symptoms in CS and reports of colonic polyps with adherent spirochetes. We performed a systematic review and meta-analysis investigating the association between CS and GI symptoms and conditions including the irritable bowel syndrome (IBS) and colonic polyps. Following PRISMA 2020 guidelines, a systematic search of Medline, CINAHL, EMBASE, and Web of Science was performed using specific keywords for CS and GI disease. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using a random-effects model. Of 75 studies identified in the search, 8 case-control studies met the inclusion criteria for meta-analysis and 67 case series studies met the inclusion criteria for pooled prevalence analysis. CS was significantly associated with diarrhea (n = 141/127, cases/controls, OR: 4.19, 95% CI: 1.72-10.21, P = 0.002) and abdominal pain (n = 64/65, OR: 3.66, 95% CI: 1.43-9.35, P = 0.007). CS cases were significantly more likely to have Rome III-diagnosed IBS (n = 79/48, OR: 3.84, 95% CI: 1.44-10.20, P = 0.007), but not colonic polyps (n = 127/843, OR: 8.78, 95% CI: 0.75-103.36, P = 0.084). In conclusion, we found evidence of associations between CS and both diarrhea and IBS, but not colonic polyps. CS is likely underestimated due to suboptimal diagnostic methods and may be an overlooked risk factor for a subset of IBS patients with diarrhea.

Circadian Rhythms and Melatonin Metabolism in Patients With Disorders of Gut-Brain Interactions.

Circadian rhythms are cyclic patterns of physiological, behavioural and molecular events that occur over a 24-h period. They are controlled by the suprachiasmatic nucleus (SCN), the brain's master pacemaker which governs peripheral clocks and melatonin release. While circadian systems are endogenous, there are external factors that synchronise the SCN to the ambient environment including light/dark cycles, fasting/fed state, temperature and physical activity. Circadian rhythms also provide internal temporal organisation which ensures that any internal changes that take place are centrally coordinated. Melatonin synchronises peripheral clocks to the external time and circadian rhythms are regulated by gene expression to control physiological function. Synchronisation of the circadian system with the external environment is vital for the health and survival of an organism and as circadian rhythms play a pivotal role in regulating GI physiology, disruption may lead to gastrointestinal (GI) dysfunction. Disorders of gut-brain interactions (DGBIs), also known as functional gastrointestinal disorders (FGIDs), are a group of diseases where patients experience reoccurring gastrointestinal symptoms which cannot be explained by obvious structural abnormalities and include functional dyspepsia (FD) and irritable bowel syndrome (IBS). Food timing impacts on the production of melatonin and given the correlation between food intake and symptom onset reported by patients with DGBIs, chronodisruption may be a feature of these conditions. Recent advances in immunology implicate circadian rhythms in the regulation of immune responses, and DGBI patients report fatigue and disordered sleep, suggesting circadian disruption. Further, melatonin treatment has been demonstrated to improve symptom burden in IBS patients, however, the mechanisms underlying this efficacy are unclear. Given the influence of circadian rhythms on gastrointestinal physiology and the immune system, modulation of these rhythms may be a potential therapeutic option for reducing symptom burden in these patients.

Physiological mechanisms of unexplained (functional) gastrointestinal disorders.

Functional gastrointestinal disorders (FGIDs) encompass a range of complex conditions with similar clinical characteristics and no overt pathology. Recent recognition of sub-clinical pathologies in FGIDs, in conjunction with physiological and biochemical abnormalities including increased intestinal permeability, microbial profile alterations, differences in metabolites and extra-intestinal manifestations of disease, call into question the designation of these conditions as 'functional'. This is despite significant heterogeneity in both symptom profile and specifics of reported physiological abnormalities hampering efforts to determine defined mechanisms that drive onset and chronicity of symptoms. Instead, the literature demonstrates these conditions are disorders of homeostatic imbalance, with disruptions in both host and microbial function and metabolism. This imbalance is also associated with extraintestinal abnormalities including psychological comorbidities and fatigue that may be a consequence of gastrointestinal disruption. Given the exploitation of such abnormalities will be crucial for improved therapeutic selection, an enhanced understanding of the relationship between alterations in function of the gastrointestinal tract and the response of the immune system is of interest in identifying mechanisms that drive FGID onset and chronicity. Considerations for future research should include the role of sex hormones in regulating physiological functions and treatment responses in patients, as well as the importance of high-level phenotyping of clinical, immune, microbial and physiological parameters in study cohorts. There is opportunity to examine the functional contribution of the microbiota and associated metabolites as a source of mechanistic insight and targets for therapeutic modulation.

Draft Genome Sequence of Streptococcus salivarius AGIRA0003, Isolated from Functional Gastrointestinal Disorder Duodenal Tissue.

Patients suffering functional dyspepsia symptoms have been shown to possess a greater relative abundance of Streptococcus compared to asymptomatic controls. Here, we describe the isolation and genomic features of a new Streptococcus isolate, from the duodenal tissue of a subject reporting dyspeptic symptoms, taxonomically assigned to Streptococcus salivarius and designated strain AGIRA0003.

Broad Purpose Vector for Site-Directed Insertional Mutagenesis in Bifidobacterium breve.

Members of the genus Bifidobacterium are notoriously recalcitrant to genetic manipulation due to their extensive and variable repertoire of Restriction-Modification (R-M) systems. Non-replicating plasmids are currently employed to achieve insertional mutagenesis in Bifidobacterium. One of the limitations of using such insertion vectors is the presence within their sequence of various restriction sites, making them sensitive to the activity of endogenous restriction endonucleases encoded by the target strain. For this reason, vectors have been developed with the aim of methylating and protecting the vector using a methylase-positive Escherichia coli strain, in some cases containing a cloned bifidobacterial methylase. Here, we present a mutagenesis approach based on a modified and synthetically produced version of the suicide vector pORI28 (named pFREM28), where all known restriction sites targeted by Bifidobacterium breve R-M systems were removed by base substitution (thus preserving the codon usage). After validating the integrity of the erythromycin marker, the vector was successfully employed to target an α-galactosidase gene responsible for raffinose metabolism, an alcohol dehydrogenase gene responsible for mannitol utilization and a gene encoding a priming glycosyltransferase responsible for exopolysaccharides (EPS) production in B. breve. The advantage of using this modified approach is the reduction of the amount of time, effort and resources required to generate site-directed mutants in B. breve and a similar approach may be employed to target other (bifido)bacterial species.