Increasing adherence to the Mediterranean diet and lifestyle is associated with reduced fecal calprotectin and intra-individual changes in microbial composition of healthy subjects.

The Mediterranean diet (MED) is associated with the modification of gut microbial composition. In this pilot study, we investigate the feasibility of a microbiota-targeted MED-based lifestyle intervention in healthy subjects. MED intervention integrating dietary counseling, a supporting mobile application, and daily physical activity measurement using step trackers was prospectively applied for 4 weeks. Blood and fecal samples were collected at baseline, after the 4-week intervention, and at 6 and 12 months. Blood counts, inflammatory markers, microbial and eukaryotic composition were analyzed. Dietary adherence was assessed using daily questionnaires. All 20 healthy participants (females 65%, median age 37), completed the 4-week intervention. Adherence to MED increased from 15.6 ± 4.1 (baseline) to 23.2 ± 3.6 points (4 weeks), p < .01, reflected by increased dietary fiber and decreased saturated fat intake (both p < .05). MED intervention modestly reduced fecal calprotectin, white blood cell, neutrophil, and lymphocyte counts, within the normal ranges (P < .05). Levels of butyrate producers including Faecalibacterium and Lachnospira were positively correlated with adherence to MED and the number of daily steps. Bacterial composition was associated with plant-based food intake, while fungal composition with animal-based food as well as olive oil and sweets. Increasing adherence to MED correlated with increased absolute abundances of multiple beneficial gut symbionts. Therefore, increasing adherence to MED is associated with reduction of fecal calprotectin and beneficial microbial alterations in healthy subjects. Microbiota targeted lifestyle interventions may be used to modify the intestinal ecosystem with potential implications for microbiome-mediated diseases.

Adherence to the Mediterranean diet is associated with decreased fecal calprotectin in patients with ulcerative colitis after pouch surgery.

BACKGROUND: Mediterranean diet (MED) is associated with health benefits, yet scarce data exist regarding the role of MED in inflammatory bowel diseases (IBD). Herein, we aimed to evaluate the association between MED and inflammatory markers in patients with IBD after pouch surgery. METHODS: Consecutive patients after pouch surgery due to ulcerative colitis (UC) were recruited at a comprehensive pouch clinic. Adherence to MED was calculated according to MED score, ranging from 0 (low adherence) to 9 (high adherence), based on food-frequency questionnaires. Pouch behavior was defined as normal pouch (NP) or pouchitis based on Pouchitis Disease Activity Index (PDAI) and disease activity was defined as active or inactive. C-reactive protein (CRP) and fecal calprotectin were assessed. RESULTS: Overall 153 patients were enrolled (male gender 47%; mean age 46 ± 14 years; mean pouch age 9.5 ± 7 years). MED scores were higher in patients with normal vs. elevated CRP and calprotectin levels (4.6 ± 1.8 vs. 4.4 ± 1.6, p = 0.28; 4.8 ± 1.8 vs. 4.07 ± 1.7, p < 0.05, respectively). In a multivariate regression, MED score was associated with decreased calprotectin levels (OR = 0.74 [0.56-0.99]). Adherence to MED was associated with dietary fiber and antioxidants intake. Finally, in a subgroup of patients with NP followed up for 8 years, higher adherence to MED trended to be inversely associated with the onset of pouchitis (log rank = 0.17). CONCLUSIONS: In patients with UC after pouch surgery, adherence to MED is associated with decreased calprotectin levels. Thus, MED may have a role in modifying intestinal inflammation in IBD.

An electrode-assisted anaerobic digestion process for the production of high-quality biogas.

Biogas is a sustainable, renewable energy source generated from organic waste degradation during anaerobic digestion (AD). AD is applied for treating different types of wastewater, mostly containing high organic load. However, AD practice is still limited due to the low quality of the produced biogas. Upgrading biogas to natural gas quality (>90% CH4) is essential for broad applications. Here, an innovative bio-electrochemically assisted AD process was developed, combining wastewater treatment and biogas upgrading. This process was based on a microbial electrolysis cell (MEC) that produced hydrogen from wastewater at a relatively high efficiency, followed by high-rate anaerobic systems for completing biodegradation of organic matter and an in situ bio-methanation process. Results showed that CH4 production yield was substantially improved upon coupling of the MEC with the AD system. Interestingly, CH4 production yield increase was most notable once circulation between AD and MEC was applied, while current density was not markedly affected by the circulation rates. The microbial community analysis confirmed that the MEC enhanced hydrogen production, leading to the enrichment of hydrogenotrophic methanogens. Thus, directing soluble hydrogen from the MEC to AD is plausible, and has great potential for biogas upgrading, avoiding the need for direct hydrogen harvesting.

Fruit Consumption is Associated with Alterations in Microbial Composition and Lower Rates of Pouchitis.

BACKGROUND: Patients with ulcerative colitis [UC] who undergo proctocolectomy with an ileal pouch-anal anastomosis commonly develop pouch inflammation [pouchitis]. Pouchitis develops in a previously normal small intestine and may involve environmental factors. We explored whether diet and microbiota alterations contributed to the pathogenesis of pouchitis. METHODS: Patients were recruited and prospectively followed at a comprehensive pouch clinic. Pouch behaviour was clinically defined as a normal pouch [NP] or pouchitis. Patients completed Food Frequency Questionnaires [FFQs]. Faecal samples were analysed for microbial composition [16S rRNA gene pyrosequencing]. RESULTS: Nutritional evaluation was performed in 172 patients [59% females], and of these, faecal microbial analysis was performed in 75 patients (microbiota cohort: NP [n = 22], pouchitis [n = 53]). Of the entire cohort, a subgroup of 39 [22.6%] patients had NP at recruitment [NP cohort]. Of these, 5 [12.8%] developed pouchitis within a year. Patients at the lowest tertile of fruit consumption [<1.45 servings/day] had higher rates of pouchitis compared with those with higher consumption [30.8% vs 3.8%, log rank, p = 0.03]. Fruit consumption was correlated with microbial diversity [r = 0.35, p = 0.002] and with the abundance of several microbial genera, including Faecalibacterium [r = 0.29, p = 0.01], Lachnospira [r = 0.38, p = 0.001], and a previously uncharacterized genus from the Ruminococcaceae family [r = 0.25, p = 0.05]. Reduction in fruit consumption over time was associated with disease recurrence and with reduced microbial diversity [Δ = -0.8 ± 0.3, p = 0.008]. CONCLUSIONS: Fruit consumption is associated with modification of microbial composition, and lower consumption was correlated with the development of pouchitis. Thus, fruit consumption may protect against intestinal inflammation via alteration of microbial composition.

Microbiome associated with denture malodour.

In the past, our inability to cultivate most of the oral microorganisms has limited our view of this complex ecosystem. In the present study, we utilized next generation deep sequencing techniques to revisit the microbiome associated with denture malodour, a growing field with the rise in life expectancy. The study population comprised 26 full dentures patients (mean age 71 ± 6.4, 10 males, 16 females) who visited the Tel Aviv University dental geriatric clinic. Denture malodour was rated organoleptically by a single odour judge, and dentures scoring 2 and above were considered malodour positive. DNA was extracted from the swab samples and analysed using next generation deep sequencing 16 s rDNA technology. Taxa identified could be classified into nine phyla, 29 genera and 117 species. Malodour positive samples showed a higher abundance of the phyla Firmicutes and Fusobacteria and the genera Leptotrichia, Atopobium, Megasphaera, Oribacterium and Campylobacter. Microbiome analysis demonstrated higher bacterial diversity within the malodourous samples and a significant difference in the microbial profile within the two groups. Taken together these results suggest a difference between the microbial populations of malodourous and non-malodourous dentures both in composition and diversity.

The correlation between Clostridium-difficile infection and human gut concentrations of Bacteroidetes phylum and clostridial species.

We aimed to assess differences in bacterial intensities of Bacteroidetes phylum and different clostridial species in the human intestines with respect to C. difficile infection. Patients with a stool assay for C. difficile toxin were identified via the microbiology laboratory in our institute. Bacterial populations were quantified from stool samples of four groups of patients: Group I-patients with C. difficile associated diarrhea (CDAD); Group II-asymptomatic C. difficile carriers; Group III-patients with non-C. difficile diarrhea; Group IV-patients with no diarrhea and negative stool samples for the C. difficile toxin (control group). Stool was examined for three genes-C. difficile toxin A gene, 16S rRNA gene from Clostridium thermocellum representing other clostridial species, and 16S rRNA gene from Bacteroides fragilis representing the Bacteroidetes phylum. Fifty-nine patients underwent analysis of the stool (CDAD group 14, carriers group 14, non-C. difficile diarrhea group 16, control group 15). C. difficile concentration was highest in the CDAD group, followed by the carriers group. Higher concentrations of both clostridial species and Bacteriodetes were observed in the control and non-C. difficile diarrhea groups compared to the CDAD and carriers groups. We demonstrated an inverse association between infection with C. difficile and the abundance of Bacteroidetes phylum and other clostridial species in human intestines. Studies with larger samples and broader diagnostic procedures are needed in order to better explore and understand this association.

In and out: an analysis of epibiotic vs periplasmic bacterial predators.

Bdellovibrio and like organisms (BALO) are obligate predators of Gram-negative bacteria, belonging to the α- and δ-proteobacteria. BALO prey using either a periplasmic or an epibiotic predatory strategy, but the genetic background underlying these phenotypes is not known. Here we compare the epibiotic Bdellovibrio exovorus and Micavibrio aeruginosavorus to the periplasmic B. bacteriovorus and Bacteriovorax marinus. Electron microscopy showed that M. aeruginosavorus, but not B. exovorus, can attach to prey cells in a non-polar manner through its longitudinal side. Both these predators were resistant to a surprisingly high number of antibiotic compounds, possibly via 26 and 19 antibiotic-resistance genes, respectively, most of them encoding efflux pumps. Comparative genomic analysis of all the BALOs revealed that epibiotic predators have a much smaller genome (ca. 2.5 Mbp) than the periplasmic predators (ca. 3.5 Mbp). Additionally, periplasmic predators have, on average, 888 more proteins, at least 60% more peptidases, and one more rRNA operon. Fifteen and 219 protein families were specific to the epibiotic and the periplasmic predators, respectively, the latter clearly forming the core of the periplasmic 'predatome', which is upregulated during the growth phase. Metabolic deficiencies of epibiotic genomes include the synthesis of inosine, riboflavin, vitamin B6 and the siderophore aerobactin. The phylogeny of the epibiotic predators suggests that they evolved by convergent evolution, with M. aeruginosavorus originating from a non-predatory ancestor while B. exovorus evolved from periplasmic predators by gene loss.

Bacterial flagella and Type III secretion: case studies in the evolution of complexity.

Bacterial flagella at first sight appear uniquely sophisticated in structure, so much so that they have even been considered 'irreducibly complex' by the intelligent design movement. However, a more detailed analysis reveals that these remarkable pieces of molecular machinery are the product of processes that are fully compatible with Darwinian evolution. In this chapter we present evidence for such processes, based on a review of experimental studies, molecular phylogeny and microbial genomics. Several processes have played important roles in flagellar evolution: self-assembly of simple repeating subunits, gene duplication with subsequent divergence, recruitment of elements from other systems ('molecular bricolage'), and recombination. We also discuss additional tentative new assignments of homology (FliG with MgtE, FliO with YscJ). In conclusion, rather than providing evidence of intelligent design, flagellar and non-flagellar Type III secretion systems instead provide excellent case studies in the evolution of complex systems from simpler components.

In vivo analysis of various substrates utilized by cystathionine gamma-synthase and O-acetylhomoserine sulfhydrylase in methionine biosynthesis.

To gain insight into the evolution of the methionine biosynthesis pathway, in vivo complementation tests were performed. The substrate specificity of three enzymes that intrinsically use different homoserine-esterified substrates and have different sulfur assimilation pathways was examined: two cystathionine gamma-synthases (the Escherichia coli enzyme that naturally utilizes O-succinylhomoserine [OSH]) and the Arabidopsis thaliana enzyme that naturally exploits O-phosphohomoserine [OPH]. Both of these act through the transsulfuration pathway. The third enzyme investigated was O-acetylhomoserine (OAH) sulfhydrylase of Leptospira meyeri, representing the enzyme that utilizes OAH and operates through the direct sulfhydrylation pathway. All the three enzymes were able to utilize OSH and OAH as substrates, with different degrees of efficiency, but only the plant enzyme was able to utilize OPH as a substrate. In addition to their inherent activity in the transsulfuration pathway, the two cystathionine gamma-synthases were also capable of acting in the direct sulfhydrylation pathway. Based on the phylogenic tree and the results of the complementation tests, we suggest that the ancestral gene was able to act as OAH or OSH sulfhydrylase. In some bacteria and plants, this ancient enzyme most probably evolved into a cystathionine gamma-synthase, thereby maintaining the ability to utilize various homoserine-esterified substrates, as well as various sulfur sources, and thus keeping the multisubstrate specificity of its ancestor. In some organisms, this ancestral gene probably underwent a duplication event, which resulted in a cystathionine gamma-synthase and a separate OAH or OSH sulfhydrylase. This led to the development of two parallel pathways of methionine biosynthesis, transsulfuration and direct sulfhydrylation, in these organisms. Although both pathways exist in several organisms, most seem to favor a single specific pathway for methionine biosynthesis in vivo.

Curli fibers mediate internalization of Escherichia coli by eukaryotic cells.

Curli fibers are adhesive surface fibers expressed by Escherichia coli and Salmonella enterica that bind several host extracellular matrix and contact phase proteins and were assumed to have a role in pathogenesis. The results presented here suggest that one such role is internalization into host cells. An E. coli K-12 strain transformed with a low-copy vector containing the gene cluster encoding curli fibers (csg operon) was internalized by several lines of eukaryotic cells. The internalization could be correlated with a high level of curli fiber expression and was abolished by disruption of the csg operon. The ability to be internalized by eukaryotic cells could be conferred even by the curli fiber gene cluster of a noninvasive K-12 strain, but the homologous csg cluster from a virulent septicemic E. coli isolate mediated a higher level of internalization. The finding that curli fibers promote bacterial internalization indicates a new role for curli fibers in pathogenesis.

Yersinia HPI in septicemic Escherichia coli strains isolated from diverse hosts.

High pathogenicity islands (HPIs), first identified in various Yersinia species, encode an iron uptake system. We have studied the occurrence of HPIs in septicemic strains of Escherichia coli isolated from a variety of hosts. The results presented in this communication indicate that most septicemic strains tested contained HPI sequences even though they already have the aerobactin encoding genes. We have also observed two types of HPI deletions, suggesting genetic instability of this element. Notable exceptions are several strains isolated from septicemia in sheep that lacked both iron acquisition systems.

Matrix-assisted refolding of single-chain Fv- cellulose binding domain fusion proteins.

We describe a method for the isolation of recombinant single-chain antibodies in a biologically active form. The single-chain antibodies are fused to a cellulose binding domain as a single-chain protein that accumulates as insoluble inclusion bodies upon expression in Escherichia coli. The inclusion bodies are then solubilized and denatured by an appropriate chaotropic solvent, then reversibly immobilized onto a cellulose matrix via specific interaction of the matrix with the cellulose binding domain (CBD) moiety. The efficient immobilization that minimizes the contact between folding protein molecules, thus preventing their aggregation, is facilitated by the robustness of the Clostridium thermocellum CBD we use. This CBD is unique in retaining its specific cellulose binding capability when solubilized in up to 6 M urea, while the proteins fused to it are fully denatured. Refolding of the fusion proteins is induced by reducing with time the concentration of the denaturing solvent while in contact with the cellulose matrix. The refolded single-chain antibodies in their native state are then recovered by releasing them from the cellulose matrix in high yield of 60% or better, which is threefold or higher than the yield obtained by using published refolding protocols to recover the same scFvs. The described method should have general applicability for the production of many protein-CBD fusions in which the fusion partner is insoluble upon expression.