Human liver microbiota modeling strategy at the early onset of fibrosis.

BACKGROUND: Gut microbiota is involved in the development of liver diseases such as fibrosis. We and others identified that selected sets of gut bacterial DNA and bacteria translocate to tissues, notably the liver, to establish a non-infectious tissue microbiota composed of microbial DNA and a low frequency live bacteria. However, the precise set of bacterial DNA, and thereby the corresponding taxa associated with the early stages of fibrosis need to be identified. Furthermore, to overcome the impact of different group size and patient origins we adapted innovative statistical approaches. Liver samples with low liver fibrosis scores (F0, F1, F2), to study the early stages of the disease, were collected from Romania(n = 36), Austria(n = 10), Italy(n = 19), and Spain(n = 17). The 16S rRNA gene was sequenced. We considered the frequency, sparsity, unbalanced sample size between cohorts to identify taxonomic profiles and statistical differences. RESULTS: Multivariate analyses, including adapted spectral clustering with L1-penalty fair-discriminant strategies, and predicted metagenomics were used to identify that 50% of liver taxa associated with the early stage fibrosis were Enterobacteriaceae, Pseudomonadaceae, Xanthobacteriaceae and Burkholderiaceae. The Flavobacteriaceae and Xanthobacteriaceae discriminated between F0 and F1. Predicted metagenomics analysis identified that the preQ0 biosynthesis and the potential pathways involving glucoryranose and glycogen degradation were negatively associated with liver fibrosis F1-F2 vs F0. CONCLUSIONS: Without demonstrating causality, our results suggest first a role of bacterial translocation to the liver in the progression of fibrosis, notably at the earliest stages. Second, our statistical approach can identify microbial signatures and overcome issues regarding sample size differences, the impact of environment, and sets of analyses. TRIAL REGISTRATION: TirguMECCH ROLIVER Prospective Cohort for the Identification of Liver Microbiota, registration 4065/2014. Registered 01 01 2014.

Takedown of Painful Ankle Arthrodesis to Total Ankle Arthroplasty: A Case Series of 77 Patients.

Treatment of painful or malaligned ankle arthrodesis can present as a challenging issue. Several published studies have demonstrated that takedown of a painful ankle arthrodesis to total ankle arthroplasty can assist in restoring some sagittal plane motion and improving functional scores. The goal of this study was to contribute to the limited body of literature with the largest cohort and longest follow-up to date. A retrospective analysis was performed on patient and surgical characteristics of those who underwent a conversion of a painful ankle arthrodesis to a total ankle arthroplasty by 1 of 3 experienced total ankle arthroplasty surgeons from February 2003 to December 2016 with ≥2 years of follow up. Seventy-seven subjects were included for evaluation, with an implant retention rate of 88% (68 of 77) and mean follow-up of 8.3 years (range 2.6 to 15.8). Of the 11 (14%) failures (defined as retrieval or exchange of metallic components), 8 (10%) were revised to a total ankle replacement, 2 (2%) underwent revision arthrodesis, and 1 (1%) elected for below-the-knee amputation. The mean time since the primary arthrodesis was 8.6 years (range 1 to 44), and the longer time interval between primary arthrodesis to takedown total ankle arthroplasty did not correlate with poorer outcome scores or increased risk of failure. The mean American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot, Buechel-Pappas, and visual analog pain scale scores improved from preoperative values, with less satisfaction noted in those who needed revision surgery. The conversion of a painful ankle arthrodesis to a total ankle implant is a viable option to obtain range of motion and improved patient satisfaction scores similar to primary total ankle replacement.

The circulating microbiome signature and inferred functional metagenomics in alcoholic hepatitis.

Intestinal dysbiosis is implicated in alcoholic hepatitis (AH). However, changes in the circulating microbiome, its association with the presence and severity of AH, and its functional relevance in AH is unknown. Qualitative and quantitative assessment of changes in the circulating microbiome were performed by sequencing bacterial DNA in subjects with moderate AH (MAH) (n = 18) or severe AH (SAH) (n = 19). These data were compared with heavy drinking controls (HDCs) without obvious liver disease (n = 19) and non-alcohol-consuming controls (NACs, n = 20). The data were related to endotoxin levels and markers of monocyte activation. Linear discriminant analysis effect size (LEfSe) analysis, inferred metagenomics, and predictive functional analysis using PICRUSt were performed. There was a significant increase in 16S copies/ng DNA both in MAH (P < 0.01) and SAH (P < 0.001) subjects. Compared with NACs, the relative abundance of phylum Bacteroidetes was significantly decreased in HDCs, MAH, and SAH (P < 0.001). In contrast, all alcohol-consuming groups had enrichment with Fusobacteria; this was greatest for HDCs and decreased progressively in MAH and SAH. Subjects with SAH had significantly higher endotoxemia (P = 0.01). Compared with alcohol-consuming groups, predictive functional metagenomics indicated an enrichment of bacteria with genes related to methanogenesis and denitrification. Furthermore, both HDCs and SAH showed activation of a type III secretion system that has been linked to gram-negative bacterial virulence. Metagenomics in SAH versus NACs predicted increased isoprenoid synthesis via mevalonate and anthranilate degradation, known modulators of gram-positive bacterial growth and biofilm production, respectively. CONCLUSION: Heavy alcohol consumption appears to be the primary driver of changes in the circulating microbiome associated with a shift in its inferred metabolic functions. (Hepatology 2018;67:1284-1302).

Rifaximin has minor effects on bacterial composition, inflammation, and bacterial translocation in cirrhosis: A randomized trial.

BACKGROUND AND AIM: Decompensated cirrhosis is characterized by disturbed hemodynamics, immune dysfunction, and high risk of infections. Translocation of viable bacteria and bacterial products from the gut to the blood is considered a key driver in this process. Intestinal decontamination with rifaximin may reduce bacterial translocation (BT) and decrease inflammation. A randomized, placebo-controlled trial investigated the effects of rifaximin on inflammation and BT in decompensated cirrhosis. METHODS: Fifty-four out-patients with cirrhosis and ascites were randomized, mean age 56 years (± 8.4), and model for end-stage liver disease score 12 (± 3.9). Patients received rifaximin 550-mg BD (n = 36) or placebo BD (n = 18). Blood and fecal (n = 15) sampling were conducted at baseline and after 4 weeks. Bacterial DNA in blood was determined by real-time qPCR 16S rRNA gene quantification. Bacterial composition in feces was analyzed by 16S rRNA gene sequencing. RESULTS: Circulating markers of inflammation, including tumor necrosis factor alpha, interleukins 6, 10, and 18, stromal cell-derived factor 1-α, transforming growth factor ß-1, and high sensitivity C-reactive protein, were unaltered by rifaximin treatment. Rifaximin altered abundance of bacterial taxa in blood marginally, only a decrease in Pseudomonadales was observed. In feces, rifaximin decreased bacterial richness, but effect on particular species was not observed. Subgroup analyses on patients with severely disturbed hemodynamics (n = 34) or activated lipopolysaccharide binding protein (n = 37) revealed no effect of rifaximin. CONCLUSION: Four weeks of treatment with rifaximin had no impact on the inflammatory state and only minor effects on BT and intestinal bacterial composition in stable, decompensated cirrhosis (NCT01769040).

Takedown of Ankle Arthrodesis With Insufficient Fibula: Surgical Technique and Intermediate-Term Follow-Up.

Conversion of ankle arthrodesis to total ankle arthroplasty has recently gained popularity. However, technical challenges are present when treating patients without a sufficient fibular buttress. We describe a technique for restoration of an adequate fibular buttress using an iliac crest bone graft or malleolar relocation. The results of 10 patients with an average follow-up period of 56 (range 24 to 123) months are presented. Of the 10 patients, 3 underwent tricortical iliac bone augmentation of the fibula, 4 underwent repositioning of the remnant fibula, and in 3, the in situ fibula was used. The average interval from fusion to takedown was 15.1 (range 5 to 35) years, and the average age at takedown was 52.8 (range 33 to 75) years. The average improvement in the American Orthopaedic Foot and Ankle Society Ankle-Hindfoot scale and Buechel-Pappas scale scores was 35.8 (range 30 to 46) and 34 (range 25 to 42), respectively. Three patients underwent a total of 7 subsequent operations related to the ankle implant. Only 1 of the patients had any residual frontal plane deformity. None of the patients exhibited any component subsidence; however, 2 patients experienced asymptomatic lateral talar component overgrowth. The improvement in the clinical scores in this group of patients suggests that takedown of an ankle arthrodesis with an insufficient fibula is a viable option to improve function. Various techniques to restore the lateral buttress can be used even with complete absence of the distal fibula.

In vitro investigation of Debaryomyces hansenii strains for potential probiotic properties.

In this study, 23 Debaryomyces hansenii strains, isolated from cheese and fish gut, were investigated in vitro for potential probiotic properties i.e. (1) survival under in vitro GI (gastrointestinal) conditions with different oxygen levels, (2) adhesion to Caco-2 intestinal epithelial cells and mucin, and (3) modulation of pro- and anti-inflammatory cytokine secretion by human monocyte-derived dendritic cells. As references two commercially available probiotic Saccharomyces cerevisiae var. boulardii (S. boulardii) strains were included in the study. Our results demonstrate that the different D. hansenii yeast strains had very diverse properties which could potentially lead to different probiotic effects. One strain of D. hansenii (DI 09) was capable of surviving GI stress conditions, although not to the same degree as the S. boulardii strains. This DI 09 strain, however, adhered more strongly to Caco-2 cells and mucin than the S. boulardii strains. Additionally, two D. hansenii strains (DI 10 and DI 15) elicited a higher IL-10/IL-12 ratio than the S. boulardii strains, indicating a higher anti-inflammatory effects on human dendritic cells. Finally, one strain of D. hansenii (DI 02) was evaluated as the best probiotic candidate because of its outstanding ability to survive the GI stresses, to adhere to Caco-2 cells and mucin and to induce a high IL-10/IL-12 ratio. In conclusion, this study shows that strains of D. hansenii may offer promising probiotic traits relevant for further study.

Combined Total Ankle Arthroplasty With Posterior Tibial Tendon Transfer for End-Stage Cavovarus Deformity.

Posterior tibial tendon transfer has been described to reduce and balance the cavovarus deformity in those patients who receive a total ankle replacement for end-stage arthritis. In this article, we discuss the indications and provide a detailed description of the technique for this powerful procedure. Case examples that demonstrate the utility of the procedure are provided.

Talolisthesis in end stage ankle arthrosis.

BACKGROUND: Sagittal displacement in patients with end stage ankle arthritis has been described as the tibiotalar ratio (TTR). Yet the incidence, distribution and predictive factors of talolisthesis are unknown. METHODS: The radiographs of 470 cases of ankle arthritis were compared with a control group of 49 normal ankles. The TTR was measured for both groups. Additional co-variables included the anterior and lateral distal tibial angles, and talar tilt. RESULTS: The mean TTR in the arthritis cohort was 34.8+9.12 compared to the normal group of 34.1+2.62. Twenty-eight percent of the ankles had anterior displacement and twenty-eight percent had posterior talolisthesis, while forty-four percent had normal tibiotalar alignment. Multivariate linear regression revealed significant predictors of anterior distal tibial angle (p<0.0001) and talar tilt (p=0.0007) for abnormal TTR. CONCLUSION: Sagittal displacement is common in end stage ankle arthritis and is affected by ligamentous laxity and joint morphology.

Articular congruency of the Salto Talaris total ankle prosthesis.

BACKGROUND: The Salto-Talaris polyethylene articulating surface was designed to allow, but limit accessory motion. This investigation examines surface characteristics between the polyethylene bearing and anatomic talar component in various positions of function. METHODS: A Salto Talaris talar prosthesis and matching polyethylene bearing were scanned to create digital solid body models and manipulated to assess surface contact during simulated gait. With computer micromanipulation of the component positions, the surface intersections were recorded for 15 different alignments. RESULTS: The Salto Talaris has limited contact congruity with four points of contact in dorsiflexion, neutral, and plantarflexion. Lateral and medial translations showed only 2-point contact. The radii of curvatures between the talar component and polyethylene surfaces do not match. There was no sulcus contact yet component separation distance was small, suggesting increased loads. CONCLUSION: Surface incongruency was measured based on computer model analysis which raises a concern of increased contact pressures.

The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology.

The food industry is constantly striving to develop new products to fulfil the ever changing demands of consumers and the strict requirements of regulatory agencies. For foods based on microbial fermentation, this pushes the boundaries of microbial performance and requires the constant development of new starter cultures with novel properties. Since the use of ingredients in the food industry is tightly regulated and under close scrutiny by consumers, the use of recombinant DNA technology to improve microbial performance is currently not an option. As a result, the focus for improving strains for microbial fermentation is on classical strain improvement methods. Here we review the use of these techniques to improve the functionality of lactic acid bacteria starter cultures for application in industrial-scale food production. Methods will be described for improving the bacteriophage resistance of specific strains, improving their texture forming ability, increasing their tolerance to stress and modulating both the amount and identity of acids produced during fermentation. In addition, approaches to eliminating undesirable properties will be described. Techniques include random mutagenesis, directed evolution and dominant selection schemes.

Gutter impingement after total ankle arthroplasty.

BACKGROUND: Symptomatic gutter impingement after ankle arthroplasty has been reported with various designs. However, the incidence and origin of impingement have not been fully elucidated. Furthermore, the outcome of patients following gutter debridement has not been reported. METHODS: A review of 489 total ankle replacements was performed to calculate the incidence of symptomatic gutter impingement in 4 different devices and between the presenting causes of arthritis. The incidence of gutter impingement was determined for patients with and without preemptive gutter resection at the time of implantation. The functional outcome of 30 of the 34 patients who had subsequent gutter debridement was determined with 3 different outcome instruments. RESULTS: Symptomatic gutter disease occurred in 34 of 489 cases (7%) followed for a minimum of 1 year (range, 1-12 years). There was a 2% incidence of gutter disease in the 194 ankles that had prophylactic gutter resection at the time of implantation and a 7% incidence in the 295 ankles that did not have gutter resection at the time of implantation. When the ankle treated with an Agility (DePuy Orthopaedics, Inc, Warsaw, IN) device was excluded, there was a 2% and an 18% incidence of gutter disease in the 2 groups, respectively (P < .05). There was no difference in the incidence of gutter disease between the various implants and origins of ankle arthritis. Postoperative outcomes were favorable in the 27 patients who did not have another procedure after the initial gutter debridement. Seven patients (21%) required reoperation. CONCLUSION: Prophylactic gutter resection should be considered at the time of implantation to reduce the incidence of postoperative symptoms. Although most patients had favorable outcomes following gutter debridement, there was a high reoperation rate. LEVEL OF EVIDENCE: Level III, retrospective comparative series.

Relationship of alignment and tibial cortical coverage to hypertrophic bone formation in Salto Talaris® total ankle arthroplasty.

A retrospective analysis of 107 Salto Talaris(®) total ankle replacements was performed to determine the effect of alignment and tibial cortical coverage on the formation of heterotopic bone. The radiographic parameters were studied for at least 18 months to detect any changes over time. The angle of insertion of the implant was most often in varus and with a positive anterior slope. A slight increase was seen in the slope over time (p < .0001) but was not clinically relevant. The mean percentage of cortical coverage of the tibial component was 89%, and only 7 patients had complete coverage of both the anterior and the posterior cortices. The percentage of patients who had hypertrophic bone growth increased over time (p < .0001). As the slope of the implant increased, there was a negative correlation with the degree of tibial coverage (p = .007). There was also an increase in the extent of hypertrophic bone as the tibial coverage decreased. None of the patients had symptoms that required an additional surgical procedure. The results of the present study indicate a high incidence of hypertrophic bone proliferation when the dimensions of the tibial component do not match the anteroposterior depth of the tibia at the plane of resection. Despite the high occurrence rate, the clinical relevance of hypertrophic bone is obscure.

Campylobacter jejuni survival within human epithelial cells is enhanced by the secreted protein CiaI.

Although it is known that Campylobacter jejuni invade the cells that line the human intestinal tract, the bacterial proteins that enable this pathogen to survive within Campylobacter-containing vacuoles (CCV) have not been identified. Here, we describe the identification and characterization of a protein that we termed CiaI for Campylobacter invasion antigen involved in intracellular survival. We show that CiaI harbours an amino-terminal type III secretion sequence and is secreted from C. jejuni through the flagellar type III secretion system. In addition, the ciaI mutant was impaired in intracellular survival when compared with a wild-type strain, as judged by the gentamicin-protection assay. Fluorescence microscopy examination of epithelial cells infected with the C. jejuni ciaI mutant revealed that the CCV were more frequently co-localized with Cathepsin D (a lysosomal marker) than the CCV in cells infected with a C. jejuni wild-type strain. Ectopic expression of CiaI-GFP in epithelial cells yielded a punctate phenotype not observed with the other C. jejuni genes, and this phenotype was abolished by mutation of a dileucine motif located in the carboxy-terminus of the protein. Based on the data, we conclude that CiaI contributes to the ability of C. jejuni to survive within epithelial cells.

Identification of potential type III secretion proteins via heterologous expression of Vibrio parahaemolyticus DNA.

We employed a heterologous secretion assay to identify proteins potentially secreted by type III secretion systems (T3SSs) in Vibrio parahaemolyticus. N-terminal sequences from 32 proteins within T3SS genomic islands and seven proteins from elsewhere in the chromosome included proteins that were recognized for export by the Yersinia enterocolitica flagellar T3SS.

A specific tongue microbiota signature is found in patients displaying an improvement of orosensory lipid perception after a sleeve gastrectomy.

Introduction: A preferential consumption of low-fat foods is reported by most of the patients after a vertical sleeve gastrectomy (VSG). The fact that a recent study shed light on a relationship between oral microbiota and fat taste sensitivity in obese patients prompted us to explore whether such a connection also exists in the context of a VSG. Methods: Thirty-two adult female patients with a severe obesity (BMI = 43.1 ± 0.7 kg/m2) and candidates for a VSG were selected. Oral microbiota composition surrounding the gustatory circumvallate papillae (CVP) and the lipid perception thresholds were explored before and 6 months after surgery. Results: VSG was found to be associated both with a qualitative (compositional changes) and quantitative (lower gene richness) remodeling of the peri-CVP microbiota. Analysis of the lipid perception allowed us to distinguish two subgroups: patients with a post-operative improvement of the fat taste sensitivity (i.e., with a lower threshold, n = 14) and unimproved patients (n = 18). Specific peri-CVP microbiota signatures also discriminated these two subgroups, unimproved patient being characterized by higher levels of Porphyromonas, Fusobacterium, and Haemophilus genera associated with lower levels of Atopobium and Prevotella genera as compared to the lipid-improved patients. Conclusion: Collectively, these data raise the possibility that the microbial environment surrounding gustatory papillae might play a role in the positive changes of fat taste sensitivity observed in some patients after VSG.

Gut microbiota dysbiosis of type 2 diabetic mice impairs the intestinal daily rhythms of GLP-1 sensitivity.

The gut-brain-beta cell glucagon-like peptide-1 (GLP-1)-dependent axis and the clock genes both control insulin secretion. Evidence shows that a keystone of this molecular interaction could be the gut microbiota. We analyzed in mice the circadian profile of GLP-1 sensitivity on insulin secretion and the impact of the autonomic neuropathy, antibiotic treated in different diabetic mouse models and in germ-free colonized mice. We show that GLP-1sensitivity is maximal during the dark feeding period, i.e., the postprandial state. Coincidently, the ileum expression of GLP-1 receptor and peripherin is increased and tightly correlated with a subset of clock gene. Since both are markers of enteric neurons, it suggests a role in the gut-brain-beta cell GLP-1-dependent axis. We evaluated the importance of gut microbiota dysbiosis and found that the abundance of ileum bacteria, particularly Ruminococcaceae and Lachnospiraceae, oscillated diurnally, with a maximum during the dark period, along with expression patterns of a subset of clock genes. This diurnal pattern of circadian gene expression and Lachnospiraceae abundance was also observed in two separate mouse models of gut microbiota dysbiosis and of autonomic neuropathy with impaired GLP-1 sensitivity (1.high-fat diet-fed type 2 diabetic, 2.antibiotic-treated/germ-free mice). Our data show that GLP-1 sensitivity relies on specific pattern of intestinal clock gene expression and specific gut bacteria. This new statement opens opportunities to treat diabetic patient with GLP-1-based therapies by using on a possible pre/probiotic co-treatment to improve the time-dependent efficiency of these therapies.

Amino-terminal residues dictate the export efficiency of the Campylobacter jejuni filament proteins via the flagellum.

Bacterial flagella play an essential role in the pathogenesis of numerous enteric pathogens. The flagellum is required for motility, colonization, and in some instances, for the secretion of effector proteins. In contrast to the intensively studied flagella of Escherichia coli and Salmonella typhimurium, the flagella of Campylobacter jejuni, Helicobacter pylori and Vibrio cholerae are less well characterized and composed of multiple flagellin subunits. This study was performed to gain a better understanding of flagellin export from the flagellar type III secretion apparatus of C. jejuni. The flagellar filament of C. jejuni is comprised of two flagellins termed FlaA and FlaB. We demonstrate that the amino-termini of FlaA and FlaB determine the length of the flagellum and motility of C. jejuni. We also demonstrate that protein-specific residues in the amino-terminus of FlaA and FlaB dictate export efficiency from the flagellar type III secretion system (T3SS) of Yersinia enterocolitica. These findings demonstrate that key residues within the amino-termini of two nearly identical proteins influence protein export efficiency, and that the mechanism governing the efficiency of protein export is conserved among two pathogens belonging to distinct bacterial classes. These findings are of additional interest because C. jejuni utilizes the flagellum to export virulence proteins.

Structure of PolC reveals unique DNA binding and fidelity determinants.

PolC is the polymerase responsible for genome duplication in many Gram-positive bacteria and represents an attractive target for antibacterial development. We have determined the 2.4-A resolution crystal structure of Geobacillus kaustophilus PolC in a ternary complex with DNA and dGTP. The structure reveals nascent base pair interactions that lead to highly accurate nucleotide incorporation. A unique beta-strand motif in the PolC thumb domain contacts the minor groove, allowing replication errors to be sensed up to 8 nt upstream of the active site. PolC exhibits the potential for large-scale conformational flexibility, which could encompass the catalytic residues. The structure suggests a mechanism by which the active site can communicate with the rest of the replisome to trigger proofreading after nucleotide misincorporation, leading to an integrated model for controlling the dynamic switch between replicative and repair polymerases. This ternary complex of a cellular replicative polymerase affords insights into polymerase fidelity, evolution, and structural diversity.

Total ankle replacement in the varus ankle.

Treatment of ankle varus with total ankle replacement (TAR) lacks consensus regarding the limits of deformity that can be managed successfully without recurrence. With newer anatomic prosthetic designs and a comprehensive surgical approach, treatment of frontal plane deformities with implant arthroplasty has gained acceptance. The purpose of this retrospective study was to determine the outcome and compare correction of large frontal plane varus deformities with TAR using 2 different replacement systems. Also, a stepwise surgical approach for consistent correction was determined. TAR was performed on 26 patients with varus ankle deformity and a mean age of 63.85 ± 9.33 (range 48-86) years. Duration of follow-up was 16.69 ± 7.26 (range 7-37) months. The difference between the immediate postoperative frontal plane radiographic alignments was compared with the preoperative deformity, and reevaluated after at least 1 year of weight-bearing function. The preoperative mortise view varus deformity was 16.8° ± 6.79° (range 6°-28°), whereas at final follow-up the degree of varus was 0° ± 2.64° (P < .0001) on the anteroposterior view and 0.5° ± 2.7° (P < .0001) on the mortise view. All but one patient was corrected to within 4° of frontal plane neutral. Overall, correction was maintained throughout the study period. In conclusion, surgical treatment of the varus ankle arthrosis with TAR can be successful.