Clinical performance study of a fecal bacterial signature test for colorectal cancer screening.

The fecal immunochemical test (FIT) is the most widely used test for colorectal cancer (CRC) screening. RAID-CRC Screen is a new non-invasive test based on fecal bacterial markers, developed to complement FIT by increasing its specificity. The test was previously clinically evaluated in FIT-positive patients (>20 µg of hemoglobin/g of feces, "FIT20"), in which it reduced the proportion of false positive results by 16.3% while maintaining most of FIT20's sensitivity. The aim of this study was to compare the sensitivity and specificity of a CRC screening program using RAID-CRC Screen in addition to FIT20 as a triage test in a European screening population undergoing screening colonoscopy with a CRC screening program with FIT20 alone in the same cohort. A cohort of 2481 subjects aged > 55 years from the German screening colonoscopy program was included. The colonoscopy findings were used as the gold standard in calculating the diagnostic capacity of the tests and included 15 CRC and 257 advanced neoplasia cases. RAID-CRC Screen added to FIT20 provided the same sensitivity as FIT20 alone (66.7%) in detecting CRC and a significantly higher specificity (97.0% vs. 96.1%, p<0.0001). The positive predictive value was 11.9% when using RAID-CRC Screen and 9.5% with FIT20 alone, and the negative predictive value was 99.8% in the two scenarios. For advanced neoplasia detection, the use of RAID-CRC Screen yielded significantly lower sensitivity than with FIT20 alone (17.5% vs. 21.8%, p = 0.0009), and the overall specificity was significantly higher when using RAID-CRC Screen compared with FIT20 alone (98.2% vs. 97.8%, p = 0.0039). Our findings confirm the results obtained in previous clinical studies in a CRC screening setting, showing the potential of RAID-CRC Screen to increase the overall specificity of FIT-based screening.

RAID Prediction: Pilot Study of Fecal Microbial Signature With Capacity to Predict Response to Anti-TNF Treatment.

BACKGROUND AND AIMS: Crohn's disease and ulcerative colitis evolve with alternate outbreaks and remissions of variable duration in both cases. Despite the advances, about 10-30% of patients do not respond to the treatment after the induction period. Besides, between 20% to 50% further patients need an optimization of the dose to respond the treatment. Recent studies have pointed gut microbiota can play a role in the anti-TNF treatment response. This study aimed to define a bacterial signature that could be used to predict the response of patients to anti-TNF treatment. METHODS: There were obtained 38 stool samples from 38 IBD patients before starting anti-TNF treatments: Adalimumab, Golimumab or Infliximab. Patients were differentiated in 2 groups: responders and non-responders to biological treatment. From each sample, DNA was purified and used in a qPCR for the quantification of the 8 microbial markers. RESULTS: In this proof of concept, the predictive ability to identify anti-TNF treatment responders was analyzed. An algorithm consisting in the combination of 4 bacterial markers showed a high capacity to discriminate between responders and non- responders. The algorithm proved high sensitivity and specificity reporting values of 93.33% and 100% respectively, with a positive predictive value of 100% and a negative predictive value of 75% for predicting response to biologic treatment. CONCLUSIONS: A specific bacterial signature could beneficiate patients with inflammatory bowel disease predicting the therapeutic effectiveness of an anti-TNF treatment, leading to a personalized therapy, improving the patients' quality of life, saving costs and gaining time in patient improvement.

This study aimed to define a microbial signature that could be used to predict the response of patients to anti-TNF treatment in inflammatory bowel disease. An algorithm consisting in the combination of 4 bacterial markers showed a high capacity to discriminate between responders and nonresponders.

In vitro Prebiotic Effect of Bread-Making Process in Inflammatory Bowel Disease Microbiome.

Inflammatory bowel disease (IBD), including its two main categories (Crohn's disease and ulcerative colitis), has been linked both to gut microbiota and to diet. Bread is a daily food that has a potential capacity as a prebiotic. Our aim was to evaluate different bread-making processes and their effect on fecal colonic microbiota in IBD patients. The microbial composition of several sourdoughs and dough samples was analyzed by high-throughput sequencing of 16S and 18S rRNA genes. Three types of bread, which followed different bread-making processes, were in vitro digested and incubated with feces from IBD patients. Changes in gut microbiota were assessed by a quantitative polymerase chain reaction using specific bacterial sequence targets. Short-chain fatty acid production was also analyzed by gas chromatography. Lactobacillus sanfranciscensis was the dominant lactic acid bacteria species found in sourdough and bread doughs prepared using sourdough, whereas Saccharomyces cerevisiae was the most dominant yeast in all groups, especially in bread doughs before baking. Differences in microbial composition in raw bread doughs were more related to the type of dough and elaboration than to fermentation time lengths. The analysis of in vitro fecal incubations with bread conditions revealed an increase in most bacterial groups analyzed and short-chain fatty acid production, both in Crohn's disease and ulcerative colitis samples. Most remarkable increases in short-chain fatty acid production mirrored higher abundances of Roseburia species. The potential prebiotic properties observed were mainly obtained when using a high quantity of bread, regardless of bread type. Overall, this study highlights the bacterial dynamics within the bread-making process and the potential prebiotic effect in IBD patients.

A Novel Grape-Derived Prebiotic Selectively Enhances Abundance and Metabolic Activity of Butyrate-Producing Bacteria in Faecal Samples.

Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) patients have different faecal microbiota profiles compared to healthy controls. Prebiotics intake influences intestinal microbiota composition which in turn influence the growth of short-chain fatty acids (SCFA) producing bacteria. This study aimed to evaluate the capacity of Previpect, a new prebiotic obtained from grapes fibre, to balance the dysbiosis found in patients with intestinal disorders. This was achieved through the analysis of specific bacterial markers and SCFA production using an in vitro fermentation system and comparing the obtained results with those obtained with other commercial prebiotics. Fresh faecal samples from patients with IBD (N = 6), IBS (N = 3), and control subjects (N = 6) were used. Previpect showed high fermentative ability enabling the growth of butyrate producing bacteria and increasing SCFA concentration up to 2.5-fold. Previpect is a promising prebiotic which may be used as a therapeutic strategy towards promotion of intestinal microbiota restoration, microbial healing, and as a preventive supplement for healthy individuals.

New fecal bacterial signature for colorectal cancer screening reduces the fecal immunochemical test false-positive rate in a screening population.

Guidelines recommend routine screening for colorectal cancer (CRC) in asymptomatic adults starting at age 50. The most extensively used noninvasive test for CRC screening is the fecal immunochemical test (FIT), which has an overall sensitivity for CRC of approximately 61.0%-91.0%, which drops to 27.0%-67.0% for advanced adenomas. These figures contain a high false-positive rate and a low positive predictive value. This work aimed to develop a new, noninvasive CRC screening tool based on fecal bacterial markers capable of decreasing FIT false-positive rates in a FIT-positive population. We defined a fecal bacterial signature (RAID-CRC Screen) in a proof-of-concept with 172 FIT-positive individuals and validated the obtained results on an external cohort of 327 FIT-positive subjects. All study participants had joined the national CRC screening program. In the clinical validation of RAID-CRC Screen, a sensitivity of 83.9% and a specificity of 16.3% were obtained for the detection of advanced neoplasm lesions (advanced adenomas and/or CRC). FIT 20 µg/g produced 184 false-positive results. Using RAID-CRC Screen, this value was reduced to 154, thus reducing the false-positive rate by 16.3%. The RAID-CRC Screen test could be implemented in CRC screening programs to allow a significant reduction in the number of colonoscopies performed unnecessarily for FIT-positive participants of CRC screening programs.

Changes in the Potential Activity of Nitrite Reducers and the Microbial Community Structure After Sediment Dredging and Plant Removal in the Empuriabrava FWS-CW.

In constructed wetlands (CW), denitrification usually accounts for > 60% of nitrogen removal and is supposedly affected by wetland management practices, such as dredging (and plant removal). These practices cause an impact in sediment properties and microbial communities living therein. We have quantified the effects of a sediment dredging event on dissimilatory nitrite reduction by analysing the structure and activities of the microbial community before and after the event. Potential rates for nitrate reduction to ammonia and denitrification were in accordance with changes in the physicochemical conditions. Denitrification was the predominant pathway for nitrite removal (> 60%) and eventually led to the complete removal of nitrate. On the contrary, dissimilatory nitrite reduction to ammonia (DNRA) increased from 5 to 18% after the dredging event. Both actual activities and abundances of 16S rRNA, nirK and nirS significantly decreased after sediment dredging. However, genetic potential for denitrification (qnirS + qnirK/q16S rRNA) remained unchanged. Analyses of the 16S rRNA gene sequences revealed the importance of vegetation in shaping microbial community structures, selecting specific phylotypes potentially contributing to the nitrogen cycle. Overall, we confirmed that sediment dredging and vegetation removal exerted a measurable effect on the microbial community, but not on potential nitrite + nitrate removal rates. According to redundancy analysis, nitrate concentration and pH were the main variables affecting sediment microbial communities in the Empuriabrava CWs. Our results highlight a high recovery of the functionality of an ecosystem service after a severe intervention and point to metabolic redundancy of denitrifiers. We are confident these results will be taken into account in future management strategies in CWs.

Reduction of faecal immunochemical test false-positive results using a signature based on faecal bacterial markers.

BACKGROUND: Colorectal cancer is the second commonest cause of cancer mortality. Some countries are implementing colorectal cancer screening to detect lesions at an early stage using non-invasive tools like the faecal immunochemical test. Despite affordability, this test shows a low sensitivity for precancerous lesions and a low positive predictive value for colorectal cancer, resulting in a high false-positive rate. AIM: To develop a new, non-invasive colorectal cancer screening tool based on bacterial faecal biomarkers, which in combination with the faecal immunochemical test, could allow a reduction in the false-positive rate. This tool is called risk assessment of intestinal disease for colorectal cancer (RAID-CRC). METHODS: We performed both the faecal immunochemical test and the bacterial markers analysis (RAID-CRC test) in stool samples from individuals with normal colonoscopy (167), non-advanced adenomas (88), advanced adenomas (30) and colorectal cancer (48). All the participants showed colorectal cancer-associated symptoms. RESULTS: Performance of the faecal immunochemical test for advanced neoplasia (ie advanced adenoma and colorectal cancer) was determined by using the cut-off value established in Catalonia (20 µg haemoglobin/g of faeces) for a population-based screening approach. Sensitivity and specificity values of 83% and 80%, respectively, and positive and negative predictive values of 56% and 94%, respectively, were obtained. When both the immunological and the biological analysis were combined, the corresponding values were 80% and 90% for sensitivity and specificity, respectively, and 70% and 94% for positive and negative predictive values, respectively, resulting in a 50% reduction of the false-positive rate. CONCLUSIONS: RAID-CRC test allows a substantial reduction in the faecal immunochemical test false-positive results (50%) in a symptomatic population. Further validation is indicated in a colorectal cancer-screening scenario.

Specific detection of "Clostridium autoethanogenum", Clostridium ljungdahlii and Clostridium carboxidivorans in complex bioreactor samples.

The high genetic similarity between some carboxydotrophic bacteria does not allow for the use of common sequencing techniques targeting the 16S rRNA gene for species identification. 16S rRNA sequencing fails to discriminate among Clostridium ljungdahlii and 'Clostridium autoethanogenum', despite this two species exhibit significant differences in CO2 assimilation and alcohol production. In this work we designed PCR primers targeting for the DNA gyrase subunit A (gyrA) and a putative formate/nitrite transporter (fnt) to specifically detect the presence of 'C. autoethanogenum', C. ljungdahlii or Clostridium carboxidivorans. We could confirm the simultaneous presence of C. ljungdahlii and 'C. autoethanogenum' in different bioreactors, and a preference of the latter for high CO2 content.

Effect of ethanol and butanol on autotrophic growth of model homoacetogens.

Research efforts aimed at increasing ethanol and butanol productivity from syngas are currently gaining attention. For most model carboxydotrophic bacteria, production rates, yields and maximum product titres have been studied in detail, but little is known on alcohol toxicity in these bacteria. The aim of this work was to investigate the inhibitory effects of ethanol and butanol on the growth of Clostridium ljungdahlii PETC, C. carboxidivorans P7, and 'Butyribacterium methylotrophicum DSM3468'. Experiments to determine inhibitory effects due to product accumulation were carried out using a synthetic mixture of CO:CO2:H2 as a substrate. These conditions were chosen to mimic gaseous effluents of biomass and waste gasification plants. Inhibition effects were recorded as changes in growth parameters. No significant inhibition was observed for ethanol at concentrations below 15 g/L. The three species exhibited higher sensitivity to butanol. Half inhibition constants for butanol could be estimated for P7 (IC50 = 4.12 g/L), DSM3468 (IC50 = 1.79 g/L), and PETC (IC50 = 9.75 g/L). In conclusion, at least for the tested strains, alcohol toxicity is not an immediate handicap for increasing alcohol production of the tested homoacetogenic strains.

Microbial electrosynthesis of butyrate from carbon dioxide: Production and extraction.

To date acetate is the main product of microbial electrosynthesis (MES) from carbon dioxide (CO2). In this work a tubular bioelectrochemical system was used to carry out MES and enhance butyrate production over the other organic products. Batch tests were performed at a fixed cathode potential of -0.8V vs SHE. The reproducibility of the results according to previous experiments was validated in a preliminary test. According to the literature butyrate production could take place by chain elongation reactions at low pH and high hydrogen partial pressure (pH2). During the experiment, CO2 supply was limited to build up pH2 and trigger the production of compounds with a higher degree of reduction. In test 1 butyrate became the predominant end-product, with a concentration of 59.7mMC versus 20.3mMC of acetate, but limitation on CO2 supply resulted in low product titers. CO2 limitation was relaxed in test 2 to increase the bioelectrochemical activity but increase pH2 and promote the production of butyrate, what resulted in the production of 87.5mMC of butyrate and 34.7mMC of acetate. The consumption of ethanol, and the presence of other products in the biocathode (i.e. caproate) suggested that butyrate production took place through chain elongation reactions, likely driven by Megasphaera sueciensis (>39% relative abundance). Extraction and concentration of butyrate was performed by liquid membrane extraction. A concentration phase with 252.4mMC of butyrate was obtained, increasing also butyrate/acetate ratio to 16.4. The results are promising for further research on expanding the product portfolio of MES.

Conversion of sewage sludge to commodity chemicals via syngas fermentation.

Gasification of sewage sludge allows the recovery of energy, and produces a mix of CO, CO2and H2called synthesis gas (or syngas), which can be fermented by acetogenic bacteria to added-value products. This work presents the conversion of syngas to organic acids and alcohols using both pure and mixed cultures. Pure culture kinetic experiments with Clostridium carboxidivorans P7 resulted in the production of high concentrations of acetate (454 mgC/L) and ethanol (167 mgC/L). The pH was the main factor driving solventogenesis, with about 50% of the products in the form of alcohols at pH 5. Conversely, laboratory-scale experiments using a carboxydotrophic mixed culture of the genus Clostridium enriched from anaerobic digester sludge of a municipal wastewater treatment plant was capable of producing mainly butyrate, with maximum concentration of 1,184 mgC/L.

Incubation at 25 °C prevents acid crash and enhances alcohol production in Clostridium carboxidivorans P7.

Incubation of carboxydotrophs at 37 °C provides optimal conditions for their growth. However, a fast accumulation of organic acids, specifically acetate, during the exponential growth phase may result in low alcohol production and substrate consumption due to a phenomenon known as "acid crash". The present work investigates growth and productivity of Clostridium carboxidivorans P7 at two incubation temperatures. At 37 °C the culture was not able to override the "acid crash", resulting in low ethanol titers (1.56 mM). On the other hand, lower metabolic rates at 25 °C enhanced ethanol and butanol production (32.1 and 14.5 mM, respectively). Moreover, at low temperatures, hexanol and caproic acid were also produced at significant concentrations, 8.21 and 9.02 mM respectively, among the highest values reported for P7. Our results demonstrate that production of biofuels with longer carbon chains molecules may be enhanced incubating syngas-fermenting acetogenic bacteria at sub-optimal temperatures.

Impact of formate on the growth and productivity of Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7 grown on syngas

The current energy model based on fossil fuels is coming to an end due to the increase in global energy demand. Biofuels such as ethanol and butanol can be produced through the syngas fermentation by acetogenic bacteria. The present work hypothesizes that formate addition would positively impact kinetic parameters for growth and alcohol production in Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7 by diminishing the need for reducing equivalents. Fermentation experiments were conducted using completely anaerobic batch cultures at different pH values and formate concentrations. PETC cultures were more tolerant to formate concentrations than P7, specially at pH 5.0 and 6.0. Complete growth inhibition of PETC occurred at sodium formate concentrations of 30.0 mM; however, no differences in growth rates were observed at pH 7.0 for the two strains. Incubation at formate concentrations lower than 2.0 mM resulted in increased growth rates for both strains. The most recognizable effects of formate addition on the fermentation products were the increase in the total carbon fixed into acids and alcohols at pH 5.0 and pH 6.0, as well as, a higher ethanol to total products ratio at pH 7.0. Taken all together, these results show the ability of acetogens to use formate diminishing the energy demand for growth, and enhancing strain productivity (AU)

No disponible

Impact of formate on the growth and productivity of Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7 grown on syngas.

The current energy model based on fossil fuels is coming to an end due to the increase in global energy demand. Biofuels such as ethanol and butanol can be produced through the syngas fermentation by acetogenic bacteria. The present work hypothesizes that formate addition would positively impact kinetic parameters for growth and alcohol production in Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7 by diminishing the need for reducing equivalents. Fermentation experiments were conducted using completely anaerobic batch cultures at different pH values and formate concentrations. PETC cultures were more tolerant to formate concentrations than P7, specially at pH 5.0 and 6.0. Complete growth inhibition of PETC occurred at sodium formate concentrations of 30.0 mM; however, no differences in growth rates were observed at pH 7.0 for the two strains. Incubation at formate concentrations lower than 2.0 mM resulted in increased growth rates for both strains. The most recognizable effects of formate addition on the fermentation products were the increase in the total carbon fixed into acids and alcohols at pH 5.0 and pH 6.0, as well as, a higher ethanol to total products ratio at pH 7.0. Taken all together, these results show the ability of acetogens to use formate diminishing the energy demand for growth, and enhancing strain productivity.

Changes of the phenol-degrading bacterial community during the decomposition of submersed Platanus acerifolia leaves.

Microorganisms are responsible for the decomposition of plant litter due to their enhanced enzyme capabilities. Among extracellular enzymes, those involved in lignin decomposition are especially relevant in leaf degradation. However, the knowledge of the bacterial contribution to the decomposition of phenol-derived compounds in submerged leaf litter is limited. We have used the large unit of the multicomponent bacterial phenol hydroxylase (LmpH) as a genetic proxy to describe changes in the phenol-degrading bacterial community during the decomposition of Platanus acerifolia leaves in a forested stream. Significant differences were found in the phenol-degrading community when three decomposition stages, initial (day 7), midterm (day 58), and late (day 112), were compared. Estimated Shannon's diversity values decreased significantly from 1.93 (initial) to 0.98 (late). According to the deduced amino acid sequences and the corresponding theoretical kinetic parameters of phenol hydroxylases, the initial community showed a low degree of specialization, presumably resulting from random colonization of leaves. At the late decomposition stage, the bacterial community became more specialized, and LmpH genes similar to high-affinity phenol hydroxylases of Comamonas sp. and Burkholderia cepacia increased. The observed changes in the bacterial community suggested an active role of bacteria during litter decomposition in aquatic environments.