A microbial signature for Crohn's disease.

OBJECTIVE: A decade of microbiome studies has linked IBD to an alteration in the gut microbial community of genetically predisposed subjects. However, existing profiles of gut microbiome dysbiosis in adult IBD patients are inconsistent among published studies, and did not allow the identification of microbial signatures for CD and UC. Here, we aimed to compare the faecal microbiome of CD with patients having UC and with non-IBD subjects in a longitudinal study. DESIGN: We analysed a cohort of 2045 non-IBD and IBD faecal samples from four countries (Spain, Belgium, the UK and Germany), applied a 16S rRNA sequencing approach and analysed a total dataset of 115 million sequences. RESULTS: In the Spanish cohort, dysbiosis was found significantly greater in patients with CD than with UC, as shown by a more reduced diversity, a less stable microbial community and eight microbial groups were proposed as a specific microbial signature for CD. Tested against the whole cohort, the signature achieved an overall sensitivity of 80% and a specificity of 94%, 94%, 89% and 91% for the detection of CD versus healthy controls, patients with anorexia, IBS and UC, respectively. CONCLUSIONS: Although UC and CD share many epidemiologic, immunologic, therapeutic and clinical features, our results showed that they are two distinct subtypes of IBD at the microbiome level. For the first time, we are proposing microbiomarkers to discriminate between CD and non-CD independently of geographical regions.

A single mutation in the gene responsible for the mucoid phenotype of Bifidobacterium animalis subsp. lactis confers surface and functional characteristics.

Exopolysaccharides (EPS) are extracellular carbohydrate polymers synthesized by a large variety of bacteria. Their physiological functions have been extensively studied, but many of their roles have not yet been elucidated. We have sequenced the genomes of two isogenic strains of Bifidobacterium animalis subsp. lactis that differ in their EPS-producing phenotype. The original strain displays a nonmucoid appearance, and the mutant derived thereof has acquired a mucoid phenotype. The sequence analysis of their genomes revealed a nonsynonymous mutation in the gene Balat_1410, putatively involved in the elongation of the EPS chain. By comparing a strain from which this gene had been deleted with strains containing the wild-type and mutated genes, we were able to show that each strain displays different cell surface characteristics. The mucoid EPS synthesized by the strain harboring the mutation in Balat_1410 provided higher resistance to gastrointestinal conditions and increased the capability for adhesion to human enterocytes. In addition, the cytokine profiles of human peripheral blood mononuclear cells and ex vivo colon tissues suggest that the mucoid strain could have higher anti-inflammatory activity. Our findings provide relevant data on the function of Balat_1410 and reveal that the mucoid phenotype is able to alter some of the most relevant functional properties of the cells.

Anal gas evacuation and colonic microbiota in patients with flatulence: effect of diet.

OBJECTIVE: To characterise the influence of diet on abdominal symptoms, anal gas evacuation, intestinal gas distribution and colonic microbiota in patients complaining of flatulence. DESIGN: Patients complaining of flatulence (n=30) and healthy subjects (n=20) were instructed to follow their usual diet for 3 days (basal phase) and to consume a high-flatulogenic diet for another 3 days (challenge phase). RESULTS: During basal phase, patients recorded more abdominal symptoms than healthy subjects in daily questionnaires (5.8±0.3 vs 0.4±0.2 mean discomfort/pain score, respectively; p=<0.0001) and more gas evacuations by an event marker (21.9±2.8 vs 7.4±1.0 daytime evacuations, respectively; p=0.0001), without differences in the volume of gas evacuated after a standard meal (262±22 and 265±25 mL, respectively). On flatulogenic diet, both groups recorded more abdominal symptoms (7.9±0.3 and 2.8±0.4 discomfort/pain, respectively), number of gas evacuations (44.4±5.3 and 21.7±2.9 daytime evacuations, respectively) and had more gas production (656±52 and 673±78 mL, respectively; p<0.05 vs basal diet for all). When challenged with flatulogenic diet, patients' microbiota developed instability in composition, exhibiting variations in the main phyla and reduction of microbial diversity, whereas healthy subjects' microbiota were stable. Taxa from Bacteroides fragilis or Bilophila wadsworthia correlated with number of gas evacuations or volume of gas evacuated, respectively. CONCLUSIONS: Patients complaining of flatulence have a poor tolerance of intestinal gas, which is associated with instability of the microbial ecosystem.

Reshaping the gut microbiome with bacterial transplantation and antibiotic intake.

The intestinal microbiota consists of over 1000 species, which play key roles in gut physiology and homeostasis. Imbalances in the composition of this bacterial community can lead to transient intestinal dysfunctions and chronic disease states. Understanding how to manipulate this ecosystem is thus essential for treating many disorders. In this study, we took advantage of recently developed tools for deep sequencing and phylogenetic clustering to examine the long-term effects of exogenous microbiota transplantation combined with and without an antibiotic pretreatment. In our rat model, deep sequencing revealed an intestinal bacterial diversity exceeding that of the human gut by a factor of two to three. The transplantation produced a marked increase in the microbial diversity of the recipients, which stemmed from both capture of new phylotypes and increase in abundance of others. However, when transplantation was performed after antibiotic intake, the resulting state simply combined the reshaping effects of the individual treatments (including the reduced diversity from antibiotic treatment alone). Therefore, lowering the recipient bacterial load by antibiotic intake prior to transplantation did not increase establishment of the donor phylotypes, although some dominant lineages still transferred successfully. Remarkably, all of these effects were observed after 1 mo of treatment and persisted after 3 mo. Overall, our results indicate that the indigenous gut microbial composition is more plastic that previously anticipated. However, since antibiotic pretreatment counterintuitively interferes with the establishment of an exogenous community, such plasticity is likely conditioned more by the altered microbiome gut homeostasis caused by antibiotics than by the primary bacterial loss.

Gut Microbiota Composition in Long-Remission Ulcerative Colitis is Close to a Healthy Gut Microbiota.

BACKGROUND: Microbiome studies report low gut microbial richness and diversity in ulcerative colitis (UC) patients. We explored whether UC patients who reach long-term clinical, endoscopic, and histological remission show a gut microbial ecosystem that is similar to healthy individuals. METHODS: We collected 184 stool samples from 111 individuals (UC patients in long remission, short remission, flare, and healthy control subjects). Microbiota was analyzed by amplicon sequencing (16S ribosomal RNA) and quantitative polymerase chain reaction for specific taxa. All UC remission patients were followed-up for 2 years. FINDINGS: A drop in species diversity and richness, underrepresentation of butyrate producers, and gain of potentially harmful bacteria were significantly detected in samples from disease-flare and short-remission patients. In contrast, Chao1 and Shannon indexes of diversity did not differ among patients in long remission and healthy control subjects. Long-remission patients also presented fecal bacterial composition closer to that in healthy control subjects. There was a positive correlation between Akkermansia muciniphila abundance and time in remission (rs = 0.53, P < .001). Logistic regression analysis showed that a high Shannon index (odds ratio, 4.83; 95% confidence interval, 1.5-20.6) or presence of A. muciniphila (odds ratio, 4.9; 95% confidence interval, 1.12-29.08) in fecal samples at entry was independently associated with clinical remission over a follow-up period of 24 months. INTERPRETATION: UC patients who achieve long-term remission show evidence of substantial recovery of the gut microbial ecosystem in terms of diversity and composition. Recovery may just reflect adequate control of inflammatory activity, but higher bacterial diversity or the presence of A. muciniphila in fecal samples predicts flare-free outcomes.

Microbiome studies have shown low gut microbial richness and diversity in ulcerative colitis patients. Patients who achieve long-term remission show evidence of substantial recovery of the gut microbial ecosystem in terms of diversity and composition.

Mutual modulation of gut microbiota and the immune system in type 1 diabetes models.

The transgenic 116C-NOD mouse strain exhibits a prevalent Th17 phenotype, and reduced type 1 diabetes (T1D) compared to non-obese diabetic (NOD) mice. A cohousing experiment between both models revealed lower T1D incidence in NOD mice cohoused with 116C-NOD, associated with gut microbiota changes, reduced intestinal permeability, shifts in T and B cell subsets, and a transition from Th1 to Th17 responses. Distinct gut bacterial signatures were linked to T1D in each group. Using a RAG-2-/- genetic background, we found that T cell alterations promoted segmented filamentous bacteria proliferation in young NOD and 116C-NOD, as well as in immunodeficient NOD.RAG-2-/- and 116C-NOD.RAG-2-/- mice across all ages. Bifidobacterium colonization depended on lymphocytes and thrived in a non-diabetogenic environment. Additionally, 116C-NOD B cells in 116C-NOD.RAG-2-/- mice enriched the gut microbiota in Adlercreutzia and reduced intestinal permeability. Collectively, these results indicate reciprocal modulation between gut microbiota and the immune system in rodent T1D models.

Dysbiosis and relapse-related microbiome in inflammatory bowel disease: A shotgun metagenomic approach.

Crohn's disease (CD) and ulcerative colitis (UC), the two main forms of inflammatory bowel disease (IBD), affect several million people worldwide. CD and UC are characterized by periods of clinical remission and relapse. Although IBD patients present chronic alterations of the gut microbiome, called dysbiosis, little attention has been devoted to the relapse-related microbiome. To address this gap, we generated shotgun metagenomic data from the stools of two European cohorts-134 Spanish (followed up for one year) and 49 Belgian (followed up for 6 months) subjects-to characterize the microbial taxonomic and metabolic profiles present. To assess the predictive value of microbiome data, we added the taxonomic profiles generated from a previous study of 130 Americans. Our results revealed that CD was more dysbiotic than UC compared to healthy controls (HC) and that strategies for energy extraction and propionate production were different in CD compared to UC and HC. Remarkably, CD and UC relapses were not associated with alpha- or beta-diversity, or with a dysbiotic score. However, CD relapse was linked to alterations at the species and metabolic pathway levels, including those involved in propionate production. The random forest method using taxonomic profiles allowed the prediction of CD vs. non-CD with an AUC = 0.938, UC vs. HC with an AUC = 0.646, and CD relapse vs. remission with an AUC = 0.769. Our study validates previous taxonomic findings, points to different relapse-related growth and defence mechanisms in CD compared to UC and HC and provides biomarkers to discriminate IBD subtypes and predict disease activity.

Mucosal microbial load in Crohn's disease: A potential predictor of response to faecal microbiota transplantation.

BACKGROUND: The remission of Crohn's disease (CD) can be accomplished by faecal microbiota transplantation (FMT). However, this procedure has a low success rate, which could be attributed to mis-communication between recipient intestinal mucosa and donor microbiota. METHODS: Here we used a human explant tissue model and an in vivo mouse model to examine changes in recipient intestinal mucosa upon contact with a faecal suspension (FS) obtained from a healthy donor. CD patients provided resected inflamed and non-inflamed mucosal tissues, whereas control colonic mucosa samples were collected from colorectal cancer patients. For the models, mucosal microbiome composition and tissue response were evaluated. FINDINGS: We show that cytokine release and tissue damage were significantly greater in inflamed compared to non-inflamed CD tissues. Moreover, mucosal samples harbouring an initial low microbial load presented a shift in composition towards that of the FS, an increase in the relative count of Faecalibacterium prausnitzii, and a higher secretion of anti-inflammatory cytokine IL-10 compared to those with a high microbial load. INTERPRETATION: Our results indicate that FMT during active inflammatory disease can compromise treatment outcome. We recommend the stratification of FMT recipients on the basis of tissue microbial load as a strategy to ensure successful colonization. FUNDING: This study was supported by grants from the Instituto de Salud Carlos III/FEDER (PI17/00614), the European Commission: (INCOMED-267128) and PERIS (SLT002/16). K.M. is a postdoctoral fellow and S.V. a senior clinical investigator of the Fund for Scientific Research Flanders, Belgium (FWO-Vlaanderen).

The gut microbiota predispose to the pathophysiology of acute postradiotherapy diarrhea.

INTRODUCTION: Severe diarrhea may complicate pelvic radiotherapy and force interruption of treatment. As there is no current clinical or experimental information on the role of the gut microbiota in this pathogenesis, we conducted a pilot observational study on the fecal microbiota in patients receiving pelvic radiotherapy. METHODS: The study involved 10 patients who underwent 5 wk of radiotherapy for abdominal tumors and 5 controls. Four fecal samples were collected from each individual: before, during, at the end, and 2 wk after treatment. Following the amplification of the bacterial 16S rRNA gene from the samples, DNA fingerprinting and cloning-sequencing techniques were used to determine their microbial profile and composition, respectively. RESULTS: Six patients suffered acute postradiotherapy diarrhea and 4 did not. In patients without diarrhea, as well as in healthy volunteers, microbial diversity was stable over a period of 7 wk. However, patients exhibiting diarrhea showed a progressive modification in their microbial diversity. A radical drop in similarity index was observed at the end (P= 0.026) and still 2 wk after radiotherapy (P= 0.014). Interestingly, cluster analysis of the microbial profile in the first sample (S1) (collected before radiotherapy) displayed a dendogram where patients that presented diarrhea clustered separately from those that did not develop diarrhea after radiotherapy. Moreover, sequence analysis of dominant bacteria in the S1 sample confirmed differences between the diarrhea and nondiarrhea groups. DISCUSSION: In this set of patients, susceptibility or protection against diarrhea after radiotherapy could be linked to different initial microbial colonization.

Activation of Immune and Defense Responses in the Intestinal Mucosa by Outer Membrane Vesicles of Commensal and Probiotic Escherichia coli Strains.

The influence of microbiota in human health is well-known. Imbalances in microbiome structure have been linked to several diseases. Modulation of microbiota composition through probiotic therapy is an attempt to harness the beneficial effects of commensal microbiota. Although, there is wide knowledge of the responses induced by gut microbiota, the microbial factors that mediate these effects are not well-known. Gram-negative bacteria release outer membrane vesicles (OMVs) as a secretion mechanism of microbial factors, which have an important role in intercellular communication. Here, we investigated whether OMVs from the probiotic Escherichia coli strain Nissle 1917 (EcN) or the commensal E. coli strain ECOR12 trigger immune responses in various cellular models: (i) peripheral blood mononuclear cells (PBMCs) as a model of intestinal barrier disruption, (ii) apical stimulation of Caco-2/PMBCs co-culture as a model of intact intestinal mucosa, and (iii) colonic mucosa explants as an ex vivo model. Stimulations with bacterial lysates were also performed. Whereas, both OMVs and lysates activated expression and secretion of several cytokines and chemokines in PBMCs, only OMVs induced basolateral secretion and mRNA upregulation of these mediators in the co-culture model. We provide evidence that OMVs are internalized in polarized Caco-2 cells. The activated epithelial cells elicit a response in the underlying immunocompetent cells. The OMVs effects were corroborated in the ex vivo model. This experimental study shows that OMVs are an effective strategy used by beneficial gut bacteria to communicate with and modulate host responses, activating signaling events through the intestinal epithelial barrier.

Transcriptional interactions suggest niche segregation among microorganisms in the human gut.

The human gastrointestinal (GI) tract is the habitat for hundreds of microbial species, of which many cannot be cultivated readily, presumably because of the dependencies between species1. Studies of microbial co-occurrence in the gut have indicated community substructures that may reflect functional and metabolic interactions between cohabiting species2,3. To move beyond species co-occurrence networks, we systematically identified transcriptional interactions between pairs of coexisting gut microbes using metagenomics and microarray-based metatranscriptomics data from 233 stool samples from Europeans. In 102 significantly interacting species pairs, the transcriptional changes led to a reduced expression of orthologous functions between the coexisting species. Specific species-species transcriptional interactions were enriched for functions important for H2 and CO2 homeostasis, butyrate biosynthesis, ATP-binding cassette (ABC) transporters, flagella assembly and bacterial chemotaxis, as well as for the metabolism of carbohydrates, amino acids and cofactors. The analysis gives the first insight into the microbial community-wide transcriptional interactions, and suggests that the regulation of gene expression plays an important role in species adaptation to coexistence and that niche segregation takes place at the transcriptional level.

Xanthine oxidoreductase and preservation injury in human liver transplantation.

BACKGROUND: Preservation injury is a major cause of primary graft dysfunction in liver transplantation (LT). Oxidative damage is considered to be the first event leading to graft damage. Xanthine oxidoreductase (XOR) and neutrophil activation, two sources of reactive oxygen species, could play a role in the development of graft dysfunction. METHODS: We determined activities of XOR forms, polymorphonuclear elastase (PMN-E), aminotransferases, and hyaluronic acid in plasma of 20 patients undergoing LT. Samples were taken from the radial artery (RA) before the anhepatic phase; from the portal vein (PV) before reperfusion; from graft caval effluent (CE) at reperfusion; and from RA, PV, and the hepatic vein (HV) 10 and 90 min postreperfusion. RESULTS: The graft, but not recipient bowel, released XOR into blood (XOR in CE, median, 61.2 mU/g protein [range, 1.9-160.4 vs. undetectable in PV before reperfusion). Circulating XOR was transformed from dehydrogenase to reversible oxidase (XOrev) (XOrev-to-XOR ratio, 48.1% in CE and 65.1% in HV 90 min postreperfusion). Neutrophil activation was detected in the recipients before reperfusion, and in liver at early post-reperfusion (median PMN-E was 0.85 microg/g protein [range, 0.01-1.58] in RA before the anhepatic phase; 2.22 microg/g protein [range, 0.20-5.88] in PV prereperfu-sion; and 3.60 microg/g protein [range, 0.48-6.78] in HV 10 min postreperfusion). XOR, but none of the other markers, was higher in the CE of patients with moderate primary graft dysfunction than in those with slight primary graft dysfunction. CONCLUSIONS: XOR release and neutrophil activation are produced during LT, and they are potentially injurious mechanisms associated with this therapy.

Aortic valve-derived calcifyng nanoparticles: no evidence of life.

INTRODUCTION AND OBJECTIVES: Calcifying nanoparticles, also known as "nanobacteria," are very small bacteria-like structures (0.1-0.5 µm) with the ability to facilitate the precipitation and growth of calcium phosphate in pathological conditions and have been associated with aortic valve calcification. The status of nanobacteria is controversial; some have proposed that they are a new class of living organism while others describe calcifying nanoparticles as mineralo-fetuin complexes. The objective of the present study is to elucidate if calcifying nanoparticles are living entities, based on whether or not they have metabolic activity, a characteristic of life, irrespective of their composition. METHODS: Calcifying nanoparticles were grown from 6 different valves randomly chosen among 84 consecutively explanted aortic valves, as described in the literature. The (1)H-NMR spectra were acquired from calcifying nanoparticles culture media to assess metabolic changes and the presence of 16sRNA in the culture media was investigated by real-time polymerase chain reaction. RESULTS: After 6 weeks in culture, calcifying nanoparticles could be seen clearly attached to the surface of culture flasks. All samples were negative for 16sRNA, discarding the presence of known bacteria. (1)H-NMR spectra showed no difference between calcifying nanoparticles and 6-week-old sterile culture media maintained under the same conditions. CONCLUSIONS: Our results show that calcifying nanoparticles cannot be considered as living organisms.

Polymorphonuclear elastase in the early diagnosis of complicated pyogenic pleural effusions.

BACKGROUND: Polymorphonuclear elastase (PMN-E) is a neutrophilic marker that has been implicated in acute inflammatory responses. OBJECTIVES: To evaluate the accuracy of PMN-E in the diagnosis of complicated pyogenic effusions. PATIENTS AND METHOD: We studied 536 patients with pleural effusion of various etiologies. There were 125 pyogenic bacterial effusions (42 typical parapneumonic, 17 borderline complicated parapneumonic and 66 complicated parapneumonic or empyema), 83 tuberculous, 91 malignant, 42 paramalignant, 95 transudates, 28 miscellaneous and 72 effusions of unknown origin. Classic markers (pH, glucose, proteins, adenosine deaminase, LDH, leukocytes and differential count) and the PMN-E level were quantified in pleural fluid. The accuracy of PMN-E as an early marker in the diagnosis of complicated pyogenic infectious effusions was evaluated among pleural effusions that were not diagnosed with classic biochemical markers, radiological findings or Gram stain. Since results of pleural fluid culture and cytological examination are generally available after a 48-hour delay, they were not included as early markers in the initial diagnosis of pleural effusions. RESULTS: Early diagnosis of complicated pyogenic bacterial effusions was achieved in only 48 of 66 cases with classic markers. Among those that were not diagnosed with these parameters, a pleural PMN-E value >3,500 microg/l discriminated between complicated and noncomplicated pyogenic bacterial effusions with a sensitivity of 67% and a specificity of 97%. CONCLUSIONS: PMN-E is useful in the early diagnosis and management of complicated pyogenic infectious effusions, which may be delayed with classic markers.

Association between inflammatory mediators and the fibrinolysis system in infectious pleural effusions.

The response of the fibrinolytic system to inflammatory mediators in empyema and complicated parapneumonic pleural effusions is still uncertain. We prospectively analysed 100 patients with pleural effusion: 25 with empyema or complicated parapneumonic effusion, 22 with tuberculous effusion, 28 with malignant effusion and 25 with transudate effusion. Inflammatory mediators, tumour necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8) and polymorphonuclear elastase, were measured in serum and pleural fluid. Fibrinolytic system parameters, plasminogen, tissue-type plasminogen activator (t-PA) and urokinase PA, PA inhibitor type 1 (PAI 1) and PAI type 2 concentrations and PAI 1 activity, were quantified in plasma and pleural fluid. The Wilcoxon signed-rank test was used to compare plasma and pleural values and to compare pleural values according to the aetiology of the effusion. The Pearson correlation coefficient was used to assess the relationship between fibrinolytic and inflammatory markers in pleural fluid. Significant differences were found between pleural and plasma fibrinolytic system levels. Pleural fluid exudates had higher fibrinolytic levels than transudates. Among exudates, tuberculous, empyema and complicated parapneumonic effusions demonstrated higher pleural PAI levels than malignant effusions, whereas t-PA was lowest in empyema and complicated parapneumonic pleural effusions. PAI concentrations correlated with TNF-alpha, IL-8 and polymorphonuclear elastase when all exudative effusions were analysed, but the association was not maintained in empyema and complicated parapneumonic effusions. A negative association found between t-PA and both IL-8 and polymorphonuclear elastase in exudative effusions was strongest in empyema and complicated parapneumonic effusions. Blockage of fibrin clearance in empyema and complicated parapneumonic effusions was associated with both enhanced levels of PAIs and decreased levels of t-PA.

Nanopartículas derivadas de válvula aórtica: sin evidencias de vida / Aortic valve-derived calcifyng nanoparticles: no evidence of life

Introducción y objetivos. Las nanopartículas calcificantes, tambien conocidas como nanobacterias, son estructuras, similares a las bacterias, de reducido tamaño (0,1-0,5 μm) que tienen la capacidad de facilitar la precipitación y el crecimiento de fosfato cálcico en condiciones patológicas y a las que se ha asociado con la calcificación de la válvula aórtica. La clasificación de las nanobacterias como organismos no está exenta de controversia; algunos autores apuntan que son una nueva clase de organismo vivo, mientras que otros las describen como complejos mineraloproteicos. El objetivo del trabajo es clarificar si las nanopartículas calcificantes son entes vivos basándonos en si tienen o no actividad metabólica, característica de los seres vivos independiente de su composición. Métodos. Las nanopartículas calcificantes se cultivaron a partir de seis válvulas aórticas seleccionadas aleatoriamente de entre 84 válvulas aórticas explantadas consecutivamente, como se ha descrito en la literatura. Se obtuvieron espectros 1H-RMN de los medios de cultivo de las nanopartículas calcificantes para evaluar posibles cambios metabólicos; mediante reacción en cadena de la polimerasa en tiempo real, se investigó la presencia de 16sRNA. Resultados. A las 6 semanas de cultivo, la presencia de nanopartículas calcificantes se observa claramente como una monocapa unida a la superficie del frasco de cultivo. Todas las muestras resultaron negativas para la presencia de 16sRNA, lo que descartó la presencia de bacterias conocidas en los cultivos. Los espectros 1H-RMN no mostraron diferencias entre los cultivos de nanopartículas calcificantes y medio de cultivo estéril en las mismas condiciones. Conclusiones. Nuestros resultados demuestran que no se puede considerar organismos vivos a las nanopartículas calcificantes (AU)

Introduction and objectives. Calcifying nanoparticles, also known as "nanobacteria", are very small bacteria-like structures (0.1-0.5μm) with the ability to facilitate the precipitation and growth of calcium phosphate in pathological conditions and have been associated with aortic valve calcification. The status of nanobacteria is controversial; some have proposed that they are a new class of living organism while others describe calcifying nanoparticles as mineralo-fetuin complexes. The objective of the present study is to elucidate if calcifying nanoparticles are living entities, based on whether or not they have metabolic activity, a characteristic of life, irrespective of their composition. Methods. Calcifying nanoparticles were grown from 6 different valves randomly chosen among 84 consecutively explanted aortic valves, as described in the literature. The 1H-NMR spectra were acquired from calcifying nanoparticles culture media to assess metabolic changes and the presence of 16sRNA in the culture media was investigated by real-time polymerase chain reaction. Results. After 6 weeks in culture, calcifying nanoparticles could be seen clearly attached to the surface of culture flasks. All samples were negative for 16sRNA, discarding the presence of known bacteria. 1H-NMR spectra showed no difference between calcifying nanoparticles and 6-week-old sterile culture media maintained under the same conditions. Conclusions. Our results show that calcifying nanoparticles cannot be considered as living organisms (AU)