Effects of Fermentation Temperature, Drying Temperature, Caliber Size, Starter Culture, and Sodium Lactate on Listeria monocytogenes Inactivation During Salami Production.

The effect of fermentation and drying temperatures, caliber, and sodium lactate on Listeria monocytogenes inactivation was studied in salami, produced in a pilot scale, inoculated with 107 CFU/g of Listeria innocua ATCC® 33090 as a surrogate microorganism for L. monocytogenes. Fermentation temperature varied between 24 and 30°C, drying temperature between 14 and 20°C, caliber between 5.1 and 13.2 cm, and sodium lactate initial concentrations in salamis were 0 and 2%. L. innocua counts, pH and water activity were determined in salamis over time. Sodium lactate (2%) decreased pH drop and Listeria inactivation during fermentation. Baranyi & Roberts equation was used to fit the experimental data and to estimate, for each test condition, inactivation rate (k), initial (Y0), and final counts of L. innocua (YEND). Total inactivation was calculated as Y0 minus YEND (Y0-YEND). Then, using a Box Benkhen experimental design, a quadratic model for k and a two-factor interaction model (2FI) for Y0 - YEND were obtained as functions of fermentation temperature, drying temperature, and caliber size. The models predicted that maximum k and Y0 -YEND, -2.62 ± 0.14 log10 CFU/g/day and 4.5 ± 0.1 log10 CFU/g, respectively, would be obtained fermenting at 30°C and drying at 20°C regardless of caliber. Drying at 14°C allowed Listeria growth until a water activity (aw) of 0.92 was reached. Therefore, if initial Listeria contamination is high (3 log10 CFU/g), drying at low temperatures will compromise product safety.

Lectin-Mimicking Aptamer as a Generic Glycan Receptor for Sensitive Detection of Glycoproteins Associated with Cancer.

The shortage of specific glycan recognition reagents has proven a significant hurdle in the development of assays to detect altered glycoforms associated with cancer. Here, a carbohydrate-binding aptamer originally selected against the glycan moiety of prostate-specific antigen (PSA) is used as a lectin-mimicking reagent. As a first proof-of-principle, this aptamer has been applied to develop a sandwich-type electrochemical biosensor for the detection of the serum amyloid P (SAP) component, a glycosylated protein whose increased sialylation has been associated with pancreatic cancer. The assay combines a specific antibody for this potential tumor biomarker and the aptamer as capture and detection receptors, respectively. Two oriented antibody immobilization approaches, protein A-based and boronic ester-based attachment to self-assembled monolayers built onto gold surfaces, were comparatively evaluated, the latter being able to circumvent the unwanted interaction between the aptamer and the glycans on the electrode-attached antibody. The resulting biosensing platform allows the detection of the SAP glycoprotein at levels of nanograms per milliliter with a reproducibility value lower than 20%, both in aqueous buffer and in serum. This work represents a proof-of-concept of a promiscuous ligand of proteins with high levels of sialylated glycans typically produced by cancer cells.

Fusarium Species and Mycotoxins Associated with Sorghum Grains in Uruguay.

Grain mold and stalk rot are among the fungal diseases that cause significant losses in sorghum worldwide and are caused by different Fusarium spp. The presence of Fusarium species in sorghum grains causes yield losses and mycotoxin contamination, which represents a risk to consumers. In this study, Fusarium graminearum species complex (FGSC) had a high incidence, followed by Fusarium fujikuroi species complex (FFSC) and F. incarnatum-equiseti species complex. Within FFSC, F. proliferatum, F. andiyazi, F. fujikuroi, F. thapsinum, F. verticillioides and F. subglutinans were identified, and this was the first report of F. fujikuroi in sorghum. The most frequent toxins found in sorghum samples were deoxynivalenol (DON) and zearalenone (ZEN). The presence of fumonisins and nivalenol (NIV) was detected at low levels. This study adds new knowledge about the occurrence of Fusarium species and mycotoxins in sorghum grains. Furthermore, this is the first report in Uruguay on fungicide sensitivity for Fusarium isolates from sorghum, which constitutes an important starting point for defining management practices to minimize fungal infection and mycotoxin contamination.

Transfer of ß-lactam and tetracycline antibiotics from spiked bovine milk to Dambo-type cheese, whey, and whey powder.

The aim of this study was to investigate the transfer of residues of five ß-lactam antibiotics (ampicillin, penicillin G, cloxacillin, dicloxacillin and cephalexin) and two tetracyclines (tetracycline and oxytetracycline) in the processing of cheese and whey powder, evaluating the effect of the processes and the final concentration in each product generated. Raw milk was fortified at two concentration levels with the seven antibiotics. The first concentration level (C1) was chosen according to the maximum residue limit (MRL) of each antibiotic (ampicillin and penicillin G: 4 µg kg-1; cloxacillin and dicloxacillin: 30 µg kg-1; cephalexin, tetracycline and oxytetracycline: 100 µg kg-1). The second concentration level (C2) was spiked as follows according to each antibiotic: 0.5 MRL (cloxacillin, dicloxacillin, cephalexin), 0.1 MRL (tetracycline and oxytetracycline) and 3 MRL (ampicillin and penicillin G). The antibiotics were analyzed by LC-MS/MS. No ampicillin or penicillin G residues were found in cheese or whey powder, although they were detected in whey at concentrations similar to those added to raw milk. Cephalexin was mostly distributed in whey between 82% and 96%, being the antibiotic that presented the highest concentration in whey powder (784 ± 98 µg kg-1) when milk was spiked at the MRL. The whey distribution of cloxacillin and dicloxacillin ranged from 57% to 59% for cloxacillin and from 46% to 48% for dicloxacillin, and both concentrated in whey powder. Tetracyclines were the antibiotics that concentrated in cheese, with retentions between 75% and 80% for oxytetracycline and between 83% and 87% for tetracycline. The distribution of antibiotics in the dissimilar stages of the cheese and whey powder production processes, as well as their concentration in the final products, depend on each type of antibiotic. Knowledge of the transfer of antibiotic residues during the process and final disposal is an input for the risk assessment of their consumption.

Phenotypic and genotypic characterization of Shiga toxin-producing Escherichia coli strains recovered from bovine carcasses in Uruguay.

Introduction: Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that cause food-borne diseases in humans. Cattle and derived foodstuffs play a known role as reservoir and vehicles, respectively. In Uruguay, information about the characteristics of circulating STEC in meat productive chain is scarce. The aim was to characterize STEC strains recovered from 800 bovine carcasses of different slaughterhouses. Methods: To characterize STEC strains we use classical microbiological procedures, Whole Genome Sequencing (WGS) and FAO/WHO risk criteria. Results: We analyzed 39 STEC isolated from 20 establishments. They belonged to 21 different O-groups and 13 different H-types. Only one O157:H7 strain was characterized and the serotypes O130:H11(6), O174:H28(5), and O22:H8(5) prevailed. One strain showed resistance in vitro to tetracycline and genes for doxycycline, sulfonamide, streptomycin and fosfomycin resistance were detected. Thirty-three strains (84.6%) carried the subtypes Stx2a, Stx2c, or Stx2d. The gene eae was detected only in two strains (O157:H7, O182:H25). The most prevalent virulence genes found were lpfA (n = 38), ompA (n = 39), ompT (n = 39), iss (n = 38), and terC (n = 39). Within the set of STEC analyzed, the majority (81.5%) belonged to FAO/WHO's risk classification levels 4 and 5 (lower risk). Besides, we detected STEC serotypes O22:H8, O113:H21, O130:H11, and O174:H21 belonged to level risk 2 associate with diarrhea, hemorrhagic colitis or Hemolytic-Uremic Syndrome (HUS). The only O157:H7 strain analyzed belonged to ST11. Thirty-eight isolates belonged to the Clermont type B1, while the O157:H7 was classified as E. Discussion: The analyzed STEC showed high genomic diversity and harbor several genetic determinants associated with virulence, underlining the important role of WGS for a complete typing. In this set we did not detect non-O157 STEC previously isolated from local HUS cases. However, when interpreting this findings, the low number of isolates analyzed and some methodological limitations must be taken into account. Obtained data suggest that cattle constitute a local reservoir of non-O157 serotypes associated with severe diseases. Other studies are needed to assess the role of the local meat chain in the spread of STEC, especially those associated with severe diseases in humans.

Experimental evaluation of ecological principles to understand and modulate the outcome of bacterial strain competition in gut microbiomes.

It is unclear if coexistence theory can be applied to gut microbiomes to understand their characteristics and modulate their composition. Through experiments in gnotobiotic mice with complex microbiomes, we demonstrated that strains of Akkermansia muciniphila and Bacteroides vulgatus could only be established if microbiomes were devoid of these species. Strains of A. muciniphila showed strict competitive exclusion, while B. vulgatus strains coexisted but populations were still influenced by competitive interactions. These differences in competitive behavior were reflective of genomic variation within the two species, indicating considerable niche overlap for A. muciniphila strains and a broader niche space for B. vulgatus strains. Priority effects were detected for both species as strains' competitive fitness increased when colonizing first, which resulted in stable persistence of the A. muciniphila strain colonizing first and competitive exclusion of the strain arriving second. Based on these observations, we devised a subtractive strategy for A. muciniphila using antibiotics and showed that a strain from an assembled community can be stably replaced by another strain. By demonstrating that competitive outcomes in gut ecosystems depend on niche differences and are historically contingent, our study provides novel information to explain the ecological characteristics of gut microbiomes and a basis for their modulation.

The RD-Connect Genome-Phenome Analysis Platform: Accelerating diagnosis, research, and gene discovery for rare diseases.

Rare disease patients are more likely to receive a rapid molecular diagnosis nowadays thanks to the wide adoption of next-generation sequencing. However, many cases remain undiagnosed even after exome or genome analysis, because the methods used missed the molecular cause in a known gene, or a novel causative gene could not be identified and/or confirmed. To address these challenges, the RD-Connect Genome-Phenome Analysis Platform (GPAP) facilitates the collation, discovery, sharing, and analysis of standardized genome-phenome data within a collaborative environment. Authorized clinicians and researchers submit pseudonymised phenotypic profiles encoded using the Human Phenotype Ontology, and raw genomic data which is processed through a standardized pipeline. After an optional embargo period, the data are shared with other platform users, with the objective that similar cases in the system and queries from peers may help diagnose the case. Additionally, the platform enables bidirectional discovery of similar cases in other databases from the Matchmaker Exchange network. To facilitate genome-phenome analysis and interpretation by clinical researchers, the RD-Connect GPAP provides a powerful user-friendly interface and leverages tens of information sources. As a result, the resource has already helped diagnose hundreds of rare disease patients and discover new disease causing genes.

Needle in a Whey-Stack: PhRACS as a Discovery Tool for Unknown Phage-Host Combinations.

The field of metagenomics has rapidly expanded to become the go-to method for complex microbial community analyses. However, there is currently no straightforward route from metagenomics to traditional culture-based methods of strain isolation, particularly in (bacterio)phage biology, leading to an investigative bottleneck. Here, we describe a method that exploits specific phage receptor binding protein (RBP)-host cell surface receptor interaction enabling isolation of phage-host combinations from an environmental sample. The method was successfully applied to two complex sample types-a dairy-derived whey sample and an infant fecal sample, enabling retrieval of specific and culturable phage hosts. IMPORTANCE PhRACS aims to bridge the current divide between in silico genetic analyses (i.e., phageomic studies) and traditional culture-based methodology. Through the labeling of specific bacterial hosts with fluorescently tagged recombinant phage receptor binding proteins and the isolation of tagged cells using flow cytometry, PhRACS allows the full potential of phageomic data to be realized in the wet laboratory.

[Experience with 24-hour ambulatory blood pressure monitoring in follow-up of patients with aortic coartacion in a pediatric hospital]. / Experiencia con monitoreo ambulatorio de presión arterial de 24 horas en seguimiento de pacientes con coartación de aorta en un hospital pediátrico.

Introduction: Hypertension (HTA) is an important comorbidity in children with aortic coarctation (COAO) and 24-hour ambulatory blood pressure monitoring (ABPM) allows an accurate diagnosis. Objective: Describe the prevalence of HTA in the office and its recategorization with ABPM Material and methods: Descriptive, observational, retrospective study; It included children between 4 and 18 years with COAO who performed ABPM. PA was registered in the office and ABPM, echocardiogram and medication. Results: 33 patients, 26 men, age 10.2 ± 3.8 years, By PA in the office: 22 normotensive; 8 HTA controlled; 2 preHTA; 1 HTA not medicated. With 32 complete MAP records, they were categorized: normotensive 11, preHTA 7, nocturnal HTA 3, masked HTA 4; HTA controlled 3; Uncontrolled HTA 3 and 1 HTA. Conclusion: The prevalence of hypertension in this population in the office was low. The ABPM recategorized and detected nocturnal HTA and masked HTA.

Introducción: La hipertensión arterial (HTA) es una comorbilidad importante en niños con coartación de aorta (COAO) y el monitoreo ambulatorio de presión arterial de 24horas (MAPA) permite un diagnóstico preciso. Objetivo: Describir la prevalencia de HTA por presión arterial (PA) en consultorio y su recategorización con MAPA Material y Método: Estudio descriptivo, observacional, retrospectivo; incluyó niños entre 4y18 años con COAO que realizaron MAPA. Se registró PA en consultorio y MAPA, ecocardiograma y medicación. Resultados: 33 pacientes, 26 varones, edad 10,2 ± 3,8 años, Por PA en consultorio: 22 normotensos; 8 HTA controlada; 2 preHTA; 1 HTA no medicado. Con 32 registros completos de MAPA, se recategorizaron: normotensos 11, preHTA 7, HTA nocturna 3, HTA enmascarada 4; HTA controlada 3; HTA no controlada 3 y 1 HTA. Conclusión: La prevalencia de HTA en esta población en consultorio fue baja. El MAPA recategorizó y detectó HTA nocturna e HTA enmascarada.

Gut microbiota modulation with long-chain corn bran arabinoxylan in adults with overweight and obesity is linked to an individualized temporal increase in fecal propionate.

BACKGROUND: Variability in the health effects of dietary fiber might arise from inter-individual differences in the gut microbiota's ability to ferment these substrates into beneficial metabolites. Our understanding of what drives this individuality is vastly incomplete and will require an ecological perspective as microbiomes function as complex inter-connected communities. Here, we performed a parallel two-arm, exploratory randomized controlled trial in 31 adults with overweight and class-I obesity to characterize the effects of long-chain, complex arabinoxylan (n = 15) at high supplementation doses (female: 25 g/day; male: 35 g/day) on gut microbiota composition and short-chain fatty acid production as compared to microcrystalline cellulose (n = 16, non-fermentable control), and integrated the findings using an ecological framework. RESULTS: Arabinoxylan resulted in a global shift in fecal bacterial community composition, reduced α-diversity, and the promotion of specific taxa, including operational taxonomic units related to Bifidobacterium longum, Blautia obeum, and Prevotella copri. Arabinoxylan further increased fecal propionate concentrations (p = 0.012, Friedman's test), an effect that showed two distinct groupings of temporal responses in participants. The two groups showed differences in compositional shifts of the microbiota (p ≤ 0.025, PERMANOVA), and multiple linear regression (MLR) analyses revealed that the propionate response was predictable through shifts and, to a lesser degree, baseline composition of the microbiota. Principal components (PCs) derived from community data were better predictors in MLR models as compared to single taxa, indicating that arabinoxylan fermentation is the result of multi-species interactions within microbiomes. CONCLUSION: This study showed that long-chain arabinoxylan modulates both microbiota composition and the output of health-relevant SCFAs, providing information for a more targeted application of this fiber. Variation in propionate production was linked to both compositional shifts and baseline composition, with PCs derived from shifts of the global microbial community showing the strongest associations. These findings constitute a proof-of-concept for the merit of an ecological framework that considers features of the wider gut microbial community for the prediction of metabolic outcomes of dietary fiber fermentation. This provides a basis to personalize the use of dietary fiber in nutritional application and to stratify human populations by relevant gut microbiota features to account for the inconsistent health effects in human intervention studies. TRIAL REGISTRATION: Clinicaltrials.gov, NCT02322112 , registered on July 3, 2015. Video Abstract.

The Kawashima Operation With Simultaneous Preparation for Transcatheter Fontan-Kreutzer Completion.

Patients with functionally single ventricle and interrupted inferior vena cava may develop progressive cyanosis soon after the Kawashima operation. Therefore, early redirection of the hepatic venous return to the pulmonary circulation is recommended. To avoid performing an early redo sternotomy, we propose to prepare these patients for the interventional Fontan-Kreutzer at the time of the Kawashima operation using a technical modification of the approach reported by Prabhu and coworkers in 2017. The technique described here uses an expanded polytetrafluoroethylene conduit interposed between the hepatic veins and the right pulmonary artery. This graft is everted and divided into two portions with a pericardial patch. The lower one is widely opened and anastomosed side-to-side to the atrium. A few months after the operation, percutaneous Fontan-Kreutzer completion can easily be performed using covered stents to open the patch and at the same time close the opening between the conduit and the atrium.

Faecal microbiota from patients with cirrhosis has a low capacity to ferment non-digestible carbohydrates into short-chain fatty acids.

BACKGROUND AND AIMS: Cirrhosis is associated with dysbiosis, but its functional consequences are still largely unknown. Short-chain fatty acids (SCFAs) account for physiological interactions between the gut microbiota and host. Our aim was to assess the impact of cirrhotic dysbiosis on the production of SCFAs. METHODS: Seventeen patients with cirrhosis and 17 controls were selected. Microbiota composition in faecal samples was assessed by next-generation 16S rRNA gene sequencing. SCFAs were measured with GC-MS in faecal samples and after in vitro batch fermentations using arabinoxylan, resistant starch, pectin, and lactulose as substrates. RESULTS: Among the 17 cirrhotic patients (mean age 58, eight males), six, nine and two were, respectively, Child-Pugh class A, B and C. Eleven patients were on oral antibiotics, 11 on lactulose and 13 on proton pump inhibitors. Cirrhotic patients showed marked differences in the composition and diversity of gut microbiome when compared to controls, that were more pronounced with increased severity. Stool samples from cirrhotic patients showed lower SCFAs content and reduced capacity to produce SCFAs in batch fermentations, with butyrate production being the most abnormal. These functional aberrancies were more pronounced with greater liver disease severity. Abundance of Ruminococcus faecis (in family Ruminococcaceae), Faecalicatena fissicatena and Fusicatenibacter saccharivorans (in family Lachnospiraceae) was positively correlated with the SCFAs production. CONCLUSION: Cirrhotic dysbiosis is associated with a decreased capacity to ferment non-digestible carbohydrates into SCFAs, especially into butyrate. These functional abnormalities are more pronounced as disease progresses. These results might inform the design of gut-targeted therapies for cirrhosis.

Inulin-type fructans improve active ulcerative colitis associated with microbiota changes and increased short-chain fatty acids levels.

The intestinal microbiota is involved in ulcerative colitis (UC) pathogenesis. Prebiotics are hypothesized to improve health through alterations to gut microbiota composition and/or activity. Our aim was therefore to determine if inulin-type fructans induce clinical benefits in UC, and identify if benefits are linked to compositional and/or functional shifts of the luminal (fecal) and mucosal (biopsy) bacterial communities. Patients (n = 25) with mild/moderately active UC received 7.5 g (n = 12) or 15 g (n = 13) daily oral oligofructose-enriched inulin (Orafti®Synergy1) for 9 weeks. Total Mayo score, endoscopic activity and fecal calprotectin were assessed. Fecal and mucosal bacterial communities were characterized by 16S rRNA tag sequencing, and short chain fatty acids (SCFA) production were measured in fecal samples. Fructans significantly reduced colitis in the high-dose group, with 77% of patients showing a clinical response versus 33% in the low-dose group (P = 0.04). Fructans increased colonic butyrate production in the 15 g/d dose, and fecal butyrate levels were negatively correlated with Mayo score (r = -0.50; P = 0.036). The high fructan dose led to an increased Bifidobacteriaceae and Lachnospiraceae abundance but these shifts were not correlated with improved disease scores. In summary, this pilot study revealed that 15 g/d dose inulin type fructans in UC produced functional but not compositional shifts of the gut microbiota, suggesting that prebiotic-induced alterations of gut microbiota metabolism are more important than compositional changes for the benefits in UC. The findings warrant future well-powered controlled studies for the use of ß-fructans as adjunct therapy in patients with active UC.

Biodiversity of Streptococcus thermophilus Phages in Global Dairy Fermentations.

Streptococcus thermophilus strains are among the most widely employed starter cultures in dairy fermentations, second only to those of Lactococcus lactis. The extensive application of this species provides considerable opportunity for the proliferation of its infecting (bacterio)phages. Until recently, dairy streptococcal phages were classified into two groups (cos and pac groups), while more recently, two additional groups have been identified (5093 and 987 groups). This highlights the requirement for consistent monitoring of phage populations in the industry. Here, we report a survey of 35 samples of whey derived from 27 dairy fermentation facilities in ten countries against a panel of S. thermophilus strains. This culminated in the identification of 172 plaque isolates, which were characterized by multiplex PCR, restriction fragment length polymorphism analysis, and host range profiling. Based on this characterisation, 39 distinct isolates representing all four phage groups were selected for genome sequencing. Genetic diversity was observed among the cos isolates and correlations between receptor binding protein phylogeny and host range were also clear within this phage group. The 987 phages isolated within this study shared high levels of sequence similarity, yet displayed reduced levels of similarity to those identified in previous studies, indicating that they are subject to ongoing genetic diversification.

Experimental evaluation of the importance of colonization history in early-life gut microbiota assembly.

The factors that govern assembly of the gut microbiota are insufficiently understood. Here, we test the hypothesis that inter-individual microbiota variation can arise solely from differences in the order and timing by which the gut is colonized early in life. Experiments in which mice were inoculated in sequence either with two complex seed communities or a cocktail of four bacterial strains and a seed community revealed that colonization order influenced both the outcome of community assembly and the ecological success of individual colonizers. Historical contingency and priority effects also occurred in Rag1-/- mice, suggesting that the adaptive immune system is not a major contributor to these processes. In conclusion, this study established a measurable effect of colonization history on gut microbiota assembly in a model in which host and environmental factors were strictly controlled, illuminating a potential cause for the high levels of unexplained individuality in host-associated microbial communities.

Dietary non-fermentable fiber prevents autoimmune neurological disease by changing gut metabolic and immune status.

The autoimmune neurological disease, Multiple Sclerosis (MS), have increased at alarming rates in the Western society over the last few decades. While there are numerous efforts to develop novel treatment approaches, there is an unmet need to identify preventive strategies. We explored whether central nervous system (CNS) autoimmunity can be prevented through dietary manipulation using a spontaneous autoimmune encephalomyelitis mouse model. We report that the nutritional supplementation of non-fermentable fiber, common components of a vegetarian diet, in early adult life, prevents autoimmune disease. Dietary non-fermentable fiber alters the composition of the gut microbiota and metabolic profile with an increase in the abundance of long-chain fatty acids. Immune assays revealed that cecal extracts and a long chain fatty acid but not cecal lysates promoted autoimmune suppressive TH2 immune responses, demonstrating that non-fermentable fiber-induced metabolic changes account for the beneficial effects. Overall, these findings identify a non-invasive dietary strategy to prevent CNS autoimmunity and warrants a focus on nutritional approaches in human MS.

To engraft or not to engraft: an ecological framework for gut microbiome modulation with live microbes.

Strategies aimed at modulating the gut microbiota by using live microbes range from single strains (probiotics or live biotherapeutics) to whole non-defined fecal transplants. Although often clinically efficacious, our understanding on how microbial-based strategies modulate gut microbiome composition and function is vastly incomplete. In this review, we present a framework based on ecological theory that provides mechanistic explanations for the findings obtained in studies that attempted to modulate the gut microbiota of humans and animals using live microbes. We argue that an ecological perspective grounded in theory is necessary to interpret and predict the impact of microbiome-modulating strategies and thus advance our ability to develop improved and targeted approaches with enhanced therapeutic efficiency.

Modified In Situ Pericardial Rerouting Technique for Scimitar Syndrome Repair.

Scimitar syndrome repair represents a challenge due to the high incidence of postoperative pulmonary venous obstruction associated with classic surgical strategies. In situ pericardial rerouting technique has been considered a promising alternative approach due to its simplicity and excellent midterm results. Access to the left atrium can be difficult in young patients with severe dextrocardia and hypoplastic right lung. We describe a modification of the original rerouting technique in which the atrial septum is repositioned in order to create a wide opening in the lateral aspect of the left atrium and ensure an adequate size of the reconstructed pathway.