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This review aims to provide a general overview to the topic of this special issue. We will first define the available categories of biotics, i.e., probiotics, prebiotics, postobiotics and synbiotics, first-actors of therapies that target the gut flora, with the aim to modify it in a given direction, generally with the goal of controlling inflammatory phenomena. We will then treat the points that must be considered when evaluating the results of these interventions, and, specifically, the changes in gut microbiota following the assumption of biotics. This context is typically that of clinical trials aimed at inducing or maintaining remission in pediatric autoimmune inflammatory diseases and that often yield conflicting results. We finally attempt to draft possible research developments for the next years.

In this work we present EasyPrimer, a user-friendly online tool developed to assist pan-PCR and High Resolution Melting (HRM) primer design. The tool finds the most suitable regions for primer design in a gene alignment and returns a clear graphical representation of their positions on the consensus sequence. EasyPrimer is particularly useful in difficult contexts, e.g. on gene alignments of hundreds of sequences and/or on highly variable genes. HRM analysis is an emerging method for fast and cost saving bacterial typing and an HRM scheme of six primer pairs on five Multi-Locus Sequence Type (MLST) genes is already available for Klebsiella pneumoniae. We validated the tool designing a scheme of two HRM primer pairs on the hypervariable gene wzi of Klebsiella pneumoniae and compared the two schemes. The wzi scheme resulted to have a discriminatory power comparable to the HRM MLST scheme, using only one third of primer pairs. Then we successfully used the wzi HRM primer scheme to reconstruct a Klebsiella pneumoniae nosocomial outbreak in few hours. The use of hypervariable genes reduces the number of HRM primer pairs required for bacterial typing allowing to perform cost saving, large-scale surveillance programs.

Severe neurological impairment (NI) is often accompanied by the need for artificial nutritional support, normally provided enterally (enteral nutrition [EN]) to ensure growth, counteract morbidity and mortality, and improve quality of life. On the other hand, long-term EN (LTEN) may contribute to the establishment, or exacerbation, of gastrointestinal disorders that may lead to malnutrition, which in turn is associated with alterations in gut microbiota (GM) composition and functional capacities. To the best of our knowledge, we investigated, for the first time in this study, the consequences of LTEN in a pediatric population in this pathological context. Using amplicon sequencing, we compared the fecal microbiota of a pediatric population suffering from severe NI and under LTEN to that of sex- and age-matched controls. The two groups presented evident differences in GM composition and a consistent differential clustering. In general, the taxonomic picture in NI children under LTEN seemed to mirror a profound dysbiotic condition, in which anti-inflammatory taxa appear severely depleted (among others, the

DNA methylation is an important mechanism involved in bacteria limiting foreign DNA acquisition, maintenance of mobile genetic elements, DNA mismatch repair, and gene expression. Changes in DNA methylation pattern are observed in bacteria under stress conditions, including exposure to antimicrobial compounds. These changes can result in transient and fast-appearing adaptive antibiotic resistance (AdR) phenotypes, e.g., strain overexpressing efflux pumps. DNA methylation can be related to DNA mutation rate, because it is involved in DNA mismatch repair systems and because methylated bases are well-known mutational hotspots. The AdR process can be the first important step in the selection of antibiotic-resistant strains, allowing the survival of the bacterial population until more efficient resistant mutants emerge. Epigenetic modifications can be investigated by third-generation sequencing platforms that allow us to simultaneously detect all the methylated bases along with the DNA sequencing. In this scenario, this sequencing technology enables the study of epigenetic modifications in link with antibiotic resistance and will help to investigate the relationship between methylation and mutation in the development of stable mechanisms of resistance.

ObjectivesSubspecies pathogen typing is a pivotal tool to detect the emergence of high-risk clones in hospital settings and to limit their spreading among patients. Unfortunately, the most used subspecies typing methods (i.e. Pulsed-field Gel Electrophoresis - PFGE, Multi-Locus Sequence Typing - MLST and Whole Genome Sequencing - WGS) are too expensive and time consuming to be suitable for real-time surveillance. Here we present Hypervariable-Locus Melting Typing (HLMT), a novel subspecies typing approach based on High Resolution Melting (HRM) analysis, which allows pathogen typing in a few hours and with ∼5 euros per sample.MethodsHLMT types the strains by clustering them using melting temperatures (HLMT-clustering) and/or by assigning them to Melting Types (MTs) on the basis of a reference dataset (HLMT-assignment). We applied HLMT (clustering and typing) to 134 Klebsiella pneumoniae strains collected during outbreaks or surveillance programs in four hospitals. Then, we compared HLMT typing results to PFGE, MLST and WGS.ResultsHLMT-clustering distinguishes most of the K. pneumoniae high-risk clones with a sensitivity comparable to PFGE and MLST. It also drawed surveillance epidemiological curves comparable to those obtained by MLST, PFGE and WGS typing. Furthermore, the results obtained by HLMT-assignment were coherent to MLST for 96% of the typed strains with a Jaccard index of 0.912.ConclusionsHLMT is a fast and scalable method for pathogen typing, suitable for real-time hospital microbiological surveillance. HLMT is also inexpensive and thus it is applicable to infection control programs in low-middle income countries.

Colombia is a country for which one of the highest biodiversity rates is reported, and one of the first in the tropical areas where an effort was made to gather information on indigenous fungi. Nevertheless, mycological data are still scarce and discontinuous, above all on soil fungi. The present study wanted to contribute to unveil the large soil fungal biodiversity in the upper Andean Colombian agro-ecosystems. The studied area is located in the department of Boyacà, considered with a notable economical value, partly devoted to subsistence agriculture. More than 150 described species were revealed in this study, belonging to 5 phyla with Ascomycota representing the dominant taxon. Basidiomycota and Zygomycota are also well represented, dominated by species of the genus Sebacina and Mortierella respectively, mainly distributed in the semi-natural plots (woodland and grassland). Most of the species are reported as first records for Colombia. Some of them are particularly interesting for their conservation significance such as Geoglossum fallax, which is the dominant species in the unimproved grassland plot. The bootstrap-based clustering analysis showed a different distribution of the species in orchards and non-cultivated areas as a possible response of the fungal community to different use of soil in the agro-environment.

Background The rapid identification of pathogen clones is pivotal for effective epidemiological control strategies in hospital settings. High Resolution Melting (HRM) is a molecular biology technique suitable for fast and inexpensive pathogen typing protocols. Unfortunately, the mathematical/informatics skills required to analyse HRM data for pathogen typing likely limit the application of this promising technique in hospital settings. Results MeltingPlot is the first tool specifically designed for epidemiological investigations using HRM data, easing the application of HRM typing to large real-time surveillance and rapid outbreak reconstructions. MeltingPlot implements a graph-based algorithm designed to discriminate pathogen clones on the basis of HRM data, producing portable typing results. The tool also merges typing information with isolates and patients metadata to create graphical and tabular outputs useful in epidemiological investigations and it runs in a few seconds even with hundreds of isolates. Availability: https://skynet.unimi.it/index.php/tools/meltingplot/. Conclusions The analysis and result interpretation of HRM typing protocols can be not trivial and this likely limited its application in hospital settings. MeltingPlot is a web tool designed to help the user to reconstruct epidemiological events by combining HRM-based clustering methods and the isolate/patient metadata. The tool can be used for the implementation of HRM based real time large scale surveillance programs in hospital settings.

Background Duodenal dysbiosis has been suggested to possibly influence the clinical manifestations of coeliac disease (CD), both at onset and when symptoms persist despite a gluten-free diet (GFD). AIMS To evaluate the relationship between duodenal microbiota composition and: i) clinical phenotype of untreated CD (UCD); ii) presence and type of persistent symptoms despite a satisfactory serological and histological response to a strict GFD. Methods Duodenal microbiota was analyzed by 16S rRNA sequencing and compared with i) clinical features in 12 adult UCD patients; ii) presence/absence and type of persistent symptoms (diarrhea-predominant vs. non-diarrhea predominant) in 25 adult treated coeliac patients (TCD) on a strict GFD. Results UCD with iron deficiency anemia (IDA) had a pro-inflammatory shift in their duodenal microbiota (reduction of Firmicutes, p = 0.03; increase of beta-Proteobacteria, p = 0.02) than those without IDA. TCD with persistent diarrhea showed a reduction of Actinobacteria (p = 0.03) and Rothia spp (p = 0.046) compared to TCD suffering from other type of persistent symptoms. Conclusion A distinctive duodenal microbiota profile is associated with IDA in UCD, and diarrhea-predominant persistent symptoms in TCD. Clinical interventions may include reconsidering patients presenting with IDA as a specific disease subtype, and dietary rebalancing if diarrhea persists despite histological response to a GFD.

To assess the effect of a fermented rice-flour obtained from Lactobacillus paracasei CBA L74 in managing infants with moderate to severe atopic dermatitis. Infants with moderate to severe atopic dermatitis, aged 6-36 months, were randomly assigned to receive once-daily consumption of rice flour containing heat-killed probiotic Lactobacillus paracasei CBA L74 or placebo for 12 weeks as supplementary approach to topical treatment. Primary outcome was SCORAD index change from baseline to 12 weeks; secondary outcomes were gut microbiota composition, as evaluated by the analysis of fecal samples, and serum cytokines at baseline and at the end of the intervention period in both groups, and steroid usage over the treatment period and one month after stopping it. V3-V4 region of the 16S ribosomal RNA gene was sequenced to evaluate changes in the gut microbiota. SCORAD index decreased over the treatment period in both groups. The difference in the SCORAD change was -2.1 (-5.5 to 1.3; p = 0.223) for the experimental vs. the placebo group, not reaching the minimal clinical difference of 8.7 units. The use of topical steroids, measured as finger tips units, decreased from 4 to 16 weeks, in both groups; the reduction was significantly higher in experimental than in placebo group (p value from Wilcoxon rank sum test = 0.031). No significant differences were observed for cytokines levels between groups. The composition of gut microbiota at the phylum and class taxonomic levels resulted very similar, at baseline and after intervention, in both groups. Similarly, no significant differences were observed in the relative abundance of bacterial genera between groups. In conclusion, though the heat-killed Lactobacillus paracaseiwas not proved to be effective in reducing the severity of atopic dermatitis, it showed a steroid sparing effect the value of which needs to be further investigated.

Clostridioides difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea, especially in hospitalized elderly patients, representing a global public health concern. Clinical presentations vary from mild diarrhea to severe pseudomembranous colitis that may progress to toxic megacolon or intestinal perforation. Antibiotic therapy is recognized as a risk factor and exacerbates dysbiosis of the intestinal microbiota, whose role in CDI is increasingly acknowledged. A clinically challenging complication is the development of recurrent disease (rCDI). In this study, using amplicon metagenomics, we compared the fecal microbiota of CDI and rCDI patients (sampled at initial and recurrent episode) and of non-infected controls. We also investigated whether CDI severity relates to specific microbiota compositions. rCDI patients showed a significantly decreased bacterial diversity as compared to controls (p &lt, 0.01). The taxonomic composition presented significant shifts: both CDI and rCDI patients displayed significantly increased frequencies of Firmicutes, Peptostreptococcaceae, Clostridium XI, Clostridium XVIII, and Enterococcaceae. Porphyromonadaceae and, within it, Parabacteroides displayed opposite behaviors in CDI and rCDI, appearing discriminant between the two. Finally, the second episode of rCDI was characterized by significant shifts of unclassified Clostridiales, Escherichia/Shigella and Veillonella. No peculiar taxa composition correlated with the severity of infection, likely reflecting the role of host-related factors in determining severity.

Inflammatory Bowel Diseases (IBDs) are gastrointestinal disorders characterized by chronic, relapsing inflammation, with growing incidence worldwide over the last decades and distinctive features in the pediatric age. An increasing body of evidence indicates that gut microbiota plays a major role in inflammatory disorders, including IBDs. In this review we will discuss the most recent evidences on dysbiotic changes associated with gut inflammation, as well as environmental and genetic factors contributing to IBD pathogenesis, with a focus on the peculiarities of the pediatric age.

MotivationGenomic investigations show that nosocomial outbreaks can be sustained by the simultaneous spreading of multiple pathogen clones. Rapid pathogen typing and real-time clones monitoring is a key strategy to control pathogen spreading in hospital settings. A novel approach to High Resolution Melting (HRM) data analysis allows pathogen typing in less than 5 hours. MeltingPlot is the first tool specifically designed for epidemiological investigations using HRM data. The tool is suitable for large real-time surveillance and rapid outbreak reconstructions.ImplementationMeltingPlot was developed in R. The web tool and the standalone versions are available.General featuresThe tool implements a graph-based algorithm designed to discriminate pathogen clones on the basis of HRM data, producing portable typing results. MeltingPlot also merges typing information with isolates and patients metadata to create graphical and tabular outputs useful in epidemiological investigations.AvailabilityThe web interface is available at https://skynet.unimi.it/index.php/tools/meltingplot, the standalone version at https://github.com/MatteoPS/MeltingPlot.

Background: Growing evidence suggests that an altered microbiota composition contributes to the pathogenesis and clinical features in celiac disease (CD). We performed a comparative analysis of the gut microbiota in adulthood CD to evaluate whether: (i) dysbiosis anticipates mucosal lesions, (ii) gluten-free diet restores eubiosis, (iii) refractory CD has a peculiar microbial signature, and (iv) salivary and fecal communities overlap the mucosal one. Methods: This is a cross-sectional study where a total of 52 CD patients, including 13 active CD, 29 treated CD, 4 refractory CD, and 6 potential CD, were enrolled in a tertiary center together with 31 controls. A 16S rRNA-based amplicon metagenomics approach was applied to determine the microbiota structure and composition of salivary, duodenal mucosa, and stool samples, followed by appropriate bioinformatic analyses. Results: A reduction of both &alpha, and &beta, diversity in CD, already evident in the potential form and achieving nadir in refractory CD, was evident. Taxonomically, mucosa displayed a significant abundance of Proteobacteria and an expansion of Neisseria, especially in active patients, while treated celiacs showed an intermediate profile between active disease and controls. The saliva community mirrored the mucosal one better than stool. Conclusion: Expansion of pathobiontic species anticipates villous atrophy and achieves the maximal divergence from controls in refractory CD. Gluten-free diet results in incomplete recovery. The overlapping results between mucosal and salivary samples indicate the use of saliva as a diagnostic fluid.

This review tackles the concept of the evolutionary mismatch, in relation with the reduction of the prevalence of the so-called "dirty old friends". These formed the variegated community of parasites and microorganisms, either prokaryotic or eukaryotic, that, over long evolutionary times, co-evolved with humans and their ancestors, inhabiting their digestive tracts, and other body districts. This community of microbial symbionts and metazoan parasites is thought to have evolved a complex network of inter-independence with the host, in particular in relation with their immune stimulating capacity, and with the consequent adaptation of the host immune response to this chronic stimulation. Strictly related to this evolutionary mismatch, the hygiene hypothesis, proposed by David Strachan in 1989, foresees that the increase in the incidence of inflammatory and autoimmune disorders during the twentieth century has been caused by the reduced exposure to parasites and microorganisms, especially in industrialized countries. Among these pathologies, inflammatory bowel diseases (IBDs) occupy a prominent role. From these premises, this review summarizes current knowledge on how variations in the composition of the gut bacterial microbiota, as well as its interactions with fungal communities, influence the overall immune balance, favouring or counteracting gut inflammation in IBDs. Additionally, the effect of worm parasites, either directly on the immune balance, or indirectly, through the modulation of bacterial and fungal microbiota, will be addressed. Finally, we will review a series of studies related to the use of molecules derived from parasitic worms and fungi, which hold the potential to be developed as postbiotics for the treatment of IBDs.

This work pictures the biodiversity of fungal consortia inhabiting real agroecosystems, sampled in one production farm in two seasons (spring, autumn), coinciding with climate gradients and key moments of the agricultural cycle. Soil was sampled from three plots differently managed in terms of fertilization, pesticide and tillage application: conventional, organic, no-tillage. Metagenomic analyses on ITS1 amplicons depicted the highest indexes of richness for organic. No-tillage resulted in inhabitation by the most divergent communities, with their own composition, prevalence and seasonal trends. Ascomycota always predominated, with the exception of conventional, that had high abundance of a single basidiomycete species. Our results showed evidence that agricultural soils under organic and no-tillage systems harbour distinct mycobiota, even in neighbouring fields. From our results, fungal consortia altered even in the first year after the management change.

The human skin microbiota is essential for maintaining homeostasis and ensuring barrier functions. Over the years, the characterization of its composition and taxonomic diversity has reached outstanding goals, with more than 10 million bacterial genes collected and cataloged. Nevertheless, the study of the skin microbiota presents specific challenges that need to be addressed in study design. Benchmarking procedures and reproducible and robust analysis workflows for increasing comparability among studies are required. For various reasons and because of specific technical problems, these issues have been investigated in gut microbiota studies, but they have been largely overlooked for skin microbiota. After a short description of the skin microbiota, the review tackles methodological aspects and their pitfalls, covering NGS approaches and high throughput culture-based techniques. Recent insights into the “core” and “transient” types of skin microbiota and how the manipulation of these communities can prevent or combat skin diseases are also covered. Finally, this review includes an overview of the main dermatological diseases, the changes in the microbiota composition associated with them, and the recommended skin sampling procedures. The last section focuses on topical and oral probiotics to improve and maintain skin health, considering their possible applications for skin diseases.

Johne's disease is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratubercolosis (MAP) which affects ruminants worldwide and has a significant economic impact. MAP has also been associated with human Crohn's disease, although this connection is not well established. MAP is highly adapted for survival within host macrophages and prevents macrophage activation, blocks phagosome acidification and maturation, and attenuates presentation of antigens to the immune system. The consequence is a very long silent infection before clinical signs are observed. The present work examined the transcriptome of bovine monocyte-derived macrophages (MDM) infected with the L1 strain of MAP at 2h, 6h and 24h post infection using RNA-seq. Pathway over-representation analysis of genes differentially expressed between infected vs. control MDM identified that immune related pathways were affected. Genes belonging to the cytokine-cytokine receptor interaction pathway and members of the JAK-STAT pathway, which is involved in the regulation of immune response, were up-regulated. However, in parallel inhibitors of immune functions were activated, including suppressor of cytokine signaling (SOCS) and cytokine-inducible SH2-containing protein (CISH), which most likely suppresses IFNγ and the JAK/STAT signaling cascade in infected MDM, which may favour MAP survival. After exposure, macrophages phagocytise pathogens, activate the complement cascade and the adaptive immune system through the antigen presentation process. However, data presented here suggest that genes related to phagocytosis and lysosome function are down regulated in MAP infected MDM. Genes of MHC class II and complement pathway were also down-regulated. This study therefore shows that MAP infection is associated with changes in expression of genes related to the host immune response that may affect its ability to survive and multiply inside the host cell.

Billions of microorganisms, or “microbiota”, inhabit the gut and affect its homeostasis, influencing, and sometimes causing if altered, a multitude of diseases. The genomes of the microbes that form the gut ecosystem should be summed to the human genome to form the hologenome due to their influence on human physiology; hence the term “microbiome” is commonly used to refer to the genetic make-up and gene−gene interactions of microbes. This review attempts to provide insight into this recently discovered vital organ of the human body, which has yet to be fully explored. We herein discuss the rhythm and shaping of the microbiome at birth and during the first years leading up to adolescence. Furthermore, important issues to consider for conducting a reliable microbiome study including study design, inclusion/exclusion criteria, sample collection, storage, and variability of different sampling methods as well as the basic terminology of molecular approaches, data analysis, and clinical interpretation of results are addressed. This basic knowledge aims to provide the pediatricians with a key tool to avoid data dispersion and pitfalls during child microbiota study.

In this work we present EasyPrimer, a user-friendly online tool developed to assist pan-PCR and High Resolution Melting (HRM) primer design. The tool finds the most suitable regions for primer design in a gene alignment and returns a clear graphical representation of their positions on the gene. EasyPrimer is particularly useful in difficult contexts, e.g. on gene alignments of hundreds of sequences and/or on highly variable genes. HRM analysis is an emerging method for fast and cost saving bacterial typing and an HRM scheme of six primer sets on five Multi-Locus Sequence Type (MLST) genes is already available for Klebsiella pneumoniae. We validated the tool designing a scheme of two HRM primer sets on the hypervariable gene wzi of Klebsiella pneumoniae and compared the two schemes. The wzi scheme resulted to have a discriminatory power comparable to the HRM MLST scheme, using only one third of primer sets. Then we successfully used the wzi HRM primer scheme to reconstruct a Klebsiella pneumoniae nosocomial outbreak in few hours. The use of hypervariable genes reduces the number of HRM primer sets required for bacterial typing allowing to perform cost saving, large-scale surveillance programs.

Obesity in childhood and adolescence is considered the most prevalent nutritional disorder, in which eating behaviours represent one important factors of influence. Many aspects influence eating behaviours, but taste is considered the main predictor. However, data concerning correlations of obesity, taste sensitivity and behavioural attitudes, such as food neophobia, in children and adolescents are inconsistent. Moreover, it has been suggested that oral bacteria could have a possible role in obesity development and, also, in taste perception. In this context, the present study focused on host related factors with a proposed link to weight gain. To this purpose, taste sensitivity, salivary microbiota composition and food neophobia were compared between children and adolescents with and without obesity in a cross-sectional study. Results showed that children with obesity presented a significantly lower ability in correctly identifying taste qualities and were characterized by a lesser number of Fungiform Papillae (reported as FP/cm2) compared to normal-weight subjects. Differences in the ecological indexes of microbial alpha-diversity was found between subjects with obesity and normal-weight ones. Moreover, independently from nutritional status, some bacterial genera seemed to differ between subjects with different sensitivity. The potentiality of this multidisciplinary approach could help to better understand and deepen the sensory-driven and microbiological factors related to weight gain.

We describe two multi drug-resistant (MDR) carbapenemase-producing Escherichia coli clinical isolates from an acute hospital in Milan. Both strains, isolated from a surgical wound sample and a surveillance rectal swab respectively, were positive for a blaNDM-type gene by Xpert Carba-R test. The whole-genome sequence characterization disclosed several resistance determinants: blaNDM-5, blaCMY-42, blaTEM-198, rmtB, mphA. The two isolates belonged to phylogenetic group A, sequence type (ST) 1702 and serotype O89:H9. PCR-based replicon typing and conjugation assay demonstrated an IncI1 plasmid localization for both blaNDM-5 and blaCMY-42 genes. This is the first report of a ST1702 NDM-5 and CMY-42- producing E. coli clone in Italy.

Celiac disease (CD), the most common chronic enteropathy worldwide, is triggered and sustained by a dysregulated immune response to dietary gluten in genetically susceptible individuals. Up to date either the role of environmental factors and the pathways leading to mucosal damage have been only partially unraveled. Therefore, we seized the unique opportunity to study a naturally-occurring experimental model of a family composed of both parents suffering from CD (one on a gluten-free diet) and two non-celiac daughters. The control group consisted in four unrelated cases, two celiac and two non-celiac subjects, all matching with family members for both disease status and genetic susceptibility. In this privileged setting, we sought to investigate gene expression in peripheral blood mononuclear cells (PBMCs), a population known to mirror the immune response state within the gut. To this purpose, PBMCs were obtained from the four biopsied-proven CD patients and the four non-celiac cases. Each group included two family members and two unrelated control subjects. After RNA purification and cDNA synthesis, each sample underwent a microarray screen on a whole-transcriptome scale, and the hybridization results were visualized by hierarchical clustering. Differentially expressed genes (DEG) were partitioned into clusters displaying comparable regulations among samples. These clusters were subjected to both functional and pathway analysis by using the Kyoto Encyclopedia of Genes and Genomes. Interestingly, on a global gene expression level, the family members clustered together, regardless of their disease status. A relevant fraction of DEG belonged to a limited number of pathways, and could be differentiated based on disease status: active CD vs. treated CD and CD vs. controls. These pathways were mainly involved in immune function regulation, cell-cell junctions, protein targeting and degradation, exosome trafficking, and signal transduction. Worth of noting, a small group of genes mapping on the male-specific region of the Y chromosome, and previously linked to cardiovascular risk, was found to be strongly upregulated in the active CD case belonging to the family, who suddenly died of a heart attack. Our results provide novel information on CD pathogenesis and may be useful in identifying new therapeutic tools and risk factors associated with this condition.

The aim of the study was to distinguish the raw milk from different farms in relation to their geographical sites within a narrowed territorial district. The goal was achieved by applying a molecular-based system for traceability that uses microbial DNA barcodes present in milk. Microbiota of milk were fingerprinted by PCR of the 16S–23S intergenic transcribed spacer using the Automated Ribosomal Intergenic Spacer Analysis (ARISA). A total of 64 markers within the range 279–756 bp were detected on the thirty-eight bulk milk samples, none of which was common to all the patterns. Overall samples did not show relevant differences across the two years of sampling. In fact, every farm maintained a specific core profile over time, thus demonstrating that the interaction between site and year of sampling is not significant and that the variability between years does not affect the distinction between grouping of farms. The system was able to trace the geographical origin of raw milk with a resolution of less than 5 km. According to the European regulations for the protection of the geographical names of foodstuffs which have a tangible link to the territory, the ARISA system described here may represent a suitable analytical tool for tracing the origin of milk integrating and reinforcing traceability processes of the dairy chain.

The hazard to human health represented by transmissible spongiform encephalopathies in sheep is one of the major reasons for implementing the genetic selection plan to break down prion diseases. The problem is particularly important because of the risk of disease transmission from ewe to lamb via milk or colostrum. In order to establish an active and convenient monitoring of the flocks already undergone genetic selection and thus, indirectly increase consumers’ security, the challenge of the work was quantifying the classical scrapie risk in bulk milk. A new quantitative real-time PCR assay for the estimation of the 171 R and Q allelic frequencies in a DNA pool representative of all the lactating ewes present in a flock was optimized and validated “in field”. The repeatability range was 3.69–5.27 for R and 4.20–5.75 for Q. The ruggedness of the allele frequencies resulted 4.26 for R and 4.77 for Q. Regarding the validation “in field”, none of the considered sources of variability (flock, month, number of genotyped animals and somatic cell count) showed a significant effect on flock and milk at the linear model. The targeted next-generation sequencing was also tested to evaluate its applicability in this context. Results show that the real-time PCR assay could represent a valid tool for the determination of 171 R/Q allele frequencies in bulk milk. The implementation of a service for breeder self-control along the production chain would aim to increase the production of high-security dairy products, while monitoring over time of the effects of genetic selection in the flocks.

Late blowing is a defect of semi-hard and hard cheeses induced by butyric clostridia that, through the formation of endospores, contaminate raw milk via silage, fresh forage and animal bedding. Generally it becomes evident during ripening, when the physical and chemical conditions of the cheese core support spores outgrowth. This causes the production of undesirable off-odors and an anomalous organization of the loaf, that seriously prejudices cheeses, with significant economic losses for the dairy industry. This study describes the development of an original application of the automated ribosomal intergenic spacer analysis (ARISA) that, relying on a multi-species-specific primer set, allows to simultaneously displaying distinctive fingerprints, diagnostic for Clostridium tyrobutyricum, generally considered the main responsible of late blowing, and for the other species so far associated with the defect: Clostridium butyricum, Clostridium sporogenes, Clostridium beijerinckii, Clostridium tertium and Clostridium perfringens, also providing relative abundance estimations. This approach has been applied to the seasonal analysis of tank raw milk and curd used for Grana Padano PDO cheese production in dairies located in different areas of the Northern Padan plain. C. beijerinckii and C. tertium, occurring both in 100% milks and curds, together with C. perfringens, retrieved in 98.7% milks and 100% curds, resulted the most abundant species in all the seasons. On the other hand, C. sporogenes always resulted the less abundant species, occurring in 77.2% of milks and 86.1% of curds. Highly significant seasonal effects were detected for C. tyrobutyricum and C. beijerinckii that displayed the most marked and opposite trends. The occurrence of a spatial pattern of variation for curd was also observed, thus envisaging relevant effects presumably linked to differential breeder practices (feeding and hygiene). This ARISA approach presents important perspectives in terms of applicability along the entire dairy production chain for diagnostic purposes and for providing an estimate of the microbiological quality of milk.

Clostridium tyrobutyricum has been identified as the main causal agent of the late blowing defect in cheese, with major effects on quality and commercial value. In this work, for the first time, we applied automated ribosomal intergenic spacer analysis (ARISA) approach to diagnose the presence of C. tyrobutyricum in raw milk before cheesemaking. A species-specific primer set was designed and used for this original application of the ARISA. Sensitivity of detection, reproducibility of the fluorescent PCR assay, and repeatability of the capillary electrophoretic analysis of amplicons were evaluated using DNA extracted from milk added with known amounts of C. tyrobutyricum genome copies, ranging from 3 × 10(6) to 3. Results indicated that the sensitivity of the technique permits to detect the bacterium in all the samples. The reproducibility, evaluated by analyzing 3 sets of serial dilutions, resulted satisfactory, with little deviation within PCR reactions amplifying the same starting amount of template (standard deviations ≤ 0.1, coefficients of variation ≤ 3%). The peaks' fluorescence displayed an evident correspondence with the number of genome copies contained in each dilution. The capillary electrophoretic analysis, tested by running a single PCR product per dilution point in 10 repeats, resulted efficient and highly repeatable, with excellent coefficients of variation ≤ 2% and standard deviations ≤ 0.1 in all the sample sets. This application of ARISA gives good estimates of the total C. tyrobutyricum DNA content allowing a specific, fine-scale resolution of this pollutant species in a complex system as milk. A further advantage linked to the automatization of the process.

The aim of this work was to characterize the mould populations of the Taleggio PDO cheese surface by a combination of culture dependent and independent techniques. DNA samples were extracted: (i) from isolates derived from brine and cheeses at various ripening midpoints, (ii) directly from the surface of commercial cheeses made in different dairies. DNA was examined by PCR at various loci (DNA barcodes). Amplified regions were both ribosomal (ITS1, ITS2 and the D1/D2 region of the 26S rRNA gene) and non-ribosomal (portions of the β-tubulin and EF1α genes). The mould community appeared to be dominated by a species that alone represents 87% of the sequences obtained using the β-tubulin barcode, and is 99% homologous to Penicillium commune . It has been obtained, always identical, from brine, from cheeses at the various ripening midpoints, and from all the commercial cheeses. It presumably represents a new variant of P. commune typical of Taleggio. Three isolates were identified as belonging to three different species of the genus Cladosporium : Cladosporiumoxysporum and two potential new species, detected on the basis of their β-tubulin sequences. Finally, 2 isolates were identified as Aureobasidium pullulans and Eutypella scoparia . This is the first characterization of the mould species for this important Italian PDO cheese production, and is one of the first reports of molecular characterization by means of DNA barcodes on mould DNA directly extracted from cheese without culturing.

A few years ago, a highly significant association between the xenotropic murine leukemia virus-related virus (XMRV) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a complex debilitating disease of poorly understood etiology and no definite treatment, was reported in Science, raising concern for public welfare. Successively, the failure to reproduce these findings, and the suspect that the diagnostic PCR was vitiated by laboratory contaminations, led to the retraction of the paper. Notwithstanding, XMRV continued to be the subject of researches and public debates. Occasional positivity in humans was also detected recently, even if the data always appeared elusive and non-reproducible. In this study, we discuss the current status of this controversial association and propose that a major role in the unreliability of the results was played by the XMRV genomic composition in itself. In this regard, we present bioinformatic analyses that show: (i) aspecific, spurious annealings of the available primers in multiple homologous sites of the human genome; (ii) strict homologies between whole XMRV genome and interspersed repetitive elements widespread in mammalian genomes. To further detail this scenario, we screen several human and mammalian samples by using both published and newly designed primers. The experimental data confirm that available primers are far from being selective and specific. In conclusion, the occurrence of highly conserved, repeated DNA sequences in the XMRV genome deeply undermines the reliability of diagnostic PCRs by leading to artifactual and spurious amplifications. Together with all the other evidences, this makes the association between the XMRV retrovirus and CFS totally unreliable.

Background Bovine Amyloidotic Spongiform Encephalopathy (BASE) is a variant of classical BSE that affects cows and can be transmitted to primates and mice. BASE is biochemically different from BSE and shares some molecular and histo-pathological features with the MV2 sub-type of human sporadic Creutzfeld Jakob Disease (sCJD). Results The present work examined the effects of BASE on gene expression in circulating immune cells. Ontology analysis of genes differentially expressed between cattle orally challenged with brain homogenate from cattle following intracranial inoculation with BASE and control cattle identified three main pathways which were affected. Within the immune function pathway, the most affected genes were related to the T cell receptor-mediated T cell activation pathways. The differential expression of these genes in BASE challenged animals at 10,12 and 24 months following challenge, vs unchallenged controls, was investigated by real time PCR. Conclusions The results of this study show that the effects of prion diseases are not limited to the CNS, but involve the immune system and particularly T cell signalling during the early stage following challenge, before the appearance of clinical signs.

The development of methods for the analysis and comparison of the nucleic acids contained in single cells is an ambitious and challenging goal that may provide useful insights in many physiopathological processes. We review here some of the published protocols for the amplification of whole genomes (WGA). We focus on the reaction known as Multiple Displacement Amplification (MDA), which probably represents the most reliable and efficient WGA protocol developed to date. We discuss some recent advances and applications, as well as some modifications to the reaction, which should improve its use and enlarge its range of applicability possibly to degraded genomes, and also to RNA via complementary DNA.

The amount of DNA available for informative genomic studies is often limiting. Thus, several methods have been developed to achieve a whole genome amplification (WGA). Particu-larly promising is a variant, called multiple displacement amplification (MDA) (1,2), which exploits the high processivity of Φ29 phage DNA polymerase and suffers an amplifi-cation bias significantly lower than previous, PCR-based WGA (3).Even though MDA-DNA has been successfully used for many applica-tions (1,4–7), some problems have been reported. For example, sequences near the ends of linear chromosomes appear underrepresented after MDA (6,8,9). Both defective priming events and abortive chain terminations could contribute to this problem, which limits the applications of MDA and jeopardizes its use on short linear DNA (e.g., cDNA and degraded genomes from forensic, archeological, and fetal origin) (10). In these cases, the terminal underrepresentation would cause the loss of substantial information.We studied this problem using λ phage DNA (48.5 kb; Promega Biosciences, San Luis Obispo, CA, USA). MDA was performed using the Genomiphi

This work applies culture-independent methods for the characterization of fungal populations (yeasts and moulds) naturally occurring in Sardinian ewe's milk sampled in the Italian areas with the largest dairy production (Sardinia and Lazio regions). Sequences of the D1/D2 variable domains at the 5′ end of the 26S rRNA gene were obtained by amplification of DNA directly isolated from milk, and this allowed identification of a total of 6 genera and 15 species of fungi. Among the 6 identified generaGeotrichumspp.,Candidaspp.,Phaeosphaeriopsisspp.,Pestalotiopsisspp. andCladosporiumspp. belong to the phylum of Ascomycota, whileCryptococcusspp. is part of the phylum of Basidiomycota. In particular, two genera (PestalotiopsisandPhaeosphaeriopsis) and two species (Plectosphaerella cucumerinaandPryceomyces carsonii) have never been reported in dairy ecosystems before. Results provide evidence that several moulds and yeasts, previously described only in ovine cheeses, are transferred directly from raw milk. The knowledge of fungal consortia inhabiting sheep raw milk is a particularly relevant issue because several species are directly involved in cheese making and ripening, determining the typical aroma. On the other hand, spoilage yeasts and moulds are involved in anomalous fermentation of cheese and may be responsible for considerable economic losses and serious risks for consumers' health.

p38α MAP kinase is activated in response to many cellular stresses and also regulates the differentiation and/or survival of various cell types in vitro, including skeletal muscle cells and cardiomyocytes. Here we show that targeted inactivation of the mouse p38α gene results in embryonic lethality at midgestation correlating with a massive reduction of the myocardium and malformation of blood vessels in the head region. However, this defect appears to be secondary to insufficient oxygen and nutrient transfer across the placenta. When the placental defect was rescued, p38α −/− embryos developed to term and were normal in appearance. Our results indicate that p38α is required for placental organogenesis but is not essential for other aspects of mammalian embryonic development.