Lower serum levels of IL-1β and IL-6 cytokines in adolescents with anorexia nervosa and their association with gut microbiota in a longitudinal study

Introduction Anorexia nervosa (AN) is an often chronic and debilitating psychiatric disease whose etiology is not completely understood. Recently, a potential role of inflammation has emerged in other psychiatric diseases, such as depression, PTSD and schizophrenia. The first results in adults with AN seemed to confirm a low-grade proinflammatory state until recent studies presented more differential findings. Studying adolescents with a shorter illness duration and fewer confounding factors might help elucidate the role of inflammation in the underlying pathophysiology of AN; however, the few available studies in adolescents remain ambiguous, and no longitudinal data are available in this age range. Results TNF-α serum levels were significantly elevated in patients with AN at admission, while IL-1β and IL-6 levels were lower at admission and discharge than in HC. After treatment, we also found significantly elevated levels of IL-6 Rα compared to HC, while IL-15 did not show significant changes. Exploratory analyses revealed positive associations of cytokine and genus-level changes between admission and discharge for IL-1β (Bacteroides) and IL-15 (Romboutsia), and negative associations for IL-15 (Anaerostipes) and TNF-α (uncultured Lachnospiraceae). Conclusion We confirmed a previous finding of elevated levels of TNF-α also in adolescents with AN; however, the reduced IL-1β and IL-6 levels differed from the mostly increased levels found in adults. A mixed pro- and anti-inflammatory state appears to be present in adolescents, potentially due to their shorter illness duration. The gut microbiota, with its regulatory function on cytokine production, might play a role in mediating these inflammatory processes in AN and could offer targets for new therapeutic approaches

Prokaryote genome fluidity is dependent on effective population size

Many prokaryote species are known to have fluid genomes, with different strains varying markedly in accessory gene content through the combined action of gene loss, gene gain via lateral transfer, as well as gene duplication. However, the evolutionary forces determining genome fluidity are not yet well understood. We here for the first time systematically analyse the degree to which this distinctive genomic feature differs between bacterial species. We find that genome fluidity is positively correlated with synonymous nucleotide diversity of the core genome, a measure of effective population size Ne. No effects of genome size, phylogeny or homologous recombination rate on genome fluidity were found. Our findings are consistent with a scenario where accessory gene content turnover is for a large part dictated by neutral evolution

Transformation and tumorigenicity testing of simian cell lines and evaluation of poliovirus replication

The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field

Agricultural by-products with bioactive effects: A multivariate approach to evaluate microbial and physicochemical changes in a fresh pork sausage enriched with phenolic compounds from olive vegetation water

The use of phenolic compounds derived from agricultural by-products could be considered as an eco-friendly strategy for food preservation. In this study a purified phenol extract from olive vegetation water (PEOVW) was explored as a potential bioactive ingredient for meat products using Italian fresh sausage as food model. The research was developed in two steps: first, an in vitro delineation of the extract antimicrobial activities was performed, then, the PEOVW was tested in the food model to investigate the possible application in food manufacturing. The in vitro tests showed that PEOVW clearly inhibits the growth of food-borne pathogens such as Listeria monocytogenes and Staphylococcus aureus. The major part of Gram-positive strains was inhibited at the low concentrations (0.375–3 mg/mL). In the production of raw sausages, two concentrates of PEOVW (L1:0.075% and L2: 0.15%) were used taking into account both organoleptic traits and the bactericidal effects. A multivariate statistical approach allowed the definition of the microbial and physicochemical changes of sausages during the shelf life (14 days). In general, the inclusion of the L2 concentration reduced the growth of several microbial targets, especially Staphylococcus spp. and LABs (2 log10 CFU/g reduction),while the increasing the growth of yeasts was observed. The reduction of microbial growth could be involved in the reduced lipolysis of raw sausages supplemented with PEOVWas highlighted by the lower amount of diacylglycerols. Moisture and aw had a significant effect on the variability of microbiological features,while food matrix (the sausages' environment) can mask the effects of PEOVW on other targets (e.g. Pseudomonas). Moreover, the molecular identification of the main representative taxa collected during the experimentation allowed the evaluation of the effects of phenols on the selection of bacteria. Genetic data suggested a possible strain selection based on storage time and the addition of phenol compounds especially on LABs and Staphylococcus spp. The modulation effects on lipolysis and the reduction of several microbial targets in a naturally contaminated product indicates that PEOVW may be useful as an ingredient in fresh sausages for improving food safety and quality

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

INTO THE BLUE: Spoilage phenotypes of Pseudomonas fluorescens in food matrices

Spoilage induced by Pseudomonas strains is commonly found in a wide range of food products as a result of the ubiquitous presence of these strains and their ability to induce alteration through different mechanisms. Particular attention has been recently paid on those P. fluorescens strains able to induce a blue discolouration on several food matrices (e.g. dairy or meat products). Actually, poor data are available about this curious event that draw the attention of European consumer from 2010. In the present manuscript a step-by-step investigation of the spoilage potential of Pseudomonas fluorescens species complex strains is reported, focusing in particular on the ability to produce an unpleasant blue pigment in food. Firstly, some general information is given to the reader to understand the P. fluorescens group as food spoiler. Then, the application of a polyphasic approach is described with the aim to investigate 136 Pseudomonas fluorescens group strains. Additionally, the achievement and the analyses of draft genomes and transcriptomes for 4 P. fluorescens strains are described to investigate the biosynthetic pathways involved in the blue pigment production. The attempt to chemically characterise the blue molecule using MALDI-TOF mass spectrometry is also reported. Finally, the execution of a transposon-mediated mutagenesis is described to confirm previously obtained genomic data and to highlight further genes involved in the blue-pigment production. The phenotypic and genotypic characterisation, based on the combination of classical microbiological tests and a MLST scheme, allowed the reconstruction of phylogenetic relationships among the isolates and the identification of a monophyletic group (named “the blue branch”) grouping all the blue-pigmenting and few uncoloured strains. The real involvement of these strains in the blue mozzarella event was confirmed by their ability to induce a blue discolouration on mozzarella cheese during a challenge test. The genomic investigation confirmed the strict phylogenetic relationship between the strains belonging to the “blue branch”. Additionally, comparative genomic tools revealed the presence of a genetic cluster unique to the blue pigmenting strains containing a second copy of five trp genes, clearly involved in the blue pigment production. The biochemical characterisation of the pigment, hampered by strong issues of solubility, led to the conclusion that the molecule is an indigo-derivative. Transposon-induced mutants confirmed the involvement of the previously identified unique cluster and the association of several genes affecting directly or indirectly the blue molecule production. Furthermore, the phenotypic characterisation of the mutants revealed a key role of iron in the production of the pigment, such as absence of any advantage of the wild-type strain in co-culture with a non-pigmenting mutant. To conclude, the present work represents an exhaustive investigation of the spoilage potential of the blue-pigmenting P. fluorescens strains, giving to food industry reliable approaches to identify, track and prevent spoilage related to the growth of these interesting bacteria.Le alterazioni causate da ceppi di Pseudomonas sono solitamente riscontrate in una grande varietà di alimenti a causa del loro essere ubiquitari e dalla loro capacità di indurre modificazioni organolettiche negli alimenti mediante diversi meccanismi. Particolare attenzione è stata posta su alcuni ceppi di P. fluorescens in grado di indurre una colorazione blu in diverse matrici alimentari (quali prodotti lattiero-caseari o carne). In realtà, poche informazioni sono ad oggi disponibili riguardo al curioso caso che ha attirato l’attenzione pubblica a partire dal 2010. In questo lavoro è riportata un’analisi a più livelli del potenziale alternate dei ceppi appartenenti allo Pseudomonas fluorescens species complex, ponendo particolare attenzione alla capacità di produrre un indesiderato pigmento blu negli alimenti. In primo luogo, ai lettori sono date delle informazioni generali per una migliore comprensione di P. fluorescens come alterante alimentare. In seguito, è descritta la messa a punto e applicazione di un approccio polifasico con l’obbiettivo di indagare 136 ceppi appartenenti al gruppo P. fluorescens. Inoltre, sono descritti l’ottenimento e le analisi dei genomi draft e dei trascrittomi di 4 ceppi di P. fluorescens con la finalità di comprendere il pathway biosintetico coinvolto nella produzione del pigmento blu. In aggiunta, è riportato il tentativo di caratterizzare chimicamente il pigmento mediante la metodica della spettrometria di massa MALDI-TOF. Infine, è riportata l’esecuzione della mutagenesi random con la finalità di confermare i risultati genomici precedentemente ottenuti e di individuare ulteriori geni coinvolti nella produzione del pigmento blu. La caratterizzazione fenotipica e genotipica, basata sulla combinazione di metodiche di microbiologia classica e di uno schema MLST, ha permesso la ricostruzione delle relazioni filogenetiche tra gli isolati e l’identificazione di un gruppo monofiletico (chiamato “ramo blu”) che raggruppa tutti i ceppi pigmentanti e pochi ceppi non-pigmentanti. Il reale coinvolgimento dei ceppi blu nei casi di mozzarella blu è stato confermato dalla possibilità degli stessi di indurre un’anomala colorazione blu su mozzarella durante un challenge test. Le analisi genomiche hanno confermato la stretta vicinanza filogenetica tra i ceppi del “ramo blu”. Inoltre, analisi di genomica comparativa hanno rivelato la presenza di un cluster genico unicamente presente nei ceppi blu, contenente una seconda copia di cinque dei sette geni per la biosintesi del triptofano, chiaramente coinvolto nella produzione del pigmento blu. La caratterizzazione biochimica del pigmento, resa difficoltosa da problemi di solubilità, ha portato alla conclusione che la molecola blu sia un derivato dell’indigo. I mutanti ottenuti mediante l’applicazione di trasposoni hanno confermato il coinvolgimento del cluster genico precedentemente identificato nella produzione del pigmento e l’associazione di ulteriori geni che influenzano direttamente o indirettamente la produzione della molecola blu. Inoltre, la caratterizzazione dei mutanti ha rivelato il ruolo importante del ferro nella produzione del pigmento e l’assenza di un effettivo vantaggio del ceppo wild-type posto in co-cultura con un mutante non pigmentante. In conclusione, questo studio rappresenta un’indagine esaustiva del potenziale alterante dei ceppi blu, dando inoltre all’industria alimentare sistemi efficaci per identificare, tracciare e prevenire l’alterazione indotta da questi interessanti ceppi

Chapter 2 – Pseudomonas and Related Genera

none2noAbstract Spoilage induced by Pseudomonas, Shewanella, and Xanthomonas is commonly found in a wide range of food products as a result of the ubiquitous presence of these genera and their ability to induce alteration through different mechanisms. In the present chapter the investigation of the spoilage potential of the strains of these three genera is reported. Historical cues, taxonomic issues, identification and isolation methods are reported, as well as the different spoilage mechanisms through which the strains are able to induce alteration of several food matrices. Finally, recent control techniques proposed in previous chapters are reported.noneAndreani, Nadia Andrea; Fasolato, LucaAndreani, NADIA ANDREA; Fasolato, Luc

Tracking the blue: MLST and phenotypic characterisation of Pseudomonas fluorescens strains isolated from food

The Pseudomonas fluorescens group consists of species involved in food contamination and spoilage. The interest on P. fluorescens increased after the \u201cblue mozzarella\u201d events occurred in Italy in 2010. 133 P. fluorescens strains isolated from food were characterised by both phenotypic and molecular approaches. In particular, a Multilocus Sequence Typing (MLST) scheme was developed and applied to characterise them at strain level. The aim of the present work was to apply the MLST scheme for tracking P. fluorescens during the production chain and to discriminate the strains producing the blue pigment

Transposon mutagenesis in Pseudomonas fluorescens reveals genes involved in blue pigment production and antioxidant protection

Pseudomonas fluorescens Ps_77 is a blue-pigmenting strain able to cause food product discoloration, causing relevant economic losses especially in the dairy industry. Unlike non-pigmenting P. fluorescens, blue pigmenting strains previously were shown to carry a genomic region that includes homologs of trpABCDF genes, pointing at a possible role of the tryptophan biosynthetic pathway in production of the pigment. Here, we employ random mutagenesis to first identify the genes involved in blue-pigment production in P. fluorescens Ps_77 and second to investigate the biological function of the blue pigment. Genetic analyses based on the mapping of the random insertions allowed the identification of eight genes involved in pigment production, including the second copy of trpB (trpB_1) gene. Phenotypic characterization of Ps_77 white mutants demonstrated that the blue pigment increases oxidative-stress resistance. Indeed, while Ps_77 was growing at a normal rate in presence of 5\u202fmM of H2O2, white mutants were completely inhibited. The antioxidative protection is not available for non-producing bacteria in co-culture with Ps_77