Ce-MBGs Loaded with Gentamicin: Characterization and In Vitro Evaluation

Mesoporous Bioactive Glasses (MBGs) are biomaterials widely used in tissue engineering, particularly for hard tissue regeneration. One of the most frequent postoperative complications following a biomaterial surgical implant is a bacterial infection, which usually requires treatment by the systemic administration of drugs (e.g., antibiotics). In order to develop biomaterials with antibiotic properties, we investigated cerium-doped MBGs (Ce-MBGs) as in situ-controlled drug delivery systems (DDSs) of gentamicin (Gen), a wide spectrum antibiotic commonly employed against bacteria responsible of postoperative infections. Here we report the optimization of Gen loading on MBGs and the evaluation of the antibacterial properties and of retention of bioactivity and antioxidant properties of the resulting materials. The Gen loading (up to 7%) was found to be independent from cerium content, and the optimized Gen-loaded Ce-MBGs retain significant bioactivity and antioxidant properties. The antibacterial efficacy was verified up to 10 days of controlled release. These properties make Gen-loaded Ce-MBGs interesting candidates for simultaneous hard tissue regeneration and in situ antibiotic release

Microbiota Survey of Sliced Cooked Ham During the Secondary Shelf Life

Sliced cooked ham packaged in a modified atmosphere is a popular ready-to-eat product, subjected to abundant microbial contamination throughout its shelf life that can lead to deterioration of both sensorial properties and safety. In this study, the microbial load and the chemical–physical features of cooked ham of five producers were monitored for a period of 12 days after the opening of the packages (i.e., the secondary shelf life), during which the products were stored in a domestic refrigerator at 5.2 ± 0.6°C. The sensorial properties presented a perceivable decay after 8 days and became unacceptable after 12 days. High-performance liquid chromatography analysis and solid-phase microextraction coupled with gas chromatography profiling of volatile metabolites indicated that lactic acid, ethanol, acetic acid, acetoin, 3-methyl-1-butanol, and 2-3 butanediol were the main metabolites that characterized the evolution of the analyzed cooked ham. The microbiota was monitored by 16S ribosomal RNA gene profiling and culture-dependent techniques. Already at the opening of packages, all the products presented high microbial load, generally dominated by lactic acid bacteria, with evident differences among the products. The increase of lactic acid bacteria somehow protected samples from abundant contamination by other bacteria, concurring with the evolution of more safe products. This role was exerted by numerous Latilactobacillus, Leuconostoc, and Carnobacterium species, among which the most frequently detected were Latilactobacillus sakei, Latilactobacillus sakei carnosum, Leuconostoc mesenteroides, and Carnobacterium divergens. Some products presented more complex communities that encompassed Proteobacteria such as Moellerella wisconsensis, Proteus hauseri, Brochothrix thermosphacta, and less frequently Pseudomonas, Erwinia, and Massilia. Opportunistic pathogenic bacteria such as Escherichia coli and Vibrio sp. were found in small quantities. The yeasts Kazachstania servazzii and Debaryomyces hansenii occurred already at 0 days, whereas various species of Candida (Candida zeylanoides, Candida sake, Candida norvegica, and Candida glaebosa) were abundant only after 12 days. These results indicated that the microbiological contaminants overgrowing during the secondary shelf life did not derive from environmental cross-contamination at the opening of the tray but were already present when the packages were opened, highlighting the phases of production up to the packaging as those crucial in managing the safety risk associated to this product

Indole and p-cresol in feces of healthy subjects: Concentration, kinetics, and correlation with microbiome

Indole and p-cresol are precursors of the most important uremic toxins, generated from the fermentation of amino acids tryptophan and tyrosine by the proteolytic community of intestinal bacteria. The present study focused on the relationship between the microbiome composition, the fecal levels of indole and p-cresol, and their kinetics of generation/degradation in fecal cultures. The concentration of indole and p-cresol, the volatilome, the dry weight, and the amount of ammonium and carbohydrates were analyzed in the feces of 10 healthy adults. Indole and p-cresol widely differed among samples, laying in the range of 1.0–19.5 μg/g and 1.2–173.4 μg/g, respectively. Higher fecal levels of indole and p-cresol were associated with lower carbohydrates and higher ammonium levels, that are markers of a more pronounced intestinal proteolytic metabolism. Positive relationship was observed also with the dry/wet weight ratio, indicator of prolonged intestinal retention of feces. p-cresol and indole presented a statistically significant negative correlation with OTUs of uncultured Bacteroidetes and Firmicutes, the former belonging to Bacteroides and the latter to the families Butyricicoccaceae (genus Butyricicoccus), Monoglobaceae (genus Monoglobus), Lachnospiraceae (genera Faecalibacterium, Roseburia, and Eubacterium ventriosum group). The kinetics of formation and/or degradation of indole and p-cresol was investigated in fecal slurries, supplemented with the precursor amino acids tryptophan and tyrosine in strict anaerobiosis. The presence of the precursors bursted indole production but had a lower effect on the rate of p-cresol formation. On the other hand, supplementation with indole reduced the net rate of formation. The taxa that positively correlated with fecal levels of uremic toxins presented a positive correlation also with p-cresol generation rate in biotransformation experiments. Moreover other bacterial groups were positively correlated with generation rate of p-cresol and indole, further expanding the range of taxa associated to production of p-cresol (Bacteroides, Alistipes, Eubacterium xylanophylum, and Barnesiella) and indole (e.g., Bacteroides, Ruminococcus torques, Balutia, Dialister, Butyricicoccus). The information herein presented contributes to disclose the relationships between microbiota composition and the production of uremic toxins, that could provide the basis for probiotic intervention on the gut microbiota, aimed to prevent the onset, hamper the progression, and alleviate the impact of nephropaties

Phylogenomic analysis of the genus Leuconostoc

Leuconostoc is a genus of saccharolytic heterofermentative lactic acid bacteria that inhabit plant-derived matrices and a variety of fermented foods (dairy products, dough, milk, vegetables, and meats), contributing to desired fermentation processes or playing a role in food spoilage. At present, the genus encompasses 17 recognized species. In total, 216 deposited genome sequences of Leuconostoc were analyzed, to check the delineation of species and to infer their evolutive genealogy utilizing a minimum evolution tree of Average Nucleotide Identity (ANI) and the core genome alignment. Phylogenomic relationships were compared to those obtained from the analysis of 16S rRNA, pheS, and rpoA genes. All the phylograms were subjected to split decomposition analysis and their topologies were compared to check the ambiguities in the inferred phylogenesis. The minimum evolution ANI tree exhibited the most similar topology with the core genome tree, while single gene trees were less adherent and provided a weaker phylogenetic signal. In particular, the 16S rRNA gene failed to resolve several bifurcations and Leuconostoc species. Based on an ANI threshold of 95%, the organization of the genus Leuconostoc could be amended, redefining the boundaries of the species L. inhae, L. falkenbergense, L. gelidum, L. lactis, L. mesenteroides, and L. pseudomesenteroides. Two strains currently recognized as L. mesenteroides were split into a separate lineage representing a putative species (G16), phylogenetically related to both L. mesenteroides (G18) and L. suionicum (G17). Differences among the four subspecies of L. mesenteroides were not pinpointed by ANI or by the conserved genes. The strains of L. pseudomesenteroides were ascribed to two putative species, G13 and G14, the former including also all the strains presently belonging to L. falkenbergense. L. lactis was split into two phylogenetically related lineages, G9 and G10, putatively corresponding to separate species and both including subgroups that may correspond to subspecies. The species L. gelidum and L. gasicomitatum were closely related but separated into different species, the latter including also L. inhae strains. These results, integrating information of ANI, core genome, and housekeeping genes, complemented the taxonomic delineation with solid information on the phylogenetic lineages evolved within the genus Leuconostoc

Draft Genome Sequences of 12 Leuconostoc carnosum Strains Isolated from Cooked Ham Packaged in a Modified Atmosphere and from Fresh Sausages

Leuconostoc carnosum is a lactic acid bacterium that preferentially colonizes meat. In this work, we present the draft genome sequences of 12 Leuconostoc carnosum strains isolated from modified-atmosphere-packaged cooked ham and fresh sausages. Three strains harbor bacteriocin genes

Effect of Rearing Temperature on Growth and Microbiota Composition of Hermetia illucens

The potential utilization of black soldier fly (Hermetia illucens) as food or feed is interesting due to the nutritive value and the sustainability of the rearing process. In the present study, larvae and prepupae of H. illucens were reared at 20, 27, and 33 °C, to determine whether temperature affects the whole insect microbiota, described using microbiological risk assessment techniques and 16S rRNA gene survey. The larvae efficiently grew across the tested temperatures. Higher temperatures promoted faster larval development and greater final biomass but also higher mortality. Viable Enterobacteriaceae, Bacillus cereus, Campylobacter, Clostridium perfringens, coagulase-positive staphylococci, Listeriaceae, and Salmonella were detected in prepupae. Campylobacter and Listeriaceae counts got higher with the increasing temperature. Based on 16S rRNA gene analysis, the microbiota of larvae was dominated by Providencia (>60%) and other Proteobateria (mainly Klebsiella) and evolved to a more complex composition in prepupae, with a bloom of Actinobacteria, Bacteroidetes, and Bacilli, while Providencia was still present as the main component. Prepupae largely shared the microbiota with the frass where it was reared, except for few lowly represented taxa. The rearing temperature was negatively associated with the amount of Providencia, and positively associated with a variety of other genera, such as Alcaligenes, Pseudogracilibacillus, Bacillus, Proteus, Enterococcus, Pediococcus, Bordetella, Pseudomonas, and Kerstersia. With respect to the microbiological risk assessment, attention should be paid to abundant genera, such as Bacillus, Myroides, Proteus, Providencia, and Morganella, which encompass species described as opportunistic pathogens, bearing drug resistances or causing severe morbidity

Improved fed-batch processes with Wickerhamomyces anomalus WC 1501 for the production of D-arabitol from pure glycerol

D-Arabitol, a five-carbon sugar alcohol, represents a main target of microbial biorefineries aiming to valorize cheap substrates. The yeast Wickerhamomyces anomalus WC 1501 is known to produce arabitol in a glycerol-based nitrogen-limited medium and preliminary fed-batch processes with this yeast were reported to yield 18.0 g/L arabitol

Longitudinal Survey of Fungi in the Human Gut: ITS Profiling, Phenotyping, and Colonization

The fungal component of the intestinal microbiota of eight healthy subjects was studied over 12 months using metagenome survey and culture-based approaches. Aspergillus, Candida, Debaryomyces, Malassezia, Penicillium, Pichia, and Saccharomyces were the most recurrent and/or dominant fungal genera, according to metagenomic analysis. The biodiversity of fungal communities was lower and characterized by greater unevenness, when compared to bacterial microbiome. The dissimilarities both among subjects and over the time within the same subject suggested that most of the fungi passed through the gastro-intestinal tract (GIT) without becoming stable colonizers. Certain genera, such as Aspergillus and Penicillium, were isolated in a minority of cases, although they recurred abundantly and frequently in the metagenomics survey, likely being environmental or food-borne fungi that do not inhabit the GIT. Candida genus was recurrently detected. Candida albicans isolates dominated among the cultivable mycobiota and longitudinally persisted, likely as commensals inhabiting the intestine or regularly reaching it from Candida-colonized districts, such as the oral cavity. Other putative colonizers belonged to Candida zeylanoides, Geotrichum candidum, and Rhodotorula mucilaginosa, with persisting biotypes being identified. Phenotyping of fungal isolates indicated that C. albicans adhered to human epithelial cells more efficiently and produced greater amounts of biofilm in vitro than non-albicans Candida (NAC) and non-Candida fungi (NCF). The C. albicans isolates also induced the highest release of HBD-2 by human epithelial cells, further differing from NAC and NCF. Nine representative isolates were administered to mice to evaluate the ability to colonize the intestine. Only two out of three C. albicans strains persisted in stools of animals 2 weeks after the end of the oral administration, whereas NAC and NCF did not. These results confirm the allochthonous nature of most the intestinal fungi, while C. albicans appears to be commonly involved in stable colonization. A combination of specific genetic features in the microbe and in the host likely allow colonization from fungi normally present solely as passengers. It remains to be established if other species identified as potential colonizers, in addition to Candida, are true inhabitants of the GIT or rather reach the intestine spreading from other body districts

Fermentative processes for the upcycling of xylose to xylitol by immobilized cells of Pichia fermentans WC1507

Xylitol is a pentose-polyol widely applied in the food and pharmaceutical industry. It can be produced from lignocellulosic biomass, valorizing second-generation feedstocks. Biotechnological production of xylitol requires scalable solutions suitable for industrial scale processes. Immobilized-cells systems offer numerous advantages. Although fungal pellet carriers have gained attention, their application in xylitol production remains unexplored. In this study, the yeast strain P. fermentans WC 1507 was employed for xylitol production. The optimal conditions were observed with free-cell cultures at pH above 3.5, low oxygenation, and medium containing (NH4)2SO4 and yeast extract as nitrogen sources (xylitol titer 79.4 g/L, YP/S 66.3%, and volumetric productivity 1.3 g/L/h). Yeast cells were immobilized using inactive Aspergillus oryzae pellet mycelial carrier (MC) and alginate beads (AB) and were tested in flasks over three consecutive production runs. Additionally, the effect of a 0.2% w/v alginate layer, coating the outer surface of the carriers (cMC and cAB, respectively), was examined. While YP/S values observed with both immobilized and free cells were similar, the immobilized cells exhibited lower final xylitol titer and volumetric productivity, likely due to mass transfer limitations. AB and cAB outperformed MC and cMC. The uncoated AB carriers were tested in a laboratory-scale airlift bioreactor, which demonstrated a progressive increase in xylitol production in a repeated batch process: in the third run, a xylitol titer of 63.0 g/L, YP/S of 61.5%, and volumetric productivity of 0.52 g/L/h were achieved. This study confirmed P. fermentans WC 1507 as a promising strain for xylitol production in both free- and entrapped-cells systems. Considering the performance of the wild strain, a metabolic engineering intervention aiming at further improving the efficiency of xylitol production could be justified. MC and AB proved to be viable supports for cell immobilization, but additional process development is necessary to identify the optimal bioreactor configuration and fermentation conditions

Tecniche genomiche e metagenomiche per la caratterizzazione di batteri e comunità microbiche in nicchie ecologiche rilevanti per la salute umana

Le moderne tecnologie di Next Generation Sequencing sono un componente cruciale nello studio di microrganismi e comunità microbiche grazie all’enorme quantità di dati fornita in breve tempo. Con queste tecnologie è possibile identificare e caratterizzare i microrganismi utilizzando un ampio numero di tool bioinformatici che possono sostituire le classiche tecniche di tipizzazione in vitro, portando un risparmio di tempo e risorse. La genomica e la metagenomica possono essere applicate a vari campi e forniscono informazioni sia riguardo un singolo organismo, sia su intere comunità microbiche. I nostri studi sono stati concentrati su due nicchie ecologiche: matrici alimentari e microbiota intestinale umano, entrambi importanti per la salute umana. Un prima parte della tesi descrive la comparazione genomica di 12 ceppi di Leuconostoc carnosum isolati da prodotti a base di carne. Questo batterio è un noto colonizzatore di queste matrici, ricopre un ruolo nel loro deterioramento, ma alcuni ceppi presentano effetti utili alla preservazione. Abbiamo eseguito un sequenziamento whole genome per tutti i ceppi, e dopo l’assembly sono state identificate le caratteristiche genomiche, la presenza di plasmidi, di geni responsabili di antibiotico-resistenza, produzione di batteriocine, sintesi di ammine biogene e abbiamo ricostruito i loro pathway metabolici. La comparazione ha rivelato che i ceppi sono strettamente correlati e condividono la maggior parte delle caratteristiche metaboliche, evidenziando l’adattamento all’ambiente di isolamento grazie alle 23 peptidasi presenti nel genoma core. Questo studio fornisce un approfondimento genomico e metabolico su questo batterio ubiquitario nei prodotti a base di carne. Un secondo progetto della tesi è stato indirizzato a indagare la presenza delle beta-glucuronidasi (GUS) nel microbiota intestinale umano attraverso una strategia metagenomica inedita. Le GUS prodotte dai batteri del microbiota sono capaci di rimuovere le porzioni di acido glucuronico da molti composti e metaboliti, come farmaci e xenobiotici. Queste molecole vengono coniugate con l’acido glucuronico nel fegato per l’escrezione attraverso il tratto gastrointestinale, quindi l’azione enzimatica potrebbe riattivarle permettendone il riassorbimento comportando un’alterazione dell’efficacia del farmaco ed effetti negativi sulla salute. Le GUS sono organizzate in classi in base alle differenze nel loro sito catalitico. Sono stati utilizzati 60 metagenomi ottenuti con shotgun sequencing provenienti da soggetti sani divisi in coorti in base alla provenienza geografica. Da questo set di dati, è stata definita la composizione batterica ed è stata sviluppata ed impiegata una nuova strategia per indagare la distribuzione delle diverse GUS. La beta diversità calcolata con gli indici di Jaccard e Bray-Curtis è stata impiegata per determinare le distanze tra i campioni e determinare le differenze tra essi in termini di distribuzione del tipo di GUS e abbondanza dei batteri che le contengono. Poiché alle differenze strutturali dell’enzima corrisponde una diversa specificità di substrato, e considerando la proporzione delle comunità batteriche contenenti GUS, possiamo valutare che la composizione del microbiota può alterare l’escrezione di alcuni farmaci o xenobioti e determinare un’ampia variabilità interindividuale in termini di risposta al farmaco. Un’ultima parte del progetto di tesi descrive l’applicazione dell’approccio metagenomico in due diversi studi: il primo incentrato a indagare la composizione microbica di campioni fecali arricchiti per identificare specie intestinali capaci di degradare la mucina, e il secondo focalizzato sulla descrizione del microbiota di larve di Hermetia illucens allevate per consumo umano o animale.The modern Next Generation Sequencing technologies represent a crucial component in the study of microorganisms and microbial communities thanks to the huge quantity of data they can provide in a short period of time. These technologies allow the identification and characterization of microorganisms exploiting a vast number of bioinformatic tools that can replace the standard in vitro typing techniques, resulting in savings of time and resources. Genomics and metagenomics can be applied in different fields and they can provide information on single microorganism or on entire microbial communities. We focused our studies on two ecological niches: food matrices and human gut microbiome, due to their relevance to human health. The first work of this thesis is a comparative genomic study of 12 Leuconostoc carnosum strains isolated from meat-based products. This bacterium is a known colonizer of meat-based food matrices, it plays a role in spoilage, but preservative effects have also been proposed for some strains. In our study we performed whole genome sequencing for all the strains, and after genome assembly we identified their genomic features, the presence of plasmids, and genes related to antibiotic resistance, bacteriocins production, biogenic amines synthesis. We also reconstructed their metabolic pathways. The comparison revealed that the strains are closely related and share most of the metabolic features, confirming the adaptation to the meat environment due to the presence of 23 peptidase genes in their core genome. With this study we provided a deeper insight into the genomic and metabolic features of this bacterium ubiquitous in meat products. The second project of the thesis aimed to investigate through an inedited metagenomic strategy the presence of beta-glucuronidases (GUS) in the human gut microbiome. Beta-glucuronidases (GUS) produced by gut microbiome bacteria can remove glucuronic acid moieties from a vast range of compounds and metabolites, like drugs and xenobiotics. These molecules are conjugated with glucuronic acid in the liver to be excreted in the gastrointestinal tract, so the action of GUS may reactivate them allowing the reabsorption, with unpredictable and different efficacy of drugs and negative effect on health. GUS are classified in classes by the differences in the catalytic site. 60 shotgun sequenced metagenomic samples from healthy subjects, ascribed to five geographically distinct cohorts, have been retrieved. From this dataset, bacterial composition has been defined and a novel pipeline to investigate distribution the different GUS has been developed and utilized. Beta-diversity calculated on Bray-Curtis dissimilarity index has been used to determine the distances among samples and determine the differences among samples in terms of GUS type distribution and abundance of bacteria containing GUS. Since the structural differences in the enzyme involve a different substrate specificity, and taking into account the ratio of bacterial community harbouring GUS genes, we can assess that the microbiota composition can alter the excretion of certain drugs or xenobiotics, and determine a wide interindividual variability in terms of response to drugs. In the third part of the thesis, I present metagenomic analysis carried out in two other different studies, the former aimed to investigate the microbial composition in enriched human faecal samples to identify gut mucin degraders, and the latter focused on the description of the microbiota of Hermetia illucens larvae reared for food or feed consumption