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The use of whole-genome sequencing (WGS) using next-generation sequencing (NGS) technology has become a widely accepted method for microbiology laboratories in the application of molecular typing for outbreak tracing and genomic epidemiology. Several studies demonstrated the usefulness of WGS data analysis through single-nucleotide polymorphism (SNP) calling from a reference sequence analysis for Brucella melitensis, whereas gene-by-gene comparison through core-genome multilocus sequence typing (cgMLST) has not been explored so far.

Background. The design of tendon biomimetic electrospun fleece with Amniotic Epithelial Stem Cells (AECs) that have shown a high tenogenic attitude may represent an alternative strategy to overcome the unsatisfactory results of conventional treatments in tendon regeneration. Methods. In this study, we evaluated AEC-engineered electrospun poly(lactide-co-glycolide) (PLGA) fleeces with highly aligned fibers (ha-PLGA) that mimic tendon extracellular matrix, their biocompatibility, and differentiation towards the tenogenic lineage. PLGA fleeces with randomly distributed fibers (rd-PLGA) were generated as control. Results. Optimal cell infiltration and biocompatibility with both PLGA fleeces were shown. However, only ha-PLGA fleeces committed AECs towards an Epithelial-Mesenchymal Transition (EMT) after 48 h culture, inducing their cellular elongation along the fibers’ axis and the upregulation of mesenchymal markers. AECs further differentiated towards tenogenic lineage as confirmed by the up-regulation of tendon-related genes and Collagen Type 1 (COL1) protein expression that, after 28 days culture, appeared extracellularly distributed along the direction of ha-PLGA fibers. Moreover, long-term co-cultures of AEC-ha-PLGA bio-hybrids with fetal tendon explants significantly accelerated of half time AEC tenogenic differentiation compared to ha-PLGA fleeces cultured only with AECs. Conclusions. The fabricated tendon biomimetic ha-PLGA fleeces induce AEC tenogenesis through an early EMT, providing a potential tendon substitute for tendon engineering research.

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Injured tendons are challenging in their regeneration; thus, tissue engineering represents a promising solution. This research tests the hypothesis that the response of amniotic epithelial stem cells (AECs) can be modulated by fiber diameter size of tendon biomimetic fleeces. Particularly, the effect of electrospun poly(lactide-co-glycolide) (PLGA) fleeces with highly aligned microfibers possessing two different diameter sizes (1.27 and 2.5 µm: ha1- and ha2-PLGA, respectively) was tested on the ability of AECs to differentiate towards the tenogenic lineage by analyzing tendon related markers (Collagen type I: COL1 protein and mRNA Scleraxis: SCX, Tenomodulin: TNMD and COL1 gene expressions) and to modulate their immunomodulatory properties by investigating the pro- (IL-6 and IL-12) and anti- (IL-4 and IL-10) inflammatory cytokines. It was observed that fiber alignment and not fiber size influenced cell morphology determining the morphological change of AECs from cuboidal to fusiform tenocyte-like shape. Instead, fleece mechanical properties, cell proliferation, tenogenic differentiation, and immunomodulation were regulated by changing the ha-PLGA microfiber diameter size. Specifically, higher DNA quantity and better penetration within the fleece were found on ha2-PLGA, while ha1-PLGA fleeces with small fiber diameter size had better mechanical features and were more effective on AECs trans-differentiation towards the tenogenic lineage by significantly translating more efficiently SCX into the downstream effector TNMD. Moreover, the fiber diameter of 1.27 µm induced higher expression of pro-regenerative, anti-inflammatory interleukins mRNA expression (IL-4 and IL-10) with favorable IL-12/IL-10 ratio with respect to the fiber diameter of 2.5 µm. The obtained results demonstrate that fiber diameter is a key factor to be considered when designing tendon biomimetic fleece for tissue repair and provide new insights into the importance of controlling matrix parameters in enhancing cell differentiation and immunomodulation either for the cells functionalized within or for the transplanted host tissue.

The key factor in the timely conclusion of this investigation was intersectoral collaboration among epidemiologists, microbiologists, veterinarians, statisticians and health and food safety authorities at national, regional and local levels.

A total of 66 Listeria monocytogenes (Lm) isolated from 2013 to 2018 in a small-scale meat processing plant and a dairy facility of Central Italy were studied. Whole Genome Sequencing and bioinformatics analysis were used to assess the genetic relationships between the strains and investigate persistence and virulence abilities. The biofilm forming-ability was assessed in vitro. Cluster analysis grouped the Lm from the meat plant into three main clusters: two of them, both belonging to CC9, persisted for years in the plant and one (CC121) was isolated in the last year of sampling. In the dairy facility, all the strains grouped in a CC2 four-year persistent cluster. All the studied strains carried multidrug efflux-pumps genetic determinants (sugE, mdrl, lde, norM, mepA). CC121 also harbored the Tn6188 specific for tolerance to Benzalkonium Chloride. Only CC9 and CC121 carried a Stress Survival Islet and presented high-level cadmium resistance genes (cadA1C1) carried by different plasmids. They showed a greater biofilm production when compared with CC2. All the CC2 carried a full-length inlA while CC9 and CC121 presented a Premature Stop Codon mutation correlated with less virulence. The hypo-virulent clones CC9 and CC121 appeared the most adapted to food-processing environments; however, even the hyper-virulent clone CC2 warningly persisted for a long time. The identification of the main mechanisms promoting Lm persistence in a specific food processing plant is important to provide recommendations to Food Business Operators (FBOs) in order to remove or reduce resident Lm.

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