Robertsonian Fusion and Centromere Repositioning Contributed to the Formation of Satellite-free Centromeres During the Evolution of Zebras.

Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated with highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and Equus asinus. Here, through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras Equus burchelli (15 of 22) and Equus grevyi (13 of 23), demonstrating that the absence of satellite DNA at the majority of centromeres is compatible with genome stability and species survival and challenging the role of satellite DNA in centromere function. Nine satellite-free centromeres are shared between the two species in agreement with their recent separation. We assembled all centromeric regions and improved the reference genome of E. burchelli. Sequence analysis of the CENP-A binding domains revealed that they are LINE-1 and AT-rich with four of them showing DNA amplification. In the two zebras, satellite-free centromeres emerged from centromere repositioning or following Robertsonian fusion. In five chromosomes, the centromeric function arose near the fusion points, which are located within regions marked by traces of ancestral pericentromeric sequences. Therefore, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. Finally, in one case, a satellite-free centromere was seeded on an inversion breakpoint. At 11 chromosomes, whose primary constrictions seemed to be associated with satellite repeats by cytogenetic analysis, satellite-free neocentromeres were instead located near the ancestral inactivated satellite-based centromeres; therefore, the centromeric function has shifted away from a satellite repeat containing locus to a satellite-free new position.

A Satellite-Free Centromere in Equus przewalskii Chromosome 10.

In mammals, centromeres are epigenetically specified by the histone H3 variant CENP-A and are typically associated with satellite DNA. We previously described the first example of a natural satellite-free centromere on Equus caballus chromosome 11 (ECA11) and, subsequently, on several chromosomes in other species of the genus Equus. We discovered that these satellite-free neocentromeres arose recently during evolution through centromere repositioning and/or chromosomal fusion, after inactivation of the ancestral centromere, where, in many cases, blocks of satellite sequences were maintained. Here, we investigated by FISH the chromosomal distribution of satellite DNA families in Equus przewalskii (EPR), demonstrating a good degree of conservation of the localization of the major horse satellite families 37cen and 2PI with the domestic horse. Moreover, we demonstrated, by ChIP-seq, that 37cen is the satellite bound by CENP-A and that the centromere of EPR10, the ortholog of ECA11, is devoid of satellite sequences. Our results confirm that these two species are closely related and that the event of centromere repositioning which gave rise to EPR10/ECA11 centromeres occurred in the common ancestor, before the separation of the two horse lineages.

Molecular Dynamics and Evolution of Centromeres in the Genus Equus.

The centromere is the chromosomal locus essential for proper chromosome segregation. While the centromeric function is well conserved and epigenetically specified, centromeric DNA sequences are typically composed of satellite DNA and represent the most rapidly evolving sequences in eukaryotic genomes. The presence of satellite sequences at centromeres hampered the comprehensive molecular analysis of these enigmatic loci. The discovery of functional centromeres completely devoid of satellite repetitions and fixed in some animal and plant species represented a turning point in centromere biology, definitively proving the epigenetic nature of the centromere. The first satellite-free centromere, fixed in a vertebrate species, was discovered in the horse. Later, an extraordinary number of satellite-free neocentromeres had been discovered in other species of the genus Equus, which remains the only mammalian genus with numerous satellite-free centromeres described thus far. These neocentromeres arose recently during evolution and are caught in a stage of incomplete maturation. Their presence made the equids a unique model for investigating, at molecular level, the minimal requirements for centromere seeding and evolution. This model system provided new insights on how centromeres are established and transmitted to the progeny and on the role of satellite DNA in different aspects of centromere biology.

Birth, evolution, and transmission of satellite-free mammalian centromeric domains.

Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Equus caballus Chromosome 11. Here, we investigated the satellite-free centromeres of Equus asinus by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regions. A subset of these centromeres is associated with DNA amplification. The location of CENPA binding domains can vary in different individuals, giving rise to epialleles. The analysis of epiallele transmission in hybrids (three mules and one hinny) showed that centromeric domains are inherited as Mendelian traits, but their position can slide in one generation. Conversely, centromere location is stable during mitotic propagation of cultured cells. Our results demonstrate that the presence of more than half of centromeres void of satellite DNA is compatible with genome stability and species survival. The presence of amplified DNA at some centromeres suggests that these arrays may represent an intermediate stage toward satellite DNA formation during evolution. The fact that CENPA binding domains can move within relatively restricted regions (a few hundred kilobases) suggests that the centromeric function is physically limited by epigenetic boundaries.

Telomeric-Like Repeats Flanked by Sequences Retrotranscribed from the Telomerase RNA Inserted at DNA Double-Strand Break Sites during Vertebrate Genome Evolution.

Interstitial telomeric sequences (ITSs) are stretches of telomeric-like repeats located at internal chromosomal sites. We previously demonstrated that ITSs have been inserted during the repair of DNA double-strand breaks in the course of evolution and that some rodent ITSs, called TERC-ITSs, are flanked by fragments retrotranscribed from the telomerase RNA component (TERC). In this work, we carried out an extensive search of TERC-ITSs in 30 vertebrate genomes and identified 41 such loci in 22 species, including in humans and other primates. The fragment retrotranscribed from the TERC RNA varies in different lineages and its sequence seems to be related to the organization of TERC. Through comparative analysis of TERC-ITSs with orthologous empty loci, we demonstrated that, at each locus, the TERC-like sequence and the ITS have been inserted in one step in the course of evolution. Our findings suggest that telomerase participated in a peculiar pathway of DNA double-strand break repair involving retrotranscription of its RNA component and that this mechanism may be active in all vertebrate species. These results add new evidence to the hypothesis that RNA-templated DNA repair mechanisms are active in vertebrate cells.

Exploring the Specificity of Rationally Designed Peptides Reconstituted from the Cell-Free Extract of Deinococcus radiodurans toward Mn(II) and Cu(II).

A series of five rationally designed decapeptides [DEHGTAVMLK (DP1), THMVLAKGED (DP2), GTAVMLKDEH (Term-DEH), TMVLDEHAKG (Mid-DEH), and DEHGGGGDEH (Bis-DEH)] have been studied for their interactions with Cu(II) and Mn(II) ions. The peptides, constructed including the most prevalent amino acid content found in the cell-free extract of Deinococcus radiodurans (DR), play a fundamental role in the antioxidant mechanism related to its exceptional radioresistance. Mn(II) ions, in complex with these peptides, are found to be an essential ingredient for the DR protection kit. In this work, a detailed characterization of Cu(II) systems was included, because Cu(II)-peptide complexes have also shown remarkable antioxidant properties. All peptides studied contain in their sequence coordinating residues that can bind effectively Mn(II) or Cu(II) ions with high affinity, such as Asp, Glu, and His. Using potentiometric techniques, NMR, EPR, UV-vis, and CD spectroscopies, ESI-MS spectrometry, and molecular model calculations, we explored the binding properties and coordination modes of all peptides toward the two metal ions, were able to make a metal affinity comparison for each metal system, and built a structural molecular model for the most stable Cu(II) and Mn(II) complexes in agreement with experimental evidence.

Insertion of Telomeric Repeats in the Human and Horse Genomes: An Evolutionary Perspective.

Interstitial telomeric sequences (ITSs) are short stretches of telomeric-like repeats (TTAGGG)n at nonterminal chromosomal sites. We previously demonstrated that, in the genomes of primates and rodents, ITSs were inserted during the repair of DNA double-strand breaks. These conclusions were derived from sequence comparisons of ITS-containing loci and ITS-less orthologous loci in different species. To our knowledge, insertion polymorphism of ITSs, i.e., the presence of an ITS-containing allele and an ITS-less allele in the same species, has not been described. In this work, we carried out a genome-wide analysis of 2504 human genomic sequences retrieved from the 1000 Genomes Project and a PCR-based analysis of 209 human DNA samples. In spite of the large number of individual genomes analyzed we did not find any evidence of insertion polymorphism in the human population. On the contrary, the analysis of ITS loci in the genome of a single horse individual, the reference genome, allowed us to identify five heterozygous ITS loci, suggesting that insertion polymorphism of ITSs is an important source of genetic variability in this species. Finally, following a comparative sequence analysis of horse ITSs and of their orthologous empty loci in other Perissodactyla, we propose models for the mechanism of ITS insertion during the evolution of this order.

Centromere sliding on a mammalian chromosome.

The centromere directs the segregation of chromosomes during mitosis and meiosis. It is a distinct genetic locus whose identity is established through epigenetic mechanisms that depend on the deposition of centromere-specific centromere protein A (CENP-A) nucleosomes. This important chromatin domain has so far escaped comprehensive molecular analysis due to its typical association with highly repetitive satellite DNA. In previous work, we discovered that the centromere of horse chromosome 11 is completely devoid of satellite DNA; this peculiar feature makes it a unique model to dissect the molecular architecture of mammalian centromeres. Here, we exploited this native satellite-free centromere to determine the precise localization of its functional domains in five individuals: We hybridized DNA purified from chromatin immunoprecipitated with an anti CENP-A antibody to a high resolution array (ChIP-on-chip) of the region containing the primary constriction of horse chromosome 11. Strikingly, each individual exhibited a different arrangement of CENP-A binding domains. We then analysed the organization of each domain using a single nucleotide polymorphism (SNP)-based approach and single molecule analysis on chromatin fibres. Examination of the ten instances of chromosome 11 in the five individuals revealed seven distinct 'positional alleles', each one extending for about 80-160 kb, were found across a region of about 500 kb. Our results demonstrate that CENP-A binding domains are autonomous relative to the underlying DNA sequence and are characterized by positional instability causing the sliding of centromere position. We propose that this dynamic behaviour may be common in mammalian centromeres and may determine the establishment of epigenetic alleles.

Genome-wide evolutionary and functional analysis of the Equine Repetitive Element 1: an insertion in the myostatin promoter affects gene expression.

BACKGROUND: In mammals, an important source of genomic variation is insertion polymorphism of retrotransposons. These may acquire a functional role when inserted inside genes or in their proximity. The aim of this work was to carry out a genome wide analysis of ERE1 retrotransposons in the horse and to analyze insertion polymorphism in relation to evolution and function. The effect of an ERE1 insertion in the promoter of the myostatin gene, which is involved in muscle development, was also investigated. RESULTS: In the horse population, the fraction of ERE1 polymorphic loci is related to the degree of similarity to their consensus sequence. Through the analysis of ERE1 conservation in seven equid species, we established that the level of identity to their consensus is indicative of evolutionary age of insertion. The position of ERE1s relative to genes suggests that some elements have acquired a functional role. Reporter gene assays showed that the ERE1 insertion within the horse myostatin promoter affects gene expression. The frequency of this variant promoter correlates with sport aptitude and racing performance. CONCLUSIONS: Sequence conservation and insertion polymorphism of ERE1 elements are related to the time of their appearance in the horse lineage, therefore, ERE1s are a useful tool for evolutionary and population studies. Our results suggest that the ERE1 insertion at the myostatin locus has been unwittingly selected by breeders to obtain horses with specific racing abilities. Although a complex combination of environmental and genetic factors contributes to athletic performance, breeding schemes may take into account ERE1 insertion polymorphism at the myostatin promoter.

Discovery and comparative analysis of a novel satellite, EC137, in horses and other equids.

Centromeres are the sites of kinetochore assembly and spindle fiber attachment and consist of protein-DNA complexes in which the DNA component is typically characterized by the presence of extended arrays of tandem repeats called satellite DNA. Here, we describe the isolation and characterization of a 137-bp-long new satellite DNA sequence from the horse genome (EC137), which is also present, even if less abundant, in the domestic donkey, the Grevy's zebra and the Burchelli's zebra. We investigated the chromosomal distribution of the EC137 sequence in these 4 species. Moreover, we analyzed its architectural organization by high-resolution FISH. The position of this sequence with respect to the primary constriction and in relation to the 2 major horse satellite tandem repeats (37 cen and 2PI) on horse chromosomes suggests that the new centromeric equine satellite is an accessory DNA element, presumably contributing to the organization of pericentromeric chromatin. FISH on combed DNA fibers reveals that the EC137 satellite is organized in relatively short stretches (2-8 kb) which are strictly intermingled within 37 cen or 2PI arrays. This arrangement suggests that interchanges between satellite families are a frequent occurrence in the horse genome.

Pathogenic profile and antimicrobial resistance of Escherichia coli, Escherichia marmotae and Escherichia ruysiae detected from hunted wild boars in Sardinia (Italy).

The objective of this study was to evaluate the occurrence of E. coli in hunted wild boars in Sardinia (Italy) and to further characterize the isolates with Whole Genome Sequencing to assess the genetic relatedness and the presence of virulence and antimicrobial resistance (AMR) genes. Samples were taken from 66 wild boars between 2020 and 2022 slaughtered in five hunting houses. A total of 181 samples were tested, including 66 samples from mesenteric lymph nodes, 66 samples from colon content and 49 samples from carcass surface. Isolates referable to Escherichia species were detected in all of the wild boars sampled. On a selection of 61 isolates, sequencing was conducted and antimicrobial susceptibility was tested. Among these, three isolates were confirmed to be two Escherichia marmotae (cryptic clade V) and one Escherichia ruysiae (cryptic clade III). E. coli pathotypes identified were UPEC (13 %), ExPEC-UPEC (5.6 %) and ETEC (3.7 %). Moreover, 3/6 E. marmotae isolates had typical ExPEC genes. Genetic similarity was observed in isolates collected from animals slaughtered in the same hunting house; this suggests epidemiological links deriving from the presence of animals infected with closely related strains or the result of cross-contamination. Antimicrobial resistance genes were detected in three non-pathogenic E. coli isolates: one isolate had sul2, tet(B), aph(6)-ld and aph(3″)-lb resistance genes and two had the fosA7 gene. This study confirmed that wild boars can act as reservoirs and spreaders of pathogenic Escherichia species and it provides information for future comparative genomic analysis in wildlife. Although isolates showed a limited resistome, the detection of resistance in non-pathogenic isolates underlines the need to monitor antimicrobial resistance in the wild boar population. To the best of our knowledge, this is the first detection of E. mamotae and E. ruysiae isolates in wild boars in Italy and the presence of this pathogen in wildlife and livestock need to be investigated further.

Uncoupling of satellite DNA and centromeric function in the genus Equus.

In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs.

Short-term performance analysis of sludge treatment reed beds.

Sludge treatment reed beds (STRBs) represent an established technology for managing sludge produced by wastewater treatment plants (WWTPs). In this study, an analysis conducted on sludge stratigraphy in a STRB serving the municipal WWTPs of Helsinge (42,000 person equivalent, Denmark) is presented. The STRB has been in operation since 1996. The analysis was conducted for four months including two consecutive loadings of one of the 10 basins. The amount of dewatering during the short initial phase was significant, and the rehydration of deeper layers was negligible. A clear reduction trend was observed for volatile solids and labile organic matter, which is the first step towards organic matter humification. This hypothesis was also confirmed by water soluble carbon, which decreased with the increasing depth and the time of resting. About the N cycle, ammonification and nitrification showed a reciprocal balancing effect, so as to cause an equilibrium between the stable trends of NH(4)(+) and NO(3)(-) which were similar along the depth and the time of monitoring. The highest values of both parameters were found at the surface, where the combined effect of plant action, direct diffusive oxygen transfer from the atmosphere and high nutrient contents of the freshly loaded sludge was likely more intense.

Use of commercial protective cultures in portioned sheep milk cheeses to control Listeria monocytogenes.

The main objective of this study was to innovate soft and semi-cooked sheep milk cheese production processes with the use of a commercial protective culture able to control Listeria monocytogenes growth. A freeze-dried commercial culture of Lactobacillus plantarum was tested in DS cheese and PS cheese, two types of pasteurized sheep milk, raw-paste cheeses aged for no less than 20 and 30 days respectively. In the first step, in vitro tests were conducted to identify the most suitable matrix for the growth of L. plantarum in order to create a subculture that could be used at industrial cheese-making plants. During the second phase of the study, L. plantarum culture was introduced in the manufacturing process of the cheeses in a production plant. Finally, a challenge test was conducted on portioned DS and PS cheeses in order to evaluate the activity of the protective culture against L. monocytogenes: the cheeses were portioned, experimentally contaminated with L. monocytogenes strains, vacuum packed and stored at +4°C (correct storage conditions) and at +10°C (thermal abuse). Cheeses were analysed at the end of the shelf-life to evaluate the presence and growth of L. monocytogenes, to enumerate lactic acid bacteria and determine chemicalphysical features. The results confirmed that protective cultures are a useful technological innovation to control L. monocytogenes growth during cheese storage without altering composition, microflora and chemical- physical characteristics of the product. However, the use of protective cultures should be applied as an integration of risk control measures and not as a substitute for preventive actions.

Comparison of Activity of Commercial Protective Cultures and Thermophilic Lactic Acid Bacteria against Listeria monocytogenes: A New Perspective to Improve the Safety of Sardinian PDO Cheeses.

Listeria monocytogenes contamination that occurs during and post-processing of dairy products is a serious concern for consumers, and bioprotective cultures can be applied to control the growth of the pathogen in sheep milk cheeses. However, to respect specifications provided for protected designation of origin (PDO) cheeses, only autochthonous microorganisms can be used as bioprotective cultures in these products. This in vitro study aimed to evaluate thermophilic lactic acid bacteria (LAB) isolated from sheep milk as bio-preservative agents to control L. monocytogenes growth in PDO cheese. Results were compared with those obtained with a commercial protective culture (cPC) composed of a Lactiplantibacillus plantarum bacteriocin producer designed to inhibit L. monocytogenes growth in cheese. The in vitro antilisterial activities of n.74 autochthonous LAB and a cPC were tested against 51 L. monocytogenes strains using an agar well diffusion assay. In addition, 16S rRNA sequencing of LAB isolates with antilisterial activity was conducted and strains of Lactobacillus helveticus, Lactobacillus delbrueckii subsp. indicus, Lactobacillus delbrueckii subsp. sunkii, Lactobacillus delbrueckii subsp. lactis and Enterococcus faecalis were identified. In this study, 33.6% (74/220) bacterial strains isolated from milk had characteristics compatible with thermophilic LAB, of which 17.6% (13/74) had in vitro antilisterial activity. These results demonstrate that raw sheep milk can be considered an important source of autochthonous thermophilic LAB that can be employed as protective cultures during the manufacturing of Sardinian PDO cheeses to improve their food safety. The use of bioprotective cultures should be seen as an additional procedure useful to improve cheese safety along with the correct application of good hygienic practices during manufacturing and the post-processing stages.

Effect of Commercial and Autochthonous Bioprotective Cultures for Controlling Listeria monocytogenes Contamination of Pecorino Sardo Dolce PDO Cheese.

The composition and physicochemical characteristics of short-aged Pecorino Sardo PDO (Protected Designation of Origin) cheese makes it permissive to Listeria monocytogenes growth. The PDO product specification stipulates that this cheese is produced with whole sheep's milk inoculated with cultures from the area of origin. Therefore, the use of bioprotective cultures for the inhibition of pathogens in PDO cheeses is allowed only if autochthonous microorganisms are used. Furthermore, bioprotective cultures are generally used on the cheese surface to prevent the outgrowth of L. monocytogenes, the application of which can be time-consuming and require specialist technical knowledge. In this study, we examine the direct addition of bioprotective cultures to the cheese vat and compare the activity of a commercial bioprotective culture (Lactiplantibacillus plantarum) and an autochthonous lactic acid bacterium with bioprotective properties (Lactobacillus delbruekii sups. sunkii), for the inhibition of L. monocytogenes in Pecorino Sardo PDO cheese. Three types of Pecorino Sardo PDO cheese were made with bioprotective cultures added directly to the cheese milk along with the starter inoculum: PSA, with the commercial bioprotective culture; PSB, with the autochthonous bioprotective culture; and a CTRL cheese with no bioprotective culture. A challenge test was performed on each of these cheeses by artificially contaminating the cheese surface with L. monocytogenes (2 Log10 CFU/g). Three batches of each cheese type were analyzed to enumerate mesophilic and thermophilic lactic acid bacteria and to investigate the growth potential of L. monocytogenes during manufacturing, at the end of ripening, at the end of shelf-life, and after 180 days from cheese production. Both bioprotective cultures tested in this study showed inhibitory action against the pathogen with 0.3-1.8 Log10 CFU/g (colony-forming unit per gram) reduction levels. The autochthonous organism, L. sunkii, was as effective as the commercially supplied culture, and the addition of the bioprotective cultures to the cheese-making procedure offered protection against L. monocytogenes. The direct addition of bioprotective cultures to the making procedure of Pecorino Sardo PDO cheese is a potentially innovative strategy to improve the safety of this product.

Preliminary data on the microbial profile of dry and wet aged bovine meat obtained from different breeds in Sardinia.

This study aimed to evaluate the influence of dry and wet aging on microbial profile and physicochemical characteristics of bovine loins obtained from four animals of two different breeds, namely two Friesian cull cows and two Sardo-Bruna bovines. During dry and wet aging aerobic colony count, Enterobacteriaceae, mesophilic lactic acid bacteria, Pseudomonas, molds and yeasts, Salmonella enterica, Listeria monocytogenes and Yersinia enterocolitica, pH and water activity (aw) were determined in meat samples collected from the internal part of the loins. Moreover, the microbial profile was determined with sponge samples taken from the surface of the meat cuts. Samples obtained from Friesian cows were analyzed starting from the first day of the aging period and after 7, 14, and 21 days. Samples obtained from the Sardo Bruna bovines were also analyzed after 28 and 35 days. Wet aging allowed better control of Pseudomonas spp. during storage that showed statistically lower levels (P>0.05) in wet-aged meats with respect to dry-aged meats during aging and particularly at the end of the period (P>0.01) in both cattle breeds. At the end of the experiment (21 days), aerobic colony count and Pseudomonas in Fresian cows' dry-aged meats showed mean levels >8 log, while lactic acid bacteria mean counts >7 log were detected in wet-aged meats of both cattle breeds. In meats submitted to dry aging, pH was significantly higher (P<0.01) with respect to wet-aged meats at all analysis times and in both cattle breeds. Aw showed a stable trend during both dry and wet aging without significant differences. These preliminary results highlight the critical importance of the strict application of good hygiene practices during all stages of production of these particular cuts of meat intended for aging.

Evaluation of vacuum packaging for extending the shelf life of Sardinian fermented sausage.

Salsiccia sarda or Sardinian fermented sausage is a traditional dry-fermented sausage included in the list of traditional food products of Sardinia (Italy). At the request of some producing plants, the possibility of extending the shelf life of the vacuum-packed product up to 120 days was evaluated. Manufacturing of 90 samples, representing 3 different batches of Sardinian fermented sausage was carried out in two producing plants (A and B). In the packaged product and subsequently every 30 days for four months (T0, T30, T60, T120), the following analyses were conducted on all samples: physicochemical characteristics, total aerobic mesophilic count, Enterobacteriaceae count, detection of Listeria monocytogenes, Salmonella spp., mesophilic lactic acid bacteria, and coagulase-positive Staphylococci. Moreover, surfaces in contact and surfaces not in contact with food were sampled in both producing plants. Sensory profile analysis was also performed for every analysis time. At the end of the extended shelf life, pH values were equal to 5.90±0.11 (producing plant A) and 5.61±0.29 (producing plant B). Water activity mean values at T120 were 0.894±0.02 (producing plant A) and 0.875±0.01 (producing plant B). L. monocytogenes was detected in 73.3% (33/45) of the samples from producing plant A, with mean levels of 1.12±0.76 log10 CFU/g. In producing plant B, L. monocytogenes was never detected. Enterobacteriaceae were detected in 91.1% (41/45) of samples in producing plant A with mean values of 3.15±1.21 log10 CFU/g, and in 35.5% (16/45) samples in producing plant B samples with mean values of 0.72±0.86 log10 CFU/g. Salmonella and Staphylococcus aureus were never detected. Regarding environmental samples, the sites that were most contaminated by L. monocytogenes were the bagging table (contact surface) and processing room floor drains (non-contact surface) with a prevalence of 50% each (8/16 positive samples for both sampling sites). Sensory analysis results showed that at T30 the overall sensory quality was at its highest;moreover, the visual-tactile aspect, the olfactory characteristics, the gustatory aspects, and the texture showed significant differences in samples throughout the shelf life, with a decreased intensity at 120 days of storage. Overall, the quality and sensory acceptance of the vacuumpacked Sardinian fermented sausage was not affected until 120 days of shelf-life. However, the possible contamination by L. monocytogenes calls attention to the hygienic management of the entire technological process. The environmental sampling was confirmed as a useful verification tool during control.

Traceability, virulence and antimicrobial resistance of Yersinia enterocolitica in two industrial cheese-making plants.

Between 2018 and 2019, 309 environmental and food samples were collected from two industrial cheese-making plants located in Sardinia, in order to investigate Y. enterocolitica presence and to characterize the isolates. Y. enterocolitica isolates were further compared with isolates detected during a previous investigation from sheep and goat raw milk samples. Y. enterocolitica was detected in 7.4 % of the samples and the prevalence was higher, even if not significantly (P > 0.05) higher in non-food contact surface samples (10.2 %) than in food contact surface samples (3.8 %). The highest prevalence was detected in floor samples (13.5 %), followed by drain samples (7.2 %), which might serve as main harborage sites for further contamination. Y. enterocolitica was also detected in food contact surfaces, namely shelves of the Ricotta cooling room and packaging room, one cheese cutting machine surface and one raw milk filter sample. The biotype 1A isolates identified in this study were classified into six different serotypes. Additionally, a bioserotype 2/O:5,27 isolate was identified in one goat milk sample. All 1A isolates possessed the virulence genes invA and ystB while the 2/O:5,27 isolate showed the presence of ail, ystA, invA and yadA genes, thus confirming a pathogenic potential. The isolates showed intrinsic resistance to amoxicillin-clavulanic acid, ticarcillin and cefoxitin due to the presence of the blaA gene. Whole genome sequencing allowed to identify seven different sequence types among the 1A isolates, thus showing a high genetic diversity. The same Y. enterocolitica sequence type (ST3) was detected from three different areas of the same cheese-making plant, indicating a possible transfer of the microorganism along the processing lines. Y. enterocolitica contamination in cheese-making plants can pose a risk to human health. Preventive measures include the hygienic design of the plant layout and equipment, in association with proper cleaning and disinfection programmes.

The localization of centromere protein A is conserved among tissues.

Centromeres are epigenetically specified by the histone H3 variant CENP-A. Although mammalian centromeres are typically associated with satellite DNA, we previously demonstrated that the centromere of horse chromosome 11 (ECA11) is completely devoid of satellite DNA. We also showed that the localization of its CENP-A binding domain is not fixed but slides within an about 500 kb region in different individuals, giving rise to positional alleles. These epialleles are inherited as Mendelian traits but their position can move in one generation. It is still unknown whether centromere sliding occurs during meiosis or during development. Here, we first improve the sequence of the ECA11 centromeric region in the EquCab3.0 assembly. Then, to test whether centromere sliding may occur during development, we map the CENP-A binding domains of ECA11 using ChIP-seq in five tissues of different embryonic origin from the four horses of the equine FAANG (Functional Annotation of ANimal Genomes) consortium. Our results demonstrate that the centromere is localized in the same region in all tissues, suggesting that the position of the centromeric domain is maintained during development.