Genomic Characterization of Listeria monocytogenes and Other Listeria Species Isolated from Sea Turtles.

Listeria monocytogenes is a ubiquitous pathogen found both in the environment and food. It can cause listeriosis in a wide range of animals as well as in humans. Investigations on presence, spread and virulence are still limited to terrestrial and human environments. Embracing the One Health Approach, investigating the presence and spread of L. monocytogenes in marine ecosystems and among wildlife, would provide us with useful information for human health. This study investigated the presence of L. monocytogenes and Listeria spp. in two species of sea turtles common in the Mediterranean Sea (Caretta caretta and Chelonia mydas). A total of one hundred and sixty-four carcasses of sea turtles (C. caretta n = 161 and C. mydas n = 3) stranded along the Abruzzo, Molise, Campania, and Calabria coasts, were collected. Brain and fecal samples were taken, enriched, and cultured for the detection of Listeria spp. From the specimens collected, strains of L. monocytogenes (brain n = 1, brain and feces n = 1, multiorgan n = 1 and feces n = 1), L. innocua (feces n = 1 and brain n = 1), and L. ivanovii (brain n = 1) were isolated. Typical colonies were isolated for Whole Genome Sequencing (WGS). Virulence genes, disinfectants/metal resistance, and antimicrobial resistance were also investigated. L. monocytogenes, L. innocua, and L. ivanovii were detected in C. caretta, whilst only L. monocytogenes and L. innocua in C. mydas. Notable among the results is the lack of significant differences in gene distribution between human and sea turtle strains. Furthermore, potentially pathogenic strains of L. monocytogenes were found in sea turtles.

Dynamic of SARS-CoV-2 variants circulation in Tunisian pediatric population, during successive waves, from March 2020 to September 2022.

The emergence of SARS-CoV-2 variants has led to several cases among children. However, limited information is available from North African countries. This study describes the SARS-CoV-2 strains circulating in Tunisian pediatric population during successive waves. A total of 447 complete sequences were obtained from individuals aged from 13 days to 18 years, between March 2020 and September 2022: 369 sequences generated during this study and 78 ones, available in GISAID, previously obtained from Tunisian pediatric patients. These sequences were compared with 354 and 274 ones obtained from Tunisian adults and a global dataset, respectively. The variant circulation dynamics of predominant variants were investigated during the study period using maximum-likelihood phylogenetic analysis. Among the studied population, adolescents were the predominant age group, comprising 55.26% of cases. Twenty-three lineages were identified; seven of which were not previously reported in Tunisia. Phylogenetic analysis showed a close relationship between the sequences from Tunisian adults and children. The connections of sequences from other countries were variable according to variants: close relationships were observed for Alpha, B1.160 and Omicron variants, while independent Tunisian clusters were observed for Delta and B.1.177 lineages. These findings highlight the pivotal role of children in virus transmission and underscore the impact of vaccination on virus spread. Vaccination of children, with booster doses, may be considered for better management of future emergences.

Genomic Characterization of a Wild-Type Bovine alphaherpesvirus 1 (BoAHV-1) Strain Isolated in an Outbreak in Central Italy.

Bovine alphaherpesvirus-1 (BoAHV-1) infection is common in cattle worldwide. However, information on the spread of BoAHV-1-circulating strains in Italy remains limited. In this study, we investigated an outbreak characterized by severe respiratory symptoms in a cattle herd (n = 30) located in Central Italy. BoAHV-1 was isolated from three cattle in a cell culture, which confirmed viral infection. Next, we characterized one (16453/07 TN) of the three isolates of BoAHV-1 using whole-genome sequencing. BLASTn and phylogenetic analysis revealed a nucleotide identity >99% with all BoAHV-1 strains belonging to subtype 1.1, highlighting the genetic stability of the virus. This study reports the first full genomic characterization of a BoAHV-1 isolate in Italy, enriching our understanding of the genetic characteristics of the circulating BoAHV-1 strain in Italy.

Identification of IncA Plasmid, Harboring blaVIM-1 Gene, in S. enterica Goldcoast ST358 and C. freundii ST62 Isolated in a Hospitalized Patient.

In the present study, we analyzed the genome of two S. enterica strains TS1 and TS2 from stool and blood cultures, respectively, and one strain of C. freundii TS3, isolated from a single hospitalized patient with acute myeloid leukemia. The S. enterica Goldcoast ST358 (O:8 (C2-C3) serogroup), sequenced by the MiSeq Illumina system, showed the presence of ß-lactamase genes (blaVIM-1, blaSHV-12 and blaOXA-10), aadA1, ant(2″)-Ia, aac(6')-Iaa, aac(6')-Ib3, aac(6')-Ib-cr, qnrVC6, parC(T57S), and several incompatibility plasmids. A wide variety of insertion sequences (ISs) and transposon elements were identified. In C. freundii TS3, these were the blaVIM-1, blaCMY-150, and blaSHV-12, aadA1, aac(6')-Ib3, aac(6')-Ib-cr, mph(A), sul1, dfrA14, ARR-2, qnrVC6, and qnrB38. IncA plasmid isolated from E.coli/K12 transconjugant and C. freundii exhibited a sequence identity >99.9%. The transfer of IncA plasmid was evaluated by conjugation experiments.

Occurrence and temporal distribution of extended-spectrum ß-lactamase-producing Escherichia coli in clams from the Central Adriatic, Italy.

The spread of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli is a major public health issue. Bivalves are filter-feeder animals capable of bioaccumulating the microorganisms present in water. This physiological characteristic makes them both good indicators of environmental contamination and possible carriers of pathogenic bacteria, including those resistant to antimicrobials. The aim of this study was to investigate the occurrence of ESBL-producing E. coli in clams (n = 308) collected from harvesting areas of the Central Adriatic Sea between 2018 and 2019. ESBL- /class C ß-lactamase (AmpC)- producing E. coli and Escherichia spp. were isolated by streaking over the surface of MacConkey agar plates supplemented with cefotaxime enriched broths of the initial shellfish suspension. E. coli and Escherichia spp. resistant to cefotaxime were screened for ESBL production by using the double disk synergy test. Susceptibility to different antimicrobials and confirmation of ESBL-production were determined by the minimum inhibitory concentration (MIC) test. Isolates were further characterized by whole genome sequencing (WGS) and bioinformatic analysis of genomes with different tools. Overall, ESBL-producing E. coli were isolated from 3% of the samples. Of 13 ESBL- and ESBL-/AmpC-producing Escherichia spp. (n = 11 E. coli, n = 1 E. marmotae, n = 1 E. ruysiae) isolates, 13 were resistant to ampicillin and cefotaxime, 9 to sulfamethoxazole, 6 to tetracycline and nalidixic acid, 4 to trimethoprim, and 3 to ceftazidime, cefoxitin, ciprofloxacin, and chloramphenicol. Moreover, the majority (8/11) of the ESBL-producing E. coli isolates were multidrug-resistant. WGS showed that the isolates predominantly carried the blaCTX-M-15 gene (3/11) and blaCTX-M-14 and blaCTX-M-1 (2/11 each). The AmpC ß-lactamase CMY-2 was found in two isolates. Phylogroup A was the most prevalent (5/11), followed by phylogroups D (4/11), F (1/11), and B2 (1/11). Ten different sequence types (STs) were identified. Occurrence at sampling sites ranged between 0 and 27%. To identify associations between the occurrence of ESBL-producing E. coli and E. coli levels, samples were divided into two groups, with E. coli at >230 MPN/100 g and E. coli at ≤230 MPN/100 g. ESBL-producing E. coli isolates were significantly more commonly recovered in samples with higher E. coli levels (14%) than in those with lower levels of E. coli (2%). Moreover, the majority (3/4) of the potentially pathogenic strains were isolated in samples with higher E. coli levels. These findings provided evidence for the bacterial indicator of fecal contamination, E. coli, as an index organism for ESBL-producing E. coli isolates in bivalves.

Evaluation of next generation sequencing approaches for SARS-CoV-2.

Within public health control strategies for SARS-CoV-2, whole genome sequencing (WGS) is essential for tracking viral spread and monitoring the emergence of variants which may impair the effectiveness of vaccines, diagnostic methods, and therapeutics. In this manuscript different strategies for SARS-CoV-2 WGS including metagenomic shotgun (SG), library enrichment by myBaits® Expert Virus-SARS-CoV-2 (Arbor Biosciences), nCoV-2019 sequencing protocol, ampliseq approach by Swift Amplicon® SARS-CoV-2 Panel kit (Swift Biosciences), and Illumina COVIDSeq Test (Illumina Inc.), were evaluated in order to identify the best approach in terms of results, labour, and costs. The analysis revealed that Illumina COVIDSeq Test (Illumina Inc.) is the best choice for a cost-effective, time-consuming production of consensus sequences.

SARS-CoV-2 excretion kinetics in nasopharyngeal and stool samples from the pediatric population.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for serious respiratory infections in humans. Even in the absence of respiratory symptoms, gastrointestinal (GI) signs were commonly reported in adults and children. Thus, oral-fecal transmission was suspected as a possible route of infection. The objective of this study was to describe RNA shedding in nasopharyngeal and stool samples obtained from asymptomatic and symptomatic children and to investigate virus viability. Methods: This study included 179 stool and 191 nasopharyngeal samples obtained from 71 children, which included symptomatic (n = 64) and asymptomatic (n = 7) ones. They were collected every 7 days from the onset of the infection until negativation. Viral RNA was detected by real-time RT-PCR, targeting the N and ORF1 genes. Whole-genome sequencing was performed for positive cases. Viral isolation was assessed on Vero cells, followed by molecular detection confirmation. Results: All cases included in this study (n = 71) were positive in their nasopharyngeal samples. SARS-CoV-2 RNA was detected in 36 stool samples obtained from 15 out of 71 (21.1%) children; 13 were symptomatic and two were asymptomatic. Excretion periods varied from 7 to 21 days and 7 to 14 days in nasopharyngeal and fecal samples, respectively. Four variants were detected: Alpha (n = 3), B.1.160 (n = 3), Delta (n = 7), and Omicron (n = 1). Inoculation of stool samples on cell culture showed no specific cytopathic effect. All cell culture supernatants were negative for RT-qPCR. Conclusion: Our study demonstrated nasopharyngeal and fecal shedding of SARS-CoV-2 RNA by children up to 21 and 14 days, respectively. Fecal shedding was recorded in symptomatic and asymptomatic children. Nevertheless, SARS-CoV-2 was not isolated from positive stool samples.

Spatial and temporal dynamics of West Nile virus between Africa and Europe.

It is unclear whether West Nile virus (WNV) circulates between Africa and Europe, despite numerous studies supporting an African origin and high transmission in Europe. We integrated genomic data with geographic observations and phylogenetic and phylogeographic inferences to uncover the spatial and temporal viral dynamics of WNV between these two continents. We focused our analysis towards WNV lineages 1 (L1) and 2 (L2), the most spatially widespread and pathogenic WNV lineages. Our study shows a Northern-Western African origin of L1, with back-and-forth exchanges between West Africa and Southern-Western Europe; and a Southern African origin of L2, with one main introduction from South Africa to Europe, and no back introductions observed. We also noticed a potential overlap between L1 and L2 Eastern and Western phylogeography and two Afro-Palearctic bird migratory flyways. Future studies linking avian and mosquito species susceptibility, migratory connectivity patterns, and phylogeographic inference are suggested to elucidate the dynamics of emerging viruses.

Harmonization of supervised machine learning practices for efficient source attribution of Listeria monocytogenes based on genomic data.

BACKGROUND: Genomic data-based machine learning tools are promising for real-time surveillance activities performing source attribution of foodborne bacteria such as Listeria monocytogenes. Given the heterogeneity of machine learning practices, our aim was to identify those influencing the source prediction performance of the usual holdout method combined with the repeated k-fold cross-validation method. METHODS: A large collection of 1 100 L. monocytogenes genomes with known sources was built according to several genomic metrics to ensure authenticity and completeness of genomic profiles. Based on these genomic profiles (i.e. 7-locus alleles, core alleles, accessory genes, core SNPs and pan kmers), we developed a versatile workflow assessing prediction performance of different combinations of training dataset splitting (i.e. 50, 60, 70, 80 and 90%), data preprocessing (i.e. with or without near-zero variance removal), and learning models (i.e. BLR, ERT, RF, SGB, SVM and XGB). The performance metrics included accuracy, Cohen's kappa, F1-score, area under the curves from receiver operating characteristic curve, precision recall curve or precision recall gain curve, and execution time. RESULTS: The testing average accuracies from accessory genes and pan kmers were significantly higher than accuracies from core alleles or SNPs. While the accuracies from 70 and 80% of training dataset splitting were not significantly different, those from 80% were significantly higher than the other tested proportions. The near-zero variance removal did not allow to produce results for 7-locus alleles, did not impact significantly the accuracy for core alleles, accessory genes and pan kmers, and decreased significantly accuracy for core SNPs. The SVM and XGB models did not present significant differences in accuracy between each other and reached significantly higher accuracies than BLR, SGB, ERT and RF, in this order of magnitude. However, the SVM model required more computing power than the XGB model, especially for high amount of descriptors such like core SNPs and pan kmers. CONCLUSIONS: In addition to recommendations about machine learning practices for L. monocytogenes source attribution based on genomic data, the present study also provides a freely available workflow to solve other balanced or unbalanced multiclass phenotypes from binary and categorical genomic profiles of other microorganisms without source code modifications.

Listeria monocytogenes in ready to eat meat products from Zambia: phenotypical and genomic characterization of isolates.

The contamination of ready to eat foods (RTE) products due to Listeria monocytogenes could compromise the products safety becoming a great risk for the consumers. The high presence of L. monocytogenes in RTE products has been described worldwide, but few data are available about these products from African countries. The aims of this study were to report the presence of L. monocytogenes in Zambian RTE products, providing genomic characterization and data on similarity with African circulating strains using whole genome sequencing (WGS). A total of 304 RTE products, produced by different Zambian manufacturers, were purchased at retail, from major supermarkets located in Lusaka, Zambia, comprising 130 dairy and 174 meat products. L. monocytogenes was detected only in 18 (10.3%) RTE meat products of the 174 samples tested. The MLST analysis grouped the 18 L. monocytogenes isolates in 7 clonal complexes (CCs): CC1 (n = 5), CC2 (n = 4), CC9 (n = 4), CC5 (n = 2), CC121 (n = 1), CC155 (n = 1), and CC3 (n = 1). According to the cgMLST results, several clusters were detected, in particular belonging to hyper-virulent clones CC1 and CC2. Regarding the virulence factors, a complete L. monocytogenes Pathogenicity Island 3 (LIPI-3) was present both in the CC1 and CC3, in addition to LIPI-1. Several resistance genes and mobile genetic elements were detected, including Stress Islands, the bcrABC cassette and Tn6188_qac transposon, plasmids and intact prophages. Despite being a first preliminary work with a limited number of samples and isolates, this study helped to increase existing knowledge on contaminated RTE products in Zambia, confirming the presence of hyper-virulent L. monocytogenes CCs, which could play an important role in human diseases, posing a public health concern for consumers.

Culicoides species responsible for the transmission of Epizootic Haemorrhagic Disease virus (EHDV) serotype 8 in Italy.

Epizootic haemorrhagic disease (EHD) is a viral disease transmitted by Culicoides biting midges that affects wild and domestic ruminants. The causative agent, EHD virus (EHDV), belongs to the family Sedoreoviridae, genus Orbivirus. The virus has never been reported in Europe until October 2022, when the virus was for the first time detected in Sicily and Sardinia. After the first clinical cases, an intensive entomological field activity was carried out in five EHD affected farms located in Sardinia, with the aim of assessing the EHDV vector competence in European species of Culicoides. EHDV­8 was detected in C. imicola, C. obsoletus/scoticus, C. newsteadi, C. pulicaris ss, and C. bysta. The first 4 species have also been demonstrated to be able to transmit bluetongue virus (BTV). According to these results, it is likely that EHDV­8, sharing the same transmission patterns of BTV, can also spread to Europe.

Multi-approach methods to predict cryptic carbapenem resistance mechanisms in Klebsiella pneumoniae detected in Central Italy.

The rapid emergence of carbapenem-resistant Klebsiella pneumoniae (Kp) strains in diverse environmental niches, even outside of the clinical setting, poses a challenge for the detection and the real-time monitoring of novel antimicrobial resistance trends using molecular and whole genome sequencing-based methods. The aim of our study was to understand cryptic resistance determinants responsible for the phenotypic carbapenem resistance observed in strains circulating in Italy by using a combined approach involving whole genome sequencing (WGS) and genome-wide association study (GWAS). In this study, we collected 303 Kp strains from inside and outside clinical settings between 2018-2022 in the Abruzzo region of Italy. The antimicrobial resistance profile of all isolates was assessed using both phenotypic and bioinformatic methods. We identified 11 strains resistant to carbapenems, which did not carry any known genetic determinants explaining their phenotype. The GWAS results showed that incongruent carbapenem-resistant phenotype was associated specifically with strains with two capsular types, KL13 and KL116 including genes involved in the capsule synthesis, encoding proteins involved in the assembly of the capsule biosynthesis apparatus, capsule-specific sugar synthesis, processing and export, polysaccharide pyruvyl transferase, and lipopolysaccharide biosynthesis protein. These preliminary results confirmed the potential of GWAS in identifying genetic variants present in KL13 and KL116 that could be associated with carbapenem resistance traits in Kp. The implementation of advanced methods, such as GWAS with increased antimicrobial resistance surveillance will potentially improve Kp infection treatment and patient outcomes.

Unveiling the immunomodulatory shift: Epithelial-mesenchymal transition Alters immune mechanisms of amniotic epithelial cells.

Epithelial-mesenchymal transition (EMT) changes cell phenotype by affecting immune properties of amniotic epithelial cells (AECs). The present study shows how the response to lipopolysaccharide of cells collected pre- (eAECs) and post-EMT (mAECs) induces changes in their transcriptomics profile. In fact, eAECs mainly upregulate genes involved in antigen-presenting response, whereas mAECs over-express soluble inflammatory mediator transcripts. Consistently, network analysis identifies CIITA and Nrf2 as main drivers of eAECs and mAECs immune response, respectively. As a consequence, the depletion of CIITA and Nrf2 impairs the ability of eAECs and mAECs to inhibit lymphocyte proliferation or macrophage-dependent IL-6 release, thus confirming their involvement in regulating immune response. Deciphering the mechanisms controlling the immune function of AECs pre- and post-EMT represents a step forward in understanding key physiological events wherein these cells are involved (pregnancy and labor). Moreover, controlling the immunomodulatory properties of eAECs and mAECs may be essential in developing potential strategies for regenerative medicine applications.

Novel Amplicon-Based Sequencing Approach to West Nile Virus.

West Nile virus is a re-emerging arbovirus whose impact on public health is increasingly important as more and more epidemics and epizootics occur, particularly in America and Europe, with evidence of active circulation in Africa. Because birds constitute the main reservoirs, migratory movements allow the diffusion of various lineages in the world. It is therefore crucial to properly control the dispersion of these lineages, especially because some have a greater health impact on public health than others. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to West Nile virus. This study was carried out on different strains from lineage 1 and 2 from Senegal and Italy. The presented protocol/approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for West Nile genomic surveillance.

Complete Genome of African Swine Fever Virus Genotype II in Central Italy.

We report here the whole-genome sequence of the African swine fever virus (ASFV) genotype II, strain 20355/RM/2022_Italy, identified in a wild boar in the city of Rome (Lazio region, Italy) in April 2022.

Severe Streptococcus equi Subspecies zooepidemicus Outbreak from Unpasteurized Dairy Product Consumption, Italy.

During November 2021-May 2022, we identified 37 clinical cases of Streptococcus equi subspecies zooepidemicus infections in central Italy. Epidemiologic investigations and whole-genome sequencing showed unpasteurized fresh dairy products were the outbreak source. Early diagnosis by using sequencing technology prevented the spread of life-threatening S. equi subsp. zooepidemicus infections.

Molecular Characterization of the First African Swine Fever Virus Genotype II Strains Identified from Mainland Italy, 2022.

African swine fever (ASF) is responsible for important socio-economic effects in the global pig industry, especially for countries with large-scale piggery sectors. In January 2022, the African swine fever virus (ASFV) genotype II was identified in a wild boar population in mainland Italy (Piedmont region). This study describes the molecular characterization, by Sanger and next-generation sequencing (NGS), of the first index case 632/AL/2022 and of another isolate (2802/AL/2022) reported in the same month, in close proximity to the first, following multiple ASF outbreaks. Phylogenetic analysis based on the B646L gene and NGS clustered the isolates 632/AL/2022 and 2802/AL/2022 within the wide and most homogeneous p72 genotype II that includes viruses from European and Asian countries. The consensus sequence obtained from the ASFV 2802/AL/2022 isolate was 190,598 nucleotides in length and had a mean GC content of 38.38%. At the whole-genome level, ASF isolate 2802/AL/2022 showed a close genetic correlation with the other representative ASFV genotype II strains isolated between April 2007 and January 2022 from wild and domestic pigs in Eastern/Central European (EU) and Asian countries. CVR subtyping clustered the two Italian ASFV strains within the major CVR variant circulating since the first virus introduction in Georgia in 2007. Intergenic region I73R-I329L subtyping placed the Italian ASFV isolates within the variant identical to the strains frequently identified among wild boars and domestic pigs. Presently, given the high sequence similarity, it is impossible to trace the precise geographic origin of the virus at a country level. Moreover, the full-length sequences available in the NCBI are not completely representative of all affected territories.

Exposure of Mytilus galloprovincialis to Microplastics: Accumulation, Depuration and Evaluation of the Expression Levels of a Selection of Molecular Biomarkers.

Microplastic contamination is a growing marine environmental issue with possible consequences for seafood safety. Filter feeders are the target species for microplastic (MPs) pollution because they filter large quantities of seawater to feed. In the present study, an experimental contamination of Mytilus galloprovincialis was conducted using a mixture of the main types of MPs usually present in the seawater column (53% filaments, 30% fragments, 3% granules) in order to test the purification process as a potential method for removing these contaminants from bivalves intended for human consumption. A set of molecular biomarkers was also evaluated in order to detect any variations in the expression levels of some genes associated with biotransformation and detoxification, DNA repair, cellular response, and the immune system. Our results demonstrate that: (a) the purification process can significantly reduce MP contamination in M. galloprovincialis; (b) a differential expression level has been observed between mussels tested and in particular most of the differences were found in the gills, thus defining it as the target organ for the use of these biomarkers. Therefore, this study further suggests the potential use of molecular biomarkers as an innovative method, encouraging their use in next-generation marine monitoring programs.

SARS-CoV-2 in Namibian Dogs.

The pandemic of coronavirus disease 19 (COVID-19) has focused the attention of researchers, and especially public opinion, on the role of the human-animal-environment interface in disease emergence. At the beginning of the COVID-19 pandemic, media reports regarding the role of pets in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused significant concern and social anxiety. Although nowadays proven negligible in developed countries, essentially no studies have been performed in low-income African areas where companion animals are often raised differently from high income countries, and the contact patterns occurring in these scenarios could affect the epidemiological scenario. An extensive molecular biology survey was performed from March 2022 to September 2022 on Namibian dogs residing in urban and rural areas, showing a low but not negligible SARS-CoV-2 prevalence (1%; 95CI: 0.33-2.32%) of 5 out of 500. In only one instance (i.e., a 4-year-old female Labrador) was there a clear association that could be established between the infections of the owner and animal. In all other cases, no evidence of human infection could be obtained and no episodes of COVID-19 were reported by the owners. Although no consistent evidence of pet-to-pet transmission was proven in the present study, a cautionary principle suggests intensive and dedicated investigation into companion animal populations, especially when animal contact is frequent and a particularly susceptible population is present.

Profiling of mitochondrial heteroplasmy in single human oocytes by next-generation sequencing.

Mitochondrial DNA (mtDNA) plays a crucial role in the development of a competent oocyte. Indeed, mtDNA alterations may predispose to chromosome nondisjunction, resulting in infertility due to a reduced vitality and quality of oocytes and embryos. In this methods paper, the multiple displacement amplification approach was applied in combination with next-generation sequencing (NGS) to amplify and sequence, in single-end, the entire mtDNA of single human oocytes to directly construct genomic NGS libraries, and subsequently, to highlight and quantify the mutations they presented. The bioinformatic workflow was carried out with a specific ad hoc developed in-house software. This approach proved to be sensitive and specific, also highlighting the mutations present in heteroplasmy, showing deletion, insertion or substitution mutations in the genes involved in the respiratory chain, even if the found variants were benign or of uncertain meaning. The analysis of mtDNA mutations in the oocyte could provide a better understanding of specific genetic abnormalities and of their possible effect on oocyte developmental competence. This study shows how this approach, based on a massive parallel sequencing of clonally amplified DNA molecules, allows to sequence the entire mitochondrial genome of single oocytes in a short time and with a single analytical run and to verify mtDNA mutations.