Microbiota profiling with long amplicons using Nanopore sequencing : full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon

Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples are prone to contain DNA from other sources (e.g. host or environment). The usual approach is sequencing short regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. To achieve an increased taxonomic resolution, we aim to develop long-amplicon PCR-based approaches using Nanopore sequencing. We assessed two different genetic markers: the full-length 16S rRNA (~1,500 bp) and the 16S-ITS-23S region from the rrn operon (4,300 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities and two pools of low-biomass samples (dog skin). Nanopore sequencing was performed on MinION™ using the 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using EPI2ME or Minimap2 with rrn database. Consensus sequences of the 16S-ITS-23S genetic marker were obtained using canu. Results: The full-length 16S rRNA and the 16S-ITS-23S region of the rrn operon were used to retrieve the microbiota composition of the samples at the genus and species level. For the Staphylococcus pseudintermedius isolate, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the16S-ITS-23S genetic marker, and in ~68%, with the 16S rRNA gene when using EPI2ME. Using mock communities, we found that the full-length 16S rRNA gene represented better the abundances of a microbial community; whereas, 16S-ITS-23S obtained better resolution at the species level. Finally, we characterized low-biomass skin microbiota samples and detected species with an environmental origin. Conclusions: Both full-length 16S rRNA and the 16S-ITS-23S of the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, targeting the 16S-ITS-23S of the rrn operon would be the best choice

Expression Analysis of MicroRNAs in FFPE samples of canine cutaneous and oral melanoma by RT-qPCR

MicroRNA (miRNA), a class of small, non-coding RNA - regulating post-transcriptionally protein expression - are emerging as clinical biomarkers in many pathologies, including cancer [1]. Since miRNA are supposed to represent fundamental key regulators, better understanding of melanoma biology is essential to improve staging and therapy. The aim of the study was to investigate whether miRNA expression can vary in canine melanoma samples derived from formalin-fixed-paraffin-embedded (FFPE) tissues. Experimental design of the study included three groups, each one composed of 7 animals: i) control healthy skin group ii) oral melanoma group iii) skin melanoma group. The histhopatology and immunoistochemistry details of dogs included in the study are previously reported  [2]. Two tissue slides were used for miRNA extraction. The expression levels of seven miRNA - miR-145, miR-146a, miR-425-5p, miR-223, miR-365, miR-155 and miR-134 - were detected and assessed by qPCR using TaqMan® probes [3-5]. Five miRNA were significantly up-(n=3) or down-(n=2) regulated. In details, miR-146a and miR-155 abundance was increased as compared with control in both oral and skin melanoma (Fig 1 B,E) (p = 0.004 and 0.014 and p = 0.043 and 0.035 respectively), while the levels of miR-145 and miR-365 were lower (Fig 1 A,D) (p = 0.018 and 0.008 and p = 0.01 and 0.028, respectively). MiR-425-5p was upregulated (p = 0.039) only in skin melanoma (Fig. 1 C). Furthermore, functional analysis, carried out using miRNet web-based tool, showed that 76 genes related to cancer-associated pathways were possible target of these five microRNA (p = 6.95E-9); in particular, 21 target genes were associated with melanoma (p = 1.47E-5), including BRAF and CDK, E2F, FGF and PIK3 families. In conclusion, miR-145, miR-146a, miR-425-5p, miR-365 and miR-155 are differentially expressed in melanoma and healthy FFPE samples, suggesting that they may play a role in canine melanoma pathogenesis and/or progression

Short communication:Intra- and inter-individual milk microbiota variability in healthy and infected water buffalo udder quarters

The concept that ruminant mammary gland quarters are anatomically and physiologically unrelated has been recently challenged by immunological evidence. How this interdependence reflects on individual quarter milk microbiota is unknown. The aim of the present study was to cover this gap by investigating the interdependence of quarters among the same mammary gland at the milk microbiota level using next-generation sequencing of the V4\u201316S rRNA gene. A total of 52 samples were included in this study and classified as healthy or affected by subclinical mastitis. Extraction of DNA, amplification of the V4\u201316S rRNA gene, and sequencing using Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, Waltham, MA) were carried out. We found that the intra-individual variability was lower than the inter-individual one. The present findings further support at milk microbiota level the hypothesis of the interdependence of quarters, as previously demonstrated following immunological studies, suggesting that individual factors (e.g., immunity, genetics) may have a role in modulating milk microbiota

Characterization of skin surface and dermal microbiota in dogs with mast cell tumor

The skin microbiota interacts with the host immune response to maintain the homeostasis. Changes in the skin microbiota are linked to the onset and the progression of several diseases, including tumors. We characterized the skin surface and dermal microbiota of 11 dogs affected by spontaneous mast cell tumor (MCT), using skin contralateral sites as intra-animal healthy controls. The microbial profile differed between healthy and tumor skin surfaces and dermis, demonstrating that the change in microbiota composition is related to the presence of MCT. The number of observed taxa between MCT and healthy skin surfaces was detected, showing a decrease in number and heterogeneity of taxa over the skin surface of MCT, at both inter- and intra-individual level. Preliminary data on bacterial population of MCT dermis, obtained only on three dogs, demonstrated an intra-individual reduction of taxa number when compared to the skin surface. Taxonomy reveals an increase of Firmicutes phylum and Corynebacteriaceae family in MCT skin surface when compared to the healthy contralateral. In conclusion, we demonstrate that microbial population of skin surface and dermis is related to mast cell tumor. Our study provides the basis for future investigations aiming to better define the interaction between mast cell tumors, microbiota and host immune response

Water buffalo microbiota and immunity during infectious diseases

La tesi té com a objectiu investigar la resposta immune dels búfals d’aigua (Bubalus bubalis) durant malalties infeccioses (per exemple, mastitis, brucel·losi i tuberculosi) i malalties de produccions, com malalties metabòliques relacionades amb el peripart o l’estrès. Tenint en compte la relació entre l’entorn microbià i el sistema immunitari, també s’ha identificat el contingut de la microbiota de llet. El sistema immunitari del búfalo d’aigua ha estat poc caracteritzat fins ara, per no parlar de la microbiota, que era desconeguda. Els búfals d’aigua són sensibles a les mateixes malalties que els remugants, com per exemple mastitis, tuberculosi, brucel·losi, però no es coneix l’impacte d’aquestes malalties sobre el sistema immune dels búfals. El búfal d’aigua presenta diferències anatòmiques (per exemple a la glàndula mamària i a nivell de la pell) i fisiològiques (malalties infeccioses i periparts) en comparació amb els remugants. Per tant, és evident que tant el sistema immune com la microbiota podrien presentar diverses diferències. A la meva tesi doctoral vaig tractar d’abordar algunes d’aquestes qüestions. En primer lloc, es va investigar la microbiota de llet dels búfals d’aigua en relació amb la mastitis. En segon lloc, l’avaluació de la resposta immune, en termes d’expressió gènica i miRNAs, es va tractar en animals afectats per brucel·losi i tuberculosi. Finalment, es va realitzar la caracterització del període de transició mesurant proteïnes de fase aguda per avaluar l’estat de la inflamació durant el període de transició.El objetivo de la tesis es investigar la respuesta inmunitaria de los búfalos de agua (Bubalus bubalis) durante las enfermedades infecciosas (por ejemplo, mastitis, brucelosis y tuberculosis) y las enfermedades de producción, como las enfermedades metabólicas relacionadas con el periparto o el estrés. Debido a que el sistema inmunitario está relacionado con el entorno microbiano, también se ha caracterizado la composición de la microbiota de la leche. Hasta el momento, el sistema inmunitario del búfalo de agua no estaba bien caracterizado y la microbiota de la leche era desconocida. Aunque los búfalos sean sensibles a las mismas enfermedades que los rumiantes (como por ejemplo mastitis, tuberculosis, brucelosis), se desconoce el impacto de estas enfermedades en el sistema inmunitario de los búfalos. En comparación con los otros rumiantes, el búfalo de agua presenta diferencias anatómicas (por ejemplo, en la glándula mamaria y en la piel) y fisiológicas (enfermedades infecciosas y peripartos). Por lo tanto, es evidente que tanto el sistema inmune como la microbiota podrían presentar varias diferencias. En mi tesis doctoral, he abordado algunos de estos problemas. En primer lugar, se investigó la microbiota de leche de los búfalos de agua durante la enfermedad de la mastitis. En segundo lugar, se evaluó la respuesta inmunitaria en animales afectados por brucelosis y tuberculosis, mediante técnicas de expresión génica y miRNAs. Finalmente, se caracterizó el período de transición midiendo las proteínas de fase aguda para evaluar el estado de inflamación.The thesis aims to investigate the immune response of water buffaloes (Bubalus bubalis) during infectious diseases (e.g. mastitis, brucellosis and tuberculosis) and productions diseases, such as peripartum related metabolic diseases or stress. Given the relationship between the microbial environment and the immune system, the microbiota content of milk has been identified as well. The immune system of water buffalo has been poorly addressed so far, not to mention the microbiota, which was unknown. Dairy water buffaloes are sensitive to the same diseases as dairy ruminants, such as for example mastitis, tuberculosis, brucellosis, but the impact of these diseases on water buffaloes’ immune system are unknown. Water buffalo presents anatomical (e.g. at mammary gland and skin level) and physiological (peripartum and infectious diseases) differences as compared to cow and other dairy ruminants. Therefore, it is evident that both the immune system and microbiota could present several differences. In my PhD thesis, I tried to address some of these issues. Firstly, the milk microbiota of water buffaloes was investigated in relation to mastitis disease. Secondly, the evaluation of the immune response, in terms of gene expression ad miRNAs, was carried out in animals affected by brucellosis and tuberculosis. Finally, the characterization of the transition period was performed measuring acute phase proteins to assess the inflammation status during the transition period

Water buffalo microbiota and immunity during infectious diseases

La tesi té com a objectiu investigar la resposta immune dels búfals d'aigua (Bubalus bubalis) durant malalties infeccioses (per exemple, mastitis, brucel·losi i tuberculosi) i malalties de produccions, com malalties metabòliques relacionades amb el peripart o l'estrès. Tenint en compte la relació entre l'entorn microbià i el sistema immunitari, també s'ha identificat el contingut de la microbiota de llet. El sistema immunitari del búfalo d'aigua ha estat poc caracteritzat fins ara, per no parlar de la microbiota, que era desconeguda. Els búfals d'aigua són sensibles a les mateixes malalties que els remugants, com per exemple mastitis, tuberculosi, brucel·losi, però no es coneix l'impacte d'aquestes malalties sobre el sistema immune dels búfals. El búfal d'aigua presenta diferències anatòmiques (per exemple a la glàndula mamària i a nivell de la pell) i fisiològiques (malalties infeccioses i periparts) en comparació amb els remugants. Per tant, és evident que tant el sistema immune com la microbiota podrien presentar diverses diferències. A la meva tesi doctoral vaig tractar d'abordar algunes d'aquestes qüestions. En primer lloc, es va investigar la microbiota de llet dels búfals d'aigua en relació amb la mastitis. En segon lloc, l'avaluació de la resposta immune, en termes d'expressió gènica i miRNAs, es va tractar en animals afectats per brucel·losi i tuberculosi. Finalment, es va realitzar la caracterització del període de transició mesurant proteïnes de fase aguda per avaluar l'estat de la inflamació durant el període de transició.El objetivo de la tesis es investigar la respuesta inmunitaria de los búfalos de agua (Bubalus bubalis) durante las enfermedades infecciosas (por ejemplo, mastitis, brucelosis y tuberculosis) y las enfermedades de producción, como las enfermedades metabólicas relacionadas con el periparto o el estrés. Debido a que el sistema inmunitario está relacionado con el entorno microbiano, también se ha caracterizado la composición de la microbiota de la leche. Hasta el momento, el sistema inmunitario del búfalo de agua no estaba bien caracterizado y la microbiota de la leche era desconocida. Aunque los búfalos sean sensibles a las mismas enfermedades que los rumiantes (como por ejemplo mastitis, tuberculosis, brucelosis), se desconoce el impacto de estas enfermedades en el sistema inmunitario de los búfalos. En comparación con los otros rumiantes, el búfalo de agua presenta diferencias anatómicas (por ejemplo, en la glándula mamaria y en la piel) y fisiológicas (enfermedades infecciosas y peripartos). Por lo tanto, es evidente que tanto el sistema inmune como la microbiota podrían presentar varias diferencias. En mi tesis doctoral, he abordado algunos de estos problemas. En primer lugar, se investigó la microbiota de leche de los búfalos de agua durante la enfermedad de la mastitis. En segundo lugar, se evaluó la respuesta inmunitaria en animales afectados por brucelosis y tuberculosis, mediante técnicas de expresión génica y miRNAs. Finalmente, se caracterizó el período de transición midiendo las proteínas de fase aguda para evaluar el estado de inflamación.The thesis aims to investigate the immune response of water buffaloes (Bubalus bubalis) during infectious diseases (e.g. mastitis, brucellosis and tuberculosis) and productions diseases, such as peripartum related metabolic diseases or stress. Given the relationship between the microbial environment and the immune system, the microbiota content of milk has been identified as well. The immune system of water buffalo has been poorly addressed so far, not to mention the microbiota, which was unknown. Dairy water buffaloes are sensitive to the same diseases as dairy ruminants, such as for example mastitis, tuberculosis, brucellosis, but the impact of these diseases on water buffaloes' immune system are unknown. Water buffalo presents anatomical (e.g. at mammary gland and skin level) and physiological (peripartum and infectious diseases) differences as compared to cow and other dairy ruminants. Therefore, it is evident that both the immune system and microbiota could present several differences. In my PhD thesis, I tried to address some of these issues. Firstly, the milk microbiota of water buffaloes was investigated in relation to mastitis disease. Secondly, the evaluation of the immune response, in terms of gene expression ad miRNAs, was carried out in animals affected by brucellosis and tuberculosis. Finally, the characterization of the transition period was performed measuring acute phase proteins to assess the inflammation status during the transition period

Impact of intramammary inoculation of inactivated Lactobacillus rhamnosus and antibiotics on the milk microbiota of water buffalo with subclinical mastitis

Water buffalo mastitis represents a major issue in terms of animal health, cost of therapy, premature culling and decreased milk yeld. The emergence of antibiotic resistance has led to investigate strategies to avoid or reduce antibiotics' based therapies, in particular during subclinical mastitis. The use of Generally Regarded As Safe bacteria (GRAS) such as Lactobacillus rhamnosus to restore the unbalance in mammary gland microbiota could provide potential corrective measures. The aim of this study was to investigate the changes in milk microbiota after the intramammary treatment with inactivated cultures of Lactobacillus rhamnosus of mammary gland quarters naturally affected by subclinical mastitis as compared to antibiotic therapy.A number of 43 quarters affected by subclinical mastitis with no signs of clinical inflammation and aerobic culture positive for pathogens were included in the study. The experimental design was as follows: 11 quarters were treated with antibiotics, 15 with inactivated cultures of Lactobacillus rhmnosus and 17 with PBS as negative control, by means of intrammary injection. Samples were collected at eight time points, pre- (T-29, T-21, T-15, T-7, T0 days) and post- treatment (T1, T2, and T6 days). Microbiological culture and Somatic Cell Count (SCC) were perfomed on all the samples, and microbiota was determined on milk samples collected at T0 and T6 by amplifying the V4 region of 16S rRNA gene by PCR and sequencing using next generation sequencing technique. Treatment with Lactobacillus rhamnosus elicited a strong chemotactic response, as determined by a significant increase of leukocytes in milk, but did not change the microbiological culture results of the treated quarters. For what concerns the analysis of the microbiota, the treatment with Lactobacillus rhamnosus induced the modification in relative abundance of some genera such as Pseudomonas and 5-7N15. As expected, antibiotic treatment caused major changes in microbiota structure with an increase of Methylobacterium relative abundance. No changes were detected after PBS treatment. In conclusion, the present findings demonstrated that the in vivo intrammmary treatment with Lactobacillus rhamnosus has a transient pro-inflammatory activity by increasing SCC and is capable to modify the microbiota of milk after six days from inoculation, albeit slightly, even when the bacterial cultures were heat inactivated. Further studies are necessary to assess the potential use of this GRAS as supportive therapy against mastitis

Gut microbiome features in COVID-19: analysis of a cohort of hospitalized patients

1 Abstract – Objective: Emerging evidence suggests a direct involvement of the gastrointestinal tract in COVID-19. Although the specific immune response is of paramount importance in the SARS-CoV-2 virus elimination process, aberrant immune activity could lead to severe disease and late inflammatory forms. In this context, the intestinal microbiota plays a primary role in the maturation and maintenance of the immune system. This study investigates whether the SARS-CoV-2 infection can be associated with alterations in the gut microbiome composition and if such variations could correlate with the severity of symptoms and disease outcomes. Patients and Methods: We performed shotgun metagenomic sequencing of stool samples of 45 patients, aged between 30 and 95 years, hospitalized with COVID-19. Patients were grouped by clinical severity (i.e., non-crit- ical or critical), type of hospitalization (non-intensive care or intensive therapy unit) and outcome (survival or deceased) to explore the impact of the gut microbiome changes on patients’ health. Results: COVID-19 severity is associated with alterations in the intestinal microbiome, reduced microbial biodiversity and increased Escherichia and Bacteroides genera. No statistical significance was found between the extent of dysbiosis and clinical severity. We found an enrichment of micro-eukaryotic species, e.g., Can- dida albicans, Candida tropicalis, Saccharomyces cerevisiae and bacterial species previously associated with diseases as well as unhealthy cardiometabolic markers, e.g., Escherichia coli, Bacteroides fragilis, Clostrid- ium bolteae, Clostridium innocuum, Clostridium symbiosum, Eggerthella lenta, Enterococcus faecium, and Flavonifractor plautii. Conclusions: Our findings suggest a trend of correlation between the degree of intestinal dysbiosis and the se - verity of the disease, likely depending on the depletion of some microorganisms with immunomodulatory effect. Furthermore, gut dysbiosis could explain the inflammatory outcome, which persists after viral negativization and might justify possible future complications