Breast cancer patient-derived organoids for the investigation of patient-specific tumour evolution.

BACKGROUND: A reliable preclinical model of patient-derived organoids (PDOs) was developed in a case study of a 69-year-old woman diagnosed with breast cancer (BC) to investigate the tumour evolution before and after neoadjuvant chemotherapy and surgery. The results were achieved due to the development of PDOs from tissues collected before (O-PRE) and after (O-POST) treatment. METHODS: PDO cultures were characterized by histology, immunohistochemistry (IHC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), confocal microscopy, flow cytometry, real-time PCR, bulk RNA-seq, single-cell RNA sequencing (scRNA-seq) and drug screening. RESULTS: Both PDO cultures recapitulated the histological and molecular profiles of the original tissues, and they showed typical mammary gland organization, confirming their reliability as a personalized in vitro model. Compared with O-PRE, O-POST had a greater proliferation rate with a significant increase in the Ki67 proliferation index. Moreover O-POST exhibited a more stem-like and aggressive phenotype, with increases in the CD24low/CD44low and EPCAMlow/CD49fhigh cell populations characterized by increased tumour initiation potential and multipotency and metastatic potential in invasive lobular carcinoma. Analysis of ErbB receptor expression indicated a decrease in HER-2 expression coupled with an increase in EGFR expression in O-POST. In this context, deregulation of the PI3K/Akt signalling pathway was assessed by transcriptomic analysis, confirming the altered transcriptional profile. Finally, transcriptomic single-cell analysis identified 11 cell type clusters, highlighting the selection of the luminal component and the decrease in the number of Epithelial-mesenchymal transition cell types in O-POST. CONCLUSION: Neoadjuvant treatment contributed to the enrichment of cell populations with luminal phenotypes that were more resistant to chemotherapy in O-POST. PDOs represent an excellent 3D cell model for assessing disease evolution.

INTEGRATE: Model-based multi-omics data integration to characterize multi-level metabolic regulation.

Metabolism is directly and indirectly fine-tuned by a complex web of interacting regulatory mechanisms that fall into two major classes. On the one hand, the expression level of the catalyzing enzyme sets the maximal theoretical flux level (i.e., the net rate of the reaction) for each enzyme-controlled reaction. On the other hand, metabolic regulation controls the metabolic flux through the interactions of metabolites (substrates, cofactors, allosteric modulators) with the responsible enzyme. High-throughput data, such as metabolomics and transcriptomics data, if analyzed separately, do not accurately characterize the hierarchical regulation of metabolism outlined above. They must be integrated to disassemble the interdependence between different regulatory layers controlling metabolism. To this aim, we propose INTEGRATE, a computational pipeline that integrates metabolomics and transcriptomics data, using constraint-based stoichiometric metabolic models as a scaffold. We compute differential reaction expression from transcriptomics data and use constraint-based modeling to predict if the differential expression of metabolic enzymes directly originates differences in metabolic fluxes. In parallel, we use metabolomics to predict how differences in substrate availability translate into differences in metabolic fluxes. We discriminate fluxes regulated at the metabolic and/or gene expression level by intersecting these two output datasets. We demonstrate the pipeline using a set of immortalized normal and cancer breast cell lines. In a clinical setting, knowing the regulatory level at which a given metabolic reaction is controlled will be valuable to inform targeted, truly personalized therapies in cancer patients.

Insights on the molecular mechanisms of cytotoxicity induced by AS1411 linked to folate-functionalized DNA nanocages in cancer cells.

Self-assembled multivalent DNA nanocages are an emerging class of molecules useful for biomedicine applications. Here, we investigated the molecular mechanisms of cytotoxicity induced by AS1411 free aptamer, AS1411-linked nanocages (Apt-NCs) and nanocages harboring both folate and AS1411 functionalization (Fol-Apt-NCs) in HeLa and MDA-MB-231 cancer cell lines. The three treatments showed different cytotoxic efficacy and Fol-Apt-NCs resulted the most effective in inhibiting cell proliferation and inducing apoptotic pathways and ROS activation in both HeLa and MDA-MB-231 cells. RNA-seq analysis allowed to identify biological functions and genes altered by the various treatments, depending on the AS1411 route of intracellular entry, highlighting the different behavior of the two cancer cell lines. Notably, Fol-Apt-NCs altered the expression of a subset of genes associated to cancer chemoresistance in MDA-MB-231, but not in HeLa cells, and this may explain the increased chemosensitivity to drugs delivered through DNA nanocages of the triple-negative breast cancer cells.

First-Void Urine Microbiome in Women with Chlamydia trachomatis Infection.

BACKGROUND: Chlamydia trachomatis (CT) is the agent of the most common bacterial sexually transmitted infection worldwide. Until now, little information is available about the microbial composition of urine samples during CT urethritis. Therefore, in this study, we characterized the microbiome and metabolome profiles of first-void urines in a cohort of women with CT urethral infection attending an STI clinic. METHODS: Based on CT positivity by nucleic acid amplification techniques on urine samples, the enrolled women were divided into two groups, i.e., "CT-negative" (n = 21) and "CT-positive" (n = 11). Urine samples were employed for (i) the microbiome profile analysis by means of 16s rRNA gene sequencing and (ii) the metabolome analysis by 1H-NMR. RESULTS: Irrespective of CT infection, the microbiome of first-void urines was mainly dominated by Lactobacillus, L. iners and L. crispatus being the most represented species. CT-positive samples were characterized by reduced microbial biodiversity compared to the controls. Moreover, a significant reduction of the Mycoplasmataceae family-in particular, of the Ureaplasma parvum species-was observed during CT infection. The Chlamydia genus was positively correlated with urine hippurate and lactulose. CONCLUSIONS: These data can help elucidate the pathogenesis of chlamydial urogenital infections, as well as to set up innovative diagnostic and therapeutic approaches.

The Genome-Wide Impact of Nipblb Loss-of-Function on Zebrafish Gene Expression.

Transcriptional changes normally occur during development but also underlie differences between healthy and pathological conditions. Transcription factors or chromatin modifiers are involved in orchestrating gene activity, such as the cohesin genes and their regulator NIPBL. In our previous studies, using a zebrafish model for nipblb knockdown, we described the effect of nipblb loss-of-function in specific contexts, such as central nervous system development and hematopoiesis. However, the genome-wide transcriptional impact of nipblb loss-of-function in zebrafish embryos at diverse developmental stages remains under investigation. By RNA-seq analyses in zebrafish embryos at 24 h post-fertilization, we examined genome-wide effects of nipblb knockdown on transcriptional programs. Differential gene expression analysis revealed that nipblb loss-of-function has an impact on gene expression at 24 h post fertilization, mainly resulting in gene inactivation. A similar transcriptional effect has also been reported in other organisms, supporting the use of zebrafish as a model to understand the role of Nipbl in gene regulation during early vertebrate development. Moreover, we unraveled a connection between nipblb-dependent differential expression and gene expression patterns of hematological cell populations and AML subtypes, enforcing our previous evidence on the involvement of NIPBL-related transcriptional dysregulation in hematological malignancies.

Unraveling gut microbiota in Parkinson's disease and atypical parkinsonism.

BACKGROUND: Although several studies have suggested that abnormalities in gut microbiota may play a critical role in the pathogenesis of PD, data are still extremely heterogeneous. METHODS: 16S gene ribosomal RNA sequencing was performed on fecal samples of 350 individuals, subdivided into idiopathic PD (n = 193, of whom 39 were drug naïve) stratified by disease duration, PSP (n = 22), MSA (n = 22), and healthy controls (HC; n = 113). Several confounders were taken into account, including dietary habits. RESULTS: Despite the fact that unadjusted comparison of PD and HC showed several differences in relative taxa abundances, the significant results were greatly reduced after adjusting for confounders. Although most of these differences were associated with disease duration, lower abundance in Lachnospiraceae was the only difference between de novo PD and HC (remaining lower across almost all PD duration strata). Decreased Lachnospiraceae and increased Lactobacillaceae and Christensenellaceae were associated with a worse clinical profile, including higher frequencies of cognitive impairment, gait disturbances, and postural instability. When compared with HC, MSA and PSP patients shared the changes in PD, with a few exceptions: in MSA, Lachnospiraceae were not lower, and Prevotellaceae were reduced; in PSP, Lactobacillaceae were similar, and Streptococcaceae were reduced. CONCLUSIONS: Gut microbiota may be an environmental modulator of the pathogenesis of PD and contribute to the interindividual variability of clinical features. Data are influenced by PD duration and several confounders that need to be taken into account in future studies. Prospective studies in de novo PD patients are needed to elucidate the net effect of dysbiosis on the progression of the disease. © 2018 International Parkinson and Movement Disorder Society.

The Typhoid Toxin Promotes Host Survival and the Establishment of a Persistent Asymptomatic Infection.

Bacterial genotoxins, produced by several Gram-negative bacteria, induce DNA damage in the target cells. While the responses induced in the host cells have been extensively studied in vitro, the role of these effectors during the course of infection remains poorly characterized. To address this issue, we assessed the effects of the Salmonella enterica genotoxin, known as typhoid toxin, in in vivo models of murine infection. Immunocompetent mice were infected with isogenic S. enterica, serovar Typhimurium (S. Typhimurium) strains, encoding either a functional or an inactive typhoid toxin. The presence of the genotoxic subunit was detected 10 days post-infection in the liver of infected mice. Unexpectedly, its expression promoted the survival of the host, and was associated with a significant reduction of severe enteritis in the early phases of infection. Immunohistochemical and transcriptomic analysis confirmed the toxin-mediated suppression of the intestinal inflammatory response. The presence of a functional typhoid toxin further induced an increased frequency of asymptomatic carriers. Our data indicate that the typhoid toxin DNA damaging activity increases host survival and favours long-term colonization, highlighting a complex cross-talk between infection, DNA damage response and host immune response. These findings may contribute to understand why such effectors have been evolutionary conserved and horizontally transferred among Gram-negative bacteria.

Gut microbiome response to short-term dietary interventions in reactive hypoglycemia subjects.

BACKGROUND: Reactive hypoglycemia is a metabolic disorder that provokes severe hypoglycemic episodes after meals. Over recent years, the gut microbiota has been recognized as potential target for the control of metabolic diseases, and the possibility to correct gut microbiota dysbioses through diet, favouring the recovery of metabolic homeostasis, has been considered. METHODS: We investigate the impact of 2 short-term (3-day) nutritional interventions, based on the macrobiotic Ma-Pi 2 diet and a control Mediterranean diet, on the structure and functionality of the gut microbiota in 12 patients affected by reactive hypoglycemia. The gut microbiota composition was characterized by next-generation sequencing of the V3 to V4 region of the 16S rRNA gene, and the ecosystem functionality was addressed by measuring the faecal concentration of short-chain fatty acids (SCFAs). In order to measure the short-term physiological gut microbiota fluctuation, the microbiomes of 7 healthy people were characterized before and after 3 days of constant diet. RESULTS: While no convergence of the gut microbiota compositional profiles was observed, a significant increase in SCFA faecal levels was induced only in the Ma-Pi 2 diet group, suggesting the potential of this diet to support a short-term functional convergence of the gut microbiota, regardless of the individual compositional layout. CONCLUSIONS: The Ma-Pi 2 diet, with its high fibre load, was effective in increasing the production of SCFAs by the gut microbiota. Because these metabolites are known for their ability to counterbalance the metabolic deregulation in persons with glucose impairment disorders, their increased bioavailability could be of some relevance in reactive hypoglycemia.

Modulation of gut microbiota dysbioses in type 2 diabetic patients by macrobiotic Ma-Pi 2 diet.

The gut microbiota exerts a role in type 2 diabetes (T2D), and deviations from a mutualistic ecosystem layout are considered a key environmental factor contributing to the disease. Thus, the possibility of improving metabolic control in T2D by correcting gut microbiome dysbioses through diet has been evaluated. Here, we explore the potential of two different energy-restricted dietary approaches - the fibre-rich macrobiotic Ma-Pi 2 diet or a control diet recommended by Italian professional societies for T2D treatment - to correct gut microbiota dysbioses in T2D patients. In a previous 21-d open-label MADIAB trial, fifty-six overweight T2D patients were randomised to the Ma-Pi 2 or the control diet. For the present study, stools were collected before and after intervention from a subset of forty MADIAB participants, allowing us to characterise the gut microbiota by 16S rRNA sequencing and imputed metagenomics. To highlight microbiota dysbioses in T2D, the gut microbiota of thirteen normal-weight healthy controls were characterised. According to our findings, both diets were effective in modulating gut microbiome dysbioses in T2D, resulting in an increase of the ecosystem diversity and supporting the recovery of a balanced community of health-promoting SCFA producers, such as Faecalibacterium, Roseburia, Lachnospira, Bacteroides and Akkermansia. The Ma-Pi 2 diet, but not the control diet, was also effective in counteracting the increase of possible pro-inflammatory groups, such as Collinsella and Streptococcus, in the gut ecosystem, showing the potential to reverse pro-inflammatory dysbioses in T2D, and possibly explaining the greater efficacy in improving the metabolic control.

Development of a microarray-based tool to characterize vaginal bacterial fluctuations and application to a novel antibiotic treatment for bacterial vaginosis.

The healthy vaginal microbiota is generally dominated by lactobacilli that confer antimicrobial protection and play a crucial role in health. Bacterial vaginosis (BV) is the most prevalent lower genital tract infection in women in reproductive age and is characterized by a shift in the relative abundances of Lactobacillus spp. to a greater abundance of strictly anaerobic bacteria. In this study, we designed a new phylogenetic microarray-based tool (VaginArray) that includes 17 probe sets specific for the most representative bacterial groups of the human vaginal ecosystem. This tool was implemented using the ligase detection reaction-universal array (LDR-UA) approach. The entire probe set properly recognized the specific targets and showed an overall sensitivity of 6 to 12 ng per probe. The VaginArray was applied to assess the efficacy of rifaximin vaginal tablets for the treatment of BV, analyzing the vaginal bacterial communities of 22 BV-affected women treated with rifaximin vaginal tablets at a dosage of 25 mg/day for 5 days. Our results showed the ability of rifaximin to reduce the growth of various BV-related bacteria (Atopobium vaginae, Prevotella, Megasphaera, Mobiluncus, and Sneathia spp.), with the highest antibiotic susceptibility for A. vaginae and Sneathia spp. Moreover, we observed an increase of Lactobacillus crispatus levels in the subset of women who maintained remission after 1 month of therapy, opening new perspectives for the treatment of BV.

Anti-chlamydial activity of vaginal fluids: new evidence from an in vitro model.

Introduction: We assessed the in vitro anti-chlamydial activity of fresh vaginal secretions, deciphering the microbial and metabolic components able to counteract Chlamydia trachomatis viability. Methods: Forty vaginal samples were collected from a group of reproductive-aged women and their anti-chlamydial activity was evaluated by inhibition experiments. Each sample underwent 16S rRNA metabarcoding sequencing to determine the bacterial composition, as well as 1H-NMR spectroscopy to detect and quantify the presence of vaginal metabolites. Results: Samples characterized by a high anti-chlamydial activity were enriched in Lactobacillus, especially Lactobacillus crispatus and Lactobacillus iners, while not-active samples exhibited a significant reduction of lactobacilli, along with higher relative abundances of Streptococcus and Olegusella. Lactobacillus gasseri showed an opposite behavior compared to L. crispatus, being more prevalent in not-active vaginal samples. Higher concentrations of several amino acids (i.e., isoleucine, leucine, and aspartate; positively correlated to the abundance of L. crispatus and L. jensenii) lactate, and 4-aminobutyrate were the most significant metabolic fingerprints of highly active samples. Acetate and formate concentrations, on the other hand, were related to the abundances of a group of anaerobic opportunistic bacteria (including Prevotella, Dialister, Olegusella, Peptostreptococcus, Peptoniphilus, Finegoldia and Anaerococcus). Finally, glucose, correlated to Streptococcus, Lachnospira and Alloscardovia genera, emerged as a key molecule of the vaginal environment: indeed, the anti-chlamydial effect of vaginal fluids decreased as glucose concentrations increased. Discussion: These findings could pave the way for novel strategies in the prevention and treatment of chlamydial urogenital infections, such as lactobacilli probiotic formulations or lactobacilli-derived postbiotics.

Chlamydia trachomatis and Neisseria gonorrhoeae rectal infections: Interplay between rectal microbiome, HPV infection and Torquetenovirus.

Men having sex with men (MSM) represent a key population, in which sexually transmitted rectal infections (STIs) caused by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and high-risk HPV (HR-HPV) are very common and linked to significant morbidity. Investigating the anorectal microbiome associated with rectal STIs holds potential for deeper insights into the pathogenesis of these infections and the development of innovative control strategies. In this study, we explored the interplay at the rectal site between C. trachomatis, N. gonorrhoeae, HR-HPV infection, and the anorectal microbiome in a cohort of 92 MSM (47 infected by CT and/or NG vs 45 controls). Moreover, we assessed the presence of Torquetenovirus (TTV), a non-pathogenic endogenous virus, considered as a possible predictor of immune system activation. We found a high prevalence of HR-HPV rectal infections (61%), especially in subjects with a concurrent CT/NG rectal infection (70.2%) and in people living with HIV (84%). In addition, we observed that TTV was more prevalent in subjects with CT/NG rectal infections than in non-infected ones (70.2% vs 46.7%, respectively). The anorectal microbiome of patients infected by CT and/or NG exhibited a reduction in Escherichia, while the presence of TTV was significantly associated with higher levels of Bacteroides. We observed a positive correlation of HR-HPV types with Escherichia and Corynebacterium, and a negative correlation with the Firmicutes phylum, and with Prevotella, Oscillospira, Sutterella. Our findings shed light on some of the dynamics occurring within the rectal environment involving chlamydial/gonococcal infections, HPV, TTV, and the anorectal microbiome. These data could open new perspectives for the control and prevention of STIs in MSM.

Effect of ospemifene on vaginal microbiome in postmenopausal women with vulvovaginal atrophy.

OBJECTIVES: This study aimed (i) to compare the vaginal microbiome profiles of women suffering from vulvovaginal atrophy with that of healthy postmenopausal women and to (ii) assess the effect of ospemifene and systemic hormone treatment on the composition of the vaginal microbiome. METHODS: Sixty-seven postmenopausal women attending the Gynecology Clinic of Azienda Ospedaliero-Universitaria of Bologna (Italy) were enrolled. Of them, 39 received a diagnosis of atrophy and 28 were considered healthy controls. In the group of atrophic women, 20 were prescribed ospemifene and 19 received hormone treatment. The vaginal health index was calculated, and a vaginal swab was collected for the assessment of vaginal maturation index and the analysis of vaginal microbiome through 16S rRNA gene sequencing. Clinical/microbiological analyses were repeated after 3 months of treatment. RESULTS: The vaginal microbiome of atrophic women was characterized by a significant reduction of Lactobacillus ( P = 0.002) and an increase of Streptococcus ( P = 0.008) and Sneathia ( P = 0.02). A positive correlation between vaginal health index/vaginal maturation index and Lactobacillus abundance was found ( P = 0.002 and P = 0.035, respectively). Both therapeutic approaches effectively improved vaginal indices. Systemic hormone treatment induced changes in minority bacterial groups of the vaginal microbiome, whereas ospemifene was able to eliminate specific bacterial taxa, such as Staphylococcus ( P = 0.04) and Clostridium ( P = 0.01). Both treatments induced a trend in the increase of bifidobacteria. CONCLUSIONS: The vaginal microbiome of atrophic women differs significantly from that of healthy postmenopausal women. Ospemifene may lead to a condition of vaginal health, likely characterized by the reduction of "potentially harmful" bacteria.

Longitudinal Changes of Ocular Surface Microbiome in Patients Undergoing Hemopoietic Stem Cell Transplant (HSCT).

PURPOSE: To evaluate changes in the ocular surface microbiome (OSM) between pre- and post-haemopoietic stem cell transplant (HSCT) in the same patient, and to assess the potential impact of these changes in ocular graft-versus-host disease (o)GVHD development. METHODS: Lower fornix conjunctival swabs of 24 patients were obtained before and after HSCT and subjected to DNA extraction for amplification and sequencing of the V3-V4 regions of the bacterial 16S rRNA gene. The obtained reads were reconstructed, filtered, and clustered into zero-radius operational taxonomic units (zOTUs) at 97% identity level before taxonomic assignment, and biodiversity indexes were calculated. Transplant characteristics were recorded, and dry eye was diagnosed and staged 1-4 according to the Dry Eye WorkShop (DEWS) score. RESULTS: No significant difference in OSM alpha diversity between pre- and post-transplant was found. A significant difference in beta diversity was observed between patients with a DEWS score of 1 versus 3 (p = 0.035). Increased corneal damage between pre- and post-HSCT was significantly associated with a decrease in alpha diversity. The changes in OSM were not associated with oGVHD, nor with any transplant parameter. CONCLUSIONS: This preliminary study is the first study to analyse changes in the OSM before and after HSCT longitudinally. No trend in OSM biodiversity, microbial profile, or overall composition changes before and after HSCT was significant or associated with oGVHD onset. The great variability in the observed OSM profiles seems to suggest the absence of a patient-specific OSM "signature".

Unbalance of intestinal microbiota in atopic children.

BACKGROUND: Playing a strategic role in the host immune function, the intestinal microbiota has been recently hypothesized to be involved in the etiology of atopy. In order to investigate the gastrointestinal microbial ecology of atopic disease, here we performed a pilot comparative molecular analysis of the faecal microbiota in atopic children and healthy controls. RESULTS: Nineteen atopic children and 12 healthy controls aged 4-14 years were enrolled. Stools were collected and the faecal microbiota was characterized by means of the already developed phylogenetic microarray platform, HTF-Microbi.Array, and quantitative PCR. The intestinal microbiota of atopic children showed a significant depletion in members of the Clostridium cluster IV, Faecalibacterium prausnitzii, Akkermansia muciniphila and a corresponding increase of the relative abundance of Enterobacteriaceae. CONCLUSION: Depleted in key immunomodulatory symbionts, the atopy-associated microbiota can represent an inflammogenic microbial consortium which can contribute to the severity of the disease. Our data open the way to the therapeutic manipulation of the intestinal microbiota in the treatment of atopy by means of pharmaceutical probiotics.

A Deep Look at the Vaginal Environment During Pregnancy and Puerperium.

A deep comprehension of the vaginal ecosystem may hold promise for unraveling the pathophysiology of pregnancy and may provide novel biomarkers to identify subjects at risk of maternal-fetal complications. In this prospective study, we assessed the characteristics of the vaginal environment in a cohort of pregnant women throughout their different gestational ages and puerperium. Both the vaginal bacterial composition and the vaginal metabolic profiles were analyzed. A total of 63 Caucasian women with a successful pregnancy and 9 subjects who had a first trimester miscarriage were enrolled. For the study, obstetric examinations were scheduled along the three trimester phases (9-13, 20-24, 32-34 gestation weeks) and puerperium (40-55 days after delivery). Two vaginal swabs were collected at each time point, to assess the vaginal microbiome profiling (by Nugent score and 16S rRNA gene sequencing) and the vaginal metabolic composition (1H-NMR spectroscopy). During pregnancy, the vaginal microbiome underwent marked changes, with a significant decrease in overall diversity, and increased stability. Over time, we found a significant increase of Lactobacillus and a decrease of several genera related to bacterial vaginosis (BV), such as Prevotella, Atopobium and Sneathia. It is worth noting that the levels of Bifidobacterium spp. tended to decrease at the end of pregnancy. At the puerperium, a significantly lower content of Lactobacillus and higher levels of Gardnerella, Prevotella, Atopobium, and Streptococcus were observed. Women receiving an intrapartum antibiotic prophylaxis for Group B Streptococcus (GBS) were characterized by a vaginal abundance of Prevotella compared to untreated women. Analysis of bacterial relative abundances highlighted an increased abundance of Fusobacterium in women suffering a first trimester abortion, at all taxonomic levels. Lactobacillus abundance was strongly correlated with higher levels of lactate, sarcosine, and many amino acids (i.e., isoleucine, leucine, phenylalanine, valine, threonine, tryptophan). Conversely, BV-associated genera, such as Gardnerella, Atopobium, and Sneathia, were related to amines (e.g., putrescine, methylamine), formate, acetate, alcohols, and short-chain fatty-acids (i.e., butyrate, propionate).

Gardnerella vaginalis clades in pregnancy: New insights into the interactions with the vaginal microbiome.

Gardnerella vaginalis (GV) is an anaerobic bacterial species involved in the pathogenesis of bacterial vaginosis (BV), a condition of vaginal dysbiosis associated with adverse pregnancy outcomes. GV strains are categorized into four clades, characterized by a different ability to produce virulence factors, such as sialidase. We investigated the distribution of GV clades and sialidase genes in the vaginal ecosystem of a cohort of pregnant women, assessing the correlations between GV clades and the whole vaginal microbiome. A total of 61 Caucasian pregnant women were enrolled. Their vaginal swabs, collected both at the first and third trimester of pregnancy, were used for (i) evaluation of the vaginal status by Nugent score, (ii) vaginal microbiome profiling by 16S rRNA sequencing, (iii) detection and quantification of GV clades and sialidase A gene by qPCR assays. DNA of at least one GV clade was detected in most vaginal swabs, with clade 4 being the most common one. GV clade 2, together with the presence of multiple clades (>2 simultaneously), were significantly associated with a BV condition. Significantly higher GV loads and sialidase gene levels were found in BV cases, compared to the healthy status. Clade 2 was related to the major shifts in the vaginal microbial composition, with a decrease in Lactobacillus and an increase in several BV-related taxa. As the number of GV clades detected simultaneously increased, a group of BV-associated bacteria tended to increase as well, while Bifidobacterium tended to decrease. A negative correlation between sialidase gene levels and Lactobacillus, and a positive correlation with Gardnerella, Atopobium, Prevotella, Megasphaera, and Sneathia were observed. Our results added knowledge about the interactions of GV clades with the inhabitants of the vaginal microbiome, possibly helping to predict the severity of BV and opening new perspectives for the prevention of pregnancy-related complications.

Torquetenovirus in pregnancy: Correlation with vaginal microbiome, metabolome and pro-inflammatory cytokines.

Torquetenovirus (TTV) is a negative sense, single-stranded DNA virus present in many body fluids of apparently healthy individuals. At present, it is considered a non-pathogenic endogenous virus. TTV can be detected in the vagina of pregnant women, its abundance being modulated with the extent of immune system activation. Until now, there is only scarce information regarding the association between TTV and the composition of the vaginal environment. Therefore, this study aimed to assess the presence of TTV in the vaginal ecosystem of a cohort of white women with a normal pregnancy (n = 60) at different gestational stages (first, second and third trimester) and in 9 subjects suffering a first trimester miscarriage. For each woman, we determined (i) the presence and titer of TTV, (ii) the vaginal bacterial composition by means of Nugent score and 16S rRNA gene sequencing, (iii) the vaginal metabolic profiles through 1H-NMR spectroscopy, and (iv) the vaginal concentration of two pro-inflammatory cytokines (IL-6 and IL-8). More than one third of women were found negative for TTV at all gestational stages. Although not statistically significant, the positivity for TTV dropped from 53.3% in the first to 36.6% in the third trimester. TTV loads varied greatly among vaginal samples, ranging between 2 × 101 and 2 × 105 copies/reaction. No difference in TTV prevalence and loads was observed between women with normal pregnancies and miscarriages. The presence of TTV was more common in women with a higher vaginal leucocyte count (p = 0.02). The levels of IL-6 (p = 0.02), IL-8 (p = 0.03), propionate (p = 0.001) and cadaverine (p = 0.006) were significantly higher in TTV-positive samples. TTV titer was positively correlated with the concentrations of 4-hydroxyphenyllactate (p < 0.0001), isoleucine (p = 0.01) and phenylalanine (p = 0.04). TTV-positive samples were characterized by a higher relative abundance of Sneathia (p = 0.04) and Shuttleworthia (p = 0.0009). In addition, a trend toward a decrease of Lactobacillus crispatus and Lactobacillus jensenii, and an increase of Lactobacillus iners was observed for TTV-positive samples. In conclusion, we found that TTV is quite common in women with normal pregnancy outcomes, representing a possible predictor of local immune status.

A tool based on Ligation Detection Reaction-Universal Array (LDR-UA) for the characterization of VTEC by identification of virulence-associated and serogroup-specific genes.

Verocytoxigenic Escherichia coli (VTEC) are zoonotic pathogens whose natural reservoir is represented by ruminants, particularly cattle. Infections are mainly acquired by consumption of undercooked contaminated food of animal origin, contact with infected animals and contaminated environment. VTEC O157 is the most frequently isolated serogroup from cases of human disease, however, other VTEC serogroups, such as O26, O111, O145 and O103, are increasingly reported as causing Hemolytic Uremic Syndrome (HUS) worldwide. The identification of VTEC is troublesome, hindering the development of effective prevention strategies. In fact, VTEC are morphologically indistinguishable from harmless E. coli and their pathogenic potential is not strictly dependent on the serogroup, but relies on the presence of a collection of virulence genes. We developed a diagnostic tool for VTEC based on the Ligation Detection Reaction coupled to Universal Array (LDR-UA) for the simultaneous identification of virulence factors and serogroup-associated genes. The method includes the investigation of 40 sites located in 13 fragments from 12 genes (sodCF1/F2, adfO, terB, ehxA, eae, vtx1, vtx2, ihp1, wzx, wbdI, rfbE, dnaK) and was evaluated by performing a trial on a collection of 67 E. coli strains, both VTEC and VT-negative E. coli, as well as on 25 isolates belonging to other related species. Results of this study showed that the LDR-UA technique was specific in identifying the target microorganism. Moreover, due to its higher throughput, the LDR-UA can be a valid and cheaper alternative to real time PCR-based (rt-PCR) methods for VTEC identification.

ORMA: a tool for identification of species-specific variations in 16S rRNA gene and oligonucleotides design.

16S rRNA gene is one of the preferred targets for resolving species phylogenesis issues in microbiological-related contexts. However, the identification of single-nucleotide variations capable of distinguishing a sequence among a set of homologous ones can be problematic. Here we present ORMA (Oligonucleotide Retrieving for Molecular Applications), a set of scripts for discriminating positions search and for performing the selection of high-quality oligonucleotide probes to be used in molecular applications. Two assays based on Ligase Detection Reaction (LDR) are presented. First, a new set of probe pairs on cyanobacteria 16S rRNA sequences of 18 different species was compared to that of a previous study. Then, a set of LDR probe pairs for the discrimination of 13 pathogens contaminating bovine milk was evaluated. The software determined more than 100 candidate probe pairs per dataset, from more than 300 16S rRNA sequences, in less than 5 min. Results demonstrated how ORMA improved the performance of the LDR assay on cyanobacteria, correctly identifying 12 out of 14 samples, and allowed the perfect discrimination among the 13 milk pathogenic-related species. ORMA represents a significant improvement from other contexts where enzyme-based techniques have been employed on already known mutations of a single base or on entire subsequences.