Serum from COVID-19 patients promotes endothelial cell dysfunction through protease-activated receptor 2.

BACKGROUND: Endothelial dysfunction plays a central role in the pathophysiology of COVID-19 and is closely linked to the severity and mortality of the disease. The inflammatory response to SARS-CoV-2 infection can alter the capacity of the endothelium to regulate vascular tone, immune responses, and the balance between anti-thrombotic and pro-thrombotic properties. However, the specific endothelial pathways altered during COVID-19 still need to be fully understood. OBJECTIVE: In this study, we sought to identify molecular changes in endothelial cells induced by circulating factors characteristic of COVID-19. METHODS AND RESULTS: To this aim, we cultured endothelial cells with sera from patients with COVID-19 or non-COVID-19 pneumonia. Through transcriptomic analysis, we were able to identify a distinctive endothelial phenotype that is induced by sera from COVID-19 patients. We confirmed and expanded this observation in vitro by showing that COVID-19 serum alters functional properties of endothelial cells leading to increased apoptosis, loss of barrier integrity, and hypercoagulability. Furthermore, we demonstrated that these endothelial dysfunctions are mediated by protease-activated receptor 2 (PAR-2), as predicted by transcriptome network analysis validated by in vitro functional assays. CONCLUSION: Our findings provide the rationale for further studies to evaluate whether targeting PAR-2 may be a clinically effective strategy to counteract endothelial dysfunction in COVID-19.

Development of an Oral IgA Response against SARS-CoV-2 Following Immunization with Different COVID-19 Vaccines.

The mucosal immune response is recognized to be important in the early control of infection sustained by viruses with mucosal tissues as the primary site of entry and replication, such as SARS-CoV-2. Mucosal IgA has been consistently reported in the mouth and eye of SARS-CoV-2 infected subjects, where it correlated inversely with COVID-19 symptom severity. Yet, there is still scarce information on the comparative ability of the diverse SARS-CoV-2 vaccines to induce local IgA responses at the virus entry site. Thus, the aim of this study was to assess the presence of anti-SARS-CoV-2 IgA in the saliva of 95 subjects vaccinated with a booster dose and different combinations of vaccines, including mRNA-1273 (Moderna), BNT162b2 (Pfizer-BioNTech), and Vaxzevria (AstraZeneca). The results showed the presence of a mucosal response in 93.7% of vaccinated subjects, with a mean IgA titer of 351.5 ± 31.77 U/mL, strongly correlating with the serum anti-SARS-CoV-2 IgG titer (p < 0.0001). No statistically significant differences emerged between the vaccine types, although the salivary IgA titer appeared slightly higher after receiving a booster dose of the mRNA-1273 vaccine (Moderna) following two doses of BNT162b2 (Pfizer-BioNTech), compared to the other vaccine combinations. These data confirm what was previously reported at the eye level and suggest that monitoring salivary IgA may be a useful tool for driving forward vaccine design and surveillance strategies, potentially leading to novel routes of vaccine administration and boosting.

Impact of C- and N-terminal protection on the stability, metal chelation and antimicrobial properties of calcitermin.

The main limitation to the use of antimicrobial peptides (AMPs) as regular drugs, against antibiotic and antifungal resistance, mainly relates to their rapid degradation by proteolytic enzymes. The introduction of suitable structural changes in the peptide chain can make the peptide less susceptible to the action of proteases, thus overcoming this problem. To improve the plasma stability of calcitermin, a metal-chelating AMP present in the human respiratory tract and investigated in the present study, C- and/or N- terminal modifications have been introduced in the native sequence. Evaluation of peptide stability has been performed to determine the half-life times in human plasma of both native calcitermin and its derivatives. However, the protection of the peptide termini can also affect its metal coordination behaviour. Thus, the characterization of Zn2+ and Cu2+ complexes has been performed by means of several techniques, including potentiometry, high-resolution mass spectrometry, UV-Vis, circular dichroism and EPR. On the basis of the obtained results, it was possible to compare the biological activity of the studied systems, taking into account both the metal-binding ability and the peptide stability to search for a link among them. A significant result of this study is that the N-terminal protection increases the calcitermin half-life over seven times and the formation of metal complexes confers resistance towards degradation almost doubling its half-life.

Characterization of the Pathogenic Potential of the Beach Sand Microbiome and Assessment of Quicklime as a Remediation Tool.

Beach sand may act as a reservoir for potential human pathogens, posing a public health risk. Despite this, the microbiological monitoring of sand microbiome is rarely performed to determine beach quality. In this study, the sand microbial population of a Northern Adriatic Sea beach sand was profiled by microbiological (CFU counts) and molecular methods (WGS, microarray), showing significant presence of potential human pathogens including drug-resistant strains. Consistent with these results, the potential of quicklime as a restoring method was tested in vitro and on-field. Collected data showed that adding 1-3% quicklime (w/w) to sand provided an up to -99% of bacteria, fungi, and viruses, in a dose- and time-dependent manner, till 45 days post-treatment. In conclusion, data suggest that accurate monitoring of sand microbiome may be essential, besides water, to assess beach quality and safety. Moreover, first evidences of quicklime potential for sand decontamination are provided, suggesting its usage as a possible way to restore the microbiological quality of sand in highly contaminated areas.

Circulating miRNAs Expression in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex multifactorial disease that causes increasing morbidity worldwide, and many individuals with ME/CFS symptoms remain undiagnosed due to the lack of diagnostic biomarkers. Its etiology is still unknown, but increasing evidence supports a role of herpesviruses (including HHV-6A and HHV-6B) as potential triggers. Interestingly, the infection by these viruses has been reported to impact the expression of microRNAs (miRNAs), short non-coding RNA sequences which have been suggested to be epigenetic factors modulating ME/CFS pathogenic mechanisms. Notably, the presence of circulating miRNAs in plasma has raised the possibility to use them as valuable biomarkers for distinguishing ME/CFS patients from healthy controls. Thus, this study aimed at determining the role of eight miRNAs, which were selected for their previous association with ME/CFS, as potential circulating biomarkers of the disease. Their presence was quantitatively evaluated in plasma from 40 ME/CFS patients and 20 healthy controls by specific Taqman assays, and the results showed that six out of the eight of the selected miRNAs were differently expressed in patients compared to controls; more specifically, five miRNAs were significantly upregulated (miR-127-3p, miR-142-5p, miR-143-3p, miR-150-5p, and miR-448), and one was downmodulated (miR-140-5p). MiRNA levels directly correlated with disease severity, whereas no significant correlations were observed with the plasma levels of seven pro-inflammatory cytokines or with the presence/load of HHV-6A/6B genome, as judged by specific PCR amplification. The results may open the way for further validation of miRNAs as new potential biomarkers in ME/CFS and increase the knowledge of the complex pathways involved in the ME/CFS development.

Potential Use of a Combined Bacteriophage-Probiotic Sanitation System to Control Microbial Contamination and AMR in Healthcare Settings: A Pre-Post Intervention Study.

Microbial contamination in the hospital environment is a major concern for public health, since it significantly contributes to the onset of healthcare-associated infections (HAIs), which are further complicated by the alarming level of antimicrobial resistance (AMR) of HAI-associated pathogens. Chemical disinfection to control bioburden has a temporary effect and can favor the selection of resistant pathogens, as observed during the COVID-19 pandemic. Instead, probiotic-based sanitation (probiotic cleaning hygiene system, PCHS) was reported to stably abate pathogens, AMR, and HAIs. PCHS action is not rapid nor specific, being based on competitive exclusion, but the addition of lytic bacteriophages that quickly and specifically kill selected bacteria was shown to improve PCHS effectiveness. This study aimed to investigate the effect of such combined probiotic-phage sanitation (PCHSφ) in two Italian hospitals, targeting staphylococcal contamination. The results showed that PCHSφ could provide a significantly higher removal of staphylococci, including resistant strains, compared with disinfectants (-76%, p < 0.05) and PCHS alone (-50%, p < 0.05). Extraordinary sporadic chlorine disinfection appeared compatible with PCHSφ, while frequent routine chlorine usage inactivated the probiotic/phage components, preventing PCHSφ action. The collected data highlight the potential of a biological sanitation for better control of the infectious risk in healthcare facilities, without worsening pollution and AMR concerns.

Shaping the subway microbiome through probiotic-based sanitation during the COVID-19 emergency: a pre-post case-control study.

BACKGROUND: The COVID-19 pandemic has highlighted the extent to which the public transportation environment, such as in subways, may be important for the transmission of potential pathogenic microbes among humans, with the possibility of rapidly impacting large numbers of people. For these reasons, sanitation procedures, including massive use of chemical disinfection, were mandatorily introduced during the emergency and remain in place. However, most chemical disinfectants have temporary action and a high environmental impact, potentially enhancing antimicrobial resistance (AMR) of the treated microbes. By contrast, a biological and eco-sustainable probiotic-based sanitation (PBS) procedure was recently shown to stably shape the microbiome of treated environments, providing effective and long-term control of pathogens and AMR spread in addition to activity against SARS-CoV-2, the causative agent of COVID-19. Our study aims to assess the applicability and impact of PBS compared with chemical disinfectants based on their effects on the surface microbiome of a subway environment. RESULTS: The train microbiome was characterized by both culture-based and culture-independent molecular methods, including 16S rRNA NGS and real-time qPCR microarray, for profiling the train bacteriome and its resistome and to identify and quantify specific human pathogens. SARS-CoV-2 presence was also assessed in parallel using digital droplet PCR. The results showed a clear and significant decrease in bacterial and fungal pathogens (p < 0.001) as well as of SARS-CoV-2 presence (p < 0.01), in the PBS-treated train compared with the chemically disinfected control train. In addition, NGS profiling evidenced diverse clusters in the population of air vs. surface while demonstrating the specific action of PBS against pathogens rather than the entire train bacteriome. CONCLUSIONS: The data presented here provide the first direct assessment of the impact of different sanitation procedures on the subway microbiome, allowing a better understanding of its composition and dynamics and showing that a biological sanitation approach may be highly effective in counteracting pathogens and AMR spread in our increasingly urbanized and interconnected environment. Video Abstract.

Coinfection of Dermal Fibroblasts by Human Cytomegalovirus and Human Herpesvirus 6 Can Boost the Expression of Fibrosis-Associated MicroRNAs.

Tissue fibrosis can affect every type of tissue or organ, often leading to organ malfunction; however, the mechanisms involved in this process are not yet clarified. A role has been hypothesized for Human Cytomegalovirus (HCMV) and Human Herpesvirus 6 (HHV-6) infections as triggers of systemic sclerosis (SSc), a severe autoimmune disease causing progressive tissue fibrosis, since both viruses and antiviral immune responses toward them have been detected in patients. Moreover, HCMV or HHV-6A infection was reported to increase the expression of fibrosis-associated transcriptional factors and miRNAs in human dermal fibroblasts. However, it is unlikely that they have separate effects in the infected host, as both viruses are highly prevalent in the human population. Thus, our study aimed to investigate, by quantitative real-time PCR microarray, the impact of HCMV/HHV-6A coinfection on the expression of pro-fibrotic miRNAs in coinfected cells, compared to the effect of single viruses. The results showed a possible synergistic effect of the two viruses on pro-fibrotic miRNA expression, thus suggesting that HCMV and HHV-6 may enhance each other and cooperate at inducing enhanced miRNA-driven fibrosis. These data may also suggest a possible use of virus-induced miRNAs as novel diagnostic or prognostic biomarkers for SSc and its clinical treatment.

Evaluation of Anti-SARS-CoV-2 IgA Response in Tears of Vaccinated COVID-19 Subjects.

Secretory IgA (sIgA), which may play an important role in the early defense against SARS-CoV-2 infection, were detected in the eye of COVID-19 patients. However, an evaluation of the sIgA response in the tears of vaccinated or non-vaccinated COVID-19 subjects is still lacking. Aimed at characterizing sIgA mucosal immunity in the eye, this study analyzed tear samples from 77 COVID-19 patients, including 63 vaccinated and 14 non-vaccinated subjects. The groups showed similar epidemiological features, but as expected, differences were observed in the percentage of asymptomatic/pauci-symptomatic subjects in the vaccinated vs. non-vaccinated cohort (46% and 29% of the total, respectively). Consistent with this, ocular sIgA values, evaluated by a specific quantitative ELISA assay, were remarkably different in vaccinated vs. non-vaccinated group for both frequency (69.8% vs. 57.1%, respectively) and titer (1372.3 U/mL vs. 143.7 U/mL, respectively; p = 0.01), which was significantly differently elevated depending on the type of administered vaccine. The data show for the first time significant differences of available vaccines to elicit sIgA response in the eye and suggest that quantitative tear-based sIgA tests may potentially serve as a rapid and easily accessible biomarker for the assessment of the development of a protective mucosal immunity toward SARS-CoV-2.

Calcitermin-Loaded Smart Gels Activity against Candida albicans: A Preliminary In Vitro Study.

Calcitermin is an antimicrobial peptide of 15 amino acids found in human nasal fluid characterized by antifungal and antibacterial properties. Candida albicans is the most common human fungal pathogen affecting many tissues, such as vaginal mucosa. In this study a formulation suitable for calcitermin administration on vaginal mucosa was developed for the treatment of fungal infections. To favor topical application, mucosal adhesion, and permanence, gels based on poloxamer 407 and xanthan gum were designed and compared with regard to their rheological behavior, erosion, and leakage. The selected gel was loaded with calcitermin, whose release kinetic was evaluated in vitro by Franz cells. An antifungal activity assay was conducted to assess the calcitermin anticandidal potential and the effect of its inclusion in the selected gel. The rheological study revealed the elastic and viscous moduli behavior as a function of poloxamer 407 and xanthan gum concentration. Xanthan gum presence decreased the transition temperature of the gel, while prolonging its erosion and leakage. Particularly, poloxamer 407, 18% and xanthan gum 0.4% were chosen. The calcitermin loading in the selected gel resulted in a transparent and homogeneous formulation and in a 4-fold decrease of the release rate with respect to the calcitermin solution, as evidenced by Franz cell study. The anticandidal activity tests demonstrated that calcitermin-loaded gel was more active against Candida albicans with respect to the peptide solution.

Efficacy of the additional use of subgingival air-polishing with erythritol powder in the treatment of periodontitis patients: a randomized controlled clinical trial. Part II: effect on sub-gingival microbiome.

OBJECTIVES: To date, scarce evidence exists around the application of subgingival air-polishing during treatment of severe periodontitis. The aim of this study was to evaluate the effect on the health-related and periodontitis-related subgingival microbiome of air-polishing during non-surgical treatment of deep bleeding pockets in stage III-IV periodontitis patients. MATERIALS AND METHODS: Forty patients with stage III-IV periodontitis were selected, and pockets with probing depth (PD) 5-9 mm and bleeding on probing were selected as experimental sites. All patients underwent a full-mouth session of erythritol powder supragingival air-polishing and ultrasonic instrumentation. Test group received additional subgingival air-polishing at experimental sites. Subgingival microbial samples were taken from the maxillary experimental site showing the deepest PD at baseline. Primary outcome of the first part of the present study was the 3-month change in the number of experimental sites. Additional analysis of periodontal pathogens and other sub-gingival plaque bacteria sampled at one experimental site at baseline and 3 months following treatment was performed through a real-time quantitative PCR microarray. RESULTS: In the test group, a statistical increase of some health-related species was observed (Abiotropha defectiva, Capnocytophaga sputigena, and Lautropia mirabilis), together with the decrease of pathogens such as of Actinomyces israelii, Catonella morbi, Filifactor alocis, Porphyromonas endodontalis, Sele-nomonas sputigena, Tannerella forsythia, Treponema denticola, and Treponema socranskii. In the control group, statistical significance was found only in the decrease of Filifactor alocis, Tannerella forsythia, and Treponema socranskii. CONCLUSIONS: The addition of erythritol-chlorhexidine powder seems to cause a shift of the periodontal micro-biome toward a more eubiotic condition compared to a conventional treatment. The study was registered on Clinical Trials.gov (NCT04264624). CLINICAL RELEVANCE: Subgingival air-polishing could help re-establishing a eubiotic microbioma in deep bleeding periodontal pockets after initial non-surgical treatment.

Next-generation sequencing and PCR technologies in monitoring the hospital microbiome and its drug resistance.

The hospital environment significantly contributes to the onset of healthcare-associated infections (HAIs), which represent one of the most frequent complications occurring in healthcare facilities worldwide. Moreover, the increased antimicrobial resistance (AMR) characterizing HAI-associated microbes is one of the human health's main concerns, requiring the characterization of the contaminating microbial population in the hospital environment. The monitoring of surface microbiota in hospitals is generally addressed by microbial cultural isolation. However, this has some important limitations mainly relating to the inability to define the whole drug-resistance profile of the contaminating microbiota and to the long time period required to obtain the results. Hence, there is an urgent need to implement environmental surveillance systems using more effective methods. Molecular approaches, including next-generation sequencing and PCR assays, may be useful and effective tools to monitor microbial contamination, especially the growing AMR of HAI-associated pathogens. Herein, we summarize the results of our recent studies using culture-based and molecular analyses in 12 hospitals for adults and children over a 5-year period, highlighting the advantages and disadvantages of the techniques used.

Human Cytomegalovirus and Human Herpesvirus 6 Coinfection of Dermal Fibroblasts Enhances the Pro-Inflammatory Pathway Predisposing to Fibrosis: The Possible Impact on Systemic Sclerosis.

Systemic sclerosis (SSc) is a severe autoimmune disease likely triggered by genetic and environmental factors, including viral infections. Human cytomegalovirus (HCMV) and human herpesvirus 6A species (HHV-6A) have been associated with SSc, based on in vivo and in vitro evidence, but the data are still inconclusive. Furthermore, despite both viruses being highly prevalent in humans and able to exacerbate each other's effects, no data are available on their joint effects. Hence, we aimed to study their simultaneous impact on the expression of cell factors correlated with fibrosis and apoptosis in in vitro coinfected fibroblasts, representing the main target cell type in SSc. The results, obtained by a microarray detecting 84 fibrosis/apoptosis-associated factors, indicated that coinfected cells underwent higher and more sustained expression of fibrosis-associated parameters compared with single-infected cells. Thus, the data, for the first time, suggest that HCMV and HHV-6A may cooperate in inducing alterations potentially leading to cell fibrosis, thus further supporting their joint role in SSc. However, further work is required to definitively answer whether ß-herpesviruses are causally linked to the disease and to enable the possible use of targeted antiviral treatments to improve clinical outcomes.

Introduction of Probiotic-Based Sanitation in the Emergency Ward of a Children's Hospital During the COVID-19 Pandemic.

Background: Antimicrobial resistance (AMR) represents a major threat to public health, especially in the hospital environment, and the massive use of disinfectants to prevent COVID-19 transmission might intensify this risk, possibly leading to future AMR pandemics. However, the control of microbial contamination is crucial in hospitals, since hospital microbiomes can cause healthcare-associated infections (HAIs), which are particularly frequent and severe in pediatric wards due to children having high susceptibility. Aim: We have previously reported that probiotic-based sanitation (PCHS) could stably decrease pathogens and their AMR in the hospital environment, reduce associated HAIs in adult hospitals, and inactivate enveloped viruses. Here, we aimed to test the effect of PCHS in the emergency room (ER) of a children's hospital during the COVID-19 pandemic. Methods: Conventional chemical disinfection was replaced by PCHS for 2 months during routine ER sanitation; the level of environmental bioburden was characterized before and at 2, 4, and 9 weeks after the introduction of PCHS. Microbial contamination was monitored simultaneously by conventional culture-based CFU count and molecular assays, including 16S rRNA NGS for bacteriome characterization and microarrays for the assessment of the resistome of the contaminating population. The presence of SARS-CoV-2 was also monitored by PCR. Results and conclusions: PCHS usage was associated with a stable 80% decrease in surface pathogens compared to levels detected for chemical disinfection (P < 0.01), accompanied by an up to 2 log decrease in resistance genes (Pc < 0.01). The effects were reversed when reintroducing chemical disinfection, which counteracted the action of the PCHS. SARS-CoV-2 was not detectable in both the pre-PCHS and PCHS periods. As the control of microbial contamination is a major issue, especially during pandemic emergencies, collected data suggest that PCHS may be successfully used to control virus spread without simultaneous worsening of the AMR concern.

Pathogen Control in the Built Environment: A Probiotic-Based System as a Remedy for the Spread of Antibiotic Resistance.

The high and sometimes inappropriate use of disinfectants and antibiotics has led to alarming levels of Antimicrobial Resistance (AMR) and to high water and hearth pollution, which today represent major threats for public health. Furthermore, the current SARS-CoV-2 pandemic has deeply influenced our sanitization habits, imposing the massive use of chemical disinfectants potentially exacerbating both concerns. Moreover, super-sanitation can profoundly influence the environmental microbiome, potentially resulting counterproductive when trying to stably eliminate pathogens. Instead, environmentally friendly procedures based on microbiome balance principles, similar to what applied to living organisms, may be more effective, and probiotic-based eco-friendly sanitation has been consistently reported to provide stable reduction of both pathogens and AMR in treated-environments, compared to chemical disinfectants. Here, we summarize the results of the studies performed in healthcare settings, suggesting that such an approach may be applied successfully also to non-healthcare environments, including the domestic ones, based on its effectiveness, safety, and negligible environmental impact.

Potential of an Eco-Sustainable Probiotic-Cleaning Formulation in Reducing Infectivity of Enveloped Viruses.

The COVID-19 pandemic has deeply influenced sanitization procedures, and high-level disinfection has been massively used to prevent SARS-CoV-2 spread, with potential negative impact on the environment and on the threat of antimicrobial resistance (AMR). Aiming to overcome these concerns, yet preserving the effectiveness of sanitization against enveloped viruses, we assessed the antiviral properties of the Probiotic Cleaning Hygiene System (PCHS), an eco-sustainable probiotic-based detergent previously proven to stably abate pathogen contamination and AMR. PCHS (diluted 1:10, 1:50 and 1:100) was tested in comparison to common disinfectants (70% ethanol and 0.5% sodium hypochlorite), in suspension and carrier tests, according with the European UNI EN 14476:2019 and UNI EN 16777:2019 standards. Human alpha- and beta-coronaviruses hCoV-229E and SARS-CoV-2, human herpesvirus type 1, human and animal influenza viruses, and vaccinia virus were included in the study. The results showed that PCHS was able to inactivate 99.99% of all tested viruses within 1-2 h of contact, both in suspension and on surface. Notably, while control disinfectants became inactive within 2 h after application, the PCHS antiviral action persisted up to 24 h post-application, suggesting that its use may effectively allow a continuous prevention of virus spread via contaminated environment, without worsening environmental pollution and AMR concern.

Insights into the knowledge of complex diseases: Environmental infectious/toxic agents as potential etiopathogenetic factors of systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease secondary to three cardinal pathological features: immune-system alterations, diffuse microangiopathy, and fibrosis involving the skin and internal organs. The etiology of SSc remains quite obscure; it may encompass multiple host genetic and environmental -infectious/chemical-factors. The present review focused on the potential role of environmental agents in the etiopathogenesis of SSc based on epidemiological, clinical, and laboratory investigations previously published in the world literature. Among infectious agents, some viruses that may persist and reactivate in infected individuals, namely human cytomegalovirus (HCMV), human herpesvirus-6 (HHV-6), and parvovirus B19 (B19V), and retroviruses have been proposed as potential causative agents of SSc. These viruses share a number of biological activities and consequent pathological alterations, such as endothelial dysfunction and/or fibroblast activation. Moreover, the acute worsening of pre-existing interstitial lung involvement observed in SSc patients with symptomatic SARS-CoV-2 infection might suggest a potential role of this virus in the overall disease outcome. A variety of chemical/occupational agents might be regarded as putative etiological factors of SSc. In this setting, the SSc complicating silica dust exposure represents one of the most promising models of study. Considering the complexity of SSc pathogenesis, none of suggested causative factors may explain the appearance of the whole SSc; it is likely that the disease is the result of a multifactorial and multistep pathogenetic process. A variable combination of potential etiological factors may modulate the appearance of different clinical phenotypes detectable in individual scleroderma patients. The in-deep investigations on the SSc etiopathogenesis may provide useful insights in the broad field of human diseases characterized by diffuse microangiopathy or altered fibrogenesis.

Oral Microbiome Dysbiosis Is Associated With Symptoms Severity and Local Immune/Inflammatory Response in COVID-19 Patients: A Cross-Sectional Study.

The human oral microbiome (HOM) is the second largest microbial community after the gut and can impact the onset and progression of several localized and systemic diseases, including those of viral origin, especially for viruses entering the body via the oropharynx. However, this important aspect has not been clarified for the new pandemic human coronavirus SARS-CoV-2, causing COVID-19 disease, despite it being one of the many respiratory viruses having the oropharynx as the primary site of replication. In particular, no data are available about the non-bacterial components of the HOM (fungi, viruses), which instead has been shown to be crucial for other diseases. Consistent with this, this study aimed to define the HOM in COVID-19 patients, to evidence any association between its profile and the clinical disease. Seventy-five oral rinse samples were analyzed by Whole Genome Sequencing (WGS) to simultaneously identify oral bacteria, fungi, and viruses. To correlate the HOM profile with local virus replication, the SARS-CoV-2 amount in the oral cavity was quantified by digital droplet PCR. Moreover, local inflammation and secretory immune response were also assessed, respectively by measuring the local release of pro-inflammatory cytokines (L-6, IL-17, TNFα, and GM-CSF) and the production of secretory immunoglobulins A (sIgA). The results showed the presence of oral dysbiosis in COVID-19 patients compared to matched controls, with significantly decreased alpha-diversity value and lower species richness in COVID-19 subjects. Notably, oral dysbiosis correlated with symptom severity (p = 0.006), and increased local inflammation (p < 0.01). In parallel, a decreased mucosal sIgA response was observed in more severely symptomatic patients (p = 0.02), suggesting that local immune response is important in the early control of virus infection and that its correct development is influenced by the HOM profile. In conclusion, the data presented here suggest that the HOM profile may be important in defining the individual susceptibility to SARS-CoV-2 infection, facilitating inflammation and virus replication, or rather, inducing a protective IgA response. Although it is not possible to determine whether the alteration in the microbial community is the cause or effect of the SARS-CoV-2 replication, these parameters may be considered as markers for personalized therapy and vaccine development.

Microbial Contamination in Hospital Environment Has the Potential to Colonize Preterm Newborns' Nasal Cavities.

Infants born before 28 weeks are at risk of contracting healthcare-associated infections (HAIs), which could be caused by pathogens residing on contaminated hospital surfaces. In this longitudinal study, we characterized by NGS the bacterial composition of nasal swabs of preterm newborns, at the time of birth and after admission to the Neonatal Intensive Care Unit (NICU), comparing it with that of the environmental wards at the time of delivery and during the hospitalization. We characterized the resistome on the samples too. The results showed that environmental microorganisms responsible for HAIs, in particular Staphylococcus spp., Streptococcus spp., Escherichia-Shigella spp., and K. pneumoniae, were detected in higher percentages in the noses of the babies after 13 days of hospitalization, in terms of the number of colonized patients, microorganism amount, and relative abundance. The analysis of nasal bacteria resistome evidenced the absence of resistance genes at the time of birth, some of which appeared and increased after the admission in the NICU. These data suggest that hospital surface microbiota might be transported to respiratory mucosae or other profound tissues. Our study highlights the importance of a screening that allows characterizing the microbial profile of the environment to assess the risk of colonization of the newborn.

Modulation of microRNome by Human Cytomegalovirus and Human Herpesvirus 6 Infection in Human Dermal Fibroblasts: Possible Significance in the Induction of Fibrosis in Systemic Sclerosis.

Human cytomegalovirus (HCMV) and Human herpesvirus 6 (HHV-6) have been reportedly suggested as triggers of the onset and/or progression of systemic sclerosis (SSc), a severe autoimmune disorder characterized by multi-organ fibrosis. The etiology and pathogenesis of SSc are still largely unknown but virological and immunological observations support a role for these beta-herpesviruses, and we recently observed a direct impact of HCMV and HHV-6 infection on the expression of cell factors associated with fibrosis at the cell level. Since miRNA expression has been found profoundly deregulated at the tissue level, here we aimed to investigate the impact on cell microRNome (miRNome) of HCMV and HHV-6 infection in in vitro infected primary human dermal fibroblasts, which represent one of the main SSc target cells. The analysis, performed by Taqman arrays detecting and quantifying 754 microRNAs (miRNAs), showed that both herpesviruses significantly modulated miRNA expression in infected cells, with evident early and late effects and deep modulation (>10 fold) of >40 miRNAs at each time post infection, including those previously recognized for their key function in fibrosis. The correlation between these in vitro results with in vivo observations is strongly suggestive of a role of HCMV and/or HHV-6 in the multistep pathogenesis of fibrosis in SSc and in the induction of fibrosis-signaling pathways finally leading to tissue fibrosis. The identification of specific miRNAs may open the way to their use as biomarkers for SSc diagnosis, assessment of disease progression and possible antifibrotic therapies.