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.

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.

Advanced Molecular and Immunological Diagnostic Methods to Detect SARS-CoV-2 Infection.

COVID-19 emerged in late 2019 in China and quickly spread across the globe, causing over 521 million cases of infection and 6.26 million deaths to date. After 2 years, numerous advances have been made. First of all, the preventive vaccine, which has been implemented in record time, is effective in more than 95% of cases. Additionally, in the diagnostic field, there are numerous molecular and antigenic diagnostic kits that are equipped with high sensitivity and specificity. Real Time-PCR-based assays for the detection of viral RNA are currently considered the gold-standard method for SARS-CoV-2 diagnosis and can be used efficiently on pooled nasopharyngeal, or oropharyngeal samples for widespread screening. Moreover, additional, and more advanced molecular methods such as droplet-digital PCR (ddPCR), clustered regularly interspaced short palindromic repeats (CRISPR) and next-generation sequencing (NGS), are currently under development to detect the SARS-CoV-2 RNA. However, as the number of subjects infected with SARS-CoV-2 continuously increases globally, health care systems are being placed under increased stress. Thus, the clinical laboratory plays an important role, helping to select especially asymptomatic individuals who are actively carrying the live replicating virus, with fast and non-invasive molecular technologies. Recent diagnostic strategies, other than molecular methods, have been adopted to either detect viral antigens, i.e., antigen-based immunoassays, or human anti-SARS-CoV-2 antibodies, i.e., antibody-based immunoassays, in nasal or oropharyngeal swabs, as well as in blood or saliva samples. However, the role of mucosal sIgAs, which are essential in the control of viruses entering the body through mucosal surfaces, remains to be elucidated, and in particular the role of the immune response in counteracting SARS-CoV-2 infection, primarily at the site(s) of virus entry that appears to be promising.

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.

Recent Advances in Unveiling the Role of Beta-Herpesviruses in Autoimmune Diseases.

A dysregulated immune response can lead to recognition of self-antigens as non-self-antigens, leading to failure of the immune tolerance toward normal cells and tissues, and the consequent development of a variety of autoimmune diseases [...].

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.

Anti-SARS-Cov-2 IgA Response in Tears of COVID-19 Patients.

The pandemic virus SARS-CoV-2 has been reported to be able to enter the body via the eye conjunctiva, but the presence of antiviral response in the eye remains poorly known. Our study was thus aimed to analyze the presence of secretory mucosal anti-SARS-CoV-2 type A immunoglobulins (IgA) in the conjunctival fluid of COVID-19 patients. The tears of 28 COVID-19 patients and 20 uninfected controls were collected by the Schirmer test and analyzed by a specific ELISA assay detecting anti-spike (S1) virus protein IgA. The results showed that 35.7% of COVID-19 subjects have specific antiviral IgA at the ocular level, persisting till 48 days post disease onset. Most of the IgA positive subjects presented mild symptoms. The collected data indicate a prolonged persistence of anti-SARS-CoV-2 IgA at the eye level and suggest that IgA detection may be extremely helpful in clarifying virus pathology and epidemiology.

COVID-19 Is a Multifaceted Challenging Pandemic Which Needs Urgent Public Health Interventions.

Until less than two decades ago, all known human coronaviruses (CoV) caused diseases so mild that they did not stimulate further advanced CoV research. In 2002 and following years, the scenario changed dramatically with the advent of the new more pathogenic CoVs, including Severe Acute Respiratory Syndome (SARS-CoV-1), Middle Eastern respiratory syndrome (MERS)-CoV, and the new zoonotic SARS-CoV-2, likely originated from bat species and responsible for the present coronavirus disease (COVID-19), which to date has caused 15,581,007 confirmed cases and 635,173 deaths in 208 countries, including Italy. SARS-CoV-2 transmission is mainly airborne via droplets generated by symptomatic patients, and possibly asymptomatic individuals during incubation of the disease, although for the latter, there are no certain data yet. However, research on asymptomatic viral infection is currently ongoing worldwide to elucidate the real prevalence and mortality of the disease. From a clinical point of view, COVID-19 would be defined as "COVID Planet " because it presents as a multifaceted disease, due to the large number of organs and tissues infected by the virus. Overall, based on the available published data, 80.9% of patients infected by SARS-CoV-2 develop a mild disease/infection, 13.8% severe pneumonia, 4.7% respiratory failure, septic shock, or multi-organ failure, and 3% of these cases are fatal, but mortality parameter is highly variable in different countries. Clinically, SARS-CoV-2 causes severe primary interstitial viral pneumonia and a "cytokine storm syndrome", characterized by a severe and fatal uncontrolled systemic inflammatory response triggered by the activation of interleukin 6 (IL-6) with development of endothelitis and generalized thrombosis that can lead to organ failure and death. Risk factors include advanced age and comorbidities including hypertension, diabetes, and cardiovascular disease. Virus entry occurs via binding the angiotensin-converting enzyme 2 (ACE2) receptor present in almost all tissues and organs through the Spike (S) protein. Currently, SARS-CoV-2 infection is prevented by the use of masks, social distancing, and improved hand hygiene measures. This review summarizes the current knowledge on the main biological and clinical features of the SARS-CoV-2 pandemic, also focusing on the principal measures taken in some Italian regions to face the emergency and on the most important treatments used to manage the COVID-19 pandemic.