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Background The microbiome of the oral cavity is the second-largest and diverse microbiota after the gut, harboring over 700 species of bacteria and including also fungi, viruses, and protozoa. With its diverse niches, the oral cavity is a very complex environment, where different microbes preferentially colonize different habitats. Recent data indicate that the oral microbiome has essential functions in maintaining oral and systemic health, and the emergence of 16S rRNA gene next-generation sequencing (NGS) has greatly contributed to revealing the complexity of its bacterial component. However, a detailed site-specific map of oral microorganisms (including also eukaryotes and viruses) and their relative abundance is still missing. Here, we aimed to obtain a comprehensive view of the healthy oral microbiome (HOM), including its drug-resistance features. Results The oral microbiome of twenty healthy subjects was analyzed by whole-genome sequencing (WGS) and real-time quantitative PCR microarray. Sampled oral micro-habitat included tongue dorsum, hard palate, buccal mucosa, keratinized gingiva, supragingival and subgingival plaque, and saliva with or without rinsing. Each sampled oral niche evidenced a different microbial community, including bacteria, fungi, and viruses. Alpha-diversity evidenced significant differences among the different sampled sites ( p  < 0.0001) but not among the enrolled subjects ( p  = 0.876), strengthening the notion of a recognizable HOM. Of note, oral rinse microbiome was more representative of the whole site-specific microbiomes, compared with that of saliva. Interestingly, HOM resistome included highly prevalent genes conferring resistance to macrolide, lincosamides, streptogramin, and tetracycline. Conclusions The data obtained in 20 subjects by WGS and microarray analysis provide for the first time a comprehensive view of HOM and its resistome, contributing to a deeper understanding of the composition of oral microbiome in the healthy subject, and providing an important reference for future studies, allowing to identify microbial signatures related to functional and metabolic alterations associated with diseases, potentially useful for targeted therapies and precision medicine.

Background The control of viral infections in the brain involves the activation of microglial cells, the macrophages of the brain that are constantly surveying the central nervous system, and the production of amyloid-beta (Aβ) as an anti-microbial molecule. Recent findings suggest a possible implication of HHV-6A in AD. We evaluated the effect of HHV-6A infection on microglial cell expression Aβ and the activation status, determined by TREM2, ApoE, cytokines, and tau expression. Methods We have infected microglial cells (HMC3, ATCC®CRL-3304), in monolayer and human peripheral blood monocyte-derived microglia (PBM-microglia) spheroid 3D model, with HHV-6A (strain U1102) cell-free virus inocula with 100 genome equivalents per 1 cell. We collected the cells 1, 3, 7, and 14 days post-infection (d.p.i.) and analyzed them for viral DNA and RNA, ApoE, Aβ (1-40, 1-42), tau, and phospho-tau (Threonine 181) by real-time immunofluorescence and cytokines by immunoenzymatic assay. Results We observed a productive infection by HHV-6A. The expression of Aβ 1-42 increased from 3 d.p.i., while no significant induction was observed for Aβ 1-40. The HHV-6A infection induced the activation (TREM2, IL-1beta, ApoE) and migration of microglial cells. The secretion of tau started from 7 d.p.i., with an increasing percentage of the phosphorylated form. Conclusions In conclusion, microglial cells are permissive to HHV-6A infection that induces the expression of Aβ and an activation status. Meanwhile, we hypothesize a paracrine effect of HHV-6A infection that activates and induces microglia migration to the site of infection.

Bacteriophages are viruses that exclusively kill bacteria and are the most ubiquitous organisms on the planet. Since their discovery, bacteriophages have been considered an important weapon to fight human and animal infections of bacterial origin due to their specific ability to attack the associated target bacteria. With the discovery of antibiotics, phage treatment was progressively abandoned in Western countries. However, due to the recent emergence of growing antimicrobial resistance (AMR) to antibiotics, interest in phage use in human therapy has once again grown. Similarly, at the environmental level, the extensive use of disinfectants based on chemicals, including biocides in agriculture, has been associated with the emergence of resistance against disinfectants themselves, besides having a high environmental impact. Due to these issues, the applications of phages with biocontrol purposes have become an interesting option in several fields, including farms, food industry, agriculture, aquaculture and wastewater plants. Notably, phage action is maintained even when the target bacteria are multidrug resistant (MDR), rendering this option extremely interesting in counteracting AMR emergence both for therapeutical and decontamination purposes. Based on this, bacteriophages have been interestingly proposed as environmental routine sanitizers in hospitals, to counteract the spread of the pathogenic MDR bacteria that persistently contaminate hard surfaces. This review summarizes the studies aimed at evaluating the potential use of phages as decontaminants, with a special focus on hospital sanitation.

Built environments (BEs) currently represent the areas in which human beings spend most of their life. Consistently, microbes populating BEs mostly derive from human occupants and can be easily transferred from BE to occupants. The hospital microbiome is a paradigmatic example, representing a reservoir for harmful pathogens that can be transmitted to susceptible patients, causing the healthcare‐associated infections (HAIs). Environmental cleaning is a crucial pillar in controlling BE pathogens and preventing related infections, and chemical disinfectants have been largely used so far towards this aim. However, despite their immediate effect, chemical‐based disinfection is unable to prevent recontamination, has a high environmental impact, and can select/increase antimicrobial resistance (AMR) in treated microbes. To overcome these limitations, probiotic‐based sanitation (PBS) strategies were recently proposed, built on the use of detergents added with selected probiotics able to displace surrounding pathogens by competitive exclusion. PBS was reported as an effective and low‐impact alternative to chemical disinfection, providing stable rebalance of the BE microbiome and significantly reducing pathogens and HAIs compared to disinfectants, without exacerbating AMR and pollution concerns. This minireview summarizes the most significant results obtained by applying PBS in sanitary and non‐sanitary settings, which overall suggest that PBS may effectively tackle the infectious risk meanwhile preventing the further spread of pathogenic and resistant microbes. Built environments microbiome derives from the human microbiome and can be transmitted to susceptible patients, causing healthcare‐associated infections (HAIs). Chemical disinfection cannot control stably the infectious risk and favors the selection of antimicrobial resistance (AMR). By contrast, probiotic‐based sanitation (PBS) was reported to provide stable rebalance of the BE microbiome, significantly reducing pathogens and HAIs compared to disinfectants, without exacerbating AMR.

Healthcare-Associated Infections (HAIs) are one of the most frequent complications occurring in healthcare facilities. Contaminated environmental surfaces provide an important potential source for transmission of many healthcare-associated pathogens, thus indicating the need for new and sustainable strategies. This study aims to evaluate the effect of a novel cleaning procedure based on the mechanism of biocontrol, on the presence and survival of several microorganisms responsible for HAIs (i.e. coliforms, Staphyloccus aureus, Clostridium difficile, and Candida albicans) on hard surfaces in a hospital setting. The effect of microbial cleaning, containing spores of food grade Bacillus subtilis, Bacillus pumilus and Bacillus megaterium, in comparison with conventional cleaning protocols, was evaluated for 24 weeks in three independent hospitals (one in Belgium and two in Italy) and approximately 20000 microbial surface samples were collected. Microbial cleaning, as part of the daily cleaning protocol, resulted in a reduction of HAI-related pathogens by 50 to 89%. This effect was achieved after 3-4 weeks and the reduction in the pathogen load was stable over time. Moreover, by using microbial or conventional cleaning alternatively, we found that this effect was directly related to the new procedure, as indicated by the raise in CFU/m2 when microbial cleaning was replaced by the conventional procedure. Although many questions remain regarding the actual mechanisms involved, this study demonstrates that microbial cleaning is a more effective and sustainable alternative to chemical cleaning and non-specific disinfection in healthcare facilities. This study indicates microbial cleaning as an effective strategy in continuously lowering the number of HAI-related microorganisms on surfaces. The first indications on the actual level of HAIs in the trial hospitals monitored on a continuous basis are very promising, and may pave the way for a novel and cost-effective strategy to counteract or (bio)control healthcare-associated pathogens.

Healthcare Associated Infections (HAI) are a global concern, further threatened by the increasing drug resistance of HAI-associated pathogens. On the other hand, persistent contamination of hospital surfaces contributes to HAI transmission, and it is not efficiently controlled by conventional cleaning, which does not prevent recontamination, has a high environmental impact and can favour selection of drug-resistant microbial strains. In the search for effective approaches, an eco-sustainable probiotic-based cleaning system (Probiotic Cleaning Hygiene System, PCHS) was recently shown to stably abate surface pathogens, without selecting antibiotic-resistant species. The aim of this study was to determine whether PCHS application could impact on HAI incidence. A multicentre, pre-post interventional study was performed for 18 months in the Internal Medicine wards of six Italian public hospitals (January 1st 2016-June 30th 2017). The intervention consisted of the substitution of conventional sanitation with PCHS, maintaining unaltered any other procedure influencing HAI control. HAI incidence in the pre and post-intervention period was the main outcome measure. Surface bioburden was also analyzed in parallel. Globally, 11,842 patients and 24,875 environmental samples were surveyed. PCHS was associated with a significant decrease of HAI cumulative incidence from a global 4.8% (284 patients with HAI over 5,930 total patients) to 2.3% (128 patients with HAI over 5,531 total patients) (OR = 0.44, CI 95% 0.35-0.54) (P<0.0001). Concurrently, PCHS was associated with a stable decrease of surface pathogens, compared to conventional sanitation (mean decrease 83%, range 70-96.3%), accompanied by a concurrent up to 2 Log drop of surface microbiota drug-resistance genes (P<0.0001; Pc = 0.008). Our study provides findings which support the impact of a sanitation procedure on HAI incidence, showing that the use of a probiotic-based environmental intervention can be associated with a significant decrease of the risk to contract a HAI during hospitalization. Once confirmed in larger experiences and other target populations, this eco-sustainable approach might be considered as a part of infection control and prevention (IPC) strategies. Trial registration-ISRCTN International Clinical Trials Registry, ISRCTN58986947.

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 Zn 2+ and Cu 2+ 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.

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.

Contamination of hospital surfaces by clinically-relevant pathogens represents a major concern in healthcare facilities, due to its impact on transmission of healthcare-associated infections (HAIs) and to the growing drug resistance of HAI-associated pathogens. Routinely used chemical disinfectants show limitations in controlling pathogen contamination, due to their inefficacy in preventing recontamination and selection of resistant strains. Recently we observed that an innovative approach, based on a cleanser added with spores of non-pathogenic probiotic Bacilli, was effective in stably counteracting the growth of several pathogens contaminating hospital surfaces. Here, we wanted to study the impact of the Bacillus-based cleanser on the drug-resistance features of the healthcare pathogens population. In parallel, the ability of cleanser-derived Bacilli to infect hospitalized patients was also investigated. Collected data showed that Bacilli spores can germinate on dry inanimate surfaces, generating the bacterial vegetative forms which counteract the growth of pathogens and effectively substitute for them on treated surfaces. Strikingly, this procedure did not select resistant species, but conversely induced an evident decrease of antibiotic resistance genes in the contaminating microbial population. Also importantly, all the six HAI-positive patients hosted in the treated areas resulted negative for probiotic Bacilli, thus adding evidences to their safety-to-use. These results indicate that this probiotic-based procedure is active not only in controlling surface microbial contamination but also in lowering drug-resistant species, suggesting that it may have relevant clinical and therapeutical implications for the management of HAIs.

Background Healthcare-associated infections (HAIs) caused by microbes that acquire antimicrobial resistance (AMR) represent an increasing threat to human health worldwide. The high use of chemical disinfectants aimed at reducing the presence of pathogens in the hospital environment can simultaneously favor the selection of resistant strains, potentially worsening AMR concerns. In the search for sustainable ways to control bioburden without affecting this aspect, probiotic-based sanitation (PBS) using Bacillus spp. was proposed to achieve stable reduction of pathogens, AMR, and associated HAIs. Although Bacillus probiotics are classified as nonpathogenic, comprehensive data about the potential genetic alterations of these probiotics following prolonged contact with surrounding pathogens are not yet available. This study aimed to assess in depth the genetic content of PBS- Bacillus isolates to evaluate any eventual variations that occurred during their usage. Results WGS analysis was used for the precise identification of PBS- Bacillus species and detailed profiling of their SNPs, resistome, virulome, and mobilome. Analyses were conducted on both the original PBS detergent and 172 environmental isolates from eight hospitals sanitized with PBS over a 30-month period. The two species B. subtilis and B. velezensis were identified in both the original product and the hospital environment, and SNP analysis revealed the presence of two clusters in each species. No virulence/resistance genes or mobile conjugative plasmids were detected in either the original PBS- Bacillus strain or any of the analyzed environmental isolates, confirming their high genetic stability and their low/no tendency to be involved in horizontal gene transfer events. Conclusions The data obtained by metagenomic analysis revealed the absence of genetic sequences associated with PBS- Bacillus and the lack of alterations in all the environmental isolates analyzed, despite their continuous contact with surrounding pathogens. These results support the safety of the Bacillus species analyzed. Further metagenomic studies aimed at profiling the whole genomes of these and other species of Bacillus , possibly during longer periods and under stress conditions, would be of interest since they may provide further confirmation of their stability and safety.