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.

Hashimoto's thyroiditis (HT) is the most common of all thyroid diseases and is characterized by abundant lymphocyte infiltrate and thyroid impairment, caused by various cell- and antibody-mediated immune processes. Viral infections have been suggested as possible environmental triggers, but conclusive data are not available. We analyzed the presence and transcriptional state of human herpesvirus 6 (HHV-6) in thyroid fine needle aspirates (FNA) and peripheral blood mononuclear cells (PBMCs) from 34 HT patients and 28 controls, showing that HHV-6 DNA prevalence (82% vs. 10%, p≤0.001) and viral load were significantly increased in FNA from HT patients, and thyrocytes from HT FNA displayed a 100-fold higher HHV-6 DNA load compared to infiltrating lymphocytes. In addition, while HHV-6 was strictly latent in positive samples from controls, a low grade acute infection was detected in HT samples. HHV-6 variant characterization was carried out in 10 HT FNA samples, determining that all specimens harbored HHV-6 Variant A.The tropism of HHV-6 for thyroid cells was verified by infection of Nthy-ori3-1, a thyroid follicular epithelial cell line, showing that thyrocytes are permissive to HHV-6 replication, which induces de novo expression of HLA class II antigens. Furthermore, HHV-6-infected Nthy-ori3-1 cells become targets for NK-mediated killing, NK cells from HT patients show a significantly more efficient killing of HHV-6 infected thyroid cells than healthy controls, and HT patients have increased T-cell responses to HHV-6 U94 protein, associated to viral latency. These observations suggest a potential role for HHV-6 (possibly variant A) in the development or triggering of HT.

BackgroundContamination 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.MethodsHere, 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.ResultsCollected 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.ConclusionsThese 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.

BackgroundHealthcare-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.AimThis 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.MethodsThe 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.ResultsMicrobial 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.ConclusionsThis 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.

To elucidate the roles of human herpesvirus (HHV)-6 primary unexplained infertile women, a prospective randomized study was conducted on a cohort of primary unexplained infertile women and a cohort of control women, with at least one successful pregnancy. HHV-6 DNA was analyzed and the percentage and immune-phenotype of resident endometrial Natural Killer (NK) cells, as the first line of defense towards viral infections, was evaluated in endometrial biopsies. Cytokine levels in uterine flushing samples were analyzed. HHV-6A DNA was found in 43% of endometrial biopsies from primary unexplained infertile women, but not in control women. On the contrary, HHV-6B DNA was absent in endometrial biopsies, but present in PBMCs of both cohorts. Endometrial NK cells presented a different distribution in infertile women with HHV6-A infection compared with infertile women without HHV6-A infection. Notably, we observed a lower percentage of endometrial specific CD56brightCD16- NK cells. We observed an enhanced HHV-6A-specific endometrial NK cell response in HHV-6A positive infertile women, with a marked increase in the number of endometrial NK cells activating towards HHV-6A infected cells. The analysis of uterine flushing samples showed an increase in IL-10 levels and a decrease of IFN-gamma concentrations in infertile women with HHV6-A infection. Our study indicates, for the first time, that HHV-6A infection might be an important factor in female unexplained infertility development, with a possible role in modifying endometrial NK cells immune profile and ability to sustain a successful pregnancy.

The results indicate that (i) gingival tissues can be considered a potential reservoir for HHV-7; (ii) when present, HHV-7 persists in a latent state in the majority of cases; (iii) the presence of EBV seems to be associated with the diseased state of the patient and site.

No abstract