1. Investigación

After 2 years of the COVID-19 pandemic, we continue to face vital challenges stemming from SARS-CoV-2 variation, causing changes in disease transmission and severity, viral adaptation to animal hosts, and antibody/vaccine evasion. Since the monitoring, characterization, and cataloging of viral variants are important and the existing information on this was scant for Sicily, this pilot study explored viral variants circulation on this island before and in the growth phase of the second wave of COVID-19 (September and October 2020), and in the downslope of that wave (early December 2020) through sequence analysis of 54 SARS-CoV-2-positive samples. The samples were nasopharyngeal swabs collected from Sicilian residents by a state-run one-health surveillance laboratory in Palermo. Variant characterization was based on RT-PCR amplification and sequencing of four regions of the viral genome. The B.1.177 variant was the most prevalent one, strongly predominating before the second wave and also as the wave downsized, although its relative prevalence decreased as other viral variants, particularly B.1.160, contributed to virus circulation. The occurrence of the B.1.160 variant may have been driven by the spread of that variant in continental Europe and by the relaxation of travel restrictions in the summer of 2020. No novel variants were identified. As sequencing of the entire viral genome in Sicily for the period covered here was restricted to seven deposited viral genome sequences, our results shed some light on SARS-CoV-2 variant circulation during that wave in this insular region of Italy which combines its partial insular isolation with being a major entry point for the African immigration.

The widespread irrational use of antibiotics in recent years has resulted in an increase in the detection of multi-resistant bacterial strains, particularly methicillin-resistant Staphylococcus aureus (MRSA). The use of natural derivatives such as flavonoids is postulated as one of the most promising avenues to solve this emerging public health problem. The objective of the present study is to characterize the antimicrobial activity of icariin, a flavonoid compound isolated from a variety of plants of the Epimedium genus, against human and animal clinical MRSA isolates. Our study found that icariin alone did not have any antimicrobial effect on S. aureus or MRSA clinical isolates. However, icariin enhanced the effect of amoxycillin-clavulanate or ampicillin, whereas no effect was seen when used in combination with vancomycin. Specifically, co-incubation of S. aureus with amoxycillin-clavulanate plus icariin resulted in an increased proportion of dead cells, suggesting that this flavonoid potentially increases antimicrobial activity when used in combination with the beta-lactam antibiotic amoxycillin-clavulanate. Furthermore, we demonstrate that co-incubation of S. aureus with AmoxyClav plus icariin resulted in increased membrane disruption and growth inhibition. This study demonstrates the potential utility of icariin in permitting lower antibiotic therapeutic doses in alignment with strategies to reduce the spread of antibiotic resistance. Further research is required to determine the optimum concentration of icariin and to define clinically relevant combinations of flavonoid and antibiotic.

Clostridioides difficile is a major cause of health-care related infections and antibiotic-associated diarrhea. High recurrence rates following antibiotic treatment, along with the emergence of hypervirulent and multidrug resistant ribotypes makes essential the development of safe, effective, novel therapies for the treatment of C. difficile infections. The primary outcome evaluated in this meta-analysis was the effectiveness of fecal microbiota transplantation (FMT). Secondary outcomes were the proportion of patients suffering adverse effects along with the most effective administration route. The mean treatment effectiveness was 82% (95% CI: 75-89). Overall, patients receiving FMT via colonoscopy experienced more adverse effects than patients whom received enema, or oral capsules (71'6% vs 40'2%, and 35'3% respectively). Comparing administration of FMT by colonoscopy versus enema resulted in a Hedges’ g of 0'74 (95% CI of 0'9 to 0'58), indicating a slight advantage in favor of colonoscopy. The comparison between colonoscopy and capsule returned a Hedges’ g of 0'44 (95% CI of 0'20–0'69), indicating that delivery of the FMT by capsule was statistically significantly more effective. FMT provides an effective and safe treatment for C. difficile diarrhea. Further research into the efficacy of different preparation protocols is needed.

Neonatal sepsis is a serious condition, where an adequate empiric antibiotic treatment is crucial. The objective of this systematic review is to assess whether the World Health Organization’s recommended treatment regime remains applicable for late-onset neonatal sepsis caused by Enterobacteriaceae, in the time of increased antimicrobial resistance. PubMed was searched for articles from 2009 to 2020. A total of 49 articles were eligible for inclusion. The review was carried out in accordance with PRISMA guidelines. For Klebsiella spp. 100, 68 and 63% of the studies found sensitivity to ampicillin, gentamicin and third-generation cephalosporin in <50% of the isolates. For Escherichia coli, the corresponding values were 88, 50 and 42% respectively, whilst for Enterobacter spp. 100, 70 and 94% of the studies found <50% sensitivity to these antibiotics. Overall, there is low sensitivity to all agents in the WHO’s recommended empiric treatment regimes (WHO recommends ampicillin plus gentamicin as first-line treatment and third-generation cephalosporin as second-line treatment). A revised guideline for empiric antibiotic treatment of neonatal sepsis is urgently needed due to the increased threat of antimicrobial resistant Enterobacteriaceae causing neonatal sepsis.

Background: Orthodontic treatment with fixed appliances is associated with changes in oral microbiota, including increased Candida colonization. The Candida fungus can cause oral lesions and infections such as candidiasis and angular cheilitis, and is harmful to both the patient and the orthodontist. Poor hygiene facilitates the colonization of these microorganisms. The key aim was to quantify the colonization of C. albicans in patients prior to beginning orthodontic treatment, and during the treatment process. Material and Methods: A total of 124 patients (43 males and 80 females) with a mean age of 19.5 years, who required treatment with metal or aesthetic (ceramic) braces, were studied. Microbiological samples were taken from the oral cavity using the swab technique throughout the treatment and cultured on a Sabouraud Dextrose Agar plate and, if positive, cultured on a CHROMagar® Candida plate. Results: In contrast to other published studies, no statistically significant increase in C. albicans colonization was observed during the orthodontic treatment. The fixed appliances had no influence on the presence, absence or level of colonization by C. albicans and there were no significant differences between the different appliances studied. Conclusions: Our study showed that frequency of oral hygiene measures by study participants did not affect the rate of oral carriage of Candida in a statistically significant manner. This observation contrasted with published literature, which suggests that thorough brushing is important to prevent the build-up of Candida species.

In the era of antimicrobial resistance, fungal pathogens are not an exception. Several strategies, including antimicrobial stewardship programs and high throughput screening of new drugs, are being implemented. Several recent studies have demonstrated the effectiveness of plant compounds with antifungal activity. In this systematic review, we examine the use of natural compounds as a possible avenue to fight fungal infections produced by Candida albicans, the most common human fungal pathogen. Electronic literature searches were conducted through PubMed/MEDLINE, Cochrane, and Science Direct limited to the 5 years. A total of 131 articles were included, with 186 plants extracts evaluated. Although the majority of the natural extracts exhibited antifungal activities against C. albicans (both in vivo and in vitro), the strongest antifungal activity was obtained from Lawsonia inermis, Pelargonium graveolens, Camellia sinensis, Mentha piperita, and Citrus latifolia. The main components with proven antifungal activities were phenolic compounds such as gallic acid, thymol, and flavonoids (especially catechin), polyphenols such as tannins, terpenoids and saponins. The incorporation of nanotechnology greatly enhances the antifungal properties of these natural compounds. Further research is needed to fully characterize the composition of all herbal extracts with antifungal activity as well as the mechanisms of action of the active compounds.

Extrinsic black dental staining is an external dental discoloration of bacterial origin, considered a special form of dental plaque. Currently, there is no definitive therapeutic option for eliminating black stain. This study employed 16S rRNA metagenomics to analyze black stain and white-plaque samples from 27 adult volunteers. Study objectives were to: describe the microbial diversity of adult black stain samples; characterize their taxonomic profile; compare the microbiomes of black stain versus white-plaque from adult volunteers and propose a functional map of the black stain microbiome using PICRUSt2. The black stain microbiome was poorer in species diversity as compared to white-plaque. The five most abundant genera in black stain were Capnocytophaga, Leptotrichia, Fusobacterium, Corynebacterium and Streptococcus. Functional analysis of microbial species revealed conserved and consistent clustering of functional pathways within and between black stain and white-plaque microbiomes. We describe enrichment of heme biosynthetic pathways in black stain. Our results suggest that the dysbiosis in black stain resembles “orally healthy” communities. The increased abundance of heme biosynthetic pathways suggests that heme-dependent iron sequestration and subsequent metabolism are key for black stain formation. Further research should decipher the regulation of heme biosynthetic genes and characterize the temporal sequence leading to colonization and dysbiosis.

Background: Resistance to antimicrobial agents has become a problem in modern society. Antibiotic resistant bacteria undermine the prevention and treatment of infections. Undergraduate dental students in Europe are required to receive information in aspects of microbiology relevant for dental practice, including oral microbial pathogens and resistance mechanisms against antimicrobial compounds. The objective of this study was to implement a research-based strategy to aid the understanding of the increase in antimicrobial resistance in undergraduate dental student training. The primary outcome of this project is the efficacious delivery of the learning objectives. Methods: Ten volunteer undergraduate student “ambassadors” were recruited to manage the project with assistance from lead academics. Student ambassadors were a source of peer learning for their colleagues. The project consisted of three phases: Pre-project preparation (in which the ambassadors received special instruction and training); Practical experience (in which the ambassadors worked with volunteer student colleagues to carry out the project); Public presentation of results (in which ambassadors presented study results at a scientific conference of their choosing). Results: A total of 1164 students volunteered for the project, corresponding to an average participation rate of 76.4% students per year of the course. Following final debriefing, student participants and ambassadors were strongly positive in their evaluation of the achievement of 8 key student learning objectives. The results demonstrate that most volunteers improved their knowledge related to antimicrobial resistance mechanisms in microbiology. Additional benefits of participation in this project included an improvement in dental knowledge and ethics in biomedical research for the student volunteers, whilst the student ambassadors reported improved knowledge about critical thinking and study design, as well as a deeper understanding about microbiological analysis methods. Conclusions: To the best of our knowledge, this the first instance of the application of project-based methodologies to the teaching of a traditionally non-laboratory component of a subject taught in the dentistry curriculum. Results from both students and ambassadors highlighted the increase in dental knowledge and an increased awareness of antimicrobial resistance as the key outcomes of this project.

More than 90% of malignant tumors of the head and neck are oral squamous cell carcinomas (OSCC). Early OSCC detection using salivary biomarkers could prevent malignant transformations and enhance patient survival. A systematic search in MEDLINE and the Central Register of Controlled Trials and meta-analysis were undertaken to identify the screening potential of 6 salivary biomarkers for early OSCC detection: IL-8, IL1-β, DUSP-1 and S100P mRNAs, miR125a and miR200a microRNAs. The sensitivities of IL-8 (0.41; 95%CI 0.19-0.99), IL1-β (0.26; 95%CI 0.19- 0.99), DUSP-1 (0.61; 95%CI 0.01-0.98), and S100P (0.67; 95%CI 0.32- 0.99) were calculated. Specificities of the biomarkers analyzed were found to be IL-8 (0.69; 95%CI 0.66-0.99), IL1-β (0.47; 95%CI 0.46 - 0.90), DUSP-1 (0.75; 95%CI 0.33-1) and S100P (0.73; 95%CI 0.18-0.99). Early detection of OSCC was best achieved by screening for salivary mRNA DUSP-1 and S100P. Further investigation is required into miRNAs as novel biomarkers

INTRODUCTION AND AIMS: This study investigates the effects of the application of antimicrobial phototherapy on black-stained tooth surfaces. Key variables were area, color and time taken to reappearance of black stain following dental prophylaxis with and without antimicrobial phototherapy. Differences in bacterial composition of black stain (specifically Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Porphyromonas gingivalis) were analyzed. MATERIAL AND METHODS: The phototherapy device used was GLO™ Science LLC, New York, America, emitting light at 475 nm and a power of 3W. Light was applied in 2 cycles of 8 min on 31 volunteers. Microbial identification was carried out on DNA extracted from black plaque. This study is registered with ClinicalTrials.gov, number NCT03309748. RESULTS: Following antimicrobial phototherapy, 64.5% of patients displayed reduced pigmentation area. Plaque color was lighter in 48.4% of subjects. Pigmentation area and depth of color returned to normal levels during the course of the study. Colonization by the three bacterial species decreased, although the changes were not statistically significant. We report a key novel finding showing elevated levels of colonization by Tannerella forsythia (83.9%) in adult black stain. CONCLUSIONS: Application of phototherapy results in a reduction in area, color and bacterial colonization of black plaque in adults. The changes were not found to be statistically significant, perhaps owing to the low illumination power of the homewhitening device. For the first time, we document the elevated presence of Tannerella forsythia in adult black stain. We also demonstrate the potential application of a commercially available home-whitening device for black plaque treatment.