Harnessing the Power of Biologic Agents on the Oral Microbiota: A Way to Promote Oral and Systemic Health?

During the long history of their evolution, higher organisms, including mammals, have learnt to take great advantage from living in close contact with selected populations of microbes.1 By living in close contact, animals and microbes underwent a progressive and mutual co-evolutive process that is believed to be a major driving force in the development of adaptive immunity of vertebrates.2.

Microbiological and FE-SEM Assessment of d-PTFE Membrane Exposed to Oral Environment after Alveolar Socket Preservation Managed with Granular nc-HA.

AIM: The aim of this study was to analyze, by the aid of microbiological analysis and the field emission scanning electron microscopical (FE-SEM) analysis, the role of high-density polytetrafluoroethylene (d-PTFE) membranes in avoiding the microbial colonization of a nanocrystalline hydroxyapatite (nc-HA) bone graft and the involvement of this colonization in the healing process. MATERIALS AND METHODS: Six patients underwent extraction of unrecoverable teeth, and a socket preservation technique was carried out with nc-HA synthetic bone graft and then covered with a d-PTFE membrane. After 28 days from surgery, FE-SEM analysis and BioTimer assay technique to assess the microbiological count of streptococci species were carried out. Data were collected and analyzed by the Student's t test (confidence interval: 95%). RESULTS: The mean amount of bacteria measured on the upper side of the membrane was 6.52 ± 0.50 CFU, while on the lower side, it was 6.59 ± 0.40 CFU. Significant differences were not found between the two sides of the membrane or between the different sectors (p > 0.05). The FE-SEM analysis revealed structured biofilms on both sides of the membrane: species of cocci, bacilli, and fusobacteria were recognizable in occasional settled vegetations. CONCLUSION: Since the amount of bacteria found was low, the improved impermeability of the d-PTFE membrane permitted the healing process to proceed uneventful and without signs of infection or inflammation. CLINICAL RELEVANCE: The infection of the graft site could lead to a failure of the socket preservation technique which could delay or compromise the rehabilitation following procedures. The use of d-PTFE can improve the bone regeneration thanks to its antimicrobial properties.

Behaviour of Bdellovibrio bacteriovorus in the presence of Gram-positive Staphylococcus aureus.

The present study aimed to characterize the behavior of Bdellovibrio bacteriovorus in the presence of Staphylococcus aureus. B. bacteriovorus was co-cultured with S. aureus or Pseudomonas aeruginosa or Streptococcus mutans, in planktonic and sessile conditions. Co-cultures were studied by Field-Emission Scanning Electron Microscopy (FESEM), Scanning Transmission Electron Microscopy (STEM), turbidimetry, quantitative PCR (qPCR), and sequencing of gene Bd0108 of B. bacteriovorus. Results indicated that B. bacteriovorus comparably inhibited planktonic growth of P. aeruginosa and S. aureus, but not of S. mutans. FESEM and STEM showed that B. bacteriovorus interacts with S. aureus affecting its cell wall and membrane. Sequencing of gene Bd0108 did not reveal any of the mutations that can arise from the host-interaction (hit) locus. Although some Gram-negative species are reported to be B. bacteriovorus prey, it seems that in case of nutrient deficiency this predatory bacterium can also take advantage of some Gram-positive species. B. bacteriovorus behaviour in the presence of S. aureus is relevant for its possible therapeutic use in several pathologies, like cystic fibrosis in which S. aureus and P. aeruginosa frequently coexist as infectious agents.

Intensity and duration of in-vitro antibacterial activity of different adhesives used in orthodontics.

This work investigated the antibacterial activity of 14 bonding agents to predict their ability to inhibit white-spot development during orthodontic treatment. Standardized, sterilized disks of each material were continuously rinsed (for up to 180 d) in a flow of sterile saline. At predetermined time points, the residual ability of each material to inhibit bacterial growth (determined by measuring the size of inhibition halos around disks placed onto appropriate culture media seeded with Streptococcus gordonii DSM6777, Streptococcus sanguinis DSM20567, Streptococcus mutans DSM20523, or Lactobacillus acidophilus DSM20079) and biofilm formation (determined by measuring the numbers of bacteria adherent to disks following incubation in appropriate broths) was tested in triplicate and compared with the baseline activities of freshly prepared materials. Overall antibacterial and anti-biofilm activities, adjusted for exposure time and strain of bacteria, were assessed. The decrease of antibacterial activity was faster (30-60 d) and complete for fluoride-enriched materials, but slower (90 d) and partial for antimicrobial-containing materials (benzalkonium chloride, zinc oxide, chlorexidine, or MDPB). Materials enriched with benzalkonium chloride, chlorexidine, or MDPB showed the highest antibacterial activities. Anti-biofilm assays yielded similar results. These data could be helpful for clinicians in the choice of the best performing bonding agent also in light of duration of the clinical application.

Microbiological and host factors are involved in promoting the periodontal failure of metaloceramic crowns.

This study was aimed at looking into the microbiological/inflammatory parameters predicting the periodontal success/failure of fixed prostheses. Microbiological and inflammatory patterns were studied at 102 sites having metaloceramic crowns in place from 3 to 6 years and divided in healthy sites (HS), gingivitis affected (MG), and periodontitis affected (PB). Total bacterial flora and selected indicator species in subgingival plaque were quantified by quantitative real-time PCR. The concentrations of IL-1ß, IL-6, and TNF-α were determined in gingival crevicular fluid (GCF) by enzyme-linked immunosorbent assays. The experimental sites showed no significant difference with respect to the age and gender of the patients and to the position of the crown margins. Poor marginal adaptation was significantly higher in MG and PB. The total amounts of bacteria per probing depth showed no significant differences among the three groups and their controls, while both MG and PB sites showed altered patterns in the distribution of specific bacteria. Both MG and PB sites showed significantly higher levels of inflammatory cytokines in GCF. The control teeth of PB subjects showed significantly higher levels of IL-1ß as compared to other control sites. Data confirm that the application of metaloceramic crowns is a factor of risk for the development of gingival/periodontal inflammation. This risk is possibly associated with microbiological and host factors that predispose to the onset of periodontal alterations at sites reconstructed with metaloceramic crowns. These factors, once their role is confirmed by longitudinal studies, could be used to set up rapid tests to early predict the onset of periodontal disease at reconstructed sites.

Viral hemagglutinin is involved in promoting the internalisation of Staphylococcus aureus into human pneumocytes during influenza A H1N1 virus infection.

Secondary pneumonia caused by Staphylococcus aureus is reemerging as a primary cause of excess mortality associated with infection by the influenza A virus. We have investigated in vitro the cellular and molecular mechanisms underlying this synergism. Experimental data show a significant increase in the efficiency of internalisation of S. aureus into cultured pneumocytes during the early phases of viral infection, while a relevant increase in the efficiency of adhesion is evident only later during viral infection, suggesting that the 2 effects are based on different molecular mechanisms. Data reported in this paper show that S. aureus cells can bind the viral hemagglutinin (HA) and that this binding promotes enhanced bacterial internalisation by 2 mechanisms: binding to HA exposed at the surface of infected cells and binding to free extracellular virions, enabling internalisation at high efficiency also in cells that are not infected by the virus. The affinity of binding that involves S. aureus and HA was shown to be enhanced by the reducing extracellular environment that the virus can generate.

Molecular and immunological characterization of Staphylococcus aureus in pediatric atopic dermatitis: implications for prophylaxis and clinical management.

S. aureus represents a critical cofactor in atopic dermatitis (AD). In this paper, the prevalence of S. aureus infection/colonization was evaluated in 117 children as well as in their cohabitants, in order to assess the value of S. aureus characterization in predicting disease onset and severity and in providing indications for prophylaxis. Results showed that children with AD as well as their cohabitants had a significantly greater incidence of S. aureus infection/colonization as compared to controls. The genetic characterization showed a virtual identity of the bacteria strains collected at different sites of the patients with those found in the cohabitants, suggesting both a direct transmission between the nasal reservoir and the lesions in the same atopic subject and a risk for reinfection within family cohabitants. These data stress the need of preliminary laboratory assessment and posttherapy control in both AD patients and their close contacts for effective S. aureus eradication.

Inflammatory Periimplant Diseases and the Periodontal Connection Question.

Implant therapy has become a widespread reality in modern dentistry. Nevertheless, dental implants can fail due to different causes, among which inflammatory peri-implant diseases (IPDs) are a major challenge, with prevalences that are much higher than previously believed.Specific searches were undertaken for each question raised between October and November 2017, in the PubMed website database (US National Library of Medicine, National Institutes of Health; Bethesda, Maryland, United States). Only articles written in English and published from 2007 onward were considered initially. The following keywords were used in the searches "periimplantitis (PI)," "periimplant mucositis (PM)," "dental implant failure," "periimplant microbiota," "periodontal microbiota," "implant failure" (no temporal limit), and "foreign body reaction" (no temporal limit). The selection process resulted in the selection of 239 articles that were analyzed in detail in elaborating this review. The reference list was limited to the 47 most relevant articles due to editorial limits of this Journal.Intrinsic differences between natural teeth and dental implants are able to give rise to inflammatory diseases that share only minor and scarcely relevant characters, and would consequently deserve different and specifically designed instruments and strategies, for both diagnosis and therapy.

Immediate or delayed retrieval of the displaced third molar: A review.

BACKGROUND: The displacement of a third molar is a rare occurrence, but it could lead to serious and/or life threatening complication. Aim of this review is to understand the most correlated causes of displacement and the possible solutions proposed in literature to avoid and solve this complication for maxillary and mandibular third molars at the appropriate time. MATERIAL AND METHODS: A search for "third molar displacement" was performed by using Pubmed database. Articles referred to soft tissues displacement, from 1957 to 2018, were included in the review. The references lists of all eligible articles were examined and additional studies were added to the review only if indexed on Pubmed. All the articles on maxillary sinus displacement and the dislocation of dental fragments or surgical equipment were excluded. RESULTS: From a total of 134 results, 68 articles were examined for satisfying inclusion criteria. 18 articles were excluded because not inherent with the topic; 19 articles on infratemporal space, 11 on sublingual space, 9 on submandibular space, 11 on lateral pharyngeal space displacement were considered congruent for the review and included. CONCLUSIONS: The displacement of the third molar in deeper tissues could be avoided by the use of proper surgical procedures and instrumentarium. If displacement occurs, and the operator could not reach the tooth in safe conditions, the patient should be immediately referred to a maxillo-facial surgeon, because of the possibility of further displacement or the onset of hazardous or potentially fatal infections in vital regions. Key words:Third molar, wisdom tooth, maxillary, mandibular, displacement.

Rebuilding the Gut Microbiota Ecosystem.

A microbial ecosystem in which bacteria no longer live in a mutualistic association is called dysbiotic. Gut microbiota dysbiosis is a condition related with the pathogenesis of intestinal illnesses (irritable bowel syndrome, celiac disease, and inflammatory bowel disease) and extra-intestinal illnesses (obesity, metabolic disorder, cardiovascular syndrome, allergy, and asthma). Dysbiosis status has been related to various important pathologies, and many therapeutic strategies aimed at restoring the balance of the intestinal ecosystem have been implemented. These strategies include the administration of probiotics, prebiotics, and synbiotics; phage therapy; fecal transplantation; bacterial consortium transplantation; and a still poorly investigated approach based on predatory bacteria. This review discusses the various aspects of these strategies to counteract intestinal dysbiosis.

Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655.

The AphA enzyme of Escherichia coli, a molecular class B periplasmic phosphatase that belongs to the DDDD superfamily of phosphohydrolases, was purified and subjected to biochemical characterization. Kinetic analysis with several substrates revealed that the enzyme essentially behaves as a broad-spectrum nucleotidase highly active on 3'- and 5'-mononucleotides and monodeoxynucleotides, but not active on cyclic nucleotides, or nucleotides di- and triphosphate. Mononucleotides are degraded to nucleosides, and AphA apparently does not exhibit any nucleotide phosphomutase activity. However, it can transphosphorylate nucleosides in the presence of phosphate donors. Kinetic properties of AphA are consistent with structural data, and suggest a role for the hydrophobic pocket present in the active site crevice, made by residues Phe 56, Leu71, Trp77 and Tyr193, in conferring preferential substrate specificity by accommodating compounds with aromatic rings. AphA was inhibited by several chelating agents, including EDTA, EGTA, 1,10-phenanthroline and dipicolinic acid, with EDTA being apparently the most powerful inhibitor.

Evaluation of microbiota associated with Herpesviruses in active sites of generalized aggressive periodontitis.

AIMS: The present study aimed to investigate microbial patterns associated with disease progression and coinfection by different Herpesviruses in generalized aggressive periodontitis (GAP). METHODS: Microbiological samples were obtained from active (AS) and non-active (n-AS) sites in 165 subjects affected by GAP and were analyzed for 40 bacterial species by the Checkerboard DNA-DNA Hybridization technique and for Herpes simplex 1 (HSV-1), Human Cytomegalovirus (CMV), and Epstein Bar virus (EBV) by PCR.Common Factor Analysis and Multiple Regression Analysis were applied to disclose specific microbial patterns associated with the three viruses. RESULTS: Herpesviruses were detected in 37.6% of subjects. Detection of each of the searched viruses was associated with specific patterns of subgingival biofilm in AS. Logistic regression analyses evidenced several virus/bacteria associations: i) EBV with Aggregatibacter actinomycetemcomitans; ii) CMV with A. actinomycetemcomitans, Veillonella parvula, Parvimonas micra and Fusobacterium nucleatum subsp. polymorphum; iii) HSV-1 with Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium periodonticum and Staphylococcus aureus. CONCLUSIONS: Microbiological data suggest that Herpesviruses are probably not mere spectators of disease progression and that specific patterns of subgingival plaque are correlated with the presence of different Herpesviruses.

Rhinoviruses promote internalisation of Staphylococcus aureus into non-fully permissive cultured pneumocytes.

Respiratory viruses, including rhinoviruses, frequently promote bacterial opportunistic infections, through mechanisms that still deserve to be investigated in detail. This work was aimed at understanding how a viral infection mostly affecting the upper respiratory tract, such as the common cold, can repeatedly promote opportunistic infections in the lower airways, a site where viral replication is limited. The adhesivity and invasivity of Staphylococcus aureus were evaluated, in permissive and non-permissive cells, infected with Rhinovirus-1b. The role of inflammatory cytokines, and of ICAM-1 overexpression in the Rhinovirus-S. aureus cooperation was evaluated. Rhinovirus-1b enhanced the efficiency of internalisation of S. aureus irrespective of cellular permissivity, even when very low viral multiplicities of infection were used. Experiments performed with UV inactivated and heat inactivated viral particles suggested that this enhancement does not depend upon viral replication, but requires viral adhesion. Experimental data suggest that Rhinovirus-1b can significantly increase the ability of S. aureus to internalise into pneumocytes with a mechanism that involves the virus induced release of IL-6 and IL-8, and the overexpression of ICAM-1. Overall data disclose a possible mechanism through which rhinoviruses can promote bacterial infections in the lower respiratory tract.

Antibacterial activity of essential oils mixture against PSA.

Pseudomonas syringae pv. actinidiae (PSA) is the causal agent of bacterial canker of kiwifruit. It is very difficult to treat pandemic disease. The prolonged treatment with antibiotics, has resulted in failure and resistance and alternatives to conventional antimicrobial therapy are needed. The aim of our study was to analyse the phenotypic characteristics of PSA, identify new substances from natural source i.e. essential oils (EOs) able to contain the kiwifruit canker and investigate their potential use when utilised in combination. Specially, we investigated the morphological differences of PSA isolates by scanning electron microscope, and the synergic action of different EOs by time-kill and checkerboard methods. Our results demonstrated that PSA was able to produce extracellular polysaccharides when it was isolated from trunk, and, for the first time, that it was possible to kill PSA with a mixture of EOs after 1 h of exposition. We hypothesise on its potential use in agriculture.

The molecular class C acid phosphatase of Chryseobacterium meningosepticum (OlpA) is a broad-spectrum nucleotidase with preferential activity on 5'-nucleotides.

The olpA gene of Chryseobacterium meningosepticum, encoding a molecular class C phosphatase, was cloned and expressed in Escherichia coli. The gene encodes a 29-kDa polypeptide containing an amino-terminal signal peptide typical of bacterial membrane lipoproteins. Expression in E. coli results in a functional product that mostly partitions in the outer membrane. A secreted soluble OlpA derivative (sOlpA) lacking the N-terminal cysteine residue for lipid anchoring was produced in E. coli and purified by means of two steps of ion exchange chromatography. Analysis of the kinetic parameters of sOlpA with several organic phosphoesters revealed that the enzyme was able to efficiently hydrolyze nucleotide monophosphates, with a strong preference for 5'-nucleotides and for 3'-AMP. The enzyme was also able to hydrolyze sugar phosphates and beta-glycerol phosphate, although with a lower efficiency, whereas it was apparently inactive against nucleotide di- and triphosphates, diesters, and phytate. OlpA, therefore, can be considered a broad-spectrum nucleotidase with preference for 5'-nucleotides. Its functional behaviour exhibits differences from that of the Haemophilus influenzae OMP P4 lipoprotein, revealing functional heterogeneity among phosphatases of molecular class C.

Low methicillin-resistant Staphylococcus aureus carriage rate among Italian dental students.

We assessed methicillin-resistant Staphylococcus aureus (MRSA) carriage rate among dental students from an Italian university. A total of 157 subjects participated (67 preclinical students and 90 clinical students); samples were collected from the nose, mouth, and skin. Five preclinical students and 0 clinical students were MRSA-positive. Carriage rates were 3.2% (95% confidence interval [CI], 0.4%-6.0%) overall, 7.5% (95% CI, 1.2%-13.8%) in preclinical students and 0% in clinical students. There were 2 MRSA clusters among the preclinical students: 3 second-year and 2 first-year students, who sat close to one another in the classroom the day of the sample. MRSA carriage was not associated with dental health care. The pooled carriage rate among dental students was assessed to obtain a reliable figure of carriage rate unaffected by local conditions. The 4 published surveys were pooled, and the fixed-effects method was used. Among the 484 dental students, the pooled carriage rate was 4.1% (95% CI, 2.4%-5.8%).

Bdellovibrio bacteriovorus directly attacks Pseudomonas aeruginosa and Staphylococcus aureus Cystic fibrosis isolates.

Bdellovibrio bacteriovorus is a predator bacterial species found in the environment and within the human gut, able to attack Gram-negative prey. Cystic fibrosis (CF) is a genetic disease which usually presents lung colonization by Pseudomonas aeruginosa or Staphylococcus aureus biofilms. Here, we investigated the predatory behavior of B. bacteriovorus against these two pathogenic species with: (1) broth culture; (2) "static" biofilms; (3) field emission scanning electron microscope (FESEM); (4) "flow" biofilms; (5) zymographic technique. We had the first evidence of B. bacteriovorus survival with a Gram-positive prey, revealing a direct cell-to-cell contact with S. aureus and a new "epibiotic" foraging strategy imaged with FESEM. Mean attaching time of HD100 to S. aureus cells was 185 s, while "static" and "flow" S. aureus biofilms were reduced by 74 (at 24 h) and 46% (at 20 h), respectively. Furthermore, zymograms showed a differential bacteriolytic activity exerted by the B. bacteriovorus lysates on P. aeruginosa and S. aureus. The dual foraging system against Gram-negative (periplasmic) and Gram-positive (epibiotic) prey could suggest the use of B. bacteriovorus as a "living antibiotic" in CF, even if further studies are required to simulate its in vivo predatory behavior.

Characterization of biofilms in drug-sensitive and drug-resistant strains of Candida albicans.

In this study, we investigated the biofilm formation in strains of Candida albicans susceptible (CO23) or resistant to fluconazole (CO23RFLC) or micafungin (CO23RFK). The effect of drug resistance on biofilm formation was investigated through the cell surface hydrophobicity and the mannan content. Moreover, biofilm formation was evaluated after 24, 48 and 72 hours with crystal violet assay, dry weight, as well as scanning electron microscopy. Our results showed an increase in hydrophobicity, polysaccharides content, metabolic activity and dry weight. Observation of sensitive and resistant strains confirmed the differences in cell morphology. Finally, the expression of genes involved in biofilm formation, such as HWP1 and EFG1, evaluated with relative real-time RT-PCR. Resistant strains proved to up- regulate the expression of HWP1. These results demonstrated the existence of important differences between drug-susceptible and drug-resistant strains biofilm of C. albicans.

Histone deacetylase inhibitors may reduce pathogenicity and virulence in Candida albicans.

Candida albicans is able to establish mucosal and invasive diseases by means of different virulence factors that are frequently regulated by epigenetic mechanisms, including the acetylation-deacetylation of histones and of other regulatory proteins. The focus of our work was on understanding the possible effects of several histone deacetylase inhibitors (HDACi) on the expression of phenotypes that are associated with virulence and pathogenicity in C. albicans, such as adhesion to epithelial cells and the yeast to hypha transition. Some of the HDACi used for experiments caused a 90% reduction in the adherence of C. albicans to human cultured pneumocytes and significantly inhibited serum-induced germination. Inhibition of germination was correlated with a significant reduction in transcription of EFG1. Inhibition appeared less evident when an HDA1-deficient strain was tested. These results suggest that selective and specific HDACi could prove to be a valid approach for selected at-risk patients in the combined treatment of infections caused by C. albicans.

Discovery of uracil-based histone deacetylase inhibitors able to reduce acquired antifungal resistance and trailing growth in Candida albicans.

Among fungal pathogens such as Candida albicans, acquired drug resistance has not been associated with plasmids or other transferable elements, but it is thought to involve primarily mutations and genetic or epigenetic phenomena. This prompted us to test some histone deacetylase inhibitors (HDACi) from our library, in combination with fluconazole, against C. albicans strains in vitro. Among the tested compounds, the two chloro-containing uracil-hydroxamates 1c and 1d showed a strong reduction of the MIC values on Candida strains that show the trailing growth effect. In this assay, 1c,d were more potent than SAHA, a well-known HDAC inhibitor, in reducing the Candida growth. More interestingly, 1c,d as well as SAHA were able to inhibit the fluconazole-induced resistance induction in Candida cultures.