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Background Dental procedures often produce aerosol and splatter which have the potential to transmit pathogens such as SARS‐CoV‐2. The existing literature is limited. Objective(s) To develop a robust, reliable and valid methodology to evaluate distribution and persistence of dental aerosol and splatter, including the evaluation of clinical procedures. Methods Fluorescein was introduced into the irrigation reservoirs of a high‐speed air‐turbine, ultrasonic scaler and 3‐in‐1 spray, and procedures were performed on a mannequin in triplicate. Filter papers were placed in the immediate environment. The impact of dental suction and assistant presence were also evaluated. Samples were analysed using photographic image analysis, and spectrofluorometric analysis. Descriptive statistics were calculated and Pearson’s correlation for comparison of analytic methods. Results All procedures were aerosol and splatter generating. Contamination was highest closest to the source, remaining high to 1–1.5 m. Contamination was detectable at the maximum distance measured (4 m) for high‐speed air‐turbine with maximum relative fluorescence units (RFU) being: 46,091 at 0.5 m, 3,541 at 1.0 m, and 1,695 at 4 m. There was uneven spatial distribution with highest levels of contamination opposite the operator. Very low levels of contamination (≤0.1% of original) were detected at 30 and 60 minutes post procedure. Suction reduced contamination by 67–75% at 0.5–1.5 m. Mannequin and operator were heavily contaminated. The two analytic methods showed good correlation (r=0.930, n=244, p

Summary Streptococcus gordonii is an oral commensal and an early coloniser of dental plaque. In vitro, S. gordonii is conditionally auxotrophic for arginine in monoculture but biosynthesises arginine when coaggregated with Actinomyces oris. Here, we investigated the arginine-responsive regulatory network of S. gordonii and the basis for conditional arginine auxotrophy. ArcB, the catabolic ornithine carbamoyltransferase involved in arginine degradation, was also essential for arginine biosynthesis. However, arcB was poorly expressed following arginine depletion, indicating that arcB levels may limit S. gordonii arginine biosynthesis. Arginine metabolism gene expression was tightly co-ordinated by three ArgR/AhrC family regulators, encoded by argR, ahrC and arcR genes. Microarray analysis revealed that > 450 genes were regulated in response to rapid shifts in arginine concentration, including many genes involved in adhesion and biofilm formation. In a microfluidic salivary biofilm model, low concentrations of arginine promoted S. gordonii growth, whereas high concentrations (> 5 mM arginine) resulted in dramatic reductions in biofilm biomass and changes to biofilm architecture. Collectively, these data indicate that arginine metabolism is tightly regulated in S. gordonii and that arginine is critical for gene regulation, cellular growth and biofilm formation. Manipulating exogenous arginine concentrations may be an attractive approach for oral biofilm control.

Field CA, Gidley MD, Preshaw PM, Jakubovics N. Investigation and quantification of key periodontal pathogens in patients with type 2 diabetes. J Periodont Res 2012; 47: 470–478. © 2012 John Wiley & Sons A/S Background and Objective: Diabetes is a recognized risk factor for periodontitis. There are conflicting data regarding whether healthy diabetic patients or diabetic patients with chronic periodontitis have an altered subgingival microbiota compared with nondiabetic individuals. The aim of the present study was to detect quantitative differences in selected periodontopathogens in the subgingival plaque of diabetic patients using TaqMan quantitative PCR. Material and Methods: Type 2 diabetes mellitus patients with (n = 9) or without chronic periodontal disease (n = 15) were recruited and matched to nondiabetic control subjects (n = 12 periodontally healthy, n = 12 chronic periodontitis). Subgingival plaque samples were collected from deep (> 4 mm probing depth) and shallow sites (≤ 3 mm probing depth) using paper points, and Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Porphyromonas gingivalis were quantified. Results: Forty-eight subjects (69 samples) were recruited. Marked differences were seen in the levels of all three bacterial species, relative to the total bacterial population, according to periodontal health status. Using real-time quantitative PCR, bacterial counts for P. gingivalis were significantly higher in deep pockets of diabetic and nondiabetic subjects compared with periodontally healthy subjects (p 0.05). F. nucleatum was abundant in all groups, with no clear significant differences between groups. P. gingivalis was found in higher quantities in periodontitis than in periodontally healthy subjects (p

The colonization of oral surfaces by micro-organisms occurs in a characteristic sequence of stages, each of which is potentially amenable to external intervention. The process begins with the adhesion of bacteria to host receptors on epithelial cells or in the salivary pellicle covering tooth surfaces. Interbacterial cell-cell binding interactions facilitate the attachment of new species and increase the diversity of the adherent microbial population. Microbial growth in oral biofilms is influenced by the exchange of chemical signals, metabolites and toxic products between neighbouring cells. Bacterial cells on tooth surfaces (dental plaque) produce extracellular polymers such as complex carbohydrates and nucleic acids. These large molecules form a protective matrix that contributes to the development of dental caries and, possibly, to periodontitis. The identification of key microbial factors underlying each step in the formation of oral biofilms will provide new opportunities for preventative or therapeutic measures aimed at controlling oral infectious diseases.

Mature dental biofilms consist of towering microcolonies in which the resident bacterial cells interact with one another and exchange messages in the form of signalling molecules and metabolites. These structures have been compared with the bustling office blocks and apartment buildings of busy cities. Social and communication networks are the lifeblood of large communities, and there is mounting evidence that mutually beneficial interactions between microbial cells are essential to the development of biofilms in the oral cavity. This review discusses the mutualistic partnerships that form between oral bacteria, and the contribution of interspecies communication to the formation of mixed microbial communities.

The ability to control intracellular levels of transition metals such as Mn(2+), Fe(2+) and Zn(2+) is critical for the virulence of many pathogenic bacteria. In this issue of Molecular Microbiology, Rosch et al. describe the first identification of a Mn(2+) efflux system in bacteria, MntE of Streptococcus pneumoniae, and demonstrate that it is required for virulence in an animal model. Disruption of the mntE gene leads to widespread transcriptional changes that are distinct from responses to extracellular Mn(2+). These findings reveal, for the first time, that a bacterial trace metal efflux system plays a role in disease. Thus, MntE represents a new lead for the development of antimicrobials specifically aimed at disrupting microbial metal ion homeostasis.

Interactions between and among bacterial species within a microcommunity occur in an environment that is distinct from the surrounding space. Interactions among species within one community are likely to be distinct from those within a different community. The context of mixed-species communities discussed in this chapter is primarily the human oral cavity, and the chapter focuses on the concept of distance-critical communication and its role in mediating commensalism as well as pathogenesis. The exhibition of extensive coaggregation partnerships by oral plaque bacteria suggests that distance-critical communication typically occurs within the tightly packed microcommunities known to characterize human dental plaque. Coaggregates formed among Methanothermobacter thermautotrophicus, butyrate-oxidizing Syntrophothermus lipocalidus, and acetate-oxidizing Thermacetogenium phaeum, indicating that coaggregations are relevant to interspecies hydrogen transfer among several syntrophic methanogenic consortia. Coaggregation and adhesion to host cells are primary characteristics of all oral fusobacteria. Fusobacteria coaggregate with all early and late oral colonizers. In place of producing strong toxins and enzymes, fusobacteria enhance their virulence functions through their ability to interact with other cell types. Many oral streptococci produce IgA1 proteases, which assist the community in evading the principal mediator of adaptive immunity.

The antigen I/II (AgI/II) family polypeptides, ranging from 1310 to 1653 amino acid (aa) residues, are cell wall anchored adhesins expressed by most indigenous species of oral streptococci. The polypeptides interact with a wide range of host molecules, in particular salivary agglutinin glycoprotein (SAG or gp340), and with ligands on other oral bacteria. To determine the receptor recognition properties of six different AgI/II family polypeptides from strains of Streptococcus gordonii, Streptococcus intermedius and Streptococcus mutans, the genes were cloned and expressed on the surface of the surrogate host Lactococcus lactis. The S. gordonii SspA and SspB polypeptides mediated higher binding levels of L. lactis cells to surface immobilized gp340 than did S. intermedius Pas protein, or S. mutans SpaP or PAc proteins. However, the AgI/II proteins were all similar in their abilities to mediate aggregation of lactococci by fluid phase gp340. The SpaP(I) polypeptide from S. mutans Ingbritt, which was C-terminally truncated by approximately 400 aa residues, did not bind gp340. Lactococci expressing AgI/II proteins, including SpaP(I), were aggregated by a synthetic 16 aa residue peptide SRCRP2 derived from the aa repeat block sequences within gp340. In coaggregation assays, SspB from S. gordonii was unique in mediating coaggregation with only group A and group E strains of Actinomyces naeslundii. All the other AgI/II polypeptides mediated coaggregation with group C and group D strains of A. naeslundii. Analysis of chimeric protein constructs revealed that coaggregation specificity was determined by sequences within the N-terminal half of AgI/II protein. A synthetic peptide (20 aa residues), which defines a putative adhesion epitope within the C-terminal region of polypeptide, inhibited AgI/II-mediated aggregation by gp340 but did not affect coaggregation with A. naeslundii. These results suggest that different mechanisms operate in interactions of AgI/II family polypeptides with native gp340, gp340 SRCR domain peptide, and A. naeslundii. Specificity of these interactions appears to be determined by discontinuous but interacting regions of the polypeptides, thus providing flexibility in receptor recognition for streptococcal colonization of the human host.

Microbial interactions with host molecules, and programmed responses to host environmental stimuli, are critical for colonization and initiation of pathogenesis. Bacteria of the genus Streptococcus are primary colonizers of the human mouth. They express multiple cell-surface adhesins that bind salivary components and other oral bacteria and enable the development of polymicrobial biofilms associated with tooth decay and periodontal disease. However, the mechanisms by which streptococci invade dentine to infect the tooth pulp and periapical tissues are poorly understood. Here we show that production of the antigen I/II (AgI/II) family polypeptide adhesin and invasin SspA in Streptococcus gordonii is specifically upregulated in response to a collagen type I signal, minimally the tri-peptide Gly-Pro-Xaa (where Xaa is hydroxyproline or alanine). Increased AgI/II polypeptide expression promotes bacterial adhesion and extended growth of streptococcal cell chains along collagen type I fibrils that are characteristically found within dentinal tubules. These observations define a new model of host matrix signal-induced tissue penetration by bacteria and open the way for novel therapy opportunities for oral invasive diseases.

The acquisition of transition metal ions by pathogenic bacteria is crucial to their growth and survival within the human host, however, the mechanisms of metal ion homeostasis in streptococci are unknown. The scaCBA operon in the human oral bacterium Streptococcus gordonii encodes the components of an ABC-type transporter for manganese (Mn2+). Production of substrate-binding lipoprotein ScaA was increased approximately fivefold in cells cultured in low Mn2+ medium (0.1 microM Mn2+), but not in iron (Fe2+/Fe3+)-limited medium, and was enhanced in the presence of human saliva or serum. mRNA analysis revealed that under low Mn2+ conditions, levels of scaCBA transcript (2.6 kb) were increased20-fold. The Mn2+-responsive transcriptional regulator of the sca operon was purified and characterized as a 215-amino-acid residue polypeptide, designated ScaR, with 26% identity to the Corynebacterium diphtheriae diphtheria toxin repressor (DtxR). Inactivation of scaR in S. gordonii DL1 (Challis) resulted in constitutive derepression of sca operon transcription. Expression of tpx, located immediately downstream of scaA and encoding a putative thiol peroxidase, was not subject to ScaR regulation. Purified ScaR protein bound to the scaC promoter region in vitro in the presence of Mn2+ (Kd approximately 80 nM) and, to a lesser extent, in the presence of Ni2+ or Zn2+. The metalloregulator protein binding region was localized by DNA protection analysis to a 46 bp sequence encompassing the -35 and -10 promoter signatures. This sequence was well conserved within the promoters of corresponding virulence-related permease operons in other streptococci. The results identify a new Mn2+-sensing regulator of Mn2+ transport in streptococci, important for Mn2+ homeostasis during infection of the human host.

Objective: The collection of biofilms on tracheo-esophageal speaking valves (TESV) detrimentally shortens their lifespan. A novel nontoxic enzyme (NucB) was discovered in the University of Newcastle and proved effective for dispersing marine biofilms. We propose to ascertain the effect of NucB on disrupting and removing mixed organism biofilms on TESVs.Method: We designed a case control study of ex-vivo TESVs, with build up of biofilms. Each TESV was divided into 2 equal parts. One half was treated with NucB and the other half with a nonactive control solution. Outcomes are measured by microscopy and culture of biofilm organisms.Results: Ten TESVs were collected for our analysis in the microbiology laboratory. The more efficient our agent is in dispersing biofilms, the more organisms are released that can be cultured. With aerobic growth mediums, more organisms were cultured in 9 samples (90%, 95% CI 57-99%). With the use of growth mediums favouring fungal organisms, more organisms were cultured in 6 samp...

Objective: 1) Characterize the microstructure of paranasal sinus biofilms. 2) Characterize the microbial composition of sinus biofilms. 3) Develop techniques to treat sinus infections by breaking up sinus biofilms using surfactants and a novel, marine derived nuclease.Method: Sinus biofilms were collected intraoperatively. Microbial composition was investigated by culturing and 16S rDNA sequencing, and the biofilm structure was analyzed by electron microscopy. Duplicate samples were treated with or without the marine nuclease (NucB) at 370C. The effect of NucB was measured by mass reduction before and after treatment.Results: 16SrDNA analysis demonstrated populations of Staphylococcus aureus, S epidermis, and Kocuria rhizophila. Anaerobic bacteria were recovered in limited numbers, and few fungal strains were observed. Transmission electron microscopy of characteristic tenacious biofilms showed an unusual layered structure. Samples consisted of endogenous mucin-rich layers with occasional pockets of bacte...

Microbial interactions involve sensing and responding to chemical signals or cues that are released from cells. In the early stages of plaque accumulation on freshly cleaned teeth, most interactions occur between the initial colonizers such as Streptococcus spp., Actinomyces spp., and Veillonella spp. However, mature dental plaque biofilms may contain up to 200 different phylotypes of bacteria, resulting potentially in almost 20,000 different pairwise interactions. This chapter focuses on just one of these interactions: that between two initial colonizers of oral biofilms, Streptococcus gordonii and Actinomyces oris. Interbacterial binding between oral bacteria has been investigated extensively in vitro using coaggregation assays. In these experiments, pure cultures of genetically distinct bacteria are mixed in test tubes and interactions are scored based on the extent of clumping (coaggregation). Coaggregation is used to model biofilm communities containing S. gordonii and A. oris and investigate gene regulation in mixed-species cultures. A powerful application of postgenomic technologies is the analysis of gene expression using DNA microarrays. The first genome-level analysis of S. gordonii-A. oris interactions employed microarrays to identify genes in S. gordonii that were regulated in response to coaggregation with A. oris. This study demonstrated that S. gordonii specifically activates a set of genes in response to cell-cell contact (coaggregation) with A. oris. In theory, microarrays and other genome-based expression technologies can be used to investigate cell-cell contact-induced gene regulation in any bacterial species for which the genome sequence is known.