Design and Initial Evaluation of a Novel Oral Hygiene Technology for a Special Needs Population: A New Way to Clean.

9.4 million People have swallowing problems in the US. In special needs populations, routine oral hygiene procedures such as tooth brushing can result in aspiration of microbial laden fluids leading to a significant systemic challenge. Aspiration may lead to pneumonia in susceptible populations. These circumstances indicate the need for innovative approaches to oral hygiene for special needs, convalescent, the elderly populations, and young children learning to brush who can ingest excess fluoride which causes mottled enamel. Methods include describing some of the design considerations of the new prototype fabrication and microbiological evaluation of this new device, as well a comparison study of the versions 2 and 3 of the oral care device. Results concluded that version 3.0 regarding patient ease of use was better in comparison to version 2, which was the major difference, and 90% in both groups said they would recommend the new toothbrush. In the microbiological evaluation no growth was seen on any plates containing samples from either the experimental or the control after 48 h of incubation.

Alternative Antibiotics in Dentistry: Antimicrobial Peptides.

The rise of antibiotic resistant bacteria due to overuse and misuse of antibiotics in medicine and dentistry is a growing concern. New approaches are needed to combat antibiotic resistant (AR) bacterial infections. There are a number of methods available and in development to address AR infections. Dentists conventionally use chemicals such as chlorohexidine and calcium hydroxide to kill oral bacteria, with many groups recently developing more biocompatible antimicrobial peptides (AMPs) for use in the oral cavity. AMPs are promising candidates in the treatment of (oral) infections. Also known as host defense peptides, AMPs have been isolated from animals across all kingdoms of life and play an integral role in the innate immunity of both prokaryotic and eukaryotic organisms by responding to pathogens. Despite progress over the last four decades, there are only a few AMPs approved for clinical use. This review summarizes an Introduction to Oral Microbiome and Oral Infections, Traditional Antibiotics and Alternatives & Antimicrobial Peptides. There is a focus on cationic AMP characteristics and mechanisms of actions, and an overview of animal-derived natural and synthetic AMPs, as well as observed microbial resistance.

Complete Genome Sequence of Aggregatibacter actinomycetemcomitans Strain CU1000N.

Here, we report the complete genome sequence of Aggregatibacter actinomycetemcomitans strain CU1000N. This rough strain is used extensively as a model organism to characterize localized aggressive periodontitis pathogenesis, the basic biology and oral cavity colonization of A. actinomycetemcomitans, and its interactions with other members of the oral microbiome.

Differential Effects of Antiseptic Mouth Rinses on SARS-CoV-2 Infectivity In Vitro.

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) is detectable in saliva from asymptomatic individuals, suggesting a potential benefit from the use of mouth rinses to suppress viral load and reduce virus spread. Published studies on the reduction of SARS-CoV-2-induced cytotoxic effects by mouth rinses do not exclude antiseptic mouth rinse-associated cytotoxicity. Here, we determined the effect of commercially available mouth rinses and antiseptic povidone-iodine on the infectivity of replication-competent SARS-CoV-2 viruses and of pseudotyped SARS-CoV-2 viruses. We first determined the effect of mouth rinses on cell viability to ensure that antiviral activity was not a consequence of mouth rinse-induced cytotoxicity. Colgate Peroxyl (hydrogen peroxide) exhibited the most cytotoxicity, followed by povidone-iodine, chlorhexidine gluconate (CHG), and Listerine (essential oils and alcohol). The potent antiviral activities of Colgate Peroxyl mouth rinse and povidone-iodine were the consequence of rinse-mediated cellular damage when the products were present during infection. The potency of CHG was greater when the product was not washed off after virus attachment, suggesting that the prolonged effect of mouth rinses on cells impacts the antiviral outcome. To minimalize mouth rinse-associated cytotoxicity, mouth rinse was largely removed from treated viruses by centrifugation prior to infection of cells. A 5% (v/v) dilution of Colgate Peroxyl or povidone-iodine completely blocked viral infectivity. A similar 5% (v/v) dilution of Listerine or CHG had a moderate suppressive effect on the virus, but a 50% (v/v) dilution of Listerine or CHG blocked viral infectivity completely. Mouth rinses inactivated the virus without prolonged incubation. The new infectivity assay, with limited impacts of mouth rinse-associated cytotoxicity, showed the differential effects of mouth rinses on SARS-CoV-2 infection. Our results indicate that mouth rinses can significantly reduce virus infectivity, suggesting a potential benefit for reducing SARS-CoV-2 spread.

Dysbiosis From a Microbial and Host Perspective Relative to Oral Health and Disease.

The significance of microbiology and immunology with regard to caries and periodontal disease gained substantial clinical or research consideration in the mid 1960's. This enhanced emphasis related to several simple but elegant experiments illustrating the relevance of bacteria to oral infections. Since that point, the understanding of oral diseases has become increasingly sophisticated and many of the original hypotheses related to disease causality have either been abandoned or amplified. The COVID pandemic has reminded us of the importance of history relative to infectious diseases and in the words of Churchill "those who fail to learn from history are condemned to repeat it." This review is designed to present an overview of broad general directions of research over the last 60 years in oral microbiology and immunology, reviewing significant contributions, indicating emerging foci of interest, and proposing future directions based on technical advances and new understandings. Our goal is to review this rich history (standard microbiology and immunology) and point to potential directions in the future (omics) that can lead to a better understanding of disease. Over the years, research scientists have moved from a position of downplaying the role of bacteria in oral disease to one implicating bacteria as true pathogens that cause disease. More recently it has been proposed that bacteria form the ecological first line of defense against "foreign" invaders and also serve to train the immune system as an acquired host defensive stimulus. While early immunological research was focused on immunological exposure as a modulator of disease, the "hygiene hypothesis," and now the "old friends hypothesis" suggest that the immune response could be trained by bacteria for long-term health. Advanced "omics" technologies are currently being used to address changes that occur in the host and the microbiome in oral disease. The "omics" methodologies have shaped the detection of quantifiable biomarkers to define human physiology and pathologies. In summary, this review will emphasize the role that commensals and pathobionts play in their interaction with the immune status of the host, with a prediction that current "omic" technologies will allow researchers to better understand disease in the future.

A Mini Review: The Potential Biomarkers for Non-invasive Diagnosis of Pulpal Inflammation.

For assessing the adequacy of vital pulp therapy for an inflamed pulp, the use of non-invasive diagnostic tools is necessary to avoid further damage to the teeth. Detection of biomarkers that are indicative of the inflammatory status in pulp can be a promising tool for this purpose. These biomarkers need to be reliably correlated with pulpal inflammation and to be easily detected without pulp exposure. This mini-review article aims to review biomarkers that are present in gingival crevicular fluid (GCF) in inflamed pulp conditions. Several studies have reported the availability of various biomarkers including cytokines, proteases, elastase, neuropeptides, and growth factors. Non-invasive pulpal diagnostic methods will be useful as well to determine reversibility, irreversibility, or necrosis of inflamed pulp. These types of molecular diagnoses via analyzing the proteome have revolutionized the medical field, and are one of the most promising empirical methodologies that a clinician can utilize for the proactive identification of pulpal disease.

Differential effects of antiseptic mouth rinses on SARS-CoV-2 infectivity in vitro.

SARS-CoV-2 is detectable in saliva from asymptomatic individuals, suggesting a potential benefit from the use of mouth rinses to suppress viral load and reduce virus spread. Published studies on reduction of SARS-CoV-2-induced cytotoxic effects by antiseptics do not exclude antiseptic-associated cytotoxicity. Here, we determined the effect of commercially available mouth rinses and antiseptic povidone-iodine on the infectivity of SARS-CoV-2 virus and of a non-pathogenic, recombinant, SARS-CoV-2 infection vector (pseudotyped SARS-CoV-2 virus). We first determined the effect of mouth rinses on cell viability to ensure that antiviral activity was not a consequence of mouth rinse-induced cytotoxicity. Colgate Peroxyl (hydrogen peroxide) exhibited the most cytotoxicity, followed by povidone-iodine, chlorhexidine gluconate (CHG), and Listerine (essential oils and alcohol). Potent anti-viral activities of povidone iodine and Colgate peroxyl mouth rinses was the consequence of rinse-mediated cellular damage. The potency of CHG was greater when the product was not washed off after virus attachment, suggesting that the prolonged effect of mouth rinses on cells impacts anti-viral activity. To minimalize mouth rinse-associated cytotoxicity, mouth rinse was largely removed from treated-viruses by centrifugation prior to infection of cells. A 5% (v/v) dilution of Colgate Peroxyl or povidone-iodine completely blocked viral infectivity. A similar 5% (v/v) dilution of Listerine or CHG had a moderate suppressive effect on the virus, but a 50% (v/v) dilution of Listerine or CHG blocked viral infectivity completely. Prolonged incubation of virus with mouth rinses was not required for viral inactivation. Our results indicate that mouth rinses can significantly reduce virus infectivity, suggesting a potential benefit for reducing SARS-CoV-2 spread.

Two Nearly Complete Nosocomial Pathogen Genome Sequences Reconstructed from Early-Middle 20th-Century Dental Calculus.

Acinetobacter baumannii and Stenotrophomonas maltophilia genomes were reconstructed from early-middle 20th-century human skeletal remains, maintained in natural history museums, using a metagenomic binning approach.

Draft Genome Sequence of Porphyromonas gingivalis Strain 381 Okayama (381OKJP) Stock Culture.

We report the draft genome sequence of Porphyromonas gingivalis strain 381 Okayama (381OKJP). The strain, obtained from the Socransky collection, has been used for experimentation since 1987. This sequence allows for comparisons to other sequenced 381 strains to observe acquisition of mutations and genome rearrangements in a commonly used laboratory strain.

Utilization of Variant and Fusion Proteins To Functionally Map the Aggregatibacter actinomycetemcomitans Trimeric Autotransporter Protein ApiA.

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans is a causative agent of localized aggressive periodontitis. Critical to its infection process is the first and essential step of attachment, which is related to the coordinated functions of surface components comprised of proteins and extracellular polysaccharides. One such protein is the outer membrane trimeric autotransporter protein ApiA, a versatile virulence factor with numerous functions, including cell binding, invasion, serum resistance, autoaggregation, and induction of cytokine release. Here we report on the use of Escherichia coli strains expressing protein variants to define the separate functions ascribed to the N terminus and those related to the C terminus. Importantly, a hybrid protein that comprised the N terminus of trimeric ApiA and the ß-barrel domain of monomeric autotransporter Aae was constructed, which allowed the expression of a monomer surface-exposed domain of ApiA. Functional and phenotypic analyses demonstrated that the C terminus of ApiA forms an independent domain that is crucial for general stability and trimer formation, which appears to be associated with autoaggregation, biofilm formation, and surface expression. Importantly, the results show that the monomeric form of the N-terminal passenger domain of ApiA, while surface exposed, is sufficient for binding to buccal epithelial cells; however, it is not sufficient to allow aggregation and biofilm formation, strengthening the importance of the role of trimerization in these phenotypes.

Draft Genome Sequences of Aggregatibacter actinomycetemcomitans Strains 310a and 310b.

We report the draft genome sequences of Aggregatibacter actinomycetemcomitans strains 310a (310-TR) and 310b (310-OS). Strain 310a is a clinical isolate with a rough phenotype. Strain 310b is a laboratory-adapted isolate derived from the passage of 310a and displays a smooth phenotype.

Complete Genome Sequence of Aggregatibacter actinomycetemcomitans Strain IDH781.

We report here the complete genomic sequence and methylome of Aggregatibacter actinomycetemcomitans strain IDH781. This rough strain is used extensively as a model organism to characterize localized aggressive periodontitis pathogenesis, the basic biology and oral cavity colonization of A. actinomycetemcomitans, and its interactions with other members of the oral microbiome.

Porphyromonas gingivalis: keeping the pathos out of the biont.

The primary goal of the human microbiome initiative has been to increase our understanding of the structure and function of our indigenous microbiota and their effects on human health and predisposition to disease. Because of its clinical importance and accessibility for in vivo study, the oral biofilm is one of the best-understood microbial communities associated with the human body. Studies have shown that there is a succession of select microbial interactions that directs the maturation of a defined community structure, generating the formation of dental plaque. Although the initiating factors that lead to disease development are not clearly defined, in many individuals there is a fundamental shift from a health-associated biofilm community to one that is pathogenic in nature and a central player in the pathogenic potential of this community is the presence of Porphyromonas gingivalis. This anaerobic bacterium is a natural member of the oral microbiome, yet it can become highly destructive (termed pathobiont) and proliferate to high cell numbers in periodontal lesions, which is attributed to its arsenal of specialized virulence factors. Hence, this organism is regarded as a primary etiologic agent of periodontal disease progression. In this review, we summarize some of the latest information regarding what is known about its role in periodontitis, including pathogenic potential as well as ecological and nutritional parameters that may shift this commensal to a virulent state. We also discuss parallels between the development of pathogenic biofilms and the human cellular communities that lead to cancer, specifically we frame our viewpoint in the context of 'wounds that fail to heal'.

The nucleoid-associated protein HUß affects global gene expression in Porphyromonas gingivalis.

HU is a non-sequence-specific DNA-binding protein and one of the most abundant nucleoid-associated proteins in the bacterial cell. Like Escherichia coli, the genome of Porphyromonas gingivalis is predicted to encode both the HUα (PG1258) and the HUß (PG0121) subunit. We have previously reported that PG0121 encodes a non-specific DNA-binding protein and that PG0121 is co-transcribed with the K-antigen capsule synthesis operon. We also reported that deletion of PG0121 resulted in downregulation of capsule operon expression and produced a P. gingivalis strain that is phenotypically deficient in surface polysaccharide production. Here, we show through complementation experiments in an E. coli MG1655 hupAB double mutant strain that PG0121 encodes a functional HU homologue. Microarray and quantitative RT-PCR analysis were used to further investigate global transcriptional regulation by HUß using comparative expression profiling of the PG0121 (HUß) mutant strain to the parent strain, W83. Our analysis determined that expression of genes encoding proteins involved in a variety of biological functions, including iron acquisition, cell division and translation, as well as a number of predicted nucleoid associated proteins were altered in the PG0121 mutant. Phenotypic and quantitative real-time-PCR (qRT-PCR) analyses determined that under iron-limiting growth conditions, cell division and viability were defective in the PG0121 mutant. Collectively, our studies show that PG0121 does indeed encode a functional HU homologue, and HUß has global regulatory functions in P. gingivalis; it affects not only production of capsular polysaccharides but also expression of genes involved in basic functions, such as cell wall synthesis, cell division and iron uptake.

Arginine deiminase inhibits Porphyromonas gingivalis surface attachment.

The oral cavity is host to a complex microbial community whose maintenance depends on an array of cell-to-cell interactions and communication networks, with little known regarding the nature of the signals or mechanisms by which they are sensed and transmitted. Determining the signals that control attachment, biofilm development and outgrowth of oral pathogens is fundamental to understanding pathogenic biofilm development. We have previously identified a secreted arginine deiminase (ADI) produced by Streptococcus intermedius that inhibited biofilm development of the commensal pathogen Porphyromonas gingivalis through downregulation of genes encoding the major (fimA) and minor (mfa1) fimbriae, both of which are required for proper biofilm development. Here we report that this inhibitory effect is dependent on enzymic activity. We have successfully cloned, expressed and defined the conditions to ensure that ADI from S. intermedius is enzymically active. Along with the cloning of the wild-type allele, we have created a catalytic mutant (ADIC399S), in which the resulting protein is not able to catalyse the hydrolysis of l-arginine to l-citrulline. P. gingivalis is insensitive to the ADIC399S catalytic mutant, demonstrating that enzymic activity is required for the effects of ADI on biofilm formation. Biofilm formation is absent under l-arginine-deplete conditions, and can be recovered by the addition of the amino acid. Taken together, the results indicate that arginine is an important signal that directs biofilm formation by this anaerobe. Based on our findings, we postulate that ADI functions to reduce arginine levels and, by a yet to be identified mechanism, signals P. gingivalis to alter biofilm development. ADI release from the streptococcal cell and its cross-genera effects are important findings in understanding the nature of inter-bacterial signalling and biofilm-mediated diseases of the oral cavity.

HU protein affects transcription of surface polysaccharide synthesis genes in Porphyromonas gingivalis.

K-antigen capsule synthesis is an important virulence determinant of the oral anaerobe Porphyromonas gingivalis. We previously reported that the locus required for synthesis of this surface polysaccharide in strain W83 (TIGR identification PG0106 to PG0120) is transcribed as a large (∼16.7-kb) polycistronic message. Through sequence analysis, we have now identified a 77-bp inverted repeat located upstream (206 bp) of the start codon of PG0106 that is capable of forming a large hairpin structure. Further sequence analysis just upstream and downstream of the capsule synthesis genes revealed the presence of two genes oriented in the same direction as the operon that are predicted to encode DNA binding proteins: PG0104, which is highly similar (57%) to DNA topoisomerase III, and PG0121, which has high similarity (72%) to DNA binding protein HU (ß-subunit). In this report, we show that these two genes, as well as the 77-bp inverted repeat region, are cotranscribed with the capsule synthesis genes, resulting in a large transcript that is ∼19.4 kb (based on annotation). We also show that a PG0121 recombinant protein is a nonspecific DNA binding protein with strong affinity to the hairpin structure, in vitro, and that transcript levels of the capsule synthesis genes are downregulated in a PG0121 deletion mutant. Furthermore, we show that this decrease in transcript levels corresponds to a decrease in the amount of polysaccharide produced. Interestingly, expression analysis of another polysaccharide synthesis locus (PG1136 to PG1143) encoding genes involved in synthesis of a surface-associated phosphorylated branched mannan (APS) indicated that this locus is also downregulated in the PG0121 mutant. Altogether our data indicate that HU protein modulates expression of surface polysaccharides in P. gingivalis strain W83.

A streptococcal effector protein that inhibits Porphyromonas gingivalis biofilm development.

Dental plaque formation is a developmental process involving cooperation and competition within a diverse microbial community, approximately 70 % of which is composed of an array of streptococci during the early stages of supragingival plaque formation. In this study, 79 cell-free culture supernatants from a variety of oral streptococci were screened to identify extracellular compounds that inhibit biofilm formation by the oral anaerobe Porphyromonas gingivalis strain 381. The majority of the streptococcal supernatants (61 isolates) resulted in lysis of P. gingivalis cells, and some (17 isolates) had no effect on cell viability, growth or biofilm formation. One strain, however, produced a supernatant that abolished biofilm formation without affecting growth rate. Analysis of this activity led to the discovery that a 48 kDa protein was responsible for the inhibition. Protein sequence identification and enzyme activity assays identified the effector protein as an arginine deiminase. To identify the mechanism(s) by which this protein inhibits biofilm formation, we began by examining the expression levels of genes encoding fimbrial subunits; surface structures known to be involved in biofilm development. Quantitative RT-PCR analysis revealed that exposure of P. gingivalis cells to this protein for 1 h resulted in the downregulation of genes encoding proteins that are the major subunits of two distinct types of thin, single-stranded fimbriae (fimA and mfa1). Furthermore, this downregulation occurred in the absence of arginine deiminase enzymic activity. Hence, our data indicate that P. gingivalis can sense this extracellular protein, produced by an oral streptococcus (Streptococcus intermedius), and respond by downregulating expression of cell-surface appendages required for attachment and biofilm development.

Candida albicans-produced farnesol stimulates Pseudomonas quinolone signal production in LasR-defective Pseudomonas aeruginosa strains.

Candida albicans has been previously shown to stimulate the production of Pseudomonas aeruginosa phenazine toxins in dual-species colony biofilms. Here, we report that P. aeruginosa lasR mutants, which lack the master quorum sensing system regulator, regain the ability to produce quorum-sensing-regulated phenazines when cultured with C. albicans. Farnesol, a signalling molecule produced by C. albicans, was sufficient to stimulate phenazine production in LasR(-) laboratory strains and clinical isolates. P. aeruginosa ΔlasR mutants are defective in production of the Pseudomonas quinolone signal (PQS) due to their inability to properly induce pqsH, which encodes the enzyme necessary for the last step in PQS biosynthesis. We show that expression of pqsH in a ΔlasR strain was sufficient to restore PQS production, and that farnesol restored pqsH expression in ΔlasR mutants. The farnesol-mediated increase in pqsH required RhlR, a transcriptional regulator downstream of LasR, and farnesol led to higher levels of N-butyryl-homoserine lactone, the small molecule activator of RhlR. Farnesol promotes the production of reactive oxygen species (ROS) in a variety of species. Because the antioxidant N-acetylcysteine suppressed farnesol-induced RhlR activity in LasR(-) strains, and hydrogen peroxide was sufficient to restore PQS production in las mutants, we propose that ROS are responsible for the activation of downstream portions of this quorum sensing pathway. LasR mutants frequently arise in the lungs of patients chronically infected with P. aeruginosa. The finding that C. albicans, farnesol or ROS stimulate virulence factor production in lasR strains provides new insight into the virulence potential of these strains.

Borrelia burgdorferi BBB07 interaction with integrin alpha3beta1 stimulates production of pro-inflammatory mediators in primary human chondrocytes.

Borrelia burgdorferi, the causative agent of Lyme disease, activates multiple signalling pathways leading to induction of pro-inflammatory mediators at sites of inflammation. Binding of B. burgdorferi to integrin alpha(3)beta(1) on human chondrocytes activates signalling leading to release of several pro-inflammatory mediators, but the B. burgdorferi protein that binds integrin alpha(3)beta(1) and elicits this response has remained unknown. A search of the B. burgdorferi genome for a canonical integrin binding motif, the RGD (Arg-Gly-Asp) tripeptide, revealed several candidate ligands for integrins. In this study we show that one of these candidates, BBB07, binds to integrin alpha(3)beta(1) and inhibits attachment of intact B. burgdorferi to the same integrin. BBB07 is expressed during murine infection as demonstrated by recognition by infected mouse sera. Recombinant purified BBB07 induces pro-inflammatory mediators in primary human chondrocyte cells by interaction with integrin alpha(3)beta(1). This interaction is specific, as P66, another integrin ligand of B. burgdorferi, does not activate signalling through alpha(3)beta(1). In summary, we have identified a B. burgdorferi protein, BBB07, that interacts with integrin alpha(3)beta(1) and stimulates production of pro-inflammatory mediators in primary human chondrocyte cells.

Farnesol, a common sesquiterpene, inhibits PQS production in Pseudomonas aeruginosa.

Farnesol is a sesquiterpene produced by many organisms, including the fungus Candida albicans. Here, we report that the addition of farnesol to cultures of Pseudomonas aeruginosa, an opportunistic human bacterial pathogen, leads to decreased production of the Pseudomonas quinolone signal (PQS) and the PQS-controlled virulence factor, pyocyanin. Within 15 min of farnesol addition, decreased transcript levels of pqsA, the first gene in the PQS biosynthetic operon, were observed. Transcript levels of pqsR (mvfR), which encodes the transcription factor that positively regulates pqsA, were unaffected. An Escherichia coli strain producing PqsR and containing the pqsA promoter fused to lacZ similarly showed that farnesol inhibited PQS-stimulated transcription. Electrophoretic mobility shift assays showed that, like PQS, farnesol stimulated PqsR binding to the pqsA promoter at a previously characterized LysR binding site, suggesting that farnesol promoted a non-productive interaction between PqsR and the pqsA promoter. Growth with C. albicans leads to decreased production of PQS and pyocyanin by P. aeruginosa, suggesting that the amount of farnesol produced by the fungus is sufficient to impact P. aeruginosa PQS signalling. Related isoprenoid compounds, but not other long-chain alcohols, also inhibited PQS production at micromolar concen-trations, suggesting that related compounds may participate in interspecies interactions with P. aeruginosa.