Manipulating the diseased oral microbiome: the power of probiotics and prebiotics.

Dental caries and periodontal disease are amongst the most prevalent global disorders. Their aetiology is rooted in microbial activity within the oral cavity, through the generation of detrimental metabolites and the instigation of potentially adverse host immune responses. Due to the increasing threat of antimicrobial resistance, alternative approaches to readdress the balance are necessary. Advances in sequencing technologies have established relationships between disease and oral dysbiosis, and commercial enterprises seek to identify probiotic and prebiotic formulations to tackle preventable oral disorders through colonisation with, or promotion of, beneficial microbes. It is the metabolic characteristics and immunomodulatory capabilities of resident species which underlie health status. Research emphasis on the metabolic environment of the oral cavity has elucidated relationships between commensal and pathogenic organisms, for example, the sequential metabolism of fermentable carbohydrates deemed central to acid production in cariogenicity. Therefore, a focus on the preservation of an ecological homeostasis in the oral environment may be the most appropriate approach to health conservation. In this review we discuss an ecological approach to the maintenance of a healthy oral environment and debate the potential use of probiotic and prebiotic supplementation, specifically targeted at sustaining oral niches to preserve the delicately balanced microbiome.

The balance of oral homeostasis requires delicate adjustments to prevent and counteract disease.The metabolic activities of the complete microbiome, not only key pathogens or commensals, are important to the maintenance of health.Metabolomics techniques can be valuable in identifying environmental niches deficient in disease that can act as targets for probiotic and prebiotic treatments.

Increased Handpiece Speeds without Air Coolant: Aerosols and Thermal Impact.

This study assessed the impact of increased speed of high-speed contra-angle handpieces (HSCAHs) on the aerosolization of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surrogate virus and any concomitant thermal impact on dental pulp. A bacteriophage phantom-head model was used for bioaerosol detection. Crown preparations were performed with an NSK Z95L Contra-Angle 1:5 (HSCAH-A) and a Bien Air Contra-Angle 1:5 Nova Micro Series (HSCAH-B) at speeds of 60,000, 100,000, and 200,000 revolutions per minute (rpm), with no air coolant. Bioaerosol dispersal was measured with Φ6-bacteriophage settle plates, air sampling, and particle counters. Heating of the internal walls of the pulp chambers during crown preparation was assessed with an infrared camera with HSCAH-A and HSCAH-B at 200,000 rpm (water flows ≈15 mL min-1 and ≈30 mL min-1) and an air-turbine control (≈23.5 mL min-1) and correlated with remaining tissue thickness measurements. Minimal bacteriophage was detected on settle or air samples with no notable differences observed between handpieces or speeds (P > 0.05). At all speeds, maximum settled aerosol and average air detection was 1.00 plaque-forming units (pfu) and 0.08 pfu/m3, respectively. Irrespective of water flow rate or handpiece, both maximum temperature (41.5°C) and temperature difference (5.5°C) thresholds for pulpal health were exceeded more frequently with reduced tissue thickness. Moderate and strong negative correlations were observed based on Pearson's correlation coefficient, between remaining dentine thickness and either differential (r = -0.588) or maximum temperature (r = -0.629) measurements, respectively. Overall, HSCAH-B generated more thermal energy and exceeded more temperature thresholds compared to HSCAH-A. HSCAHs without air coolant operating at speeds of 200,000 rpm did not increase bioaerosolization in the dental surgery. Thermal risk is variable, dependent on handpiece design and remaining dentine thickness.

Dental Mitigation Strategies to Reduce Aerosolization of SARS-CoV-2.

Limiting infection transmission is central to the safety of all in dentistry, particularly during the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Aerosol-generating procedures (AGPs) are crucial to the practice of dentistry; it is imperative to understand the inherent risks of viral dispersion associated with AGPs and the efficacy of available mitigation strategies. In a dental surgery setting, crown preparation and root canal access procedures were performed with an air turbine or high-speed contra-angle handpiece (HSCAH), with mitigation via rubber dam or high-volume aspiration and a no-mitigation control. A phantom head was used with a 1.5-mL min-1 flow of artificial saliva infected with Φ6-bacteriophage (a surrogate virus for SARS-CoV-2) at ~108 plaque-forming units mL-1, reflecting the upper limits of reported salivary SARS-CoV-2 levels. Bioaerosol dispersal was measured using agar settle plates lawned with the Φ6-bacteriophage host, Pseudomonas syringae. Viral air concentrations were assessed using MicroBio MB2 air sampling and particle quantities using Kanomax 3889 GEOα counters. Compared to an air turbine, the HSCAH reduced settled bioaerosols by 99.72%, 100.00%, and 100.00% for no mitigation, aspiration, and rubber dam, respectively. Bacteriophage concentrations in the air were reduced by 99.98%, 100.00%, and 100.00% with the same mitigations. Use of the HSCAH with high-volume aspiration resulted in no detectable bacteriophage, both on nonsplatter settle plates and in air samples taken 6 to 10 min postprocedure. To our knowledge, this study is the first to report the aerosolization in a dental clinic of active virus as a marker for risk determination. While this model represents a worst-case scenario for possible SARS-CoV-2 dispersal, these data showed that the use of HSCAHs can vastly reduce the risk of viral aerosolization and therefore remove the need for clinic fallow time. Furthermore, our findings indicate that the use of particle analysis alone cannot provide sufficient insight to understand bioaerosol infection risk.

Investigating the transient and persistent effects of heat on Clostridium difficile spores.

Purpose. Clostridium difficile spores are extremely resilient to high temperatures. Sublethal temperatures are associated with the 'reactivation' of dormant spores, and are utilized to maximize C. difficile spore recovery. Spore eradication is of vital importance to the food industry. The current study seeks to elucidate the transient and persisting effects of heating C. difficile spores at various temperatures.Methods. Spores of five C. difficile strains of different ribotypes (001, 015, 020, 027 and 078) were heated at 50, 60 and 70-80 °C for 60 min in phosphate-buffered saline (PBS) and enumerated at 0, 15, 30, 45 and 60 min. GInaFiT was used to model the kinetics of spore inactivation. In subsequent experiments, spores were transferred to enriched brain heart infusion (BHI) broths after 10 min of 80 °C heat treatment in PBS; samples were enumerated at 90 min and 24 h.Results. The spores of all strains demonstrated log-linear inactivation with tailing when heated for 60 min at 80 °C [(x̄=7.54±0.04 log10 vs 4.72±0.09 log10 colony-forming units (c.f.u.) ml- 1; P<0.001]. At 70 °C, all strains except 078 exhibited substantial decline in recovery over 60 min. Interestingly, 50 °C heat treatment had an inhibitory effect on 078 spore recovery at 0 vs 60 min (7.61±0.06 log10 c.f.u. ml- 1 vs 6.13±0.05 log10 c.f.u. ml- 1; P<0.001). Heating at 70/80 °C inhibited the initial germination and outgrowth of both newly produced and aged spores in enriched broths. This inhibition appeared to be transient; after 24 h vegetative counts were higher in heat-treated vs non-heat-treated spores (x̄=7.65±0.04 log10 c.f.u. ml- 1 vs 6.79±0.06 log10 c.f.u. ml- 1; P<0.001).Conclusions. The 078 spores were more resistant to the inhibitory effects of higher temperatures. Heat initially inhibits spore germination, but the subsequent outgrowth of vegetative populations accelerates after the initial inhibitory period.

Effect of fluoroquinolone resistance mutation Thr-82→Ile on Clostridioides difficile fitness.

OBJECTIVES: Fluoroquinolone resistance is common among epidemic Clostridioides difficile PCR ribotype (RT) 027 and may have contributed to outbreaks of C. difficile infection (CDI). We investigated the impact of fluoroquinolone mutations on the bacterial fitness (BF) of C. difficile RT027 isolates. METHODS: The BF of seven RT027 mutants with reduced susceptibility to moxifloxacin (moxifloxacin MIC 4-32 mg/L) was compared with their susceptible (moxifloxacin MIC 1-2 mg/L) progenitor strains in competitive batch culture (CBC), cell cytotoxicity and maximal growth rate assays. Comparative fitness dynamics of one gyrA Thr-82→Ile-harbouring isolate (CD3079M) versus the parent strain (CD3079) were also investigated in a continuous co-culture (CC) chemostat model. Mutant and parent strain populations were assessed every 24 h over 8 days using selective and non-selective agars. Sequencing was performed using NEBNext® Ultra™ chemistry and Illumina® HiSeq 3000 technologies. RESULTS: BF was significantly increased in all Thr-82→Ile isolates (w = 1.08-1.22) in CBC assays (P = 0.002). Gly-429→Val and Gln-434→Lys (gyrB) also showed no burden to fitness (w = 1.24 and 1.18, respectively), but Asp-71→Tyr conferred reduced fitness (w = 0.80). CC results for strains CD3079 and CD3079M (Thr-82→Ile) supported CBC findings; mutant-to-parent ratios differed significantly by 96 h (x¯=1.80, P = 0.025). CONCLUSIONS: The absence of a fitness cost associated with the most prevalent fluoroquinolone resistance mutations may have contributed to the success of RT027. Furthermore, a demonstrable in vitro advantage over fluoroquinolone-susceptible parent strains in CC may contribute to the maintenance of RT027, even in the absence of fluoroquinolone selection pressure.

Investigating the effect of supplementation on Clostridioides (Clostridium) difficile spore recovery in two solid agars.

BACKGROUND: A variety of supplemented solid media are used within Clostridium difficile research to optimally recover spores. Our study sought to investigate different media and additives, providing a method of optimised C. difficile spore recovery. Additionally, due to the results observed in the initial experiments, the inhibitory effects of three amino acids (glycine, l-histidine &l-phenylalanine) on C. difficile spore outgrowth were investigated. METHODS: Spores of five C. difficile strains (PCR ribotypes 001,015,020,027,078) were recovered on two commonly used solid media (BHI & CCEY, or cycloserine-cefoxitin egg yolk) supplemented with various concentrations of germinants (taurocholate, glycine & lysozyme). Agar-incorporation minimum inhibitory concentration (MIC) testing was carried out for glycine and taurocholate on vegetative cells and spores of all five strains. Additionally a BHI broth microassay method was utilised to test the growth of C. difficile in the presence of increasing concentrations (0,1,2,3,4%) of three amino acids (glycine,l-histidine,l-phenyalanine). RESULTS: CCEY agar alone and BHI supplemented with taurocholate (0.1/1%) provided optimal recovery for C. difficile spores. Glycine was inhibitory to spore recovery at higher concentrations, although these varied between the two media used. In agar-incorporated MIC testing, glycine concentrations higher than 2% (20 g/L) were inhibitory to both C. difficile spore and vegetative cell growth versus the control (mean absorbance = 0.33 ±â€¯0.02 vs 0.12 ±â€¯0.01) (P < 0.001). This indicates a potential mechanism whereby glycine interferes with vegetative cell growth. Further microbroth testing provided evidence of inhibition by two amino acids other than glycine, l-histidine and l-phenylalanine. CONCLUSIONS: We provide two media for optimal recovery of C. difficile spores (CCEY alone and BHI supplemented with 0.1/1% taurocholate). CCEY is preferred for isolation from faecal samples. For pure cultures, either CCEY or supplemented BHI agar are appropriate. The inhibitory nature of three amino acids (glycine,l-histidine,l-phenylalanine) to C. difficile vegetative cell proliferation is also highlighted.