Evaluation of the long-term protection conferred by an organosilicon-based disinfectant formulation against bacterial contamination of surfaces.

AIMS: The aim of this work was to evaluate the efficacy of an organosilicon-based, commercially available antimicrobial formulation in the My-shield® product line against bacterial surface contamination. METHODS AND RESULTS: The antimicrobial product was tested in vitro for its long-term persistence on surfaces and effectiveness against Staphylococcus aureus biofilms in comparison to 70% ethanol and 0.1% or 0.6% sodium hypochlorite. Field testing was also conducted over 6 weeks at a university athletic facility. In vitro studies demonstrated the log reductions achieved by the test product, 70% ethanol, and 0.1% sodium hypochlorite were 3.6, 3.1, and 3.2, respectively. The test product persisted on surfaces after washing and scrubbing, and pre-treatment with this product prevented S. aureus surface colonization for up to 30 days. In comparison, pre-treatment with 70% ethanol or 0.6% sodium hypochlorite was not protective against S. aureus biofilm formation after seven days. The field test demonstrated that weekly applications of the test product were more effective at reducing surface bacterial load than daily applications of a control product. CONCLUSIONS: The test product conferred greater long-term protection against bacterial growth and biofilm formation by S. aureus than ethanol and sodium hypochlorite. Even with less frequent applications, the test product maintained a high level of antimicrobial activity.

Development of a small shuttle plasmid for use in oral Veillonella and initial appraisal of potential for fluorescence-based applications.

Oral Veillonella species are among the early colonizers of the human oral cavity. We constructed a small, single-selectable-marker shuttle plasmid, examined its ability to be transformed into diverse oral Veillonella strains, and assessed its potential use for expressing a gene encoding an oxygen-independent fluorescent protein, thus generating a fluorescent Veillonella parvula strain. Because tetracycline resistance is common in Veillonella, we replaced genes encoding ampicillin- and tetracycline-resistance in a previously described shuttle plasmid (pBSJL2) with a chloramphenicol acetyltransferase gene. The resulting plasmid pCF1135 was successfully introduced into four strains representing V. parvula and V. atypica by either natural transformation or electroporation. We then modified this plasmid to express a gene encoding an oxygen-independent fluorescent protein in V. parvula SKV38. The resulting strain yielded a fluorescence signal intensity ∼16 times higher than the wild type in microplate-based fluorimetry experiments. While fluorescence microscopy demonstrated that planktonic cells, colonies, and biofilms of fluorescent V. parvula could also be imaged, photobleaching was a significant issue. In conclusion, we anticipate this genetic system and information provided here will facilitate expanded studies of oral Veillonella species' properties and behavior.

Time-lapse confocal microscopy to study in vitro Streptococcus mutans surface colonization.

The cariogenicity of Streptococcus mutans relates to its ability to form biofilms on dental surfaces. The aim of this work was to develop a flowcell system compatible with time-lapse confocal microscopy to compare the adhesion and accumulation of S. mutans cells on surfaces in unsupplemented media against media containing sucrose or sucralose (a non-metabolized sweetener) over a short period of time. Fluorescent S. mutans 3209/pVMCherry was suspended in unsupplemented media or media supplemented with 1% sucrose or 1% sucralose and passed through a 3D-printed flowcell system. Flowcells were imaged over 60 minutes using a confocal microscope. Image analysis was performed, including a newly developed object-movement-based method to measure biomass adhesion. Streptococcus mutans 3209/pVMCherry grown in 1% sucrose-supplemented media formed small, dense, relatively immobile clumps in the flowcell system measured by biovolume, surface area, and median object centroid movement. Sucralose-supplemented and un-supplemented media yielded large, loose, mobile aggregates. Architectural metrics and per-object movement were significantly different (P < 0.05) when comparing sucrose-supplemented media to either unsupplemented or sucralose-supplemented media. These results demonstrate the utility of a flowcell system compatible with time-lapse confocal microscopy and image analysis when studying initial biofilm formation and adhesion under different nutritional conditions.

In vitro model systems for exploring oral biofilms: From single-species populations to complex multi-species communities.

Numerous in vitro biofilm model systems are available to study oral biofilms. Over the past several decades, increased understanding of oral biology and advances in technology have facilitated more accurate simulation of intraoral conditions and have allowed for the increased generalizability of in vitro oral biofilm studies. The integration of contemporary systems with confocal microscopy and 16S rRNA community profiling has enhanced the capabilities of in vitro biofilm model systems to quantify biofilm architecture and analyse microbial community composition. In this review, we describe several model systems relevant to modern in vitro oral biofilm studies: the constant depth film fermenter, Sorbarod perfusion system, drip-flow reactor, modified Robbins device, flowcells and microfluidic systems. We highlight how combining these systems with confocal microscopy and community composition analysis tools aids exploration of oral biofilm development under different conditions and in response to antimicrobial/anti-biofilm agents. The review closes with a discussion of future directions for the field of in vitro oral biofilm imaging and analysis.

Association between metabolic syndrome and periodontitis: The role of lipids, inflammatory cytokines, altered host response, and the microbiome.

Periodontitis has been associated with many systemic diseases and conditions, including metabolic syndrome. Metabolic syndrome is a cluster of conditions that occur concomitantly and together they increase the risk of cardiovascular disease and double the risk of type 2 diabetes. In this review, we focus on the association between metabolic syndrome and periodontitis; however, we also include information on diabetes mellitus and cardiovascular disease, since these two conditions are significantly intertwined with metabolic syndrome. With regard to periodontitis and metabolic syndrome, to date, the vast majority of studies point to an association between these two conditions and also demonstrate that periodontitis can contribute to the development of, or can worsen, metabolic syndrome. Evaluating the effect of metabolic syndrome on the salivary microbiome, data presented herein support the hypothesis that the salivary bacterial profile is altered in metabolic syndrome patients compared with healthy patients. Considering periodontitis and these three conditions, the vast majority of human and animal studies point to an association between periodontitis and metabolic syndrome, diabetes, and cardiovascular disease. Moreover, there is evidence to suggest that metabolic syndrome and diabetes can alter the oral microbiome. However, more studies are needed to fully understand the influence these conditions have on each other.

Introducing BAIT (Biofilm Architecture Inference Tool): a software program to evaluate the architecture of oral multi-species biofilms.

Biofilm model systems are used to study biofilm growth and predict the effects of anti-biofilm interventions within the human oral cavity. Many in vitro biofilm model systems use a confocal laser scanning microscope (CLSM) in conjunction with image analysis tools to study biofilms. The aim of this study was to evaluate an in-house developed image analysis software program that we call BAIT (Biofilm Architecture Inference Tool) to quantify the architecture of oral multi-species biofilms following anti-biofilm interventions using a microfluidic biofilm system. Differences in architecture were compared between untreated biofilms and those treated with water (negative control), sodium gluconate ('placebo') or stannous fluoride (SnF2). The microfluidic system was inoculated with pooled human saliva and biofilms were developed over 22 h in filter-sterilized 25 % pooled human saliva. During this period, biofilms were treated with water, sodium gluconate, or SnF2 (1000, 3439 or 10 000 p.p.m. Sn2+) 8 and 18 h post-inoculation. After 22 h of growth, biofilms were stained with LIVE/DEAD stain, and imaged by CLSM. BAIT was used to calculate biofilm biovolume, total number of objects, surface area, fluffiness, connectivity, convex hull porosity and viability. Image analysis showed oral biofilm architecture was significantly altered by 3439 and 10 000 p.p.m. Sn2+ treatment regimens, resulting in decreased biovolume, surface area, number of objects and connectivity, while fluffiness increased (P<0.01). In conclusion, BAIT was shown to be able to measure the changes in biofilm architecture and detects possible antimicrobial and anti-biofilm effects of candidate agents.

Critical roles of arginine in growth and biofilm development by Streptococcus gordonii.

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.

Effect of Fluoride-Containing Toothpastes on Enamel Demineralization and Streptococcus mutans Biofilm Architecture.

This study aimed to explore the effect of fluoridated toothpastes on biofilm architecture and enamel demineralization in an in vitro biofilm model. Streptococcus mutans was grown on enamel and treated with slurries of commercial toothpastes, containing SnF2 or NaF. Water and chlorhexidine were used as negative and positive controls, respectively. The developed biofilms were imaged and enamel demineralization was measured. SnF2 and NaF toothpaste treatments significantly reduced enamel demineralization, but SnF2 toothpaste was more effective. Only SnF2 toothpaste and chlorhexidine treatments caused reductions on biofilm mass and thickness. In conclusion, this biofilm model was able to differentiate the effects of the SnF2 and NaF toothpastes on biofilm architecture and enamel demineralization.

A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405.

Oral pathogens, including Treponema denticola, initiate the dysregulation of tissue homeostasis that characterizes periodontitis. However, progress of research on the roles of T. denticola in microbe-host interactions and signaling, microbial communities, microbial physiology, and molecular evolution has been hampered by limitations in genetic methodologies. This is typified by an extremely low transformation efficiency and inability to transform the most widely studied T. denticola strain with shuttle plasmids. Previous studies have suggested that robust restriction-modification (R-M) systems in T. denticola contributed to these problems. To facilitate further molecular genetic analysis of T. denticola behavior, we optimized existing protocols such that shuttle plasmid transformation efficiency was increased by >100-fold over prior reports. Here, we report routine transformation of T. denticola ATCC 35405 with shuttle plasmids, independently of both plasmid methylation status and activity of the type II restriction endonuclease encoded by TDE0911. To validate the utility of this methodological advance, we demonstrated expression and activity in T. denticola of a flavin mononucleotide-based fluorescent protein (FbFP) that is active under anoxic conditions. Addition of routine plasmid-based fluorescence labeling to the Treponema toolset will enable more-rigorous and -detailed studies of the behavior of this organism.

Coaggregation between Rhodococcus and Acinetobacter strains isolated from the food industry.

In this study, coaggregation interactions between Rhodococcus and Acinetobacter strains isolated from food-processing surfaces were characterized. Rhodococcus sp. strain MF3727 formed intrageneric coaggregates with Rhodococcus sp. strain MF3803 and intergeneric coaggregates with 2 strains of Acinetobacter calcoaceticus (MF3293, MF3627). Stronger coaggregation between A. calcoaceticus MF3727 and Rhodococcus sp. MF3293 was observed after growth in batch culture at 30 °C than at 20 °C, after growth in tryptic soy broth than in liquid R2A medium, and between cells in exponential and early stationary phases than cells in late stationary phase. The coaggregation ability of Rhodococcus sp. MF3727 was maintained even after heat and Proteinase K treatment, suggesting its ability to coaggregate was protein independent whereas the coaggregation determinants of the other strains involved proteinaceous cell-surface-associated polymers. Coaggregation was stable at pH 5-9. The mechanisms of coaggregation among Acinetobacter and Rhodococcus strains bare similarity to those displayed by coaggregating bacteria of oral and freshwater origin, with respect to binding between proteinaceous and nonproteinaceous determinants and the effect of environmental factors on coaggregation. Coaggregation may contribute to biofilm formation on industrial food surfaces, protecting bacteria against cleaning and disinfection.

Functional amyloids composed of phenol soluble modulins stabilize Staphylococcus aureus biofilms.

Staphylococcus aureus is an opportunistic pathogen that colonizes the skin and mucosal surfaces of mammals. Persistent staphylococcal infections often involve surface-associated communities called biofilms. Here we report the discovery of a novel extracellular fibril structure that promotes S. aureus biofilm integrity. Biochemical and genetic analysis has revealed that these fibers have amyloid-like properties and consist of small peptides called phenol soluble modulins (PSMs). Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress. Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not. This work suggests the PSMs' aggregation into amyloid fibers modulates their biological activity and role in biofilms.

Community interactions of oral streptococci.

It is now clear that the most common oral diseases, dental caries and periodontitis, are caused by mixed-species communities rather than by individual pathogens working in isolation. Oral streptococci are central to these disease processes since they are frequently the first microorganisms to colonize oral surfaces and they are numerically the dominant microorganisms in the human mouth. Numerous interactions between oral streptococci and other bacteria have been documented. These are thought to be critical for the development of mixed-species oral microbial communities and for the transition from oral health to disease. Recent metagenomic studies are beginning to shed light on the co-occurrence patterns of streptococci with other oral bacteria. Refinements in microscopy techniques and biofilm models are providing detailed insights into the spatial distribution of streptococci in oral biofilms. Targeted genetic manipulation is increasingly being applied for the analysis of specific genes and networks that modulate interspecies interactions. From this work, it is clear that streptococci produce a range of extracellular factors that promote their integration into mixed-species communities and enable them to form social networks with neighboring taxa. These "community integration factors" include coaggregation-mediating adhesins and receptors, small signaling molecules such as peptides or autoinducer-2, bacteriocins, by-products of metabolism including hydrogen peroxide and lactic acid, and a range of extracellular enzymes. Here, we provide an overview of various types of community interactions between oral streptococci and other microorganisms, and we consider the possibilities for the development of new technologies to interfere with these interactions to help control oral biofilms.

A high-throughput microfluidic dental plaque biofilm system to visualize and quantify the effect of antimicrobials.

OBJECTIVES: Few model systems are amenable to developing multi-species biofilms in parallel under environmentally germane conditions. This is a problem when evaluating the potential real-world effectiveness of antimicrobials in the laboratory. One such antimicrobial is cetylpyridinium chloride (CPC), which is used in numerous over-the-counter oral healthcare products. The aim of this work was to develop a high-throughput microfluidic system that is combined with a confocal laser scanning microscope (CLSM) to quantitatively evaluate the effectiveness of CPC against oral multi-species biofilms grown in human saliva. METHODS: Twenty-four-channel BioFlux microfluidic plates were inoculated with pooled human saliva and fed filter-sterilized saliva for 20 h at 37°C. The bacterial diversity of the biofilms was evaluated by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). The antimicrobial/anti-biofilm effect of CPC (0.5%-0.001% w/v) was examined using Live/Dead stain, CLSM and 3D imaging software. RESULTS: The analysis of biofilms by bTEFAP demonstrated that they contained genera typically found in human dental plaque. These included Aggregatibacter, Fusobacterium, Neisseria, Porphyromonas, Streptococcus and Veillonella. Using Live/Dead stain, clear gradations in killing were observed when the biofilms were treated with CPC between 0.5% and 0.001% w/v. At 0.5% (w/v) CPC, 90% of the total signal was from dead/damaged cells. Below this concentration range, less killing was observed. In the 0.5%-0.05% (w/v) range CPC penetration/killing was greatest and biofilm thickness was significantly reduced. CONCLUSIONS: This work demonstrates the utility of a high-throughput microfluidic-CLSM system to grow multi-species oral biofilms, which are compositionally similar to naturally occurring biofilms, to assess the effectiveness of antimicrobials.

Coaggregation occurs amongst bacteria within and between biofilms in domestic showerheads.

Showerheads support the development of multi-species biofilms that can be unsightly, produce malodor, and may harbor pathogens. The outer-surface spray-plates of many showerheads support visible biofilms that likely contain a mixture of bacteria from freshwater and potentially from human users. Coaggregation, a mechanism by which genetically distinct bacteria specifically recognize one another, may contribute to the retention and enrichment of different species within these biofilms. The aim of this work was to describe the bacterial composition of outer spray-plate biofilms of three domestic showerheads and to determine the intra- and inter-biofilm coaggregation ability of each culturable isolate. The bacterial composition of the three biofilms was determined by using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) and by culturing on R2A medium. An average of 31 genera per biofilm were identified using bTEFAP and a total of 30 isolates were cultured. Even though the microbial diversity of each showerhead biofilm differed, every cultured isolate was able to coaggregate with at least one other isolate from the same or different showerhead biofilm. Promiscuous coaggregating isolates belonged to the genera Brevundimonas, Micrococcus, and Lysobacter. This work suggests that coaggregation may be a common feature of showerhead biofilms. Characterization of the mechanisms mediating coaggregation, and the inter-species interactions they facilitate, may allow for novel strategies to inhibit biofilm development.

Bacterial contamination on used face masks among nursing home healthcare personnel.

Objectives: Since the beginning of the COVID-19 pandemic, face masks have been worn by many in public areas and for prolonged periods by healthcare workers (HCWs). This may facilitate bacterial contamination and transmission to and from patients in nursing homes where clinical care areas with strict precautions and residential and activity areas are interconnected. We assessed and compared bacterial mask colonization in HCWs belonging to different demographic categories and professions (clinical and nonclinical) and among HCWs who had worn the mask for different periods of time. Design setting and participants: We conducted a point-prevalence study of 69 HCW masks at the end of a typical work shift in a 105-bed nursing home serving postacute care and rehabilitation patients. Information collected about the mask user included profession, age, sex, length of time the mask was worn, and known exposure to patients with colonization. Results: In total, 123 distinct bacterial isolates were recovered (1-5 isolates per mask), including Staphylococcus aureus from 11 masks (15.9%) and gram-negative bacteria of clinical importance from 22 masks (31.9%). Antibiotic resistance rates were low. There were no significant differences in the number of clinically important bacteria among masks worn more or less than 6 hours, and there were no significant differences among HCWs with different job functions or exposure to colonized patients. Conclusions: Bacterial mask contamination was not associated with HCW profession or exposure and did not increase after 6 hours of mask wearing in our nursing home setting. Bacteria contaminating HCW masks may differ from those colonizing patients.

Autoinducer-2 influences interactions amongst pioneer colonizing streptococci in oral biofilms.

Streptococcus gordonii and Streptococcus oralis are among the first bacterial species to colonize clean tooth surfaces. Both produce autoinducer-2 (AI-2): a family of inter-convertible cell-cell signal molecules synthesized by the LuxS enzyme. The overall aim of this work was to determine whether AI-2 alters interspecies interactions between S. gordonii DL1 and S. oralis 34 within dual-species biofilms in flowing human saliva. Based upon AI-2 bioluminescence assays, S. gordonii DL1 produced more AI-2 activity than S. oralis 34 in batch culture, and both were able to remove AI-2 activity from solution. In single-species, saliva-fed flowcell systems, S. oralis 34 formed scant biofilms that were similar to the luxS mutant. Conversely, S. gordonii DL1 formed confluent biofilms while the luxS mutant formed architecturally distinct biofilms that possessed twofold greater biovolume than the wild-type. Supplementing saliva with 0.1-10 nM chemically synthesized AI-2 (csAI-2) restored the S. gordonii DL1 luxS biofilm phenotype to that which was similar to the wild-type; above or below this concentration range, biofilms were architecturally similar to that formed by the luxS mutant. In dual-species biofilms, S. gordonii DL1 was always more abundant than S. oralis 34. Compared with dual-species, wild-type biofilms, the biovolume occupied by S. oralis 34 was reduced by greater than sevenfold when neither species produced AI-2. The addition of 1 nM csAI-2 to the dual-species luxS-luxS mutant biofilms re-established the biofilm phenotype to resemble that of the wild-type pair. Thus, this work demonstrates that AI-2 can alter the biofilm structure and composition of pioneering oral streptococcal biofilms. This may influence the subsequent succession of other species into oral biofilms and the ecology of dental plaque.

Unsweetened and sucrose-sweetened black and green tea modifies the architecture of in vitro oral biofilms.

OBJECTIVE: It is unclear whether tea infusions with or without sucrose supplementation alter oral biofilm development, so we evaluated the effect of unsweetened and sucrose-sweetened black and green tea infusions on in vitro saliva-derived biofilms. DESIGN: Biofilms were developed from human saliva for 20 h in cell-free 25% human saliva within static glass-bottom microplates. During biofilm development, biofilms were treated with either (i) unsweetened black tea, (ii) unsweetened green tea, (iii) 10% sucrose-sweetened black tea, (iv) 10% sucrose-sweetened green tea (v) deionized water (negative control), or (vi) 10% sucrose (positive control). Biofilms were incubated at 37 °C in 5% CO2. After 20 h of development, biofilms were imaged using a CLSM, and biofilm architecture and viability were evaluated. RESULTS: All the tea infusions reduced biofilm biomass and altered some other biofilm architectural outcomes (e.g., biofilm surface area) compared to the control groups. Statistically significant differences in biofilm biomass, number of objects, surface area, and convex-hull porosity were observed between biofilms treated with green and black tea. The addition of sugar to tea did not significantly modify the ability of tea to alter biofilm architecture. Only the treatment of biofilms with unsweetened black tea significantly reduced bacterial viability. CONCLUSIONS: While both teas reduced biofilm biomass and altered biofilm architecture, black tea had an enhanced effect that may relate to this tea's observed antimicrobial activity. The addition of sucrose to tea infusions did not appear to reduce the impact of either tea in modifying oral biofilm architecture.

The importance of a multifaceted approach to characterizing the microbial flora of chronic wounds.

Chronic wounds contain complex polymicrobial communities of sessile organisms that have been underappreciated because of limitations of standard culture techniques. The aim of this work was to combine recently developed next-generation investigative techniques to comprehensively describe the microbial characteristics of chronic wounds. Tissue samples were obtained from 15 patients with chronic wounds presenting to the Johns Hopkins Wound Center. Standard bacteriological cultures demonstrated an average of three common bacterial species in wound samples. By contrast, high-throughput pyrosequencing revealed increased bacterial diversity with an average of 17 genera in each wound. Data from microbial community profiling of chronic wounds were compared with published sequenced analyses of bacteria from normal skin. Increased proportions of anaerobes, Gram-negative rods and Gram-positive cocci were found in chronic wounds. In addition, chronic wounds had significantly lower populations of Propionibacterium compared with normal skin. Using epifluorescence microscopy, wound bacteria were visualized in highly organized thick confluent biofilms or as scattered individual bacterial cells. Fluorescent in situ hybridization allowed for the visualization of Staphylococcus aureus cells in a wound sample. Quorum-sensing molecules were measured by bioassay to evaluate signaling patterns among bacteria in the wounds. A range of autoinducer-2 activities was detected in the wound samples. Collectively, these data provide new insights into the identity, organization, and behavior of bacteria in chronic wounds. Such information may provide important clues to effective future strategies in wound healing.

Efficacy of an alcohol-free CPC-containing mouthwash against oral multispecies biofilms.

OBJECTIVE: The aim of this work was to develop two static-model multispecies oral biofilm systems to compare the efficacy of a placebo mouthwash to an alcohol-free mouthwash containing 0.075% CPC. METHODS: Two model biofilm systems were used: a 24-well glass-bottom microplate (GM) system and a chamber slide (CS) system. These were inoculated with Schaedler media containing pooled, unfiltered saliva. During incubation at 37 degrees C in 5% CO2, Schaedler media was replaced every 24 hours. Five-day and 10-day multispecies biofilms in the GM and CS systems were then exposed to phosphate buffered saline, the placebo mouthwash, or the alcohol-free 0.075% CPC-containing mouthwash. Biofilms were visualized in three-dimensions by Confocal Laser Scanning Microscopy (CLSM), and fluorometric analyses were performed on biofilms in the GM system. RESULTS: CLSM demonstrated that regardless of the model system used, the alcohol-free 0.075% CPC-containing mouthwash solution increased the number of damaged biofilm cells. The efficacy of CPC was inversely related to the age of the biofilm. A contrariety between the two biofilm systems was that the CS system indicated that alcohol-free 0.075% CPC-containing mouthwash partially disrupted biofilms. Fluorometric analysis ofGM biofilms also demonstrated that the alcohol-free 0.075% CPC-containing mouthwash damaged biofilm cells. CONCLUSION: Two static oral multispecies model biofilms systems demonstrated that an alcohol-free 0.075% CPC-containing mouthwash had greater antimicrobial efficacy than a placebo mouthwash. The alcohol-free 0.075% CPC-containing formulation is effective against multispecies oral biofilms.

Deciphering Endodontic Microbial Communities by Next-generation Sequencing.

INTRODUCTION: Biofilms are present in more than 70% of endodontically diseased teeth. Through the advancements in the next-generation sequencing (NGS) technologies, microbiome research has granted a deeper analysis of the microbial communities living in human hosts. Here, we reviewed previous studies that used NGS to profile the microbial communities of root canals. METHODS: A total of 12 peer-reviewed articles from PubMed were identified and critically reviewed. The study criteria were as follows: NGS platforms, sequenced bacterial hypervariable regions, teeth diagnosis with available patient information, sample characteristics, collection method, and microbial signatures. RESULTS: The most common NGS platforms used were 454 pyrosequencing (Roche Diagnostic Corporation, Risch-Rotkreuz, Switzerland) and Illumina-based technology (Illumina Inc, San Diego, CA). The hypervariable regions sequenced were between the V1 and V6 regions. The patient and sample population ranged from ages 12-76 years and asymptomatic and symptomatic teeth diagnosed with pulp necrosis with or without apical periodontitis. Microbial sampling was conducted directly from the infected pulp or the extracted teeth. The most abundant phyla were Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, and Fusobacteria. The most frequently detected genera were Prevotella, Fusobacterium, Porphyromonas, Parvimonas, and Streptococcus. Other notable microbial signatures at different taxa levels were identified but were widely variable between studies. CONCLUSIONS: Technologies based on high-throughput 16S ribosomal RNA NGS can aid in deciphering the complex bacterial communities of root canal biofilms. Thus far, only a few studies have been published with relatively small sample sizes, variable sample collection protocols, and community analyses methods. Future larger clinical studies are essential with validated standardized protocols for improved understanding of the pathogenic nature of bacterial biofilm communities in root canals.