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.

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.

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.

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.

A Sensitive Thresholding Method for Confocal Laser Scanning Microscope Image Stacks of Microbial Biofilms.

Biofilms are surface-attached microbial communities whose architecture can be captured with confocal microscopy. Manual or automatic thresholding of acquired images is often needed to help distinguish biofilm biomass from background noise. However, manual thresholding is subjective and current automatic thresholding methods can lead to loss of meaningful data. Here, we describe an automatic thresholding method designed for confocal fluorescent signal, termed the biovolume elasticity method (BEM). We evaluated BEM using confocal image stacks of oral biofilms grown in pooled human saliva. Image stacks were thresholded manually and automatically with three different methods; Otsu, iterative selection (IS), and BEM. Effects on biovolume, surface area, and number of objects detected indicated that the BEM was the least aggressive at removing signal, and provided the greatest visual and quantitative acuity of single cells. Thus, thresholding with BEM offers a sensitive, automatic, and tunable method to maintain biofilm architectural properties for subsequent analysis.

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.

Association of Escherichia coli ST131 lineage with risk of urinary tract infection recurrence among young women.

OBJECTIVES: The aim of this study was to test the hypothesis that urinary tract infections (UTIs) caused by Escherichia coli of the sequence type 131 (ST131) lineage are more likely to recur than UTIs caused by other E. coli lineages. METHODS: Isolates from 221 young women with UTI caused by E. coli participating in a randomised controlled trial were used. Participants were followed for 6 months or until UTI recurrence. RESULTS: Sequence type was not associated with risk of recurrence. Isolates in the ST131 lineage were more resistant than other STs to quinolones (6.2% vs. 1.3%) but not trimethoprim/sulfamethoxazole (15.4% vs. 15.0%). CONCLUSIONS: These results do not support an increased risk of recurrent UTI among otherwise healthy women with UTI caused by E. coli ST131.

Microbial Communities Associated with Primary and Metastatic Head and Neck Squamous Cell Carcinoma - A High Fusobacterial and Low Streptococcal Signature.

Given the potential relationship between head and neck squamous cell carcinoma (HNSCC) and microbial dysbiosis, we profiled the microbiome within healthy normal and tumorous (primary and metastatic) human tissues from the oral cavity, larynx-pharynx, and lymph nodes using 16S rRNA sequencing. Alpha and beta diversity analyses revealed that normal tissues had the greatest richness in community diversity, while the metastatic populations were most closely related to one another. Compared to the normal, the microbiota associated with tumors supported altered abundances in the phyla Fusobacteria, Firmicutes, Actinobacteria and Proteobacteria. Most notably, the relative abundance of Fusobacterium increased whereas Streptococcus decreased in both primary and metastatic samples. Principal coordinate analysis indicated a separation and clustering of samples by tissue status. However, random forest analysis revealed that the microbial profiles alone were a poor predictor for primary and metastatic HNSCC samples. Here, we report that the microbial communities residing in the tumorous tissues are compositionally distinct compared to the normal adjacent tissues. However, likely due to the smaller sample size and sample-to-sample heterogeneity, our prediction models were not able to distinguish by sample types. This work provides a foundation for future studies aimed at understanding the role of the dysbiotic tissue microbiome in HNSCC.

An in silico evaluation of treatment regimens for recurrent Clostridium difficile infection.

BACKGROUND: Clostridium difficile infection (CDI) is a significant nosocomial infection worldwide, that recurs in as many as 35% of infections. Risk of CDI recurrence varies by ribotype, which also vary in sporulation and germination rates. Whether sporulation/germination mediate risk of recurrence and effectiveness of treatment of recurring CDI remains unclear. We aim to assess the role of sporulation/germination patterns on risk of recurrence, and the relative effectiveness of the recommended tapered/pulsing regimens using an in silico model. METHODS: We created a compartmental in-host mathematical model of CDI, composed of vegetative cells, toxins, and spores, to explore whether sporulation and germination have an impact on recurrence rates. We also simulated the effectiveness of three tapered/pulsed vancomycin regimens by ribotype. RESULTS: Simulations underscored the importance of sporulation/germination patterns in determining pathogenicity and transmission. All recommended regimens for recurring CDI tested were effective in reducing risk of an additional recurrence. Most modified regimens were still effective even after reducing the duration or dosage of vancomycin. However, the effectiveness of treatment varied by ribotype. CONCLUSION: Current CDI vancomycin regimen for treating recurrent cases should be studied further to better balance associated risks and benefits.

High-purity Nisin Alone or in Combination with Sodium Hypochlorite Is Effective against Planktonic and Biofilm Populations of Enterococcus faecalis.

INTRODUCTION: Nisin, a broad-spectrum bacteriocin, has recently been highlighted for its biomedical applications. To date, no studies have examined the antimicrobial and antibiofilm properties of high-purity (>95%) nisin (nisin ZP) on Enterococcus faecalis and biofilms formed by this species. We hypothesize that nisin can inhibit E. faecalis and reduce biofilm biomass, and combinations of nisin and sodium hypochlorite (NaOCl) will enhance the antibiofilm properties against E. faecalis biofilms. METHODS: Using broth cultures, disc diffusion assays, and biofilm assays, we examined the effects of nisin on various E. faecalis growth parameters and biofilm properties (biovolume, thickness, and roughness). Confocal microscopy was used in conjunction with Imaris and Comstat2 software (Kongens Lyngby, Copenhagen, Denmark) to measure and analyze the biofilm properties. RESULTS: Nisin significantly decreased the growth of planktonic E. faecalis dose dependently. The minimum inhibitory concentrations against E. faecalis strains OG-1 and ATCC 29212 were 15 and 50 µg/mL, and the minimum bactericidal concentrations were 150 and 200 µg/mL, respectively. A reduction in biofilm biovolume and thickness was observed for biofilms treated with nisin at ≥10 µg/mL for 10 minutes. In addition, the combination of nisin with low doses of NaOCl enhanced the antibiofilm properties of both antimicrobial agents. CONCLUSIONS: Nisin alone or in combination with low concentrations of NaOCl reduces the planktonic growth of E. faecalis and disrupts E. faecalis biofilm structure. Our results suggest that nisin has potential as an adjunctive endodontic therapeutic agent and as an alternative to conventional NaOCl irrigation.

Metabolomics of Head and Neck Cancer: A Mini-Review.

Metabolomics is used in systems biology to enhance the understanding of complex disease processes, such as cancer. Head and neck cancer (HNC) is an epithelial malignancy that arises in the upper aerodigestive tract and affects more than half a million people worldwide each year. Recently, significant effort has focused on integrating multiple "omics" technologies for oncological research. In particular, research has been focused on identifying tumor-specific metabolite profiles using different sample types (biological fluids, cells and tissues) and a variety of metabolomic platforms and technologies. With our current understanding of molecular abnormalities of HNC, the addition of metabolomic studies will enhance our knowledge of the pathogenesis of this disease and potentially aid in the development of novel strategies to prevent and treat HNC. In this review, we summarize the proposed hypotheses and conclusions from publications that reported findings on the metabolomics of HNC. In addition, we address the potential influence of host-microbe metabolomics in cancer. From a systems biology perspective, the integrative use of genomics, transcriptomics and proteomics will be extremely important for future translational metabolomic-based research discoveries.

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.

Inhibition of multispecies biofilms by a fluoride-releasing dental prosthesis copolymer.

OBJECTIVES: This study aimed to develop a new mixed-species acidogenic biofilm model and use it to assess the antimicrobial properties of a novel fluoride-releasing copolymer. METHODS: Stubs composed of a copolymer of methyl methacrylate (MMA) and 2-hydroxyethyl methacrylate (HEMA) with polymethyl methacrylate (PMMA) were produced by chemically-activated free radical polymerization. A fluoride-releasing copolymer was developed by incorporating sodium fluoride in place of a portion of the PMMA. Samples were mounted in polysulfone Modified Robbins Devices (MRDs) and were optimized for single- and mixed-species biofilm formation by Candida albicans, Lactobacillus casei and Streptococcus mutans. RESULTS: Fluoride release was sustained for at least 48h in flowing conditions. Fluoride did not affect the colonization and biofilm growth of any of the microorganisms in monocultures. However, in mixed-species biofilms, cell densities of all three species were reduced approximately ten-fold (p<0.05) on the fluoridated material compared with the non-fluoridated copolymer. CONCLUSIONS: These data demonstrate that intermicrobial interactions in mixed-species acidogenic biofilms are sensitive to fluoride, and that the inclusion of fluoride in a denture lining copolymer reduces the formation of polymicrobial biofilms. CLINICAL SIGNIFICANCE: The growth of acidogenic microorganisms on denture materials is associated with denture stomatitis and dental caries on surrounding teeth. A fluoride-releasing copolymer that inhibits acidogenic mixed-species biofilms, such as the material described in this study, has the potential to control these diseases by limiting biofilm growth.

Coaggregation occurs between microorganisms isolated from different environments.

Coaggregation, the specific recognition and adherence of different microbial species, is thought to enhance biofilm formation. To date, no studies have focused on the ability of microorganisms isolated from a broad range of environments to coaggregate with each other and it is unclear whether coaggregation promotes the transmission of microorganisms between environmental niches. We aimed to evaluate the coaggregation ability of 29 bacteria and one fungus, isolated from a range of different environments, and to characterize the cell-surface polymers that mediate coaggregation between selected pairs. Strains were categorized as belonging to one of the four microbial archetypes: aquatic, broad environment, human opportunistic pathogen or human oral. A total of 23 of the 30 strains (77%) coaggregated with at least one other and 21/30 (70%) coaggregated with strains belonging to other archetypes. Nasopharyngeal bacteria belonging to the human opportunistic pathogen archetype showed the least number of coaggregations, and five Haemophilus influenzae strains did not coaggregate. Protease and sugar treatments indicated that coaggregation between strains of different archetypes was often mediated by lectin-saccharide interactions (9 of 15 evaluated pairs). In conclusion, coaggregation can occur between taxonomically disparate species isolated from discrete environments. We propose that these organisms be labeled as 'cross-environment coaggregating organisms'. The ability to coaggregate may aid species to colonize non-indigenous biofilms.

Antimicrobial nisin acts against saliva derived multi-species biofilms without cytotoxicity to human oral cells.

OBJECTIVES: Nisin is a lantibiotic widely used for the preservation of food and beverages. Recently, investigators have reported that nisin may have clinical applications for treating bacterial infections. The aim of this study was to investigate the effects of ultra pure food grade Nisin ZP (>95% purity) on taxonomically diverse bacteria common to the human oral cavity and saliva derived multi-species oral biofilms, and to discern the toxicity of nisin against human cells relevant to the oral cavity. METHODS: The minimum inhibitory concentrations and minimum bactericidal concentrations of taxonomically distinct oral bacteria were determined using agar and broth dilution methods. To assess the effects of nisin on biofilms, two model systems were utilized: a static and a controlled flow microfluidic system. Biofilms were inoculated with pooled human saliva and fed filter-sterilized saliva for 20-22 h at 37°C. Nisin effects on cellular apoptosis and proliferation were evaluated using acridine orange/ethidium bromide fluorescent nuclear staining and lactate dehydrogenase activity assays. RESULTS: Nisin inhibited planktonic growth of oral bacteria at low concentrations (2.5-50 µg/ml). Nisin also retarded development of multi-species biofilms at concentrations ≥1 µg/ml. Specifically, under biofilm model conditions, nisin interfered with biofilm development and reduced biofilm biomass and thickness in a dose-dependent manner. The treatment of pre-formed biofilms with nisin resulted in dose- and time-dependent disruption of the biofilm architecture along with decreased bacterial viability. Human cells relevant to the oral cavity were unaffected by the treatment of nisin at anti-biofilm concentrations and showed no signs of apoptotic changes unless treated with much higher concentrations (>200 µg/ml). CONCLUSION: This work highlights the potential therapeutic value of high purity food grade nisin to inhibit the growth of oral bacteria and the development of biofilms relevant to oral diseases.

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.

L-arginine destabilizes oral multi-species biofilm communities developed in human saliva.

The amino acid L-arginine inhibits bacterial coaggregation, is involved in cell-cell signaling, and alters bacterial metabolism in a broad range of species present in the human oral cavity. Given the range of effects of L-arginine on bacteria, we hypothesized that L-arginine might alter multi-species oral biofilm development and cause developed multi-species biofilms to disassemble. Because of these potential biofilm-destabilizing effects, we also hypothesized that L-arginine might enhance the efficacy of antimicrobials that normally cannot rapidly penetrate biofilms. A static microplate biofilm system and a controlled-flow microfluidic system were used to develop multi-species oral biofilms derived from pooled unfiltered cell-containing saliva (CCS) in pooled filter-sterilized cell-free saliva (CFS) at 37° C. The addition of pH neutral L-arginine monohydrochloride (LAHCl) to CFS was found to exert negligible antimicrobial effects but significantly altered biofilm architecture in a concentration-dependent manner. Under controlled flow, the biovolume of biofilms (µm(3)/µm(2)) developed in saliva containing 100-500 mM LAHCl were up to two orders of magnitude less than when developed without LAHCI. Culture-independent community analysis demonstrated that 500 mM LAHCl substantially altered biofilm species composition: the proportion of Streptococcus and Veillonella species increased and the proportion of Gram-negative bacteria such as Neisseria and Aggregatibacter species was reduced. Adding LAHCl to pre-formed biofilms also reduced biovolume, presumably by altering cell-cell interactions and causing cell detachment. Furthermore, supplementing 0.01% cetylpyridinium chloride (CPC), an antimicrobial commonly used for the treatment of dental plaque, with 500 mM LAHCl resulted in greater penetration of CPC into the biofilms and significantly greater killing compared to a non-supplemented 0.01% CPC solution. Collectively, this work demonstrates that LAHCl moderates multi-species oral biofilm development and community composition and enhances the activity of CPC. The incorporation of LAHCl into oral healthcare products may be useful for enhanced biofilm control.