No man's land: Species-specific formation of exclusion zones bordering Actinomyces graevenitzii microcolonies in nanoliter cultures.

To survive within complex environmental niches, including the human host, bacteria have evolved intricate interspecies communities driven by competition for limited nutrients, cooperation via complementary metabolic proficiencies, and establishment of homeostatic relationships with the host immune system. The study of such complex, interdependent relationships is often hampered by the challenges of culturing many bacterial strains in research settings and the limited set of tools available for studying the dynamic behavior of multiple bacterial species at the microscale. Here, we utilize a microfluidic-based co-culture system and time-lapse imaging to characterize dynamic interactions between Streptococcus species, Staphylococcus aureus, and Actinomyces species. Co-culture of Streptococcus cristatus or S. salivarius in nanoliter compartments with Actinomyces graevenitzii revealed localized exclusion of Streptococcus and Staphylococcus from media immediately surrounding A. graevenitzii microcolonies. This community structure did not occur with S. mitis or S. oralis strains or in co-cultures containing other Actinomycetaceae species such as S. odontolyticus or A. naeslundii. Moreover, fewer neutrophils were attracted to compartments containing both A. graevenitzii and Staphylococcus aureus than to an equal number of either species alone, suggesting a possible survival benefit together during immune responses.

A Cell-based Screen in Actinomyces oris to Identify Sortase Inhibitors.

Sortase enzymes are attractive antivirulence drug targets that attach virulence factors to the surface of Staphylococcus aureus and other medically significant bacterial pathogens. Prior efforts to discover a useful sortase inhibitor have relied upon an in vitro activity assay in which the enzyme is removed from its native site on the bacterial surface and truncated to improve solubility. To discover inhibitors that are effective in inactivating sortases in vivo, we developed and implemented a novel cell-based screen using Actinomyces oris, a key colonizer in the development of oral biofilms. A. oris is unique because it exhibits sortase-dependent growth in cell culture, providing a robust phenotype for high throughput screening (HTS). Three molecules representing two unique scaffolds were discovered by HTS and disrupt surface protein display in intact cells and inhibit enzyme activity in vitro. This represents the first HTS for sortase inhibitors that relies on the simple metric of cellular growth and suggests that A. oris may be a useful platform for discovery efforts targeting sortase.

Evaluation of in vitro Streptococcus mutans and Actinomyces naeslundii attachment and growth on restorative materials surfaces.

BACKGROUND: Restorative materials have varying surface characteristics from natural tooth, which may affect oral-bacterial surface attachment/growth. This study examined 48-h Streptococcus mutans (Sm) or Actinomyces naeslundii (An) growth on various restorative materials and tooth surfaces. METHODS: The quantity and viability of 48-hour-old Sm and An growth on polished (180- or 1200-grits), saliva-coated resin composite (RC), glass ionomer cements (GIC), resin-modified GIC (R-GIC), GIC containing casein phosphopeptide-amorphous calcium phosphate (3% (w/w), CPP-ACP GIC), amalgam or tooth blocks (5 × 5 × 1 mm3 ) were examined. RESULTS: Rough-polished (arithmetical mean deviation of the assessed surface roughness profile (Ra): 1.50-1.75 µm) material surfaces revealed relatively higher proportion of inorganic, positively charged surface components ((Si + Al)/C) and greater quantity of surface attached bacteria than smooth polished (Ra: 0.20-0.35 µm) material groups (P < 0.001). Less Sm and An were observed on tooth, and smooth polished GIC and CPP-ACP GIC surfaces than on resin-based materials (RC, R-GIC) and amalgam (P ≤ 0.003). Viability of Sm was found to be lower on amalgam surfaces (P < 0.001), whereas that of An appeared lower on both amalgam surfaces and rough CPP-ACP GIC surfaces (P ≤ 0.033). CONCLUSION: Surface roughness exerted a pronounced effect on in vitro growth/attached Sm/An quantity but may not have an impact on bacteria viability. Interestingly, despite smoother surfaces of various materials tested, fewer Sm/An were observed attaching on tooth surfaces.

Intrageneric and Intergeneric Interactions Developed by Oral Streptococci: Pivotal Role in the Pathogenesis of Oral Diseases.

Oral streptococci are among the most abundant genera present in the oral cavity. They are usually the first colonizers of oral surfaces and they develop extensive microbial interactions, playing a fundamental role in the pathogenesis of oral diseases such as dental caries and periodontitis. In addition to physical adherence, streptococcal cells also exchange messages with cells from another Streptococcus spp. and other microorganisms in the form of metabolites and signaling molecules. In this review, we focused on these intrageneric and intergeneric interactions, and their association with oral diseases.

Rapid evolution of decreased host susceptibility drives a stable relationship between ultrasmall parasite TM7x and its bacterial host.

Around one-quarter of bacterial diversity comprises a single radiation with reduced genomes, known collectively as the Candidate Phyla Radiation. Recently, we coisolated TM7x, an ultrasmall strain of the Candidate Phyla Radiation phylum Saccharibacteria, with its bacterial host Actinomyces odontolyticus strain XH001 from human oral cavity and stably maintained as a coculture. Our current work demonstrates that within the coculture, TM7x cells establish a long-term parasitic association with host cells by infecting only a subset of the population, which stay viable yet exhibit severely inhibited cell division. In contrast, exposure of a naïve A. odontolyticus isolate, XH001n, to TM7x cells leads to high numbers of TM7x cells binding to each host cell, massive host cell death, and a host population crash. However, further passaging reveals that XH001n becomes less susceptible to TM7x over time and enters a long-term stable relationship similar to that of XH001. We show that this reduced susceptibility is driven by rapid host evolution that, in contrast to many forms of phage resistance, offers only partial protection. The result is a stalemate where infected hosts cannot shed their parasites; nevertheless, parasite load is sufficiently low that the host population persists. Finally, we show that TM7x can infect and form stable long-term relationships with other species in a single clade of Actinomyces, displaying a narrow host range. This system serves as a model to understand how parasitic bacteria with reduced genomes such as those of the Candidate Phyla Radiation have persisted with their hosts and ultimately expanded in their diversity.

Optimizing resin-dentin bond stability using a bioactive adhesive with concomitant antibacterial properties and anti-proteolytic activities.

Secondary caries and hybrid layer degradation are two major challenges encountered in long-term resin-dentin bond stability. As a link between resin and dentin, adhesives that possess both antimicrobial and anti-proteolytic activities are in demand for eliminating bacteria-induced secondary caries and preventing hybrid layers from degradation. In the present study, a new quaternary ammonium methacryloxy silane (QAMS) prepared from sol-gel chemistry was incorporated into experimental adhesives to examine their antimicrobial effect and anti-proteolytic potential. This functional methacrylate resin monomer contains polymerizable methacryloxy functionalities as well as a positively-charged quaternary ammonium functionality with a long, lipophilic -C18H37 alkyl chain for puncturing the cell wall/membrane of surface-colonizing organisms. Antibacterial testing performed using agar diffusion test, live/dead bacterial staining and colony-forming unit counts all indicated that the QAMS-containing adhesives killed Streptococcus mutans and Actinomyces naeslundii in a dose-dependent manner via a predominant contact-killing mechanism. Gelatinolytic activity within the hybrid layers created by these adhesives was examined using in-situ zymography. Hybrid layers created with 0% QAMS-containing adhesive exhibited intense green fluorescence emitted by the hydrolyzed fluorescein-conjugated gelatin, with 4-fold increase in enzymatic activity compared with an experimental adhesive containing 5% QAMS. Taken together, incorporation of 5% QAMS in the experimental adhesive provides simultaneous antimicrobial and anti-proteolytic activities that are crucial for the maintenance of long-term resin-dentin bond integrity. STATEMENT OF SIGNIFICANCE: Durability of resin-dentin interfacial bond remains a clinically-significant challenge. Secondary caries caused by bacteria and the degradation of hybrid layers via endogenous dentin proteases are two important contributors to the poor resin-dentin bond durability. The present study developed a new 5% QAMS-containing adhesive that provides simultaneous antimicrobial and dentin protease inhibition functions to extend the longevity of resin-dentin bonds.

A Linear Plasmid-Like Prophage of Actinomyces odontolyticus Promotes Biofilm Assembly.

The human oral cavity is home to a large number of bacteria and bacteriophages (phages). However, the biology of oral phages as members of the human microbiome is not well understood. Recently, we isolated Actinomyces odontolyticus subsp. actinosynbacter strain XH001 from the human oral cavity, and genomic analysis revealed the presence of an intact prophage named xhp1. Here, we demonstrated that xhp1 is a linear plasmid-like prophage, which is a newly identified phage of A. odontolyticus The prophage xhp1 genome is a 35-kb linear double-stranded DNA with 10-bp single-stranded, 3' cohesive ends. xhp1 exists extrachromosomally, with an estimated copy number of 5. Annotation of xhp1 revealed 54 open reading frames, while phylogenetic analysis suggests that it has limited similarity with other phages. xhp1 phage particles can be induced by mitomycin C and belong to the Siphoviridae family, according to transmission electron microscopic examination. The released xhp1 particles can reinfect the xhp1-cured XH001 strain and result in tiny blurry plaques. Moreover, xhp1 promotes XH001 biofilm formation through spontaneous induction and the release of host extracellular DNA (eDNA). In conclusion, we identified a linear plasmid-like prophage of A. odontolyticus, which enhances bacterial host biofilm assembly and could be beneficial to the host for its persistence in the oral cavity.IMPORTANCE The biology of phages as members of the human oral microbiome is understudied. Here, we report the characterization of xhp1, a novel linear plasmid-like prophage identified from a human oral isolate, Actinomyces odontolyticus subsp. actinosynbacter strain XH001. xhp1 can be induced and reinfect xhp1-cured XH001. The spontaneous induction of xhp1 leads to the lysis of a subpopulation of bacterial hosts and the release of eDNA that promotes biofilm assembly, thus potentially contributing to the persistence of A. odontolyticus within the oral cavity.

Streptococcus oralis maintains homeostasis in oral biofilms by antagonizing the cariogenic pathogen Streptococcus mutans.

Bacteria residing in oral biofilms live in a state of dynamic equilibrium with one another. The intricate synergistic or antagonistic interactions between them are crucial for determining this balance. Using the six-species Zürich "supragingival" biofilm model, this study aimed to investigate interactions regarding growth and localization of the constituent species. As control, an inoculum containing all six strains was used, whereas in each of the further five inocula one of the bacterial species was alternately absent, and in the last, both streptococci were absent. Biofilms were grown anaerobically on hydroxyapatite disks, and after 64 h they were harvested and quantified by culture analyses. For visualization, fluorescence in situ hybridization and confocal laser scanning microscopy were used. Compared with the control, no statistically significant difference of total colony-forming units was observed in the absence of any of the biofilm species, except for Fusobacterium nucleatum, whose absence caused a significant decrease in total bacterial numbers. Absence of Streptococcus oralis resulted in a significant decrease in Actinomyces oris, and increase in Streptococcus mutans (P < .001). Absence of A. oris, Veillonella dispar or S. mutans did not cause any changes. The structure of the biofilm with regards to the localization of the species did not result in observable changes. In summary, the most striking observation of the present study was that absence of S. oralis resulted in limited growth of commensal A. oris and overgrowth of S. mutans. These data establish highlight S. oralis as commensal keeper of homeostasis in the biofilm by antagonizing S. mutans, so preventing a caries-favoring dysbiotic state.

Cymbopogon citratus essential oil: effect on polymicrobial caries-related biofilm with low cytotoxicity.

The objective of this study was to evaluate the effects of Cymbopogon citratus essential oil and its main compound (citral) against primary dental colonizers and caries-related species. Chemical characterization of the essential oil was performed by gas chromatography/mass spectroscopy (GC/MS), and the main compound was determined. Antimicrobial activity was tested against Actinomyces naeslundii, Lactobacillus acidophilus, S. gordonii, S. mitis, S. mutans, S. sanguinis and S. sobrinus. Minimum inhibitory and bactericide concentrations were determined by broth microdilution assay for streptococci and lactobacilli reference, and for clinical strains. The effect of the essential oil on bacterial adhesion and biofilm formation/disruption was investigated. Negative (without treatment) and positive controls (chlorhexidine) were used. The effect of citral on preformed biofilm was also tested using the same methodology. Monospecies and microcosm biofilms were tested. ANOVA or Kruskal-Wallis tests were used (α=0.05). Cytotoxicity of the essential oil to human keratinocytes was performed by MTT assay. GC/MS demonstrated one major component (citral). The essential oil showed an inhibitory effect on all tested bacterial species, including S. mutans and L. acidophilus. Essential oil of C. citratus (10X MIC) reduced the number of viable cells of lactobacilli and streptococci biofilms (p < 0.05). The essential oil inhibited adhesion of caries-related polymicrobial biofilm to dental enamel (p < 0.01). Citral significantly reduced the number of viable cells of streptococci biofilm (p < 0.001). The essential oil showed low cytotoxicity to human keratinocytes. Based on these findings, this study can contribute to the development of new formulations for products like mouthwash, against dental biofilms.

[Respiratory infections caused by slow-growing bacteria: Nocardia, Actinomyces, Rhodococcus]. / Infections respiratoires à bactéries à croissance lente : Nocardia, Actinomyces, Rhodococcus.

INTRODUCTION: Pneumonia caused by slow-growing bacteria is rare but sometimes severe. STATE OF THE ART: These infections share many similarities such as several differential diagnoses, difficulties to identify the pathogen, the importance of involving the microbiologist in the diagnostic investigation and the need for prolonged antibiotic treatment. However, major differences distinguish them: Nocardia and Rhodococcus infect mainly immunocompromised patients while actinomycosis also concerns immunocompetent patients; the severity of nocardioses is related to their hematogenous spread while locoregional extension by contiguity makes the gravity of actinomycosis. PROSPECTIVE: For these diseases, molecular diagnostic tools are essential, either to obtain a species identification and guide treatment in the case of nocardiosis or to confirm the diagnosis from a biological sample. Treatment of these infections is complex due to: (1) the limited data in the literature; (2) the need for prolonged treatment of several months; (3) the management of toxicities and drug interactions for the treatment of Nocardia and Rhodococcus. CONCLUSION: Close cooperation between pneumonologists, infectious disease specialists and microbiologists is essential for the management of these patients.

Electron Transport Chain Is Biochemically Linked to Pilus Assembly Required for Polymicrobial Interactions and Biofilm Formation in the Gram-Positive Actinobacterium Actinomyces oris.

The Gram-positive actinobacteria Actinomyces spp. are key colonizers in the development of oral biofilms due to the inherent ability of Actinomyces to adhere to receptor polysaccharides on the surface of oral streptococci and host cells. This receptor-dependent bacterial interaction, or coaggregation, requires a unique sortase-catalyzed pilus consisting of the pilus shaft FimA and the coaggregation factor CafA forming the pilus tip. While the essential role of the sortase machine SrtC2 in pilus assembly, biofilm formation, and coaggregation has been established, little is known about trans-acting factors contributing to these processes. We report here a large-scale Tn5 transposon screen for mutants defective in Actinomyces oris coaggregation with Streptococcus oralis We obtained 33 independent clones, 13 of which completely failed to aggregate with S. oralis, and the remainder of which exhibited a range of phenotypes from severely to weakly defective coaggregation. The former had Tn5 insertions in fimA, cafA, or srtC2, as expected; the latter were mapped to genes coding for uncharacterized proteins and various nuo genes encoding the NADH dehydrogenase subunits. Electron microscopy and biochemical analyses of mutants with nonpolar deletions of nuo genes and ubiE, a menaquinone C-methyltransferase-encoding gene downstream of the nuo locus, confirmed the pilus and coaggregation defects. Both nuoA and ubiE mutants were defective in oxidation of MdbA, the major oxidoreductase required for oxidative folding of pilus proteins. Furthermore, supplementation of the ubiE mutant with exogenous menaquinone-4 rescued the cell growth and pilus defects. Altogether, we propose that the A. oris electron transport chain is biochemically linked to pilus assembly via oxidative protein folding.IMPORTANCE The Gram-positive actinobacterium A. oris expresses adhesive pili, or fimbriae, that are essential to biofilm formation and Actinomyces interactions with other bacteria, termed coaggregation. While the critical role of the conserved sortase machine in pilus assembly and the disulfide bond-forming catalyst MdbA in oxidative folding of pilins has been established, little is known about other trans-acting factors involved in these processes. Using a Tn5 transposon screen for mutants defective in coaggregation with Streptococcus oralis, we found that genetic disruption of the NADH dehydrogenase and menaquinone biosynthesis detrimentally alters pilus assembly. Further biochemical characterizations determined that menaquinone is important for reactivation of MdbA. This study supports the notion that the electron transport chain is biochemically linked to pilus assembly in A. oris via oxidative folding of pilin precursors.

Biofilm-induced changes to the composite surface.

OBJECTIVES: Composites may undergo biodegradation in the oral cavity. The objective was to investigate the effect of single- and multi-species biofilms on the surface roughness and topography of two composites. METHODS: Disk-shaped specimens of a paste-like, Bis-GMA-free (Gradia Direct Anterior, GC), and a flowable, Bis-GMA-based composite (Tetric EvoFlow, Ivoclar-Vivadent) were prepared. After ethylene-oxide sterilization (38°C), specimens (n=3) were incubated with Streptococcus mutans or mixed bacterial culture (Streptococcus mutans, Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum). As negative controls, unexposed specimens and specimens exposed to sterile medium (BHI) were used. Specimens exposed to acidified BHI medium (pH=5) and enzymatic solution of cholesterol esterase served as positive control. Following 6-week incubation, the attached biofilms were collected for real-time PCR assessment, after which the surface roughness and topography of the specimens were analyzed with atomic force microscopy. Surface hydrophilicity/hydrophobicity was determined by contact angle measurements. Biofilm structure was analyzed with scanning electron microscopy. RESULTS: Even though multi-species biofilms were thicker, with more cells attached, they did not significantly affect the surface roughness of the composites. On the other hand, S. mutans alone significantly increased the roughness of Tetric by 40.3%, while its effect on Gradia was lower (12%). The total amount of attached bacteria, however, did not differ between the composites. CONCLUSIONS: S. mutans can increase the surface roughness of composites, depending on their composition. This ability of S. mutans is, however, mitigated in co-culture with other species. In particular, bacterial esterases seem to be responsible for the increased composite surface roughness upon biofilms exposure. CLINICAL SIGNIFICANCE: Cariogenic bacteria can degrade composites, thereby increasing the surface roughness. Increased roughness and subsequent improved bacterial accumulation may facilitate the development of secondary caries around composites, which is the most common reason for the restoration failure.

Consumption of apple-boysenberry beverage decreases salivary Actinomyces naeslundii and their adhesion in a multi-species biofilm model.

We hypothesised that consumption of beverage rich in both fibre and polyphenols, rather than each bioactive alone, will modulate populations of selected salivary bacteria, and their adhesion characteristics and that some of these effects may be due to the anti-microbial activity of the beverage bioactives. We investigated the effect of 4 weeks' consumption of beverages, rich in apple fibre, boysenberry polyphenols, or both on salivary bacteria in healthy subjects. In this placebo-controlled crossover study, saliva samples were collected at the beginning and end of each treatment period, and used for qPCR quantitation of Lactobacillus spp., Actinomyces naeslundii and Streptococcus mutans. The counts of salivary A. naeslundii decreased after the consumption of the apple-boysenberry beverage (P<0.05, Student's t-test). We also examined the effect of the subjects' saliva on bacterial adhesion using a mixed species biofilm model. The salivary pellicles prepared before and after each treatment were inoculated with laboratory strains of A. naeslundii, Lactobacillus rhamnosus and S. mutans and tested for biofilm formation. The post appleboysenberry beverage salivary pellicle significantly decreased the adhesion of A. naeslundii at the end of both 3 and 24 h, in the in vitro biofilm. A 1/16 dilution of the apple-boysenberry beverage itself decreased the proliferation of test strains of A. naeslundii and S. mutans by 51 and 55%, respectively (P<0.005), indicating the antimicrobial activity of its bioactives. This study demonstrated that consumption of apple-boysenberry beverage, rather than apple or the boysenberry beverage alone or the placebo, decreased salivary A. naeslundii and their adhesion under laboratory conditions. These changes are factors that influence oral microecology and potentially oral health.

Novel bioactive root canal sealer to inhibit endodontic multispecies biofilms with remineralizing calcium phosphate ions.

OBJECTIVE: The objectives of this study were to: (1) develop a bioactive endodontic sealer via dimethylaminohexadecyl methacrylate (DMAHDM), 2-methacryloyloxyethyl phosphorylcholine (MPC) and nanoparticles of amorphous calcium phosphate (NACP) for the first time; and (2) evaluate inhibition of early-stage and mature multispecies endodontic biofilm, bond strength to root canal dentine, and calcium (Ca) and phosphate (P) ion release. METHODS: A series of bioactive endodontic sealers were formulated with DMAHDM, MPC, and NACP. Root dentine bond strength was measured via a push-out test. Three endodontic strains, Enterococcus faecalis, Actinomyces naeslundii, and Fusobacterium nucleatum, were grown on endodontic sealer disks to form multispecies biofilms. Biofilms were grown for 3 days (early) and 14 days (mature). Colony-forming units (CFU), live/dead assay, metabolic activity and polysaccharide were determined. Ca and P ion release from endodontic sealer was measured. RESULTS: Incorporating DMAHDM, MPC and NACP did not decrease the push-out bond strength (p>0.1). Adding DMAHDM and MPC reduced endodontic biofilm CFU by 3 log. DMAHDM or MPC each greatly decreased the biofilm CFU (p<0.05). Endodontic sealer with DMAHDM+MPC had much greater killing efficacy than DMAHDM or MPC alone (p<0.05). Endodontic sealer with DMAHDM+MPC had slightly lower, but not significantly lower, Ca and P ion release compared to that without DMAHDM+MPC (p>0.1). CONCLUSIONS: A novel bioactive endodontic sealer was developed with potent inhibition of multispecies endodontic biofilms, reducing biofilm CFU by 3 log, while containing NACP for remineralization and possessing good bond strength to root canal dentine walls. CLINICAL SIGNIFICANCE: The new bioactive endodontic sealer is promising for endodontic applications to eradicate endodontic biofilms and strengthen root structures. The combination of DMAHDM, MPC and NACP may be applicable to other preventive and restoration resins.

Three-species biofilm model onto plasma-treated titanium implant surface.

In this study, titanium (Ti) was modified with biofunctional and novel surface by micro-arc oxidation (MAO) and glow discharge plasma (GDP) and we tested the development of a three-species periodontopatogenic biofilm onto the treated commercially-pure titanium (cpTi) surfaces. Machined and sandblasted surfaces were used as control group. Several techniques for surface characterizations and monoculture on bone tissue cells were performed. A multispecies biofilm composed of Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum was developed onto cpTi discs for 16.5h (early biofilm) and 64.5h (mature biofilm). The number of viable microorganisms and the composition of the extracellular matrix (proteins and carbohydrates) were determined. The biofilm organization was analyzed by scanning electron microscopy (SEM) and Confocal laser scanning microscopy (CLSM). In addition, MC3T3-E1 cells were cultured on the Ti surfaces and cell proliferation (MTT) and morphology (SEM) were assessed. MAO treatment produced oxide films rich in calcium and phosphorus with a volcano appearance while GDP treatment produced silicon-based smooth thin-film. Plasma treatments were able to increase the wettability of cpTi (p<0.05). An increase of surface roughness (p<0.05) and formation of anatase and rutile structures was noted after MAO treatment. GDP had the greatest surface free energy (p<0.05) while maintaining the surface roughness compared to the machined control (p>0.05). Plasma treatment did not affect the viable microorganisms counts, but the counts of F. nucleatum was lower for MAO treatment at early biofilm phase. Biofilm extracellular matrix was similar among the groups, excepted for GDP that presented the lowest protein content. Moreover, cell proliferation was not significantly affected by the experimental, except for MAO at 6days that resulted in an increased cell proliferative. Together, these findings indicate that plasma treatments are a viable and promising technology to treat bone-integrated dental implants as the new surfaces displayed improved mechanical and biological properties with no increase in biofilm proliferation.

Interactions between Streptococcus oralis, Actinomyces oris, and Candida albicans in the development of multispecies oral microbial biofilms on salivary pellicle.

The fungus Candida albicans is carried orally and causes a range of superficial infections that may become systemic. Oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque and on oral mucosa. The aims of this study were to determine the mechanisms by which S. oralis and A. oris interact with each other and with C. albicans in biofilm development. Spatial distribution of microorganisms was visualized by confocal laser scanning microscopy of biofilms labeled by differential fluorescence or by fluorescence in situ hybridization (FISH). Actinomyces oris and S. oralis formed robust dual-species biofilms, or three-species biofilms with C. albicans. The bacterial components tended to dominate the lower levels of the biofilms while C. albicans occupied the upper levels. Non-fimbriated A. oris was compromised in biofilm formation in the absence or presence of streptococci, but was incorporated into upper biofilm layers through binding to C. albicans. Biofilm growth and hyphal filament production by C. albicans was enhanced by S. oralis. It is suggested that the interkingdom biofilms are metabolically coordinated to house all three components, and this study demonstrates that adhesive interactions between them determine spatial distribution and biofilm architecture. The physical and chemical communication processes occurring in these communities potentially augment C. albicans persistence at multiple oral cavity sites.

Cymbopogon citratus essential oil: effect on polymicrobial caries-related biofilm with low cytotoxicity

Abstract The objective of this study was to evaluate the effects of Cymbopogon citratus essential oil and its main compound (citral) against primary dental colonizers and caries-related species. Chemical characterization of the essential oil was performed by gas chromatography/mass spectroscopy (GC/MS), and the main compound was determined. Antimicrobial activity was tested against Actinomyces naeslundii, Lactobacillus acidophilus, S. gordonii, S. mitis, S. mutans, S. sanguinis and S. sobrinus. Minimum inhibitory and bactericide concentrations were determined by broth microdilution assay for streptococci and lactobacilli reference, and for clinical strains. The effect of the essential oil on bacterial adhesion and biofilm formation/disruption was investigated. Negative (without treatment) and positive controls (chlorhexidine) were used. The effect of citral on preformed biofilm was also tested using the same methodology. Monospecies and microcosm biofilms were tested. ANOVA or Kruskal-Wallis tests were used (α=0.05). Cytotoxicity of the essential oil to human keratinocytes was performed by MTT assay. GC/MS demonstrated one major component (citral). The essential oil showed an inhibitory effect on all tested bacterial species, including S. mutans and L. acidophilus. Essential oil of C. citratus (10X MIC) reduced the number of viable cells of lactobacilli and streptococci biofilms (p < 0.05). The essential oil inhibited adhesion of caries-related polymicrobial biofilm to dental enamel (p < 0.01). Citral significantly reduced the number of viable cells of streptococci biofilm (p < 0.001). The essential oil showed low cytotoxicity to human keratinocytes. Based on these findings, this study can contribute to the development of new formulations for products like mouthwash, against dental biofilms.

High-throughput quantitative method for assessing coaggregation among oral bacterial species.

UNLABELLED: This paper describes a high-throughput method that relies upon a microplate reader to score coaggregation 60 min postmixing, and use of a high-speed real-time imaging technology to describe the rate of coaggregation over time. The results of visual, microplate, and FlowCam(™) aggregation scores for oral bacteria Streptococcus gordonii, Streptococcus oralis, and Actinomyces oris, whose ability to coaggregate are well characterized, are compared. Following mixing of all possible pairs, the top fraction of the supernatant was added to a microplate to quantify cell-density. Pairs were also passed through a flow cell within a FlowCam(™) to quantify the rate of coaggregation of each pair. Results from both the microplate and FlowCam(™) approaches correlated with corresponding visual coaggregation scores and microscopic observations. The microplate-based assay enables high-throughput screening, whereas the FlowCam(™) -based assay validates and quantifies the extent that autoaggregation and coaggregation occur. Together these assays open the door for future in-depth studies of autoaggregation and coaggregation among large panels of test strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Coaggregation between bacterial species is integral to multi-species biofilm development. Difficulties in rapidly and reproducibly identifying and quantifying coaggregation have limited mechanistic studies. This paper demonstrates two complementary quantitative methods to screen for coaggregation. The first approach uses a microplate-based high-throughput approach and the other uses a FlowCam(™) device. The microplate-based approach enables rapid detection of coaggregation between candidate coaggregating pairs of strains simultaneously while controlling for variation between replicates. The FlowCam(™) approach allows for in-depth analysis of the rates of coaggregation and size of aggregates formed.

Autochthonous microbial community associated with pine needle forest litterfall influences its degradation under natural environmental conditions.

The slow natural degradation of chir pine (Pinus roxburghii) needle litterfall and its accumulation on forest floors have been attributed to its lignocellulosic complexities of the biomass. The present study offers a microbiological insight into the role of autochthonous microflora associated with pine needle litterfall in its natural degradation. The denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated actinomycetes (Saccharomonospora sp., Glycomyces sp., Agrococcus sp., Leifsonia sp., Blastocatella sp., and Microbacterium sp.) as a dominant microbial community associated with pine needle litterfall with the absence of fungal decomposers. On exclusion of associated autochthonous microflora from pine litterfall resulted in colonization by decomposer fungi identified as Penicillium chrysogenum and Aspergillus sp., which otherwise failed to colonize the litterfall under natural conditions. The results, therefore, indicated that the autochthonous microbial community of pine needle litterfall (dominated by actinomycetes) obstructs the colonization of litter-degrading fungi and subsequently hinders the overall process of natural degradation of litterfall.