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Comparative Biofilm Assays Using Enterococcus faecalis OG1RF Identify New Determinants of Biofilm Formation.
Willett, JLE, Dale, JL, Kwiatkowski, LM, Powers, JL, Korir, ML, Kohli, R, Barnes, AMT, Dunny, GM
mBio. 2021;(3):e0101121
Abstract
Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar discs, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by six Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA, tig, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions and identifies multiple new genetic determinants of biofilm formation. IMPORTANCE E. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.
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Polar flagellum of the alphaproteobacterium Azospirillum brasilense Sp245 plays a role in biofilm biomass accumulation and in biofilm maintenance under stationary and dynamic conditions.
Shelud'ko, AV, Filip'echeva, YA, Telesheva, EM, Yevstigneeva, SS, Petrova, LP, Katsy, EI
World journal of microbiology & biotechnology. 2019;(2):19
Abstract
Bacteria Azospirillum brasilense may swim and swarm owing to the rotation of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf). They also construct sessile biofilms on various interfaces. As compared to the wild-type strain Sp245, the previously characterized Fla- Laf- flhB1 mutant Sp245.1063 accumulated less biomass in mature biofilms, which also were susceptible to the forces of hydrodynamic shear. In this study, we compared biofilms formed by strain Sp245 and its previously constructed derivatives on the interfaces between a minimal (malate-salt medium, or MSM) or rich (LB) liquid growth medium and a hydrophilic (glass) or hydrophobic (polystyrene) solid surface under static or dynamic conditions. In all experimental settings, the alterations in Sp245.1063's mature biofilm traits were partially (in MSM) or completely (in LB) rescued in the complemented mutant Sp245.1063 (pRK415-150177), which received the pRK415-borne coding sequence for the putative FlhB1 protein of the flagellar type III secretion system. Although Laf were not found in the biofilms of azospirilla, Fla was present on the biofilm cells of the complemented mutant Sp245.1063 (pRK415-150177) and other studied strains, which had normal flagellation on liquid and solid nutritional media. Accordingly, mature biofilms of these strains contained more biomass and were significantly more resistant to shaking at 140 rpm, as compared to the biofilms of the flagella-free mutant bacteria. These data proved that the polar flagellum of A. brasilense Sp245 plays a significant positive role in biofilm biomass increase and in biofilm stabilization.
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Impact of dental cement on the peri-implant biofilm-microbial comparison of two different cements in an in vivo observational study.
Korsch, M, Marten, SM, Walther, W, Vital, M, Pieper, DH, Dötsch, A
Clinical implant dentistry and related research. 2018;(5):806-813
Abstract
BACKGROUND The type of cement used in cemented fixed implant-supported restorations influences formation of undetected excess cement and composition of the peri-implant biofilm. Excess cement and dysbiosis of the biofilm involve the risk of peri-implant inflammation. PURPOSE The aim of the study was to investigate the impact of two different cements on the peri-implant biofilm and inflammation. MATERIALS AND METHODS In an observational study, the suprastructures of 34 patients with cemented fixed implant-supported restorations were revised. In 20 patients, a methacrylate cement (Premier Implant cement [PIC]) and in 14 patients, a zinc oxide eugenol cement (Temp Bond [TB]) were used. After revision, TB was used for recementation. During revision and follow-up after 1 year, microbial samples were obtained. RESULTS Excess cement was found in 12 (60%) of the 20 patients with PIC. Suppuration was observed in two (25%) implants with PIC without excess cement (PIC-) and in all 12 (100%) implants with PIC and excess cement (PIC+). Implants cemented with TB had neither excess cement nor suppuration. The taxonomic analysis of the microbial samples revealed an accumulation of periodontal pathogens in the PIC patients independent of the presence of excess cement. Significantly, fewer oral pathogens occurred in patients with TB compared to patients with PIC. TB was used in all cases (PIC and TB) for recementation. In the follow-up check, suppuration was not found around any of the implants with PIC-, only around one implant with PIC+ and around one implant with TB. Bacterial species associated with severe periodontal infections that were abundant in PIC- and PIC+ samples before the revision were reduced after 1 year to levels found in the TB samples. CONCLUSIONS The revision and recementation with TB had a positive effect on the peri-implant biofilm in cases with PIC. The cementation of suprastructures on implants with TB is an alternative method to be considered.
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Effect of mouthwashes on the composition and metabolic activity of oral biofilms grown in vitro.
Fernandez Y Mostajo, M, Exterkate, RAM, Buijs, MJ, Crielaard, W, Zaura, E
Clinical oral investigations. 2017;(4):1221-1230
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Abstract
OBJECTIVE The aim of this study was to determine the effect of an oxygenating mouthwash compared to two other established mouthwash products on bacterial composition and metabolic activity of oral biofilms in vitro. MATERIAL AND METHODS Twelve healthy subjects participated as donors. Plaque-saliva mixture inoculated biofilms were grown and treated with 3 different chemotherapeutic mouthwashes [amine fluoride/stannous fluoride (MD), oxygenating agent (AX), chlorhexidine 0.12 % (PA), and water (W)]. Effects of treatments were assessed on biofilm composition (16S rRNA gene amplicon sequencing), production of organic acids (formate, acetate, lactate, propionate, butyrate using capillary electrophoresis), and viability of the remaining biofilm (CFUs). RESULTS Microbial profiles of biofilms clustered per inoculum donor and were dominated by the genera Veillonella, Streptococcus, and Prevotella. Microbial diversity was only reduced after PA treatment. Significant changes in composition occurred after treatment with AX, resulting in lower proportions of Veillonella and higher proportions of non-mutans streptococci. Production of all organic acids after PA and lactate after MD was significantly lower as compared to W. AX resulted in reduction of acetate, butyrate, and propionate and increase in lactate production (p < 0.05). Viable counts were significantly lower after PA and AX treatments compared to W, while no significant reduction was observed after MD. CONCLUSIONS All studied mouthwashes affected the in vitro biofilms differently. The effects of the AX treatment were the most prominent which resulted in changes of the bacterial composition and metabolism. CLINICAL IMPLICATIONS Awareness by the dental team that mouthwashes can change the bacterial composition and metabolism is important when advising its use.
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Inoculation density and nutrient level determine the formation of mushroom-shaped structures in Pseudomonas aeruginosa biofilms.
Ghanbari, A, Dehghany, J, Schwebs, T, Müsken, M, Häussler, S, Meyer-Hermann, M
Scientific reports. 2016;:32097
Abstract
Pseudomonas aeruginosa often colonises immunocompromised patients and the lungs of cystic fibrosis patients. It exhibits resistance to many antibiotics by forming biofilms, which makes it hard to eliminate. P. aeruginosa biofilms form mushroom-shaped structures under certain circumstances. Bacterial motility and the environment affect the eventual mushroom morphology. This study provides an agent-based model for the bacterial dynamics and interactions influencing bacterial biofilm shape. Cell motility in the model relies on recently published experimental data. Our simulations show colony formation by immotile cells. Motile cells escape from a single colony by nutrient chemotaxis and hence no mushroom shape develops. A high number density of non-motile colonies leads to migration of motile cells onto the top of the colonies and formation of mushroom-shaped structures. This model proposes that the formation of mushroom-shaped structures can be predicted by parameters at the time of bacteria inoculation. Depending on nutrient levels and the initial number density of stalks, mushroom-shaped structures only form in a restricted regime. This opens the possibility of early manipulation of spatial pattern formation in bacterial colonies, using environmental factors.
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Antibacterial orthodontic cement to combat biofilm and white spot lesions.
Wang, X, Wang, B, Wang, Y
American journal of orthodontics and dentofacial orthopedics : official publication of the American Association of Orthodontists, its constituent societies, and the American Board of Orthodontics. 2015;(6):974-81
Abstract
INTRODUCTION White spot lesions are an undesired side effect of fixed orthodontic treatment. The objective of this research was to develop an antibacterial resin-modified glass ionomer cement (RMGIC) containing nanoparticles of silver (NAg) for prevention of white spot lesions. METHODS NAg was incorporated into a commercial RMGIC. The NAg-enhanced cement was compared with the unaltered RMGIC and with a commercially available composite that does not release fluoride. The experimental and control products were used to bond brackets to 80 extracted maxillary first premolars. Enamel shear bond strength and the adhesive remnant index scores were determined. A dental plaque microcosm biofilm model with human saliva as the inoculum was used to investigate biofilm viability. Bacteria on the sample surface and bacteria in the culture medium away from the sample surface were tested for metabolic activity, colony-forming units, and lactic acid production. RESULTS Adding NAg to RMGIC and aging in water for 30 days did not adversely affect the shear bond strength compared with the commercial RMGIC control (P >0.1). The RMGIC with 0.1% NAg achieved the greatest reductions in colony-forming units, metabolic activity, and lactic acid production. The RMGIC with 0.1% NAg inhibited not only the bacteria on the surface, but also the bacteria away from the surface in the culture medium. Incorporation of NAg into RMGIC greatly reduced biofilm activity. CONCLUSIONS This novel RMGIC reduced biofilm formation and plaque buildup and could inhibit white spot lesions around brackets. The method of using NAg may apply in a wide range of dental adhesives, cements, sealants, and composites to inhibit biofilm and caries.
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Effect of desensitising paste containing 8% arginine and calcium carbonate on biofilm formation of Streptococcus mutans in vitro.
Fu, D, Pei, D, Huang, C, Liu, Y, Du, X, Sun, H
Journal of dentistry. 2013;(7):619-27
Abstract
OBJECTIVES To evaluate the influence of desensitising paste containing 8% arginine and calcium carbonate (Ar-Ca) on biofilm formation on dentine. METHODS Dentine discs were cut from extracted third molars and divided into the following three groups: no treatment, pumice treatment and Ar-Ca treatment. Surface topography and roughness were examined using scanning electron microscopy (SEM) and non-contact 3D surface profiler. After sterilisation, samples were incubated with Streptococcus mutans (S. mutans) for 4 h, 24 h and 72 h. Bacterial adhesion and biofilm formation were analysed using SEM, whereas MTT and lactic acid production assays were used to analyse the metabolic activity of S. mutans. RESULTS After polishing with either pumice or Ar-Ca, the surfaces of the samples became smoother than in the control group. The Ra values of the three experimental groups decreased significantly to 0.43 μm, 0.3 μm and 0.26 μm, respectively. Compared to the control group, fewer bacteria adhered to the dentine surface in the Ar-Ca group, while biofilm thickness decreased significantly for both groups after incubating for 24 h and 72 h. MTT and lactic acid production levers also showed a significant reduction in the Ar-Ca group. CONCLUSIONS Ar-Ca appears to present antibiofilm efficacy and may provide a promising approach to combat bacterial infection in hypersensitive dentinal lesions. CLINICAL SIGNIFICANCE As a clinical application of desensitising polishing paste, the paste containing 8% arginine and calcium carbonate could also inhibit the biofilm formation effectively.
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Exopolysaccharide matrix of developed Candida albicans biofilms after exposure to antifungal agents.
da Silva, WJ, Gonçalves, LM, Seneviratne, J, Parahitiyawa, N, Samaranayake, LP, Del Bel Cury, AA
Brazilian dental journal. 2012;(6):716-22
Abstract
This study aimed to evaluate the effects of fluconazole or nystatin exposure on developed Candida albicans biofilms regarding their exopolysaccharide matrix. The minimal inhibitory concentration (MIC) against fluconazole or nystatin was determined for C. albicans reference strain (ATCC 90028). Poly(methlymethacrylate) resin (PMMA) specimens were fabricated according to the manufacturer's instructions and had their surface roughness measured. Biofilms were developed on specimens surfaces for 48 h and after that were exposed during 24 h to fluconazole or nystatin prepared in a medium at MIC, 10 x MIC or 100 x MIC. Metabolic activity was evaluated using an XTT assay. Production of soluble and insoluble exopolysaccharide and intracellular polysaccharides was evaluated by the phenol-sulfuric method. Confocal laser scanning microscope was used to evaluate biofilm architecture and percentage of dead/live cells. Data were analyzed statistically by ANOVA and Tukey's test at 5% significance level. The presence of fluconazole or nystatin at concentrations higher than MIC results in a great reduction of metabolic activity (p<0.001). At MIC or 10 x MIC, fluconazole showed high amounts of intracellular polysaccharides (p<0.05), but did not affect the exopolysaccharide matrix (p>0.05). The exposure to nystatin also did not alter the exopolysaccharide matrix at all the tested concentrations (p>0.05). Biofilm architecture was not affected by either of the antifungal agents (p>0.05). Nystatin promoted higher proportion of dead cells (p<0.05). It may be concluded that fluconazole and nystatin above the MIC concentration reduced the metabolic activity of C. albicans biofilms; however, they were not able to alter the exopolysaccharide matrix and biofilm architecture.
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Effect of brushing with conventional versus whitening dentifrices on surface roughness and biofilm formation of dental ceramics.
Azevedo, SM, Kantorski, KZ, Valandro, LF, Bottino, MA, Pavanelli, CA
General dentistry. 2012;(3):e123-30
Abstract
The aim of this study was to evaluate the effect of conventional and whitening dentifrices on the weight loss, surface roughness, and early in situ biofilm formation on the surface of dental ceramics. Standardized feldspar ceramic specimens (Vita VM7 and Vita VM13) were submitted to the following experimental conditions: no brushing; brushing without a dentifrice; brushing with a conventional dentifrice; and brushing with a whitening dentifrice. A brushing machine was used to simulate brushing. The mass and surface roughness of all specimens from the test groups were evaluated prior to and after brushing. Ten participants used an oral device for eight hours to evaluate the biofilm formed in situ on the specimens. Scanning electron microscopy was used for qualitative and quantitative analysis of the biofilm. ANOVA and Tukey tests were used to analyze the results of weight loss, surface roughness, and presence of bacteria. A one-way Kruskal-Wallis test was used for bacterial colonization results. For both ceramics, brushing with a whitening dentifrice resulted in weight loss that was significantly greater when compared to brushing without a dentifrice or with a conventional dentifrice. Increased surface roughness was noticed on VM13 ceramic samples with both dentifrices, whereas only conventional dentifrice had a significant effect on the surface roughness of VM7 samples. For both VM7 and VM13, no difference was found between the experimental conditions with regard to the presence or number of bacteria. Cocci and short rods were the predominant microbial morphotypes. Granular or fibrillar acellular material partially covered the specimens. Brushing with a whitening dentifrice resulted in significant weight loss of ceramic restorations, while brushing with both conventional and whitening dentifrices can roughen ceramic surfaces. The increase in roughness was not clinically significant to contribute to increased biofilm formation.
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In situ effects of restorative materials on dental biofilm and enamel demineralisation.
Sousa, RP, Zanin, IC, Lima, JP, Vasconcelos, SM, Melo, MA, Beltrão, HC, Rodrigues, LK
Journal of dentistry. 2009;(1):44-51
Abstract
OBJECTIVES Since secondary caries is one of the main reasons for replacing restorations, this study assessed the effects of different restorative materials on the microbiological composition of dental biofilm and on enamel demineralisation around the restoration. METHODS A randomized, double-blind, split-mouth in situ design was conducted in one phase of 14 days, during which, 20 volunteers wore palatal devices containing five human dental enamel slabs. Each slab was randomly restored with one of the following materials: Filtek-Z-250/Single Bond, control group (composite resin), Permite (amalgam), Fuji II (encapsulated resin-modified glass ionomer), Vitremer (resin-modified glass ionomer) and Ketac Molar (conventional glass ionomer). The volunteers used fluoride dentifrice, 3x/day and a 20% sucrose solution was dripped onto the slabs 8x/day. The biofilm formed on the slabs was analyzed to determine the counts of total streptococci, mutans streptococci and lactobacilli. Enamel demineralisation was determined by cross-sectional microhardness (CSMH) at 20 and 70 microm from the margin of the restoration. Kruskal-Wallis and analysis of variance, followed by least mean squares (LMS) test, were used to evaluate microbiota and CSMH among the groups. The significance level used was 5%. RESULTS No statistically significant differences were found in the cariogenic microbiota grown on the slabs. At a 20-mum distance, only Fuji II statistically differed from the other groups, showing the lowest demineralisation. At 70 microm, Fuji II significantly inhibited demineralisation when compared to Permite, Filtek-Z-250 and Ketac Molar. CONCLUSIONS In the context of fluoride dentifrice and under the cariogenic exposure conditions of this study, only the encapsulated resin-modified glass ionomer material provided additional protection against secondary caries.